Mechanism of release of norepinephrine f

Life Sai ces Vol 14, pp 1705-1719 PYiated ~ U.S A Pergamon Press MBCHAHISM OF RELEASE OF HOREP~~~~ FROM PERIPHERAL ADREer~olc aEUROa88 Bx THE caLCIVM IoaoPHORES a 537A AxD A 23187 Bguyen B Thos, Jonathan L costs, Jonathan Moes, (l) and Irvin J Supin Laborstory of Clinical Science Aatioael Institute of Mental Health Bethesdn, Maryland 20014 (USA) (Received in final form 26 March 1974) The effects of the calcium ioaophores, % 537A gad A 23187, in causing rüease of norepiaephrine (HE) were ezami.ned in isolated guinea-pig vas deferens and rat atrial segments Examination of calcite dependence, dopamine-ß-hydroxylase (DBH) rüease and rüease of deamdnated metabolites of 1fE suggest that while A 23187 rüeases by both as emoytotic end non-exocytotic mechanism, X 537A rü~ees FE predominantly thxrough a aoa-ezocytotic mechanism The rüative impotence of A 23187 is effecting rüease of HE may explain its inactivity in alteration of cardiac responses The antibiotic ioaophores X 537A (Hoffmann-LaRoche) and A 23187 (ELi Lilly) have been shown to rüease accumulated calcium Prom isolated sarcoplsamic reticulum (1-3) " In addition, they rüease histamine from mast cells (4), serotonin from platüets (5) x 537A, but sot A 23187 (6), increases the rate and force of contraction of isolated perflised rabbit hearts and contracts aortic strips presumably by its action on rüease of aorepinephrine (7,8) Rüease by A 23187 of histamine from mast cells may be secondary to an ioaophore-induced influz of eztracülular calcium (4) Amine rü~ee by X 537A from both mast cells and cardiac sympathetic nerves may represent leakage from intro-vesicular stores (4,7,8) (1)Medical Sciertist Training Program, Fellowship Ao S-TO-S-OM-01678 1705 Ionophore-Induced Catecholamine Release 1706 Vol 14, No A calcium dependent exocytotic release of norepinephrine from adrenergic terminals in the isolated guinea-pig vas deferena can be elicited by electrical stimulation of the adrenergic nerve to the organ and by depolar izing drugs such as hypertonic FCC] and veratridine Such a release is characterized by a concomitant proportional release of soluble dopamine-ß~ydrosylase (DHH), a marker of the soluble contents of the norepinephrine vesicle (9-11) A calcium independent non-exocytotic release of aorepinephrine without release of DBH can be induced by aympathomimetic drugs such ae tyramine or reserpine (12) The studies reported here document release of norepinephrine by % 53TA sad A 2318T from peripheral sympathetic nerves in the isolated guinea-pig vea deferena sad the isolated rat atrium The results suggest that % 53TA releases the amines primarily through a non-exocytotic mechanic, while A 2318T appeera to induce both ea exocytotic and tram-cytopleamic release of norepinephrine Methods Studies with Isolated Ouinea-Pig Vasa Deferentia Male albino guinea pigs (500-T00 g) were killed by a blow on the head and exeanguinated The abdomen xas opened and both uses defereatie xere excised sad placed in cold normal or calcium-free medium The normal medium had the folloxing composition (mM) : ftaCl, 138 ; RC1, 69; CaC1 " 2H20, 04 ; Mg804 " TH2, 1.45 ; KH 2P04 , 19 ; and dextrose, 11 10 The calcium-free medium vas composed of normal medium from xhich CaC1 " 2H2 xae omitted The media xere adjusted to pH - T before each experiment Folloxing eaci- eioa, the vase defereatie were xeahed five times with cold medium, then preincubated for five minutes in medium containing 25x bovine serum albumin Vol 14, No Ionophore-induced Catecholamine Release (Sigma Chemical Compeay, 8t Louis, àlo ) 1707 When used, tetrodotozin (5 Wd), xae already present in the preincubation medium Folloxing