Role of oestrogens in male erectile function 3

Chapter PHARMACOLOGICAL EFFECTS OF OESTRADIOL AND PHYTOESTROGEN (DAIDZEIN) ON RABBIT CORPUS CAVERNOSAL SMOOTH MUSCLE IN VITRO 108 6. PHARMACOLOGICAL EFFECTS OF OESTRADIOL AND PHYTOESTROGEN (DAIDZEIN) ON RABBIT CORPUS CAVERNOSAL SMOOTH MUSCLE IN VITRO 6.1. Objectives Pharmacological studies on penile cavernosum have greatly improved our understanding of the erectile process and the pathophysiology of erectile dysfunction (Adaikan et al., 1991a). During penile erection, the two haemodynamic processes viz., increased arterial inflow and restricted venous outflow are well-coordinated with the corpus cavernosal smooth muscle (CCSM) relaxation and inhibited contraction. This system is generally accepted to be under neuroregulatory control and involves the adrenergic, cholinergic and non-adrenergic and non-cholinergic systems as described earlier. However, biochemical substances including hormonal activity can modify these functions in their own specific ways. Therefore, the primary objective of this series of investigation was to characterise the direct in vitro tissue responses of oestradiol and phytoestrogen daidzein on the receptoral affinities to corpus cavernosal contractile and relaxant agents and to compare these effects with the response of tissue strips from different treatment groups of rabbits to these agents. To more completely evaluate the cavernosal smooth muscle reactivity in these experimental states, isometric tension studies were performed on isolated strips of CC. This is in an attempt to determine if some of the effects observed in vivo could be explained more clearly from changes at the receptoral level in vitro. As such, it is conceivable that a detailed pharmacological analysis of corporal tissue is required to reveal significant hormone-mediated alterations on these pathways. 6.2. Materials and Methods Genetically pure-bred strain of New Zealand White (NZW) healthy male rabbits were utilised in this investigation. All the animals used in the investigation were supplied and 109 maintained by the Laboratory Animal Centre, Animal Holding Unit, National University of Singapore, Singapore. They were housed in a stable air-conditioned atmosphere with adequate daily care and ready access to standard rabbit diet (pellets) and water. 6.2.1. Animal Groups Two groups of rabbits (n=6, each) acted as untreated controls for acute incubation studies with oestradiol and phytoestrogen. They were housed under stable conditions for week prior to experimentation. Rabbits belonging to the oral treatment groups (also n=6 each) received 0.1mg of oestradiol valerate (Progynova, Schering) or phytoestrogen, soybean isoflavone daidzein (Sigma) 0.01mg or 0.1mg as suspensions in water (fixed volume of 1ml) through oral gavage daily for 12 weeks. Control rabbits of this study consumed the same volume of the vehicle by gavage daily. The different treatment groups have been described in Table 2. 6.2.2. In Vitro Experimental Design 6.2.2.1. Collection and Storage of Penile Tissue The rabbits from different experimental groups were sacrificed with an inhalational overdose of carbon dioxide in a sealed gas chamber connected to carbon dioxide tank. The penile tissue removed in toto from the surrounding tissues was transferred immediately into freshly prepared Tyrode’s solution. The storage time was kept to the minimum and in vitro experiments begun within one hour after removal of the specimen. The tissue from each animal yielded four strips of corpora cavernosa which were adequate for testing various parameters including responses to agonists and electrical stimulation. In order to have adequate time for conducting in vitro experiments in the laboratory, the experimental protocol was adjusted in such a way that the treatment of different groups of animals was done at intervals, over a period of time. 110 6.2.2.2. Preparation of Isolated Strips of the CCSM Through fine dissection, the penile tissue was separated from the surrounding tissue remnants. The corpus spongiosum was removed and discarded and the tunica albuginea slit open through a midline incision. The corpora cavernosa were carefully dissected from the closely adherent tunica and the surrounding fascia. During the dissection, adequate care was taken to bathe the tissue in fresh Tyrode in order to prevent apoptosis. 6.2.2.3. The Organ Bath Four 0.4-0.6cm long strips were cut from the corpora cavernosa and secured vertically in 25ml organ baths containing freshly prepared Tyrode’s solution. The physiological solution was adequately aerated with carbogen (95% oxygen and 5% carbon dioxide). The buffer in the solution (sodium dihydrogen phosphate 2H2O) maintained the pH constant at 7.4 whereas the thermostatic control in the circulator pump (Churchill) minimised fluctuations and maintained the temperature constant at 37°C. The Tyrode’s solution provided the milieu required for optimal tissue responses and had the following composition (Table 10). The organ bath set up used in the study is shown in Figure 37. Constituent Quantity (mM) sodium chloride 136.9 glucose 5.6 sodium bicarbonate 11.9 potassium chloride 42g/L 199.9 magnesium sulphate 7H2O 38g/L 259.9 calcium chloride 2H2O 32g/L 264.9 sodium dihydrogen phosphate 2H2O 6.5g/L 0.04 Table 10: Chemical Composition of Tyrode’s Solution 111 Figure 37: The Organ Bath Set up for In Vitro Pharmacological Studies 6.2.2.4. G Tension and Equilibration The smooth muscle strips were allowed to equilibrate for 60 minutes at the organ bath conditions including an initial tension of 1.5g. During this equilibration period, the resting tone decreased progressively to stabilise between and 1.2g. In addition, the bath fluid was replenished periodically. 