British journal of pharmacology 2015 volume 172 part 8

BJP British Journal of Pharmacology DOI:10.1111/bph.13119 www.brjpharmacol.org Themed Section: Conditioning the Heart – Pathways to Translation EDITORIAL ‘Conditioning the heart’ – lessons we have learned from the past and future perspectives for new and old conditioning ‘drugs’ Correspondence Nina C Weber, PhD, Academic Medical Centre (AMC), Department of Anaesthesiology University of Amsterdam, Laboratory of Experimental and Clinical Experimental Anaesthesiology (L.E.I.C.A.), Academic Medical Centre (AMC), Meibergdreef 9, 1100 DD Amsterdam, the Netherlands, Tel.: +31 20 5668215, Fax: +31 20 6979441 E-mail: N.C.Hauck@amc.uva.nl Nina C Weber, PhD Department of Anaesthesiology, Laboratory of Experimental Intensive Care and Anaesthesiology (L.E.I.C.A) Academic Medical Centre (AMC), Meibergdreef 9, 1100 DD Amsterdam, The Netherlands LINKED ARTICLES This article is part of a themed section on Conditioning the Heart – Pathways to Translation To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-8 Introduction Almost a year ago my 6-year-old son asked me: ‘Mom, what is it you are doing every day in your laboratory?’ First of all I started explaining in children’s terms what the noble gas helium is, what it can do, and why we think that it is not only good to blow up balloons but might also be good for the heart of some patients However, he was not satisfied by this answer and proceeded asking things like: ‘ and why are you doing this? What will you next? How long have you been doing this?’ Finally, I ended up saying something that I had already realized for quite a while: ‘Actually, I not know why I am still doing this “conditioning” research.’ After having said so, I was quite upset as I have never been honest enough to myself to admit that most of the research others and we is still not translated to the clinical situation Do we really help patients to get better? Thus, despite several decades of research on preconditioning, postconditioning and pharmacological conditioning of the heart, we have yet to see therapeutic realization of the potential powerful protective effects of conditioning on infarction, mechanical dysfunction and arrhythmias associated with acute myocardial ischaemia and reperfusion © 2015 The British Pharmacological Society Eventually, I was searching for supportive views to get back on track with my own research, and I must admit that the overwhelming participation of excellent researchers in this themed issue gave me a lot of motivation to hang on to the pathway of getting cardioprotective strategies by conditioning translated from the experimental laboratory to the patient This is where the idea was born to bring together researchers from all over the world to share their thoughts about this question in the themed issue: ‘Conditioning the heartPathways to translation-scope for drug discovery’ Summary of content Cohen and Downey are pioneers in the area of ‘conditioning’ the heart and their review takes us through a brilliant historical journey going back to 1971 when Maroko and co-workers suggested that ST-segment shifts might be used as a marker for infarct size reduction, and that infarct size reduction would be a therapeutic option to prevent cardiac damage by ischaemia/reperfusion (I/R) injury Four decades later, passing pioneers like Hearse and Murry, Cohen et al review several pathways of pre- and post-conditioning that have been elucidated (Cohen & Downey, 2015) The endogenous mediators of the trigger phase include adenosine, bradykinin, opioids British Journal of Pharmacology (2015) 172 1909–1912 1909 BJP N C Weber and other extracellular signalling molecules All these triggers have been shown to bind to G-protein coupled receptors, but surprisingly they then activate different pathways that converge upon PKC Additionally, the mediator phase of conditioning, including enzymes from the so ‘called’ reperfusion injury salvage kinase (RISK) pathway, is discussed All signalling finally seems to lead to inhibition of the mitochondrial permeability transition pore (mPTP), which is currently suggested to be the end-effector of ischaemic preconditioning Several years after the discovery of pre-conditioning it became clear that the obviously strong endogenous protective intervention had to occur before an ischaemic insult, making the clinical application very difficult, as ischemia often already has taken place, for example, in patients presenting with an acute myocardial infarction This is when the idea of protective pharamacological interventions during reperfusion, possibly achieved by an intervention as ischaemic post-conditioning, was born However, as Cohen and Downey (2015) point out, irrespective of the logical implications of the successful interventions seen in animal experiments, several clincial trials using ischaemic mimicking agents like, adenosine, atrial natriuretic peptide, or cyclosporine A, have yet remained little successful In the last part of their review, Cohen and Downey focus on the role of platelets, platelet activating factor (PAF) and platelet P2Y2 inhibitors in cardioprotection (Cohen & Downey, 2015) It is nowadays quite accepted that in patients presenting with acute myocardial infarction (AMI), complications occurring during and after stenting can in fact be significantly improved by the use of antiplatelet drugs like aspirin, thienopyridines (clopidogrel and prasugrel) or triazolopyrimidins (tricagrelor, Cohen & Downey, 2015) So far, the anti-aggregation properties are suggested to be responsible for the protective effect against re-occlusion of a stent, but Cohen and Downey shed light upon a complete different aspect They suggest that especially the P2Y2 ADP receptor inhibitors (thienopyridines and triazolopyrimidines), in fact (already) activate the post-conditioning pathway: This means that the attempt to add another ‘conditioning’ stimulus by e.g ischaemic or pharmacological conditioning might fail (Cohen & Downey, 2015) Cohen and Downey (2015) lead the reader to a very critical but also future-oriented view on ‘conditioning’ of the heart They support further laboratory research for elucidating alternative pathways that may extend the spectrum of the anti-ischaemic armamentarium beyond the platelet inhibitors used in standard care Baxter and Bice take a closer look into the phenomenon of ‘post-conditioning’ of the heart (Bice & Baxter, 2015) This more recently described form of ‘conditioning’ has been in the focus of research over the last decade Bice and Baxter (2015) distinctively introduce the mechanisms that have been implicated in ischaemic post-conditioning and which are quite similar to those known from pre-conditioning Again, inhibition of the mPTP resembles the potential endeffector in post-conditioning and pathways like the RISK, SAFE, GSK-3 beta and cGMP/PKG are involved (Bice & Baxter, 2015) Bice and Baxter (2015) come to the conclusion, that even though promising results from initial clinical studies using both, ischaemic and pharmacological postconditioning exist, here the final translation to the clinical 1910 British Journal of Pharmacology (2015) 172 1909–1912 situation fails They suggest that study design, timing, drug administration, technical limitations with respect to the end-point measurements and – most importantly – existing co-morbidities in our patients might limit the translation of protection by conditioning to the clinic (Bice & Baxter, 2015) However, as Cohen and Downey, they end with a very promising view on the dead end some of the ‘conditioning’ researchers might feel to be stranded in In their view the variability within the group of AMI patient makes it probably impossible to find a one-size-fits-all cure for each patient, but taking into account the enormous amount of patients with cardiovascular diseases, any standard drug in a single dose might make a huge difference at least for some of the respective patients (Bice & Baxter, 2015) Another more clinically applicable form of ‘conditioning’ the heart, remote ischaemic conditioning (RIC), is described in the review of Schmidt et al (2015) RIC is defined as shortlasting periods of ischaemia applied to a distant organ form the heart, which eventually lead to the protection of the heart itself against ischaemia reperfusion injury Schmidt et al draw a picture of the most recent and promising mediators and targets involved in remote ischaemic conditioning Among these, especially the recently extensively investigated dialysate from plasma, containing most probably not one but several potent cardioprotective factors (factor X), is discussed Also MicroRNA (especially miR-144) and exosome release during RIC have recently entered the focus of RIC research Schmidt et al suggest that these might be very promising strategies to develop future therapies mimicking RIC (Schmidt et al., 2015) In addition, Pzyklenk points out that among all forms of ‘conditioning’ without doubt ‘postconditioning’ and RIC are the most promising strategies to be translated to the clinic In her view, especially the extremely complex and time sensitive signalling network involving all conditioning forms limits translation from promising preclinical trials to larger clinical trials She also critically questions the study design (patient population) of recent clinical trials examining conditioning the heart as well as the choice of the experimental protocol (Przyklenk, 2015) In her review, the reader will be confronted with the hypothesis that despite the assumption that translatability of preclinical data to the clinic would implicate a study design as close as possible to the meanwhile well-established pathways of cardioprotection, there is an enormous heterogeneity among and within the clinical studies that have been performed so far (Przyklenk, 2015) The reviews of Pagliaro and Penna (2015) and Inserte and Garcia-Dorado (2015) deal with the complex mechanism underlying reperfusion injury In this phase of I/R injury, redox signalling (Pagliaro & Penna, 2015) and cGMP/PKG signalling (Inserte & Garcia-Dorado, 2015) are critically involved in the development of cardiac damage Pagliaro and Penna (2015) point out to the fact that undifferentiated diminishment of redox signalling [reactive oxygen species (ROS) and reactive nitrogen species (RNS)] by antioxidants cannot be the future therapy in I/R damage, as in fact redox signalling is vital to several physiological processes (Pagliaro & Penna, 2015) The authors suggest a more site- and timespecific inhibition of ROS/RNS without affecting survival pathways relying on ROS/RNS, however, clinical data are again sparse regarding this topic (Pagliaro & Penna, 2015) ‘Conditioning the heart’ Next to ROS signalling, a pivotal role for cGMP/PKG signalling during reperfusion injury and cardioprotection by ‘conditioning’ has been described (Inserte & Garcia-Dorado, 2015) According to Inserte and Garcia-Dorado (2015) extensive amounts of pre-clinical data definitively support the role of cGMP/PKG in cardioprotection Moreover, the authors are convinced that targeting these key players of the signal transduction cascade in the early phase of reperfusion is a valuable and very promising therapeutic option toward diminished cardiac damage (Inserte & Garcia-Dorado, 2015) Unfortunately, the narrow dose-response curve that has to be followed when cGMP levels are increased might dampen the positive view, as too high cGMP levels have recently been shown to be rather harmful than protective (Inserte & Garcia-Dorado, 2015) The role of the extracellular signalling molecules (autacoids): adenosine, bradykinin and opioids, is extensively described in the review by Kleinbongard and Heusch (2015) and for opioids by Headrick et al (2015) These endogenous signalling molecules are un-doubtfully all involved in the different forms of ‘conditioning’ however, once again translations to clinical trials were disappointing Although