preincubation, the organs xere transferred to 20 ml beakers to xhich had been added ml of medium containing bovine serum albumin (0 25x), 50 uM pheaozybenzemine-HC1 to block reuptake of released norepiaephrine, and one of the ioaophores studied Ionophores xere added as mM (% 53ỴA) or mM (A 23187) solutions in ethanol and similar quantities of the ethanol vehicle were added to the control media Unless mentioned, the incubation continued for eizty minutes at 37 ° C in a Dubnoff incubator xith slight shaking under an atmosphere of 95x 02 and 5f C0 During the incubation, the pH xas maintained at q 2-7 Folloxing the sizty minute incubation, norepinephrine concentration end dopamine-ßhydrozylase activity xere determined in the medium Portions (200 W-) of each incubation medium xere used to assay DBH by the method of Moliaoff, et al using tyramine as a substrate (13) Opt i+~l DHH activity in the bath medium xas obtained in the presence of 20 uM copper sulfate to inactivate endogenous inhibitors DBH activity xas espreeeed ee nM of octapamiae formed per gram of tissue incubated in the bath per hour The remaining ml of each incubation medium xas acidified xith ml of 10 H perchloric acid and centrifuged at 4°C for ten minutes at 27,000 x g in a Sorvall refrigerated centrifuge Sodium metabiaulfite 100 mg, domina 400 mB, and 10 ml of 2x F9TA xere added to each supernatant and the pH xae ad.~uated to xith AeOH The norepinephriae xas adeorbed on to 400 mB alumina columns by the method of Anton and Sayre (14) The columns xere xashed once xith 10 ml of sodium acetate 0.2N (pH 8.6) and then txice xith 10 ml of glass distilled xater The catecholamines xere eluted xith ml of 0.2 A acetic acid Oae ml portions of each elutee xere used for the flnorometric determiner tion of norepinephrine (15) 1708 Vol 14, No Ionophore-Induced Catecholamine Release Studies xith Isolated Rat Atria Adult male SpregurDaxley rate (Hormone Assay Laboratories, Chicago, I11 ) neighing from 180-220 grams were killed by e blow on the head The right and left atria were removed, classed and divided into sections neighing about 10 mg each Each segment vas preincubated for thirty minutes at 37 ° C in separate vials containing ml of a modified Kreba-Ringer bicarbonate solutioâ (pH " 35) supplemented xith 200 mg/liter ascorbic acid and grams/liter of dextrose sad containing 24 1tM D,L-norepinephrinr7- 3H (6 48 ci/mmol) England ftuclear Corp , Boston, Mass ) (Aew The atriel segments were xashed for ten minutes is vials containing ml of Ringer's solution (with or xithout drug present) at 37° C, following nhich the tissues xere placed in vials containing ml Ringer's solution at 37 °C with the ioaophoree and/or drug at the indicated concentrations (see above) Ionophorea xere added as solution is ethanol After ten minutes, the tissue vas removed sad Bozen until homogenized and the media assayed for released tritium In those ezperime~s in which the effects of drugs on ioaophorrinduced release of 3H-AE were assayed, the drug xas present in the washing medium as well as in the ionophoxecontaining teat medium 0.4 A perchloric acid Each atrial segment vas homogenized is 500 ul of After centrifugation at 8000 x g for ten minutes, aliquots of the clear supernatant riuid xere aseavyed for radioactivity by liquid scintillation apectramietry Deeminated metabolites were measured by separation on Dovez AG-50 as previously described (16) The following drugs xere used : bovine serum albumin from Sigma Chemical Compaq (St Louie, Missouri) ; pheno~benzamine HC1 Pram with Kline & French Laboratories (Philadelphia, Pennsylvanie) ; veratridine from Aldrich Chemical Vol 14, No Ionophore-Induced Catecholamine Release 1709 CaorpaRy (Milwaukee, Wisconsin) ; tetrodotozin and tyremine from Calbiochem (Los Angeles, Calif.) ; ethylene bis (o~grethylenenitrilo) tetraecetic acid (EGTA) from Ciba~Geigy Pharmaceuticals (Ardsley, Acv York) ; cocaine from Mallinkroft Chemical (St Louis, Missouri) A 23187 was the kind gift of Dr R Hosley (Eli Lilly) and X 537A was the kind gift of Dr J Herger (Hoffmana-LaRoche) Statistical Analysis The statistical significance of the differences between means of various treatments was determined by 8tudeat's t test (17) Results Rüease of endogenous norepinephrine (NE) from the vas deferens (Fig 1) Both a 537A (lo uM) sad A 23187 (loo uM) caused a rüease of fE from vesa deferentia The release wes proportional with the time of incubation (15,30 and 60 min.} X 537A rüeased about three times as much AE as did A 23187, although its concentration was 10-fold lover Rüease of ~ from atrial segmente Rüease of tritium Pram atrial segments was dependent on the coacentration of the ioaophore present in the medium (Fig 2) more potent than A 23187 X 537A was 100-fold M.=i^^n^ rüease of ~ by X 537A was approzi- mately 70x of the ~-NE taken up by atrial segments Significant release of tritium could be observed at coace~rations of X 537A as lev as 0.3 1~M Rüease by A 23187 could not be ascertained at concentrations above 200 uM because of the rüative insolubility of the ionophare Significant release of tritium by A 23187 could be seen at concentrations above 30 uM Spontaneous rüease during a ten minute incubation xas alwawys less than lOK of the tritium taken up by the atrial tissue X 537A (3 titèi) sad A 23187 (100 }tM) both produced a time dependent rüease of tritium (Fig 3) 171 Ionophore-Induced Catecholamine Release Vol 14, No  0.8 P w z P 0.4 0.2 13 30 45 60 MIN FIG Time dependent release of norepinephrine from isolated vase deferentis by campounde 7C 53TA (10 yM) and A 23187 (100 uM) Groupe of vara deferentia xere incubated xith either drug for 15 ; 30 ; or 60 minutes Each point represeate the mean + (S E Di.) of at least organe 70 60 90 W N Q W ~ 40 w ;t 20 10 FIG The effect of ioaophore concentration on release of tritium from atrial segments Data are presented as the per cent of total radioactivity present prior to incubation that ie released during the ten minute incubation Values Spontaneous release represeats repreeeat mean + (S E M ) for 5-18 samples lees than lOx of radioactivity Vol 14, No IonophorrInduced Catecholamine Release 1711 100 so W ao i WC W 201 i i i 10 20 MINUTES i SO FIG The effect of incubation time on ioaophore induced release of tritium from Data are presented as the percentage of total radiosetiatriel segments vity retained during indicated incubation times Ionophores are present ea follows : A 23187 (100 uèi) and % 537A (3 11M) " Values represe~ mean _+ (S E M.) for 5-18 samples Rüease of 1PE and dopaminrß-l~ydrozyleae (DHH) from the vas deferens (Table 1) Vela deferentia were incubated for 60 with either ionophore and the incubation media examined concomitantly Hoth ionophdres released lYS sad DBH However, when the ratio of NS/DHH was ezemined, only A 23187 released these two substances is a proportion similar to that seen with hypogaetric nerve stimulation or to that found is the soluble fraction of vase defereatia homogenates of DBH % 537A released HE in ezceas The ratio of AE/DBH wen fold greater than that observed with hypogastric stimulation Increasing the dose of % 537A to 40 ~ caused a further increase in the RL/DBH ratio in the release media because there was increased HE rüeese without e significant change in release of the easyme 1712 Ionophore-Induced Catecholamine Release Vol 14, No TARi F Release of Aorepinephrine (NE) and Dapemiae-ß-hydro~rlaee (DBÜ) from Vasa Deferentia by X 537A and A 23187 Drug A 23187 (loo uM) aE (ng/g) DHH (nM/g/hr) ~/DBIi 45 8.98 + 27 x 537A (lo ti.