e.g adenosine has been tested in several trials of AMI, elective PCI or CABG patients, and some studies are positive and promising, there is still no consensus on whether adenosine reduces infarct size in the clinical scenario (Kleinbongard & Heusch, 2015) Headrick et al point out that although small clinical trials showed a benefit of morphine and remifentanil in CABG surgery patients, targeting the opioid receptors (OPR) more specifically (e.g δ-OPR agonists) in order to avoid cardiorespiratory effects of unspecific OPR agonists would be a more promising approach (Headrick et al., 2015) Especially for opioids one has to take into account that maintaining anaesthesia during surgical procedures might in itself already be cardioprotective and thus stocking up on cardioprotective interventions might be difficult in this setting (Kleinbongard & Heusch, 2015) Both expert groups agree upon the fact that ageing, co-morbidities and – most importantly – relevant drugs during the surgical procedure are the challenge that has to be overcome before autacoid mimicking drugs can find their way into the clinic (Kleinbongard & Heusch, 2015) (Headrick et al., 2015) In this context, sustained ligand-activated protection (SLP) might have a future role in clinical applications as it has been shown to be effective also in diseased animal models (Headrick et al., 2015) Kleinbongard and Heusch (2015) end with a conclusion that might frustrate those of us working on ‘pharmacological’ induced conditioning The authors argue that probably the search for more ‘drugs’ to induce cardioprotection should stop soon, and the development of more reliable RIC models in the clinic could be the future (Kleinbongard & Heusch, 2015) The two reviews dealing with the probable most easily translatable conditioning strategies using anaesthetics or noble gases extensively describe the mechanisms underlying such cardioprotection (Kikuchi et al., 2015, Smit et al., 2015) Protection induced by volatile anaesthetics (Kikuchi et al., 2015) and later on noble gases, like xenon and helium (Smit et al., 2015), has been recognized for the last two decades Unfortunately, these two reviews come to the disappointing conclusion that the application of these substances, although BJP already clinically used, is still limited and advances in this field are minimal (Kikuchi et al., 2015, Smit et al., 2015) Ageing, diabetes, hyperglycaemia and drugs frequently used in AMI patients (beta blockers, glibenclamide) have been shown to diminish this form of conditioning in animals as well as in humans (Kikuchi et al., 2015) Thus once again, more laboratory research using adapted animal models and models that properly mimic the clinical anaesthesia models are needed (Kikuchi et al., 2015, Smit et al., 2015) In the last four reviews some of the key targets involved in orchestrating different conditioning forms are excellently reviewed (Ong et al., 2015, Halestrap et al., 2015, Martin et al., 2015, Schilling et al., 2015) For the sake of brevity I will only be able to highlight some snap shots from these reviews, hereby emphasizing that under no circumstances I wish to undermine the importance of these contributions to the completion of this themed issue Regarding the aforementioned crucial involvement of mPTP in cardioprotection by different conditioning forms, the reviews by Ong et al (2015) and by Halestrap et al (2015) give an excellent detailed overview on the mechanisms by which the mPTP is regulated and in which way hexokinase (HK2) might be of importance for maintenance of the opening of this pore The opening of the mPTP at the onset of reperfusion leads to cell death Trials over the past years using cyclosporine A, an inhibitor of mPTP opening, have proven that when administered right at the beginning of reperfusion, it in fact reduces MI (Ong et al., 2015) Larger multicentre trials as the CYCLE and CIRUS study are currently running and will reveal very important results regarding the clinical use of cyclosporine A (Ong et al., 2015) The glycolytic enzyme HK2 has been found to bind to the outer membrane of the mitochondria, thereby stabilizing the contact sites of outer and inner mitochondrial membranes Dissociation of HK2 from the membrane during the ischaemic phase leads to an increased loss of cytochrome C from the mitochondria, which in turn results in opening of the mPTP during reperfusion (Halestrap et al., 2015) Ischaemic preconditioning has been shown to involve increased HK2 binding to the mitochondrial membrane, thereby inducing cardioprotection Halstrap et al critically evaluate the potential development of drugs that might increase binding of HK2 to the mitochondria, as these are yet unavailable (Halestrap et al., 2015) Also caveolins, reviewed by Schilling et al (2015), have more recently been associated with the mitochondria and it has been convincingly shown that they play a pivotal role in different forms of conditioning Caveolins are structural proteins that are essential for the formation of so called ‘caveolae’, small plasma membrane invaginations enriched with cholesterol and sphingolipids (Schilling et al., 2015) These proteins are thought to regulate protective signalling within a multiprotein (signalosome) complex Interestingly, many of the above-discussed key players (e.g opioids, adenosine, PKC) of ischaemic and pharmacological conditioning have been shown to be associated with caveolae/caveolins in a dynamic process Schilling et al distinctively focus on the fact that caveolins might be key players in overcoming the pathophysiological limits for conditioning (ageing, diabetes), hereby leaving us with the hope that future studies might identify drugs that can specifically increase caveolin expresBritish Journal of Pharmacology (2015) 172 1909–1912 1911 BJP N C Weber sion, thereby protecting the heart against ischaemia/ reperfusion damage (Schilling et al., 2015) Last but not least, Martin et al provide us with an excellent review over the role of a member of the ‘stressactivated’ kinases family, the p38 MAPK in cardiovascular disease The activation of p38 during ischaemic preconditioning cycle has been shown to attenuate the detrimental activation of the same enzyme during the lethal ischaemic period Thus, p38 MAPK activation also might trigger protective effects in the heart, thereby making the inhibition of p38 MAPK quite unpredictable in clinical trials (Martin et al., 2015) However, very recent clinical trials with the selective, potent, and orally active p38 MAPK inhibitor Losmapimod (GlaxoSmithKline, Brentford, London, UK) show promising results in the settings of myocardial infarction, and a much larger trial implementing Losmapimod is on its way (Martin et al., 2015) Concluding remarks This themed issue summarizes a very important portion of all the research ongoing in the area of cardioprotection by several ‘conditioning’ forms Of course, it cannot be complete as there is still so much to learn The main goal of this themed issue was not only to review the current state of the art in this field, but also to provide a critical overview upon the opportunities that lie ahead for strong endogenous mechanisms of conditioning to gain broader translatability into the clinical situation With regard to this, we hope that studying these review articles will convince the readership that there is indeed a future for ‘conditioning the heart’ against ischaemic damage By combining the enormous amount of knowledge we have gained from animal and preclinical studies, and applying this knowledge to the existing co-morbidities and peculiarities occurring during the perioperative period, we will hopefully succeed in our venture/conquest to identify novel candidate drugs for conditioning the heart and for testing in clinical trials Cohen MV, Downey JM (2015) Signalling pathways and mechanisms of protection in pre- and postconditioning: historical perspective and lessons for the future Br J Pharmacol 172: 1913–1932 Halestrap AP, Pereira GC, Pasdois P (2015) The role of hexokinase in cardioprotection – mechanism and potential for translation Br J Pharmacol 172: 2085–2100 Headrick JP, See Hoe LE, Du Toit EF, Peart JN (2014) Opioid receptors and cardioprotection – ‘opioidergic conditioning’ of the heart Br J Pharmacol 172: 2026–2050 Inserte J, Garcia-Dorado D (2015) cGMP/PKG pathway as a common mediator of cardioprotection Translatability and mechanism Br J Pharmacol 172: 1996–2009 Kikuchi C, Dosenovic S, Bienengraeber M (2015) Anaesthetics as cardioprotectants – translatability and mechanism Br J Pharmacol 172: 2051–2061 Kleinbongard P, Heusch G (2015) Extracellular signalling molecules in the ischaemic/reperfused heart – druggable and translatable for cardioprotection? Br J Pharmacol 172: 2010–2025 Martin ED, Bassi R, Marber MS (2015) p38 MAPK in cardioprotection – are we there yet? Br J Pharmacol 172: 2101–2113 Ong S, Dongworth RK, Cabrera-Fuentes HA, Hausenloy DJ (2015) Role of the MPTP in conditioning the heart – translatability and mechanism Br J Pharmacol 172: 2074–2084 Pagliaro P, Penna C (2015) Redox signaling and cardioprotection – translatability and mechanism Br J Pharmacol 172: 1974–1995 Przyklenk K (2015) Ischemic conditioning: pitfalls on the path to clinical translation Br J Pharmacol 172: 1961–1973 Schilling JM, Roth DM, Patel HH (2015) Caveolins in cardioprotection – translatability and mechanism Br J Pharmacol 172: 2114–2125 Schmidt MR, Redington A, Botker HE (2015) Remote conditioning the heart overview: translatability and mechanism Br J Pharmacol 172: 1947–1960 Smit KF, Weber NC, Hollmann MW, Preckel B (2015) Noble gases as cardio-protectants – translatability and mechanism Br J Pharmacol 172: 2062–2073 References Bice JS, Baxter GF (2015) Postconditioning signalling in the heart: mechanisms and translatability Br J Pharmacol 172: 1933–1946 1912 British Journal of Pharmacology (2015) 172 1909–1912 BJP British Journal of Pharmacology DOI:10.1111/bph.12903 www.brjpharmacol.org Themed Section: Conditioning the Heart – Pathways to Translation REVIEW Signalling pathways and mechanisms of protection in pre- and postconditioning: historical perspective and lessons for the future Correspondence Michael V Cohen, Department of Physiology, MSB 3050, University of South Alabama, College of Medicine, Mobile, AL 36688, USA E-mail: mcohen@southalabama.edu Received July 2014 Revised 22 August 2014 Accepted 29 August 2014 Michael V Cohen1,2 and James M Downey1 Departments of Physiology and 2Medicine, University of South Alabama College of Medicine, Mobile, AL, USA Ischaemic pre- and postconditioning are potent cardioprotective interventions that spare ischaemic myocardium and decrease infarct size after periods of myocardial ischaemia/reperfusion They are dependent on complex signalling pathways involving ligands released from ischaemic myocardium, G-protein-linked receptors, membrane growth factor receptors, phospholipids, signalling kinases, NO, PKC and PKG, mitochondrial ATP-sensitive potassium channels, reactive oxygen species, TNF-α and sphingosine-1-phosphate The final effector is probably the mitochondrial permeability transition pore and the signalling produces protection by preventing pore formation Many investigators have worked to produce a roadmap of this signalling with the hope that it would reveal where one could intervene to therapeutically protect patients with acute myocardial infarction whose hearts are being reperfused However, attempts to date to show efficacy of such an intervention in large clinical trials have been unsuccessful Reasons for this inability to translate successes in the experimental laboratory to the clinical arena are evaluated in this review It is suggested that all patients with acute coronary syndromes currently presenting to the hospital and being treated with platelet P2Y12 receptor antagonists, the current standard of care, are indeed already benefiting from protection from the conditioning pathways outlined earlier If that proves to be the case, then future attempts to further decrease infarction will have to rely on interventions which protect by a different mechanism LINKED ARTICLES This article is part of a themed section on Conditioning the Heart – Pathways to Translation To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-8 Abbreviations AMI, acute myocardial infarction; Cx43, connexin 43; GP, glycoprotein; GSK-3β, glycogen synthase kinase-3β; HB-EGF, heparin-binding EGF-like growth factor; IPC, ischaemic preconditioning; IPoC, ischaemic postconditioning; KATP, ATP-sensitive K+ channel; mPTP, mitochondrial permeability transition pore; mtKATP, mitochondrial KATP; NHE, Na+/H+ exchanger; PAF, platelet-activating factor; PCI, percutaneous coronary intervention; PDK, 3′-phosphoinositidedependent kinase; PTCA, percutaneous transluminal coronary angioplasty; RISK, reperfusion injury survival kinases; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; SAFE, survivor activating factor enhancement; SPHK1, sphingosine kinase; Src, sarcoma; STEMI, ST-segment-elevation myocardial infarction; TRAF-2, TNF receptor-associated factor © 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 1913–1932 1913 BJP M V Cohen and J M Downey Tables of Links TARGETS LIGANDS GPCRsa Transportersd Adenosine Isoflurane β-adrenoceptor Na+/Ca2+ exchangers AG490 L-NAME A1 receptor Na+/H+ exchangers (NHE) Aspirin Metoprolol e A2A receptor Enzymes Atenolol Nitric oxide (NO) A2B receptor Akt BAY 58-2667 PAF A3 receptor COX Bradykinin PD98059 S1P1 receptor eNOS Cangrelor Sevoflurane S1P2 receptor ERK cGMP Sphingosine S1P3 receptor GSK-3β Chelerythrine Sphingosine 1-phosphate Ion channelsb Guanylyl cyclase Clopidogrel TGFβ1 Connexin 43 (Cx43) PI3K Cyclosporin A Ticagrelor KATP channel PKCδ Desflurane Tirofiban Catalytic receptorsc PKCε Erythropoietin Tyrosine EGFR PKG Exenatide Urocortin GPIIa MMP Glibenclamide Wortmannin GPIIb SPHK1 Insulin PAF receptor Src kinase P2Y12 receptor TNFR1 TNFR2 These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d,eAlexander et al., 2013a,b,c,d,e) Introduction With the exception of revascularization, ischaemic preconditioning (IPC) is undeniably the most powerful cardioprotective intervention targeting ischaemia/reperfusion injury yet to be identified All scientists agree that this intervention can salvage ischaemic myocardium following a period of ischaemia and reperfusion and reduce infarct size, the original observation and time-honored parameter of cardioprotection Yet this success in the experimental laboratory has yet to be translated into a clinical procedure that has produced equally satisfying results Thus, both scientists and clinicians continue to search for the miraculous intervention that can be applied to the patient with an acute myocardial infarction (AMI) to decrease infarct size and diminish the clinical sequelae of coronary artery occlusion and reperfusion The closest we have come to this is revascularization therapy In patients with coronary occlusion and AMI, current standards demand that the coronary artery be opened to reperfuse the ischaemic myocardium Although tissue salvage understandably is dependent on reflow, this revascularization paradoxically creates injury of its own, so-called ‘reperfusion injury’ It is the latter which most recent cardioprotective interventions purport to target To appreciate why identification of an appropriate cardioprotective agent has largely failed to date and to provide hope that we may be close to developing an 1914 British Journal of Pharmacology (2015) 172 1913–1932 effective approach, it is necessary to understand the history and science of cardioprotection Pioneering studies of infarct size modification ST-segment shifts as a marker of infarct size In 1971, Maroko, working in Braunwald’s laboratory, proposed strategies for limiting necrosis following acute coronary occlusion (Maroko et al., 1971) Maroko et al recognized the importance of infarct size on outcomes following infarction and were the first to suggest that infarction might be therapeutically reduced These investigators mapped the degree of ST-segment shifts on the anterior myocardial surface at the end of 10-min coronary occlusions in dogs After the heart was reperfused and had recovered, the occlusion and mapping were repeated after an experimental intervention The sum of the ST-segment shifts was thought to reflect the severity of the ischaemia If the sum of ST-segment shifts was attenuated, it was concluded that the intervention had protected the heart The concept was brilliant in that it allowed each animal to serve as its own control, but it relied on the assumption that the ST-segment sum represented true infarct size which, unfortunately, turned out to not always be the case Maroko et al as well as subsequent investigators Pre- and postconditioning signalling were also greatly handicapped by a lack of scientific information as to how ischaemia/reperfusion actually kills heart tissue The Braunwald laboratory focused on a supply– demand relationship Demand could be reduced by β-blockers and supply could be increased by interventions thought to promote oxygen delivery such as hyaluronidase (Maroko et al., 1972) As it turned out, the supply–demand relationship was but only one determinant of cell death Yet their pioneering efforts started a field of research that still thrives today Reperfusion injury: a paradox Hearse et al (1973) introduced the ‘oxygen paradox’ Perfusion of a rat heart with hypoxic buffer for a prolonged period seemed to have little consequence, but switching back to oxygenated perfusate caused immediate cell destruction While the reintroduction of oxygen was needed for recovery, at the same time it was associated with an injury That was the paradox The concept of reperfusion injury was very attractive because at that time it was recognized that AMI was caused by a coronary thrombus that could be dissolved with a thrombolytic agent If much of the injury occurred at reperfusion, it would not be too late to prevent it with some intervention despite presentation of the patients with ischaemia in progress It was hypothesized that reintroduction of oxygen produced a burst of free radicals that in turn led to membrane damage, interference with ion pumps and volume dysregulation A closely associated hypothesis was that leukocytes would invade reperfused tissue and attack viable myocytes by releasing free radicals Personnel in Lucchesi’s laboratory concentrated on the role of free radicals in myocardial infarction (Jolly et al., 1984) Thus, free radical scavengers appeared to decrease infarction in a canine model of ischaemia/reperfusion Although these studies were championed by local advocates, the inconsistent results obtained in other independent laboratories suggested problems with this approach (Reimer et al., 1989) The same held for investigations of anti-inflammatory agents (Tissier et al., 2007a) The reason for divergent results among the many studies of antioxidant and anti-inflammatory agents have never been resolved, but even in the most supportive studies salvage was hardly greater than 10%, probably too modest to have meaningful clinical impact One began to wonder if it was even possible to alter the vulnerability of ischaemic myocardium to infarction IPC Then, in 1986, Charles Murry, in the laboratory of Reimer and Jennings, made a seminal observation (Murry et al., 1986) It was reported that preceding a 40 coronary occlusion in dogs with four cycles of coronary occlusion/5 reperfusion would decrease the amount of infarction of the risk area subtended by the occluded vessel from 28 to 7% That was a 75% reduction in infarct size despite the fact that those hearts endured an additional 20 of ischaemia They called this phenomenon IPC Perhaps because of Murry’s frankly antithetical observation that more ischaemia was better, confirmation of the observation was not immediate Three years passed before scientific papers dealing with IPC began to appear But when they did, confirmation was BJP overwhelming Those studies noted that the intervention uniformly protected canine (Murry et al., 1986; Gross and Auchampach, 1992), rodent (Liu and Downey, 1992; Yellon et al., 1992), porcine (Schott et al., 1990), rabbit (Van Winkle et al., 1991; Toombs et al., 1993), primate (Yang et al., 2010) and even avian (Rischard and McKean, 1998) hearts from myocardial infarction At last there was conclusive proof that infarct size could be modified, at least by this singular intervention of IPC Of course, therapeutic IPC of a heart would be impossible to implement clinically in any setting except, perhaps, open heart surgery Translation of IPC into clinical practice would have to wait until its mechanism was better understood before a treatment could be identified that could be administered after ischaemia had begun Mechanism of IPC: triggering phase Surface receptors trigger IPC The first insight into IPC’s mechanism was reported by Liu et al (1991) They announced that IPC is triggered by receptor occupancy Activation of the Gi-coupled adenosine A1 receptor in rabbits triggered IPC’s protection Thus, an adenosine receptor antagonist blocked IPC’s protection, while infusion of adenosine or an A1-selective agonist in lieu of brief ischaemia duplicated IPC’s protection Liu et al proposed that net dephosphorylation of ATP during ischaemia results in production and release of adenosine which then would bind to A1 adenosine receptors leading to a preconditioned phenotype So had these investigators defined IPC’s mechanism and were they ready to propose an intervention that could be used clinically? Hardly They had identified a pharmacological trigger, but unfortunately the trigger, like IPC, had to be given prior to the onset of ischaemia Identification of more parts of IPC’s signal transduction pathway and of the overall mechanism would be required Opioid and bradykinin’s signalling Two other endogenously released trigger substances, bradykinin (Wall et al., 1994) and opioids (Schultz et al., 1995), were also found to be involved in IPC’s protective action Inhibition of any of these three receptors aborted protection from a single preconditioning cycle However, simply increasing the number of preconditioning cycles could restore protection suggesting that the three receptors had an additive effect which was required to reach a protective threshold (Goto et al., 1995) Thus, the additional cycles of ischaemia/ reperfusion produced increased stimulation of the two uninhibited receptors so that the protective threshold could finally be reached All three of these triggers, adenosine, bradykinin and opioids, bind to Gi-coupled receptors The proposed multiple trigger theory implies that all triggers converge on a common target Ytrehus et al (1994) reported that PKC seemed to play a major role in IPC and it was found that protection triggered by any of the three receptors could be blocked by PKC inhibitors (Goto et al., 1995; Sakamoto et al., 1995; Baines et al., 1997; Miki et al., 1998a) Thus, PKC is believed to be this common target Hence, adenosine, bradykinin and opioids bind to their respective receptors and the second messenger British Journal of Pharmacology (2015) 172 1913–1932 1915 BJP M V Cohen and J M Downey Figure Proposed signalling scheme for conditioning Abbreviations: Brady, bradykinin; eNOS, endothelial NOS; KATP, ATP-dependent potassium channel; MEK, MAPK kinase; MMP, matrix metalloproteinase; p70S6K, p70S6 kinase; PDK1/2, 3′-phosphoinositide-dependent kinase-1/-2; PI3,4,5P3, phosphatidylinositol trisphosphate; PI4,5P2, phosphatidylinositol bisphosphate; Pro, pro-HB-EGF; Sphingo 1-P, sphingosine 1-phosphate; Src, sarcoma tyrosine kinase; TNF, TNF-α; Tyr, tyrosine Modified from Tissier et al (2007a) G-protein is cleaved into active α and βγ subunits which then result in activation of PKC It would seem intuitive that the various agonists coupled to common