~M) u45 + l28 95 + 0.35 137 + 15 x 537A (4o uM) 1696 + 17 + 1.26 191 + 23 399 ± 36 44 + Values represent the mean _+ (S E M ) from groups of at least organs Ratio of AE/DBH released by electrical stimulation was 49 _+ Ratio of AE/DHH is soluble fraction of vase deferentie (9) homogenates was 28 + (9) " Effect of calcium on the release of ftE and DHH iram the vas deferene (Fig 4) Incubation of vase deferentie for 60 in a medium fry s~hich calcium was emitted produced a slight decrease in spontaneous release of NE but not of DBH In calcium-free medium, the DBR releasing effect of A 23187 was completely blocked and ire AE releasing effect was markedly decreased Oa the other hand, both DBH and NE releasing effects of X 537A remained unaltered when calcium was not present in the medium Vol 14, No Ionophore-induced Cntecholamine Release NO DRUG A Y31é1 1713 X 6b7A FIG Effect of % 53TA (10 uM) sad A 2318 (100 uèi) on DHH and AE release from guinearpig vase deferentia in calcium-tree incubation media Each bar represents the mesa + (S E M ) of at least or8ans " NE is expressed as hB/g/hr and DBH as nM octopamine (unite)/g/hr a = p < 05 1Po Ca++ group versus Cam group b = p < 01 No Cn~ group versus Cam group c = p < 01 drug treated group versus corresponding control group Metabolites Z 537A caused e release of tritiated metabolites similar to that of reserpine With both these agents, (Table 2) a high percentage of the increment in tritium release wes present as deami.nnted metabolites The percentage of deaminated metabolites in the increment of tritium released by A 2318T was significantly less they that found after release produced by veratridine or tyremine Reserpiae or lC 537A, hovever, resulted in release of a greater proportion of deeminated metabolites than A 23182 1714 Ionophore-Induced Catecholamine Release Vol 14, No TABLE Effect of Drugs on Release of Total Tritium and Amines from Atrial Segments x Radioactivity Released Released Amine Radioactivity is Increment x No Drug 6.9 _+ x 537A (2 UM) 50 + a.5 - + l 46 _ 50 + _ 1.3d A 23187 (100 uM) 28 _+ 47 _+ 2.8 63 _+ Reserpine 23 _+ a=5 31 _+ 1.3 52 _+ 6c Veratridine (5 WQ) 50 _+ 1.0 n~5 81 _+ 2.4 101 _+ 8e Tyremine (10-4M) 29 + 1.8 a=5 - 77 + _ l.7 100 _ + 2.8e (5 x 10-~M) n~5 Amine x Total Radioactivity a=5 21 _+ 4 (a) Samples were incubated with [~H]norepinephrine, and release of total radioactivity and of deaminated products vas determined ae described in Methods The differences betveen total tritium ~ad deeminated products vas assumed to reflect release of amines The increment in total radioactivity vas expressed as a percentage oP the release of triti~ in the absence of drugs, and the amines released were expressed The increment is amine release as a percentage of the total released vas calculated by subtracting the amount of tritiated amine released in the absence of the drug from that released in the presence of the drug This difference was then expressed as a percentage of the amine Values above 1002 were achieved released in the absence of the drug by an absolute reduction in deeminated metabolites during induced releasa (b) Data ere expressed as mean ±(S E M ) for indicated number of samples (c) p < O1 reserpine induced release versus A 23187 induced release of amines (d) p < 001 reserpine induced release versus X 537A induced release of amine (e) p < 001 veratridine and tyramine induced release versus reserpine, A 23187, and x 537A induced release of amines 1715 Ionophore-induced Catecholamine Release Vol 14, No f Effect of cocaine and tetrodotozin on