Gi-proteins should trigger identical signalling However, mysteriously this is not the case Adenosine, bradykinin and opioids activate very divergent pathways; however, all three pathways eventually converge on PKC Opioid cardioprotection is dependent on downstream metalloproteinase and EGF receptor (Cohen et al., 2007a) This part of the signalling pathway was first mapped by studying ACh-stimulated receptors, Gi-coupled receptors, whose downstream protection is governed by signalling similar to that of opioids (Krieg et al., 2002; 2004; Oldenburg et al., 2002; 2003) While ACh is a potent trigger of preconditioning’s protection, it is not a physiological trigger as transient ischaemia does not cause its release ACh binds to its receptor resulting in cleavage of Gi and subsequent metalloproteinase-dependent cleavage of heparin-binding EGF-like growth factor (HB-EGF) from membrane-associated pro-HB-EGF (Figure 1) The liberated HB-EGF then activates membrane-bound EGFR by binding to its ectodomain resulting in EGFR dimerization which in turn leads to autophosphorylation of tyrosine residues on both EGFRs and binding 1916 British Journal of Pharmacology (2015) 172 1913–1932 of sarcoma (src) tyrosine kinase to form a signalling module The latter attracts and activates PI3K Bradykinin’s signalling is comparable, although a different metalloproteinase is involved Methner et al (2009) demonstrated involvement of metalloproteinase-8 and the EGFR Downstream steps are similar to those for ACh and opioids (Cohen et al., 2007a) NO PI3K-produced phosphorylated lipid metabolites phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4bisphosphate induce Akt to translocate to the plasma membrane (Andjelkovic´ et al., 1997) where it is phosphorylated by PDK1 and (Stephens et al., 1998) and this initiates a signalling cascade Akt activates ERK and endothelial NOS (Dimmeler et al., 1999) The latter enzyme catalyzes production of NO which stimulates guanylyl cyclase (GC) GC catalyzes the production of cGMP which itself activates PKG (Figure 1) NO is a gaseous free radical and important biological regulator and cellular signalling molecule In 1992, Vegh et al (1992) proposed that endogenous NO might be involved in preconditioning This announcement triggered a significant Pre- and postconditioning signalling controversy regarding the precise role of NO in IPC (Weselcouch et al., 1995) to which we unwittingly contributed In a study in in vitro rabbit hearts published in 2000, we noted that Nω-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, had no effect on the dramatic protection induced by IPC, whereas the NO donor S-nitroso-Nacetylpenicillamine administered before the index ischaemia in lieu of the repeated brief coronary occlusions mimicked IPC and protected hearts (Nakano et al., 2000) We concluded that exogenously administered NO could trigger the preconditioned state, but that endogenous production of NO was not involved in IPC This conundrum was not resolved for several years until we repeated studies with L-NAME in IPC in in vivo rabbit hearts (Cohen et al., 2006) L-NAME blocked the protection of IPC Our earlier observations, although accurate, were dependent on the in vitro model used As seen in Figure 1, bradykinin, opioids and adenosine are released by the ischaemic heart But in the isolated, buffer-perfused heart, the absence of circulating kininogens would minimize release of bradykinin In addition, opioid release would be attenuated because of the absence of cardiac innervation Therefore, virtually all triggering would be the result of adenosine release which bypasses the NO-dependent trigger pathway (Figure 1) As noted earlier, classical signalling dogma indicates that NO stimulates GC leading to generation of cGMP which in turn activates PKG (Figure 1) Studies with activators and inhibitors of PKG and cGMP analogues (Han et al., 2002; Oldenburg et al., 2004; Qin et al., 2004; Kuno et al., 2008) clearly demonstrated the involvement of PKG in IPC, and Baxter’s laboratory (D’Souza et al., 2003; Burley et al., 2007) demonstrated increased myocardial levels of cGMP after protection by B-type natriuretic peptide Additionally, BAY 58-2667, a NO-independent GC activator, conditioned rat and rabbit hearts (Krieg et al., 2009) Thus, in addition to proven involvement of endogenous NO, there is much evidence to support participation of GC and PKG in conditioning’s protection Several investigators have also demonstrated involvement of a NO-mediated, PKG-independent signalling pathway (Sun et al., 2013; Penna et al., 2014) NO can directly modify sulfhydryl residues by S-nitrosylation The latter is an important post-translational protein modification in signalling IPC increases S-nitrosylation and IPC cardioprotection can be aborted by treatment with ascorbate which is a reducing agent resulting in specific degradation of S-nitrosylated compounds Additionally, in isolated mouse hearts, pharmacologic inhibition of the soluble GC/cGMP/PKG pathway failed to block IPC-induced cardioprotection Thus, in at least some models, this alternative pathway of NO signalling may be important and it is possible that each pathway may contribute to cardioprotection and the ischaemic stimulus itself or other unidentified factors may determine whether one or the other pathway is utilized Once again, there is redundancy to the response to ischaemia which may be an adaptive change insuring a maximal cardioprotective result ATP-sensitive potassium channels and redox signalling The next critical step in this signalling cascade is opening of an ATP-sensitive K+ channel (KATP) At the same time that we BJP were uncovering the importance of adenosine in IPC, Garret Gross’ laboratory was doing studies with KATP channels Those investigators found that glibenclamide, a blocker of the channel, could also selectively block IPC’s protection (Gross and Auchampach, 1992) Similarly, pretreatment with a KATP channel opener mimicked the protection (Gross and Auchampach, 1992) For a while it seemed that it had to be either adenosine or potassium channels which governed protection, but it turned out that they were simply two links in the same chain; both were involved Several subsequent studies revealed that the KATP channel in question was located not on the sarcolemma but in the inner mitochondrial membrane (Garlid et al., 1997; Liu et al., 1998) Mitochondrial KATP channels (mtKATP) are not triggers for IPC but rather are a critical link in the signalling pathway between surface receptors and PKC (Pain et al., 2000) Opening of mtKATP is PKG-dependent (Costa et al., 2005; 2008), but the channels are obviously not accessible to cytosolic PKG There are intermediate steps involving PKCε in the mitochondria which transmits the signal from cytosolic PKG to the mtKATP channel (Costa et al., 2005; 2008; Jabu˚rek et al., 2006) One theory proposes that PKG reaches the mitochondria via signalosomes that bud off of sarcolemmal caveolae and contain critical signalling enzymes (Garlid et al., 2009) Channel opening permits K+ to enter the matrix along its electrochemical gradient K+ influx is balanced by electrogenic H+ efflux driven by the respiratory chain An important link in this signalling is the redox coupling of mtKATP channel opening and PKC activation Forbes et al (2001) were the first to recognize this link when they noticed that either of the antioxidants N-acetylcysteine or N-2mercaptopropionylglycine could block the protection from the mtKATP opener, diazoxide It is not known exactly how mtKATP opening causes production of free radicals, but one theory is that mtKATP-dependent matrix alkalinization affects complex I and/or III which are poised to generate increased amounts of superoxide and its products H2O2 and hydroxyl radical (Costa and Garlid, 2008) All of the signalling steps to this point occur in ischaemic cells However, generation of this burst of reactive oxygen species (ROS) must await reintroduction of oxygen into the myocardium which occurs during the reflow phase of the preconditioning cycle of ischaemia/reflow There are many PKC isozymes It appears that activation of PKCε is necessary and sufficient to achieve cardioprotection, while activation of PKCδ specifically blocks protection (Dorn et al., 1999; Ping et al., 2002; Inagaki et al., 2003a,b) Thus, activation of PKC continues the signalling cascade The relationship among mtKATP, ROS and PKC is poorly understood While ROS can directly activate PKC by causing release of Zn++ from the regulatory domain (Korichneva et al., 2002), connexin 43 (Cx43) appears to be a vital link in redox signalling Cx43 which makes most of the gap junctions between cardiomyocytes was noted to be necessary for preconditioning’s protection (Schwanke et al., 2002) It was later noted that protection depended on a mitochondrial population of Cx43 hemichannels located on the inner membrane Depletion of these channels attenuates both protection and ROS production from an mtKATP opener (Heinzel et al., 2005) Most recently, it was shown that an mtKATP opener causes phosphorylation of Cx43 by PKC and that phosphorylation is British Journal of Pharmacology (2015) 172 1913–1932 1917 BJP M V Cohen and J M Downey required for protection (Srisakuldee et al., 2009) This suggests some sort of circular signalling circuit as phosphoCx43 is needed for ROS production and ROS cause PKC activation, but PKC phosphorylates Cx43 The role actually played by Cx43 in the protective process (e.g a channel, a signalling molecule or a scaffold) is still a mystery The redox signalling step explains one of the mysteries of IPC Why is the heart protected when a prolonged ischaemic period is preceded by a short coronary occlusion followed by reperfusion but yet is not protected during a single prolonged insult? All of the trigger receptors are activated during the single prolonged ischaemic insult, but signalling stops at the step requiring redox coupling to PKC because of the lack of oxygen which is supplied during IPC’s short reperfusion A ROS scavenger blocks protection from IPC and it can easily be seen that the critical time for that blockade is during IPC’s reperfusion phase (Dost et al., 2008) While ROS-sensitive dyes indicate that radical production can occur during ischaemia (Becker et al., 1999), apparently the ROS species generated is not one capable of the redox signalling We also have found that reperfusing with hypoxic perfusate during the preconditioning protocol abrogates IPC’s protection (Dost et al., 2008) The identity of the ROS species involved has not been positively identified but seems to be a downstream product of HO· and is likely a product of phospholipid oxidation (Garlid et al., 2013) Adenosine signalling Signalling initiated by the third endogenous agonist that triggers IPC, adenosine, is different Adenosine’s cardioprotective effect is not dependent on Src tyrosine kinase or PI3K (Qin et al., 2003) Adenosine signalling seems to completely bypass mtKATP and ROS production (Cohen et al., 2001) and it more directly activates PKC (see Figure 1) which is where all of the trigger signalling converges The adenosine A1 receptor is coupled through Gi to PLC and PLD After the ligand binds to the receptor, Gi is cleaved into α and βγ moieties which activate PLC in the sarcolemma This enzyme catalyzes the hydrolysis of membrane inositol-containing phospholipids, including phosphatidylinositol 4,5-bisphosphate The resulting DAG stimulates translocation and activation of PKC PLD also increases DAG levels by degrading phosphatidylcholine into choline and phosphatidic acid and the latter is transformed by a phosphohydrolase into DAG These phospholipid activators of PKC also trigger release of zinc from PKC’s regulatory domain (Korichneva et al., 2002) The diversity of signalling among the triggers is confusing, but also reassuring The redundancy ensures cardioprotection even if one or more elements in the triggering cascade are blocked A2B receptors It had been noted that adenosine receptors were required for IPC’s protection in the mediator phase One hypothesis suggested that preconditioning is protective by increasing tissue adenosine levels through activation of ecto-5′-nucleotidase (Kitakaze et al., 1993) However, measurements of myocardial adenosine levels revealed that tissue adenosine concentration actually falls in IPC hearts (Goto et al., 1996; Martin et al., 1997) Our studies have indicated that the initial step of the mediator phase is activation of adenosine A2B receptors (Philipp et al., 2006) This receptor has a very low affinity for adenosine such that even during ischaemia when tissue adenosine levels reach 1–4 μM, this level would still be well below the A2B adenosine receptor’s KD of 5–15 μM However, PKC activation appears to raise the affinity of the A2B receptor permitting the adenosine concentration in ischaemic myocardium to be sufficient for occupation of this receptor (Kuno et al., 2007) It had already been shown that PKC activity can sensitize A2B signalling, although no physiologic significance was attributed to the observation (Nordstedt et al., 1989; Nash et al., 1997; Trincavelli et al., 2004) Although the details of this sensitization are still unknown, it would appear A2B receptors can respond to the heart’s endogenous adenosine only after this sensitization Thus, we proposed that the affinity state of the A2B receptor is the determinant that distinguishes the preconditioned from the non-preconditioned phenotype Our observation of involvement of the A2B receptor in IPC was supported by Eckle et al (2007) who studied mice genetically modified to lack one of the four adenosine receptor subtypes While A1, A2A and A3 adenosine receptor knockout mice could be preconditioned, A2B knockout mice could not However, there is evidence suggesting a cooperative role of A2A and A2B adenosine receptors in some forms of cardioprotection (Xi et al., 2009; Methner et al., 2010) The reperfusion injury survival kinases (RISK) pathway A kinase cascade involving PI3K, Akt and ERK has been proposed to occur in the first minutes of reperfusion following the index ischaemia (Hausenloy and Yellon, 2004; Hausenloy et al., 2005) These kinases have collectively been termed RISK (Hausenloy and Yellon, 2004) Although RISK are clearly involved in cardioprotection in rat (Hausenloy et al., 2005) and rabbit (Yang et al., 2004b; 2005) hearts, their involvement may not be universal In pig hearts, RISK are less important (Skyschally et al., 2009a) A distinct alternate pathway utilizing membrane TNF-α receptors and cytoplasmic JAK and STAT has been proposed (see succeeding text), although the end-effector for this and the RISK pathways appears to be identical The mediator phase IPC’s end-effector All signalling to this point occurs during the preconditioning cycles of ischaemia and reflow These steps are collectively called the trigger phase Subsequent steps, and there are several, are part of the mediator phase which occurs following termination of the prolonged period of ischaemia (the index ischaemia) with reperfusion (Figure 1) IPC’s end-effector appears to be the mitochondrial permeability transition pore (mPTP) and its inhibition is considered to be the final step in the protective signal transduction pathway (Griffiths and Halestrap, 1993; 1995; Squadrito et al., 1999; Di Lisa et al., 2001; Hausenloy et al., 2002) Although the molecular structure of mPTP is controversial, when 1918 British Journal of Pharmacology (2015) 172 1913–1932 Supraspinal antinociceptive mechanisms of oxycodone BJP Figure Inhibition of GABAergic eIPSCs by oxycodone in VLPAG neurons was increased in slices from the FBC model (A) A representative trace showing that oxycodone (10 μM) potently reduced GABAergic eIPSCs in VLPAG neurons in the FBC model, compared with its efficacy in slices of sham-operated mice (B) A representative trace showing that morphine (10 μM) moderately reduced GABAergic eIPSCs in a VLPAG neuron in the FBC model to a similar extent as in slices of sham-operated mice (C) Summary graphs showing that oxycodone (10 μM) elicited a statistically significant potent inhibition of GABAergic eIPSCs in the FBC model compared with that in slices of sham-operated mice In contrast, the efficacy of morphine (10 μM) was unchanged between the FBC model and sham-operated mice Each column represents means ± SEM (n = 10) The asterisk indicates a significant difference between the FBC model and sham-operated mice, as determined by Student’s t-test (two-tailed, *P < 0.05) blockade of Kir3.1 channels on the antinociceptive effects of these two opioids Injection of oxycodone or morphine at dose of nmol (i.c.v.) ameliorated mechanical hypersensitivity in the FBC model (Figure 1) Oxycodone-induced antinociception was abolished by i.c.v pretreatment with 30 pmol of the Kir3.1 channel inhibitor tertiapin-Q (oxycodone pretreated with tertiapin-Q, 0.014 ± 0.003 g, n = 11 vs oxycodone pretreated with saline, 0.054 ± 0.013 g, n = 13, F(13,11) = 24.16; P < 0.05) In contrast, morphine-induced antinociception was not blocked by tertiapin-Q (morphine pretreated with tertiapin-Q, 0.033 ± 0.006 g, n = 12 vs morphine pretreated with saline, 0.035 ± 0.005 g, n = 12) Thus, Kir3.1 channels are critical for the supraspinal antinociceptive effects of oxycodone, but not those of morphine, on mechanical hypersensitivity in the FBC model In the following experiments, we evaluated the neuronal changes and supraspinal antinociceptive mechanisms of oxycodone and morphine in the FBC model by focusing on GABAergic synaptic transmission at VLPAG neurons In the following electrophysiological assessment, we used single concentra- tion of oxycodone and morphine to elicit maximal effects on activation of μ-opioid receptors (Bradaia et al., 2005; Nakamura et al., 2013) and investigated possible alteration of the effects of oxycodone and morphine between the FBC model and sham-operated mice Leftward shift of the I–O relation curve of GABAergic eIPSCs and increased inhibitory effect of oxycodone on GABAergic eIPSCs in the FBC model Based on the disinhibition mechanism underlying supraspinal opioid-induced antinociception in VLPAG area (Vaughan and Christie, 1997; Budai and Fields, 1998; Park et al., 2010), we evaluated the amplitude of GABAergic eIPSCs in slices from the FBC model and sham-operated mice When the stimulus intensity was gradually increased, electrical stimulation gave rise to larger-amplitude eIPSCs at a lower intensity in the FBC model than the sham-operated mice, indicating a leftward shift of the I–O relationship curve of eIPSCs in British Journal of Pharmacology (2015) 172 2148–2164 2153 BJP K Takasu et al Figure The μ-opioid receptor-selective antagonist β-FNA, but not κ-opioid receptor-selective antagonist nor-BNI abolished the inhibitory effects of oxycodone and morphine, on GABAergic eIPSCs in VLPAG neurons of the FBC model (A) Left panel, a representative time course, which was shown every by averaging six IPSCs traces, indicated that oxycodone caused potent inhibition of GABAergic eIPSCs in a VLPAG neuron from the FBC model, which was abolished by β-FNA On the right panel, a summary of the effects of β-FNA on the oxycodone-induced reduction of GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 6) The asterisk indicates a statistically significant difference between the oxycodone-only and oxycodone with β-FNA-treated groups, as determined by Student’s t-test (two-tailed, ***P < 0.001) (B) Summary of the effects of nor-BNI on oxycodone-induced reduction in GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 6) for the oxycodone-only and oxycodone with nor-BNI-treated groups (C) On the left panel, a representative time course showing that morphine caused inhibition of GABAergic eIPSCs in a VLPAG neuron from the FBC model, which was abolished by β-FNA On the right panel, a summary of the effects of β-FNA on the morphine-induced reduction of GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 5) The asterisk indicates a statistically significant difference between the morphine-only and morphine with β-FNA-treated groups, as determined by Student’s t-test (two-tailed, **P < 0.01) (D) Summary of the effects of nor-BNI on morphine-induced reduction in GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 5) of the morphine-only and morphine with nor-BNI-treated groups slices of the FBC model (Figure 2, F(1,32) = 9.098; P < 0.05 vs sham-operated mice) At stimulus intensities of 10 and 15 V, the GABAergic synaptic responses in the FBC model were significantly larger than those in sham-operated mice (Figure 2) These results indicate that the GABAergic synaptic influence on VLPAG neurons was enhanced in the FBC model When the effects of oxycodone and morphine on GABAergic eIPSCs in VLPAG neurons were evaluated, 10 μM oxycodone elicited potent inhibition of eIPSCs in the FBC model compared with that in sham-operated mice (Figure 3A and C; the FBC model, 38.5 ± 4.6% inhibition vs sham operation, 24.2 ± 2.5% inhibition, n = 10, t(18) = 2.686; P < 0.05) In contrast, 10 μM morphine inhibited GABAergic eIPSCs in 2154 British Journal of Pharmacology (2015) 172 2148–2164 VLPAG neurons of the FBC model to the same extent as those from sham-operated mice (Figure 3B and C; FBC model, 28.8 ± 3.5% inhibition vs sham operation, 29.2 ± 3.0% inhibition, n = 10) The eIPSCs amplitude before drug application was not significantly different between the FBC model and sham-operated mice (FBC model, 820.1 ± 86.8 pA vs sham-operated mice, 977.8 ± 127.2 pA) because we evaluated the effects of oxycodone and morphine on eIPSCs induced by electrical stimulation at supra-threshold intensity (2× the threshold) The threshold was different (FBC model, 2.5 ± 0.3 V vs sham-operated mice, 7.1 ± 1.0 V, n = 10, t(18) = 4.334; P < 0.05) Thus, these results showed that the inhibitory effect of oxycodone on elevated eIPSCs was potentiated in the FBC model Supraspinal antinociceptive mechanisms of oxycodone BJP Figure The PPR constructed by two successive GABAergic eIPSCs was increased in the FBC model as compared with sham-operated mice, and oxycodone potently increased the PPR in the FBC model (A) A representative trace showing that oxycodone (10 μM) potently reduced the first amplitude of GABAergic eIPSCs, resulting in an increase in the PPR in slices of the FBC model This effect was greater in the FBC model compared with that in sham-operated mice (B) A representative trace showing that morphine (10 μM) reduced the first amplitude of GABAergic eIPSCs, resulting in a slight increase in the PPR in slices of the FBC model and sham-operated mice (C) Summary graphs showing a significant increment of the PPR in the FBC model compared with sham-operated mice, indicated by sharp Each column represents means ± SEM (n = or 7) Oxycodone (10 μM) significantly changed the PPR in the FBC model while morphine (10 μM) induced slight changes in PPR, indicated by asterisk (D) The increment of PPR by oxycodone (10 μM) was enhanced in the FBC model while that by morphine (10 μM) was not significantly different between the FBC model and sham-operated mice The sharp indicates a statistically significant difference between the sham-operated mice and the FBC model groups, as determined by Student’s t-test (two-tailed, #P < 0.05) The asterisk indicates a statistically significant difference between pre-treatment (control; cont) and post-treatment with each drug in the FBC model and sham-operated mice, as determined by paired t-test (two-tailed, *P < 0.05, **P < 0.01) Involvement of μ-opioid receptors but not κ-opioid receptors in the inhibitory effects of oxycodone and morphine on GABAergic eIPSCs in the FBC model We next explored the type of opioid receptors that mediate the inhibitory effects of oxycodone and morphine on GABAergic eIPSCs in VLPAG neurons in slices of the FBC model It is known that the antinociceptive effects of