ionophore-induced release of tritium from atrial segments Neither cocaine (10 ug/ml) nor tetrodotozin (2 UM) ezerted a significant effect on X 53TA- or A 23l8ỵ-induced release of tritium (Table 3) Tetrodotoain (5 W~) also failed to block ionophore-induced release of endogenous NE Pram vase deferentia (unpublished observations) TABLE Effects of Tetrodotozin and Cocaine on Ionophore Induced Release fry Rat Atrial Segmente Control X 53TA (3 lam) A2318T (100 utQ) ao ~ + 0.6 a=9 51 + 1.6 34 ± a~18 T17C (2 x 10-6M) 8.6 _+ 0.6 n~5 54 _+ 3.5 n~5 32 _+ 2.1 n=5 Cocaine (10 ug/ml) 9"1 _+ n-5 49 _+ 1" n~5 29 _+ 2.4 n~5 n=5 Data are expressed as the mean _+ (S E M ) for the indicated number of samples Discussion The ianophores X 53TA and A 2318T are ]mavn to form lipid soluble camplazas with monovalent and divalent rations, facilitating their passage across lipid barriers They were shove to be effective in transporting calcium across biological membranes (8) Because of the importance of Cam in the phenomenon of ezcitation-accretion coupling at autoaomi.c nerve endings (18-21), the effect of these ionophorea on release of biogenic amines has been studied Both X 53TA and A 23187 have been shown to release histamine from meat cells (4) 1716 Vol 14, No Ionophore-Induced Catecholamine Release Of interest, hoxever, ie the fact that the rüease induced by A 23187 is calcium dependent while % 53TA causes calcium independent rüease Hoth ioaophores have also been shown to ezert norepiaephrine-like effects in isolated cardiovascular tissues (8) We have studied the effect of both % 53TA and A 23187 on release of norepinephrine from peripheral adrenergic nerve terminals The model systems employed were the isolated vas deferens of guinea pig which has been used effectively es a modü of release of norepinephrine by ezocytosis (9-12 ; 2223) and the rat atriel segments preloaded with tritiated norepinephrine (24) % 53TA and A 23187 release 1PB and ~-HE from these preparations It is clear from the dose-response curves that peripheral adreaergic neurons differ considerably fre~m mast cells in their susceptibility to the ionophores A 23187 was much leas potent than % 53TA in causing RE rüease from the heart, strial tissue or vas deferens, which mqy ezplaia the relative inactivity of A 23187 on cardiac coatraction(6~) This contrasts with the mast cell where A 23187 is a more effective releasing agent Whether this difference reflects differences in affinity for the ionophore between the mast cell sad neuronal membrane ie not clear A 23187 releases both HE and DBH Prom the vas deferens The ratio of HS/DBH rüeaeed ie similar to that found following electrical stimulation of the hyypogastric nerve to the vas deferens and observed in the soluble fraction of uses deferentia hamogeaates (9) Since a proportional release of liE sad DBH is an iodez of release by ezocytosis (9,25,26), it appears that one of the mechanisms of AE release by A 23187 is by ezocytoeia dependence is also an iodez of release by ezocytosis (10,23) Calcium The rüease of both AS and DHH Pram vase deferentia by A 23187 and the release of ~-AE Vol 14, No Ionop~hore-Induced Catecholanina Release 1717 Pram atrial segme~s (unpublished observations) were found to be markedly reduced when calcium wee omitted tram the incubation medium Oa the other hand, % 537A releases H~ and DBH from the vas deferens sad 3H-HE from atrial segments even when calcium is omitted 81nce the ratio of 1D~/DBH increases with increasing dose of ionaphore, it appears that Z 53TA rüeeaes PE predominantly through a non-ezocytotic mechanism Reser- pine hoe been shown to enuee rüease of a high percentage of deamiaated metabolites from both central and peripheral adrenergic