oxycodone and morphine are mainly mediated by μ-opioid receptors (Lemberg et al., 2006; Peckham and Traynor, 2006), but the involvement of κ-opioid receptors is also reported (Ross and Smith, 1997; Nielsen et al., 2007) Therefore, we evaluated the effects of μ-opioid receptor-selective antagonist and κ-opioid receptor-selective antagonist on inhibition of eIPSCs by oxycodone and morphine in slices of FBC model In the presence of μ-opioid receptor-selective antagonist β-FNA (1 μM), the inhibitory effect of 10 μM oxycodone on eIPSCs was completely abolished (Figure 4A; oxycodone administered after pretreatment with β-FNA, 2.7 ± 3.8% inhibition vs oxycodone alone: 45.5 ± 2.3% inhibition, n = 6, t(10) = 9.569; P < 0.001) By contrast, the κ-opioid receptor-selective antagonist nor-BNI (1 μM) did not change the effects of oxycodone (Figure 4B; oxycodone administered after pretreatment with nor-BNI, 37.3 ± 3.2% inhibition vs oxycodone alone: 37.6 ± 3.5% inhibition, n = 6) The inhibitory effect of 10 μM morphine on eIPSCs was also abolished following pretreatment with β-FNA (Figure 4C; morphine administered after pretreatment with β-FNA, 4.6 ± 3.0% inhibition vs morphine alone: 32.3 ± 4.2% inhibition, n = 5, t(8) = 5.254; P < 0.01) while it was not blocked by nor-BNI (Figure 4D; morphine administered after pretreatment with nor-BNI, 33.5 ± 4.8% inhibition vs morphine alone: 31.0 ± 7.4% inhibition, n = 5) Thus, both British Journal of Pharmacology (2015) 172 2148–2164 2155 BJP K Takasu et al Figure The Kir3.1 channel inhibitor tertiapin-Q abolished the inhibitory effect of oxycodone, but not that of morphine, on GABAergic eIPSCs in VLPAG neurons of the FBC model (A) Left panel, a representative time course (plot of average amplitude of six IPSCs traces for min) showing that oxycodone caused potent inhibition of GABAergic eIPSCs in a VLPAG neuron from the FBC model, which was abolished by tertiapin-Q In the middle panel, each trace was recorded during the times indicated on the graph Right panel, a summary of the effects of tertiapin-Q on the oxycodone-induced reduction of GABAergic eIPSCs in slices from the FBC model and sham-operated mice Each column represents means ± SEM (n = or 7) The asterisk indicates a statistically significant difference between the oxycodone-only and oxycodone with tertiapin-Q-treated groups, as determined by Student’s t-test (two-tailed, ***P < 0.001) (B) Left panel, a representative time course showing that morphine caused inhibition of GABAergic eIPSCs in a VLPAG neuron from the FBC model, which was not abolished by tertiapin-Q In the middle panel, each trace was recorded during the times indicated on the graph Right panel, summary of the effects of tertiapin-Q on morphine-induced reduction in GABAergic eIPSCs in slices from the FBC model and sham-operated mice Each column represents means ± SEM (n = or 6), constituted of the morphine-only and morphine with tertiapin-Q-treated groups morphine and oxycodone cause inhibition of eIPSCs via activation of μ-opioid receptors, but not κ-opioid receptors This result is consistent with our previous studies demonstrating that oxycodone and morphine-induced GTPγS activity in the PAG area is mediated by μ-opioid receptors (Nakamura et al., 2013) Changes in the PPR and increased effects of oxycodone on PPR in slices of the FBC model We then analysed the PPR to evaluate whether presynaptic GABA release or the sensitivity of postsynaptic GABAA receptors was involved in the enhancement of the GABAergic synaptic response and the increased inhibitory effect of oxycodone on GABAergic eIPSCs in slices of the FBC model A change in the PPR of eIPSCs has been attributed to a presynaptic change in probability of action potentialdependent GABA release (Manabe et al., 1993; Zucker and 2156 British Journal of Pharmacology (2015) 172 2148–2164 Regehr, 2002) As seen in Figure 5A–C, the PPR recorded in the FBC model was increased along with an increase in the second eIPSC amplitude (Figure 5C; FBC model, 1.8 ± 0.2, n = vs sham-operated mice, 1.1 ± 0.1, n = 7, t(11) = 2.431; P < 0.05), indicating enhancement of presynaptic GABA release in the FBC model In slices from the FBC model, 10 μM oxycodone reduced the initial eIPSC amplitude and significantly increased PPR (Figure 5A and C) The degree of PPR change by oxycodone was significantly greater in the FBC model (Figure 5D; FBC model, 149.5 ± 45.2% increase, n = vs sham-operated mice, 30.8 ± 6.8% increase, n = 7, t(11) = 2.810; P < 0.05) In contrast, morphine reduced the initial eIPSC amplitude and slightly changed the PPR in the FBC model (Figure 5B and C) The degree of change in PPR caused by morphine did not differ significantly between the FBC model and sham-operated mice (Figure 5D; FBC model, 58.8 ± 39.8% increase, n = vs Supraspinal antinociceptive mechanisms of oxycodone BJP Figure The frequency, but not the amplitude, of mIPSCs was enhanced in VLPAG neurons from the FBC model Oxycodone and morphine decreased the frequency, but not the amplitude, of mIPSCs in slices from the FBC model and sham-operated mice (A and D) A representative trace showing that the frequency of mIPSCs in VLPAG neurons in slices from the FBC model was enhanced compared with that in slices of sham-operated mice Oxycodone at 10 μM (A–C) and morphine at 10 μM (D–F) reduced the frequency of mIPSCs without altering the amplitude in slices of the FBC model and sham-operated mice (B and E) Left panel, summary graphs showing that the frequency of mIPSCs in VLPAG neurons was increased in slices of the FBC model compared with sham-operated mice, indicated by sharp Oxycodone at 10 μM (B) and morphine at 10 μM (E) caused a statistically significant inhibition of the mIPSCs frequency in slices from the FBC model and sham-operated mice, indicated by asterisk Right panel, the extent of inhibition of mIPSCs frequency by oxycodone at 10 μM (B) and morphine at 10 μM (E) was not different between the FBC model and sham-operated mice The sharp indicates a significant difference between the FBC model and sham-operated mice, as determined by Student’s t-test (two-tailed, #P < 0.05) The asterisk indicates a statistically significant difference between pre- and post-treatment with oxycodone or morphine within the FBC model and sham-operated mice groups, as determined by paired t-test (two-tailed, *P < 0.05, **P < 0.01) Each column represents means ± SEM (n = 5) (C and F) Summary graphs showing that the amplitude of mIPSCs in VLPAG neurons was not changed in slices of the FBC model compared with sham-operated mice, and the mIPSCs’ amplitude in both the FBC model and sham-operated mice was not changed by oxycodone and morphine sham-operated mice: 32.4 ± 10.6% increase, n = 7) These results showed that inhibition of presynaptic GABA release by oxycodone, but not morphine was increased in the FBC model Involvement of Kir3.1 channels in the inhibitory effects of oxycodone on GABAergic eIPSCs in both the FBC model and sham-operated mice We next explored the involvement of the Kir3.1 channels in the inhibitory effects of oxycodone and morphine on GABAergic eIPSCs in VLPAG neurons in slices of the FBC model In the presence of the Kir3.1 channel inhibitor tertiapin-Q (100 nM), the inhibitory effect of 10 μM oxycodone on eIPSCs was completely abolished (Figure 6A; oxycodone pretreated with tertiapin-Q, 0.7 ± 4.9% inhibition vs oxycodone alone: 38.6 ± 5.0% inhibition, n = 7, t(12) = 5.383; P < 0.001) Tertiapin-Q (100 nM) alone did not have significant effects on eIPSCs (3.0 ± 4.9% inhibition, n = 7) The inhibitory effects of tertiapin-Q on the oxycodone-induced reduction of eIPSCs were reversible because a second application of oxycodone alone again reduced eIPSCs Also, in sham-operated mice, the inhibition of eIPSCs by oxycodone British Journal of Pharmacology (2015) 172 2148–2164 2157 BJP K Takasu et al Figure The Kir3.1 channel inhibitor tertiapin-Q abolished the inhibitory effect of oxycodone, but not that of morphine, on GABAergic mIPSCs frequency in VLPAG neurons of the FBC model (A) A representative trace showing that oxycodone (10 μM) caused inhibition of mIPSCs frequency in a VLPAG neuron from the FBC model, which was completely abolished by tertiapin-Q (B) Left panel, a summary of inhibition of mIPSCs frequency by oxycodone (10 μM) in the FBC model and its disruption in the presence of tertiapin-Q Each column represents means ± SEM (n = 7) The asterisk indicates a statistically significant difference between pre- and post-treatment with oxycodone alone groups, as determined by paired t-test (two-tailed, **P < 0.01, ***P < 0.001) Right panel, the extent of inhibition of mIPSCs frequency by oxycodone in the presence of tertiapin-Q and oxycodone alone The inhibitory effects of tertiapin-Q on the oxycodone-induced reduction of mIPSCs frequency were reversible because a second (2nd) application of oxycodone alone again reduced mIPSCs frequency, to a similar extent as the first (1st) application of oxycodone (D) A representative trace showing that morphine (10 μM) caused inhibition of mIPSCs frequency in a VLPAG neuron from the FBC model, which was not abolished by tertiapin-Q (E) Left panel, a summary of inhibition of mIPSCs frequency by morphine (10 μM) and no disruption of morphine-induced inhibition in the presence of tertiapin-Q Each column represents means ± SEM (n = 6) The asterisk indicates a statistically significant difference between pre- and post-treatment with morphine in the presence of tertiapin-Q and morphine alone, as determined by paired t-test (two-tailed, **P < 0.01, ***P < 0.001) Right panel, extent of inhibition of mIPSCs frequency by morphine in the presence of tertiapin-Q and morphine alone (C) and (F) Summary graphs showing that the amplitude of mIPSCs in VLPAG neurons was not changed by oxycodone and morphine in the absence and presence of tertiapin-Q was blocked by pretreatment with tertiapin-Q (Figure 6A; oxycodone pretreated with tertiapin-Q, 3.4 ± 1.5% inhibition vs oxycodone alone: 23.0 ± 2.1% inhibition, n = 5, t(8) = 7.267; P < 0.001) In contrast, the inhibitory effects of morphine on eIPSCs were not abolished by tertiapin-Q in both the FBC model and sham-operated mice (Figure 6B) The concentration of tertiapin-Q used was sufficient to 2158 British Journal of Pharmacology (2015) 172 2148–2164 block Kir3.1 channels as it was reported to effectively block opioid or GABAB receptor agonist baclofen-activated Kir3.1 channels in a brain slice (Nassirpour et al., 2010; Wu et al., 2011) In the present study, Kir3.1 channel activity is important for the inhibitory effects on eIPSCs of oxycodone, but not morphine, in both the FBC model and sham-operated mice Supraspinal antinociceptive mechanisms of oxycodone BJP Figure The PKA inhibitor H-89 had negligible effects on the inhibition by oxycodone and morphine of GABAergic eIPSCs in VLPAG neurons of the FBC model (A) Left panel, a representative time course (plot of average amplitude of six IPSCs traces for min) showing that oxycodone caused potent inhibition of GABAergic eIPSCs in VLPAG neurons from the FBC model, which was not affected by H-89 Middle panel, each trace was recorded during the times indicated on the graph Right panel, a summary of the effects of H-89 on oxycodone-induced reduction in GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 7), from the oxycodone-only and oxycodone with H-89-treated groups (B) Left panel, a representative time course showing that morphine caused moderate inhibition of GABAergic eIPSCs in a VLPAG neuron from the FBC model, which was not affected by H-89 Middle panel, each trace was recorded during the times indicated on the graph Right panel, a summary of the effects of H-89 on morphine-induced reduction in GABAergic eIPSCs in slices from the FBC model Each column represents means ± SEM (n = 8), from the morphine-only and morphine with H-89-treated groups Enhancement of GABA release in VLPAG neurons in the FBC model and presynaptic inhibition of GABA release by oxycodone and morphine In this experiment, we evaluated changes in spontaneous GABAergic mIPSC, rather than action potential-dependent GABA release by neuronal firing, following pharmacological elimination of the influence of the neuronal activities of other neurons In the presence of TTX, which blocks action potential-driven neuronal activity, the frequency of mIPSCs in slices from the FBC model was significantly increased, compared with that in sham-operated mice, whereas the amplitude of mIPSCs in VLPAG neurons was not different between the two groups (Figure 7) This showed that presynaptic, rather than postsynaptic, GABA release in VLPAG neurons was enhanced in the FBC model When the effects of oxycodone and morphine were tested, 10 μM oxycodone reduced the frequency of mIPSCs in slices of both the FBC model and sham-operated mice without changing the amplitude (Figure 7A–C) As shown in Figure 7B right, the degree of inhibition of mIPSCs frequency by oxycodone did not differ significantly between the FBC model and shamoperated mice (the FBC model, 66.0 ± 4.1% inhibition, n = vs sham-operated mice, 60.8 ± 7.0% inhibition, n = 5) Morphine also reduced the frequency of mIPSCs in slices of the FBC model and sham-operated mice without changing the amplitude (Figure 7D–F) As shown in Figure 7E right, the degree of inhibition of mIPSCs frequency by morphine did not differ significantly between the FBC model and sham-operated mice (the FBC model, 67.2 ± 5.4% inhibition, n = vs shamoperated mice, 52.8 ± 11.0% inhibition, n = 5) These results showed that both oxycodone and morphine reduced spontaneous GABA release in VLPAG neurons by acting on presynaptic GABAergic terminals under these experimental conditions British Journal of Pharmacology (2015) 172 2148–2164 2159 BJP K Takasu et al Involvement of Kir3.1 channels in the inhibitory effects of oxycodone on GABAergic mIPSCs frequency in the FBC model To further clarify whether presynaptic inhibition of action potential-independent GABA release by oxycodone and morphine was mediated by Kir3.1 channels, we next explored the effects of tertiapin-Q on the inhibitory effects of oxycodone and morphine on GABAergic mIPSCs in VLPAG neurons in slices of the FBC model In the presence of tertiapin-Q (100 nM), the inhibitory effect of 10 μM oxycodone on mIPSCs was completely abolished (Figure 8A and B; oxycodone pretreated with tertiapin-Q, 2.4 ± 6.7% inhibition vs oxycodone applied firstly: 54.1 ± 5.5% inhibition, n = 7, t(12) = 5.950; P < 0.001) The inhibitory effects of tertiapin-Q on the oxycodone-induced reduction of mIPSCs were reversible because a second application of oxycodone alone again reduced mIPSCs (Figure 8B) By contrast, the inhibitory effect of morphine on mIPSCs was not abolished by tertiapin-Q (Figure 8D and E; morphine pretreated with tertiapin-Q, 57.0 ± 3.3% inhibition vs morphine applied firstly, 53.4 ± 4.2% inhibition, n = 6) Both oxycodone and morphine had no effects on mIPSCs amplitude in the FBC model in the absence and the presence of tertiapin-Q (Figure 8C and F) Thus, Kir3.1 channel activity is important for the inhibitory effects on action potential-independent GABA release of oxycodone, but not morphine, in the FBC model No involvement of PKA activity in the inhibitory effects of oxycodone on GABAergic eIPSCs in the FBC model A previous study on β-arrestin2 knock-out mice demonstrated that an elevation of PKA activity in presynaptic terminals causes an enhancement of GABAergic synaptic transmission in PAG neurons and increases the inhibitory effects of morphine on eIPSCs (Bradaia et al., 2005) To investigate whether an elevation of PKA activity underlies the enhancement of GABAergic synaptic transmission in the FBC model, we evaluated the effects of the PKA inhibitor H-89 on GABAergic eIPSC and on the efficacy of oxycodone and morphine H-89 (10 μM) alone had no significant effect on GABAergic eIPSCs in slices of the FBC model (0.1 ± 2.7% inhibition, n = 15) before application of oxycodone or morphine (Figure 9) In the presence of H-89, the inhibitory effect of 10 μM oxycodone on GABAergic eIPSCs was not altered (Figure 9A; oxycodone pretreated with H-89, 39.1 ± 6.3% inhibition vs oxycodone applied firstly, 44.6 ± 1.3% inhibition, n = 7) The inhibitory effect of 10 μM morphine on eIPSCs was also unchanged in the presence of H-89 (Figure 9B; morphine pretreated with H-89, 21.2 ± 4.6% inhibition vs morphine applied firstly, 28.7 ± 3.6% inhibition, n = 8) Our results indicate that an elevation of PKA activity was not involved in either the enhancement of GABAergic synaptic transmission in VLPAG neurons or the effects of oxycodone and morphine in the FBC model Discussion Our study demonstrated that supraspinally administered oxycodone and morphine ameliorated mechanical 2160 British Journal of Pharmacology (2015) 172 2148–2164 hypersensitivity in the FBC model, and that oxycodoneinduced antinociception was dependent on Kir3.1 channels while that of morphine was not To clarify these supraspinal antinociceptive effects, we focused on the VLPAG area as a primary site of opioid-induced analgesia, and we evaluated GABAergic synaptic transmission and its modulation by oxycodone and morphine We found that GABAergic synaptic transmission was enhanced in the FBC model, implying that enhanced GABA release leads to greater inhibition of VLPAG neurons for descending pain control and might be involved in the development of pain hypersensitivity in the FBC model Furthermore, we found that inhibition of eIPSCs by oxycodone was enhanced in FBC model and inhibitory effects of oxycodone on eIPSCs and mIPSCs frequency were abolished by the Kir3.1 channel inhibitor tertiapin-Q, while the inhibition of eIPSCs by morphine was not altered and inhibitory effects of morphine on eIPSCs and mIPSCs frequency were independent of Kir3.1 channels in the FBC model This might be responsible for the distinct sensitivity to Kir3.1 channel inhibition of the antinociceptive effects of oxycodone and morphine on mechanical hypersensitivity in the FBC model Although we cannot rule out the possibility that oxycodone and morphine act on sites other than the VLPAG area because of the methodological limitations of i.c.v administration of drugs, we provided evidence that the enhanced GABAergic synaptic transmission at VLPAG neurons in the FBC model is an important site for supraspinal antinociception by oxycodone via Kir3.1 channel activation The present study provided evidence for an increase in GABAergic influence on VLPAG neurons in the FBC model, indicated by a leftward shift of the I–O relationship curve of GABAergic eIPSCs, facilitation of GABAergic eIPSCs by paired stimuli, and the enhanced frequency of spontaneous mIPSCs These phenomena respectively provided evidence for the enhancement of GABAergic synaptic transmission, as reported by previous studies (Caillard et al., 2000; Park et al., 2010; Kobayashi et al., 2012; Goitia et al., 2013) It was reported that a concurrent leftward shift of the I–O relationship curve and increase in mIPSC frequency reflect an enhancement of GABA release (Herman et al., 2013) By contrast, the observed concurrent increase in PPR and mIPSC frequency contrasts with the general concept that an increase in the initial probability of transmitter release decreases the magnitude of synaptic enhancement (Zucker and Regehr, 2002; Herman et al., 2013) However, it was also reported that synaptic enhancement by electrical stimulation was accompanied by an increase in the neurotransmitter release rate by the mechanism that residual Ca2+ concentration is capable of influencing transmitter release (Felmy et al., 2003; Goitia et al., 2013) The relationship between basal release probability and the direction of paired-pulse synaptic plasticity remains debatable and may vary according to synapse type or pathological state Based on the relationship between presynaptic release machinery and synaptic facilitation (Wu and Saggau, 1994; Caillard et al., 2000; Xu et al., 2008), our results indicate that the change in presynaptic release machinery might be attributed to the enhancement of GABAergic synaptic transmission in VLPAG neurons in the FBC model A similar enhanced GABA release mechanism has been reported in rats subjected to peripheral nerve ligation (Hahm et al., 2011) and chronic morphine administration Supraspinal antinociceptive mechanisms of oxycodone (Ingram et al., 1998; DuPen et al., 2007), both of which resulted in pain hypersensitivity Together with these studies, several possible mechanisms to explain the enhanced GABAregic synaptic transmission in VLPAG neurons of the FBC model have been suggested A previous study demonstrated that the GABAA receptor agonist muscimol increased neurotransmitter release in PAG neurons, possibly by mechanisms involving presynaptic or extrasynaptic GABAA receptors, which might cause propagation of presynaptic action potentials (Jang, 2011; Stell, 2011) Thus, GABA release might be triggered by GABAA activation As another possible mechanism, the enhancement of GABA release in the FBC model might be attributed to diminished inhibition of GABA release by endogenous ligands, such as endocannabinoids or adenosines These tonically activate VLPAG neurons by modulating GABA release via Gi-coupled receptors with intracellular signal factor and/or ion channels (Hack et al., 2003; Drew et al., 2009) The reduction in tonic inhibition of GABA release by such endogenous ligands might enhance presynaptic GABA release in VLPAG neurons Thus, an elevation in GABAergic inhibition to VLPAG neurons might elicit strong inhibition of VLPAG neuron activity and weaken descending pain inhibitory control This might be involved in the development and/or maintenance of chronic pain We observed a distinct inhibition of eIPSCs by oxycodone and morphine in slices from the FBC model; the inhibitory effect of oxycodone on eIPSCs was enhanced and dependent on Kir3.1 channels whereas that of morphine was neither changed nor sensitive to these channels This finding might account for the different sensitivity to Kir3.1 channels inhibition in the antinociceptive effects of oxycodone and morphine in the FBC model To date, the mechanisms mediating the potent antinociceptive effects of oxycodone, despite its weaker affinity for the μ-opioid receptors compared with morphine, have not yet been clarified In the present study using the FBC model, we have demonstrated the importance of Kir3.1 channel activity in the supraspinal antinociceptive effects and inhibition of GABAergic transmission in VLPAG neurons by oxycodone As the inhibitory effects of oxycodone and morphine on eIPSCs were completely abolished by the selective μ-opioid receptor antagonist β-FNA, but not the κ-opioid receptor antagonist nor-BNI, these two opioids act on μ-opioid receptors The distinct sensitivity to Kir3.1 channel inhibition in the inhibitory effects of oxycodone and morphine on IPSCs might be due to the distinct receptor conformations and intracellular signalling imposed by binding of different ligands A previous study of liganddirected signalling demonstrated that oxycodone, compared with morphine, induced less μ-opioid receptor desensitization and elicited prolonged activation of Kir3.1 channels in locus coeruleus neurons of midbrain slices (Virk and Williams, 2008) The inhibitory effects of oxycodone on eIPSCs, in slices of both the FBC model and sham-operated mice, were dependent on Kir3.1 channels while those of morphine were not Thus, the Kir3.1 channels dependency of oxycodone was observed not only in the FBC model, but also in the sham-operated mice, and oxycodone might act primarily on a homogeneous population of GABAergic synapses where high-density oxycodone-sensitive μ-opioid receptors coupled with Kir3.1 channels could modulate GABA release These action sites of oxycodone might be mainly presynaptic BJP μ-opioid receptors coupled with Kir3.1 channels in