neurone (24,2q) Drugs which cause depolarization of neuronal membranes, such m veratridine and scorpion ta~zin cause rüease primarily of amine (24,2T) presumably because they generate release of the vesicular contents directly to the ezterior of the cell rithout ezpoeure to monoamine ozidase Tyramine, which releases AT indirectly, is as ezception to this hypothesis,pres~ably because it inhibits monoamine ozidase(28) R 537A is much like reserpiae in causing rüeaee of deamiaated metabolites When one subtracts spontaneous release, 50x of the tritium released by Z 537A ie present as deamiaated metabolites This contrasts with verntridiae and tyramine which release almost ezclusivüy amines A 2318q causes a release of deaminated metabolites midweyy betreen reserpine and veratridiae Furthermore, absence of calcium does not camplete]y inhibit its HE releasing effect These observations suggest that A 23187 has camponeate of direct and indirect rüease The 3H-FE and NE releasing effect of neither % 53TA nor A 2318T were affected by tetrodotozin, which blocis the treasie~ sodium coaductance(29) veratridine and scorpion tasin(11,24) and bloc]ce rüease by The fact that both ionophores were also unaffected by cocaine, an effective bloc]cer of 1fE reuptake, suggests that the primary effect of the drugs is on release rather than uptake of AE Ionophore-induced Catecholamine Release 1718 Vol 14, No While the exact molecular mechanism by which these ionophores cause adreaergic discharge is not ]moan, it is clear that they act by different mechanisms A 23187 causes release of norepinephrine main]y by a calcium dependent e:ocytotic mechanism while X 537A releases the amine predaminant]y by an indirect, reserpine-liàe mechanism Ac]cnor~led~enta We thanà Mrs P Cover and T Cridlin for their ca~mpetent technical assistance with the experiments References A SCARPA, J BALDASSAitE, and G INEâI, J Gea siol 60, 735-749 (1972) A.H CASWELL and H.C PRESSMAN, Biochem Hio s Res Commun 4~, 292-298 (1972) M.L ENTMAN, J C ALLEN, E.P BORNET, P.C GILLETTE, E.T WALLICK, and A SCHWARTZ, J Mol Cell Cardiol 4, 681-687 (1972) J C FOREMAN, J.L MOHGAR, and B.D GOMPERTS, Rature 2~, 249-251 (1973) J.L C03TA and D.L MURPHY (in preparation) J.V LEVY, J A CORED, sad G INESI, Rature 242, 461-463 (1973) A SCHWARTZ, R.M LEWIS, H G HAlYLEY, R.G MUNSON, F D DIAL 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389-428 (1970) 21 S M SIFtPEBAR and Y MISU, J 22 A B THOA, G F WOOTSA, J A70;GROD, and I J IflOPIA, Proc Aat Acad Sỗi siol ( Londoa )~ 20~, 689-706 (1969) siol ( London ) 188, 219-234 (1967) U S A 6Q, 520-522 (1972) 23 G F WOO'l'EA, A B THOA, I J SIDPIA and J A7Q:LROD, Mol Pharmacol Q, 178-183 (1973) 24 J MDSS, A B THOA, and I J ~PIA, J Pharmacol ~ Ther (ia press) 25 " O H VIPEROS, L ARQUER08, R J COAAÛ'1T, aad A KIRSHAER, Mol Pharmacol ~, 69-82 (1969) 26 L B GBFFEA, B G LIVLRiT, and R A RUSH, J siol ( London ) 204, 58P-59P (1969) 27 J MOSS, R COLHURA, and I J KOPIA, J Aeurochem (in press) 28 C B SMITH, J Pharmacol ~ Ther 151, 207-220 (1966) 29 T, NeaeRe~gl, J W MOORS, and W R SCOTT, J Gea (1964) siol ~, 965-974 ... ) from groups of at least organs Ratio of AE/DBH released by electrical stimulation was 49 _+ Ratio of AE/DHH is soluble fraction of vase deferentie (9) homogenates was 28 + (9) " Effect of. .. by The fact that both ionophores were also unaffected by cocaine, an effective bloc]cer of 1fE reuptake, suggests that the primary effect of the drugs is on release rather than uptake of AE Ionophore-induced... the release of ftE and DHH iram the vas deferene (Fig 4) Incubation of vase deferentie for 60 in a medium fry s~hich calcium was emitted produced a slight decrease in spontaneous release of