GABAergic neurons because the inhibitory effect of oxycodone on mIPSCs frequency was completely abolished by tertiapin-Q Thus, Kir3.1 channels at presynapitc GABAergic neurons, which alter GABA release, are an important target for oxycodone-induced reduction of GABA release and the potent antinociceptive effects in the FBC model In the present study, nevertheless, the inhibitory effects of oxycodone on the frequency of both eIPSCs and mIPSCs were mediated by Kir3.1 channels, it was demonstrated that the enhanced efficacy of oxycodone was observed in only the frequency of eIPSCs, but not mIPSC in the FBC model This appears to be due to the fact that different mechanisms mediate spontaneous and action potential-dependent GABA release, and it is suggested that oxycodone mainly activates Kir3.1 channels located at presynaptic sites In central synapses, GABAergic eIPSCs induced by electrical stimulation, are dependent on activation of voltage-dependent Ca2+ channels via depolarization followed by propagation of action potentials in cells near the recording sites, while the frequency of mIPSCs reflects the quantal release machinery in presynaptic terminals via PK or the exocytosis step itself, independent of propagation of action potentials by neuronal activity in cells near the recording sites (Scanziani et al., 1992; Sillar and Simmers, 1994; Trudeau et al., 1996, but see Patel et al., 2000) Activation of Kir3.1 channels causes hyperpolarization and sequentially reduces action potential-dependent and -independent GABA release by reducing the release probability and neuronal spike firing in GABAergic neurons (Virk and Williams, 2008; Yum et al., 2008; Liu et al., 2012) The influence of hyperpolarization induced by Kir3.1 channels on GABA release might be greater when these channels are activated in more sites or in cells with higher excitability, as indicated by a previous study (Kohno et al., 2005; Xu et al., 2008) In the present study, oxycodone, but not morphine, elicited Kir3.1 channel-dependent inhibition of eIPSCs and mIPSCs frequency in VLPAG neurons The inhibitory effects of oxycodone on eIPSCs were enhanced in the FBC model while those on mIPSCs frequency were not changed Thus, oxycodone more effectively acts on μ-opioid receptors coupled with Kir3.1 channels at presynaptic terminals and/or near the site of generation of the action potentials in the enhanced GABAergic neurons of VLPAG area and reduces the enhanced GABA release in the FBC model As other possible mechanism, we could not exclude the possibility that oxycodone acts on μ-opioid receptors coupled with Kir3.1 channels at postsynaptic GABAergic neuron and reduces its neuronal firing, because eIPSCs were dependent on postsynaptic neuronal activity and Kir3.1 channels are expressed in postsynaptic neurons (Liu et al., 2012) The possible mechanisms remain to be clarified in future studies It was demonstrated that GABAergic synaptic transmission in VLPAG neurons was enhanced in the FBC model and the inhibitory effects of oxycodone on eIPSCs were increased; a previous study with β-arrestin2 knock-out mice demonstrated that an elevation of PKA activity in presynaptic terminals caused an enhancement of GABAergic synaptic transmission in PAG neurons and increased the inhibitory effects of morphine on eIPSCs (Bradaia et al., 2005) To investigate whether the elevation of PKA activity might underlie the enhancement of GABAergic synaptic transmission in the British Journal of Pharmacology (2015) 172 2148–2164 2161 BJP K Takasu et al FBC model, we evaluated the effects of PKA inhibitor H-89 However, H-89 had negligible effects on both GABAergic eIPSCs and the efficacy of morphine and oxycodone in the FBC model Our results indicate that elevation of PKA activity was not involved in either the enhancement of GABAergic synaptic transmission in the FBC model or the effects of the two opioids Furthermore, in contrast to enhanced inhibitory effects of morphine on GABAergic eIPSCs in the β-arrestin2 knock-out mice, the present study demonstrated that the inhibition of GABAergic eIPSCs by morphine was not increased in the FBC model This might be explained by a distinct mechanism of enhancement of GABAergic synaptic transmission between the FBC model and the β-arrestin2 knock-out mice in terms of elevation of PKA activity In the previous study, we demonstrated that the effects of oxycodone on [3H]-DAMGO binding and [35S]-GTPγS activation were attenuated in the FBC model (Nakamura et al., 2013) This seems to be opposite to the enhanced inhibition of eIPSCs by oxycodone in the FBC model In the intracellular signalling, μ-opioid receptors coupled to Kir3.1 activation could be modified by GPCR kinase 2, PKC and PKA (Chen and Yu, 1994; Johnson et al., 2006) The effects of oxycodone on GABA release might be enhanced in the intracellular signalling pathway after the activation of μ-opioid receptors The intracellular mechanisms in the FBC model underlying the modulation of GABA release by μ-opioid receptors coupled with Kir3.1 channels remain to be clarified in future studies In conclusion, we demonstrated that Kir3.1 channels are important for the supraspinal antinociceptive effects of oxycodone, but not those of morphine, in the FBC model As supraspinal mechanisms of oxycodone associated with supraspinal neuronal functional changes, we found that GABAergic synaptic transmission in VLPAG neurons was enhanced in the FBC model and that the inhibitory effects of oxycodone, but not those of morphine, on eIPSCs, were enhanced and dependent on Kir3.1 channels This is consistent with data that the supraspinal antinociceptive effects of oxycodone were dependent on Kir3.1 channels while those of morphine were not Considered together with the wellestablished disinhibition mechanisms underlying supraspinal opioid-induced antinociception (Vaughan and Christie, 1997), the enhanced GABAergic synaptic transmission at VLPAG neurons in the FBC model is an important site of supraspinal antinociception by oxycodone via Kir3.1 channel activation Author contributions K T carried out all electrophysiological studies and wrote the paper K O participated in study design and helped to prepare the paper A N and M H helped to prepare the paper T K and T T carried out the preparation of the FBC model H O carried out behavioural tests in the FBC model Y M conceived of the study and participated in its design M S., T M and T S conceived of the study and helped to prepare the paper G S performed study design and is a corresponding author of this study All authors read and approved the final paper 2162 British Journal of Pharmacology (2015) 172 2148–2164 Conflict of interest K T., K O., A N., T K., H O., T T., Y M., M H and G S are employees of Shionogi Co., Ltd., the manufacturer of oxycodone and morphine References Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013a) The Concise Guide to PHARMACOLOGY 2013/14: G protein-coupled receptors Br J Pharmacol 170: 1459–1581 Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013b) The Concise Guide to PHARMACOLOGY 2013/14: Ligand-gated ion channels Br J Pharmacol 170: 1582–1606 Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013c) The Concise Guide to PHARMACOLOGY 2013/14: Ion channels Br J Pharmacol 170: 1607–1651 Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013d) The Concise Guide to PHARMACOLOGY 2013/14: Enzymes Br J Pharmacol 170: 1797–1867 Banning A, Sjogren P, Henriksen H (1991) Pain causes in 200 patients referred to a multidisciplinary cancer pain clinic Pain 45: 45–48 Basbaum AI, Fields HL (1984) Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry Annu Rev Neurosci 7: 309–338 Becker R, Jakob D, Uhle EI, Riegel T, Bertalanffy H (2000) The significance of intrathecal opioid therapy for the treatment of neuropathic cancer pain conditions Stereotact Funct Neurosurg 75: 16–26 Bercovitch M, Adunsky A (2006) High dose controlled-release oxycodone in hospice care J Pain Palliat Care Pharmacother 20: 33–39 Bradaia A, Berton F, Ferrari S, Lüscher C (2005) β-Arrestin2, interacting with phosphodiesterase 4, regulates synaptic release probability and presynaptic inhibition by opioids Proc Natl Acad Sci U S A 102: 3034–3039 Budai D, Fields HL (1998) Endogenous opioid peptides acting at μ-opioid receptors in the dorsal horn contribute to midbrain modulation of spinal nociceptive neurons J Neurophysiol 79: 677–687 Buvanendran A, Ali A, Stoub TR, Kroin JS, Tuman KJ (2010) Brain activity associated with chronic cancer pain Pain Physician 13: E337–E342 Caillard O, Moreno H, Schwaller B, Llano I, Celio MR, Marty A (2000) Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity Proc Natl Acad Sci U S A 97: 13372–13377 Chen Y, Yu L (1994) Differential regulation by cAMP-dependent protein kinase and protein kinase C of the μ opioid receptor coupling to a G protein-activated K+ channel J Biol Chem 269: 7839–7842 Coleman RE (1998) How can we improve the treatment of bone metastases further? 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Wei-Li Yu and Yun Sun Department of Intensive Care Unit, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China We read with great interest the article by Pastukhov et al (2014a) which reported that NVP-231, the inhibitor of ceramide kinase (CERK), could reduce cell viability, DNA synthesis, colony formation and induced apoptosis of the breast and lung cancer cell lines MCF-7 and NCI-H358 The results suggested that CERK inhibition might be a good potential therapeutic target for breast and lung cancer Moreover, Pastukhov et al (2014b) had demonstrated that proliferation was reduced via CERK inhibition in renal mesangial cells and fibroblasts It suggested that CERK inhibition was a potential target for treating mesangioproliferative kidney diseases Furthermore, Payne et al (2014) had indicated that CERK was required for tumour recurrence and survival and CERK was up-regulated in tumour cells and during tumour recurrence in mouse breast cancer Furthermore, gene expression profiles showed that up-regulated CERK was related to an increased risk of recurrence in women for breast cancer These results suggested that CERK played a vital role in breast cancer recurrence and CERK inhibition might be a potential target for tumour recurrence In addition, Bini et al (2012) had showed that CERK inhibition reduced cell proliferation in human neuroblastoma cells Together, these findings indicated that CERK played a functional role in diseases We read with considerable interest and thought that CERK inhibition might be a good potential therapeutic target for diseases, including cancer, mesangioproliferative kidney diseases and neuroblastoma cells project of Anhui Medical University (Grant No 2015XKJ031) and the doctoral research fund project of the Second Affiliated Hospital of Anhui Medical University (Grant No.2014BKJ034) Conflict of interest The authors declare no conflicts of interest to disclose References Bini F, Frati A, Garcia-Gil M, Battistini C, Granado M, Martinesi M et al (2012) New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells A role for ceramide kinase Neuropharmacology 63: 524–537 Pastukhov O, Schwalm S, Zangemeister-Wittke U, Fabbro D, Bornancin F, Japtok L et al (2014a) The ceramide kinase inhibitor NVP-231 inhibits breast and lung cancer cell proliferation by inducing M phase arrest and subsequent cell death Br J Pharmacol 171: 5829–5844 Acknowledgements Pastukhov O, Schwalm S, Römer I, Zangemeister-Wittke U, Pfeilschifter J, Huwiler A (2014b) Ceramide kinase contributes to proliferation but not to prostaglandin E2 formation in renal mesangial cells and fibroblasts Cell Physiol Biochem 34: 119–133 This work was supported by Anhui Provincial Natural Science Foundation (Grant No 1508085QC49), the school fund Payne AW, Pant DK, Pan TC, Chodosh LA (2014) Ceramide kinase promotes tumor cell survival and mammary tumor recurrence Cancer Res 74: 1–12 © 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