6.2.2.5. Contact Time and Dose Cycle for Drugs The contact time and dose cycle for drugs were kept constant for all experiments. In case of noradrenaline, the drug contact time consisted of 2-5 minutes and the cycle time, 45-60 minutes. For acetylcholine and nitroglycerine, they were 1-2 minutes and 30 minutes respectively. The duration of contact (incubation) for the oestradiol and daidzein was 60 minutes and that required for unmasking nitrergic relaxation, 30 minutes, for all the experiments. The tissue strips were washed twice with Tyrode’s solution after each drug. 112 6.2.2.6. Electrical Field Stimulation Transmural stimulation of rabbit corpus cavernosal smooth muscle strips evoked nerve action potential that was observed as a relaxant response in a precontracted tissue. Through a preliminary study on CC strips from control animals, the ideal frequency, duration and voltage that brought about the optimal response in the tissue were ascertained and then adhered to. Isolated strips of corpora cavernosa were mounted between two platinum wire electrodes (28mm long and 8mm apart) in 25ml organ baths containing the Tyrode’s solution. A Grass S88 stimulator was used to provide square wave pulses that stimulated the CC strips for 10 seconds at minutes intervals. 6.2.2.7. Drugs and Dilutions Stock solutions of the drugs were stored at -20°C. They were diluted to the required concentration with distilled water / physiological saline (0.9% w/v) just prior to use, on the day of experimentation. All the concentrations of drugs were expressed as the final concentration of the agent in the organ bath fluid. The chemicals and drugs used in the study and their sources are listed in Appendix 3. 6.2.3. Isolated Tissue Experiments 6.2.3.1. Response to Noradrenaline Adrenergic (contractile, excitatory and anti-erectile) response of CC strips was obtained through cumulative additions of increasing concentrations of noradrenaline (NA). Complete concentration-response graphs were obtained in all the strips with the drug concentration ranging from 0.24µM to 1052µM. The effective concentration that produced 50% of the maximum contractile response (EC50) was calculated for each strip. The contractile effects of NA at different concentrations were measured as the peak 113 tension of the tonic contraction developed when the strip was exposed to the neurotransmitter and the response expressed as a percentage of the maximum contraction. 6.2.3.2. Responses to Acetylcholine and Nitroglycerine The relaxant, inhibitory (pro-erectile) responses to acetylcholine (ACh) and the directly acting NO donor, nitroglycerine (NTG) were recorded after adequate precontraction of the CC strips with the EC50 of noradrenaline. The effects of different concentrations in the range of 0.22µM to 659µM of ACh and 0.18µM to 78µM in case of NTG were studied and compared with those obtained in different experimental groups. The relaxant response to both the agents was expressed as the percentage of the tone secondary to the EC50 of noradrenaline. 6.2.3.3. Nitrergic Neurotransmission Nonadrenergic, noncholinergic (relaxant, inhibitory and pro-erectile) responses (NANC) of the CC strips to electrical field stimulation (EFS) were recorded at frequencies 2, 5, 10, 20 and 40hz with 90v square waves for 1ms duration and 10 seconds exposure. Prior to this, the CC strips were incubated with 3.5µM of atropine and 5.1µM of guanethidine for 30 minutes in order to block the contractile cholinergic and adrenergic responses and to unmask the relaxant response. 6.2.3.4. Incubation with Oestrogen and Phytoestrogen In order to characterise the direct response of the CC strips to oestradiol and the naturally occurring phytoestrogen daidzein, the CC strips were incubated with 2.5µg of the respective agent (100ng/ml) for 60 minutes and the full concentration-response to each neurotransmitter and modulator repeated and recorded. The time taken for tissue recovery after thorough clearance of the agent through repeated washing with Tyrode’s solution was also recorded at the end of each experiment. Further to this, the direct concentration114 response to ascending doses of 10ng/ml, 100ng/ml, 1µg/ml and 10µg/ml of oestradiol and phytoestrogen were studied after adequate precontraction of the CC strips with the EC50 of noradrenaline. 6.2.4. Data Analysis Data was analyzed by univariate analysis of variance and Tukey’s test for multiple comparisons. All the results were expressed as mean ± SEM and levels of P[...]... of these earlier studies involving castration or low testosterone looked at E2 levels or the independent changes brought about by E2 increase Phytoestrogens vary from oestradiol in their receptoral affinities (Kostelac et al., 20 03) , for eg daidzein binds more readily than 133 oestradiol or even genistein to ERβ Additionally, they have multiple nongenomic effects including inhibition of tyrosine kinase... stimulation in the chronic study Taking into consideration the relative importance of nNOS compared to eNOS as the critical 134 isoform in the mediation of penile erectile function (Cashen et al., 2002), the results in this investigation of oestradiol and daidzein induced impairment of NO-NANC response and the reduced sensitivity of erectile tissue to nitroglycerine (NO donor) for the final common... incubation effect of increasing concentration of oestradiol valerate on the noradrenaline precontracted tone of cavernosal strips from normal rabbits in 4 organ bath chambers 124 NA 9.46µM 1 minute PE:250ng 2.5µg 25µg 250µg Figure 48: Concentration – Response to Daidzein Sample tracing from the study on acute incubation effect of increasing concentration of phytoestrogen daidzein on the noradrenaline... strips (Figure 40) NA 9.46µM 1 minute 0.22 0.88 3 10 32 97 220 659 Figure 40: Typical Tracing of Rabbit Cavernosal Response to Acetylcholine Sample tracing of the relaxant (pro -erectile) response of noradrenaline precontracted cavernosal strips from control rabbit to increasing concentrations of acetylcholine (µM) The relaxant response to directly acting NO donor, nitroglycerine was studied on CC strips... dominant role in mediating penile erection; this sequentially involves sexual arousal, NO release 131 from NANC neurons and endothelium within the CC, NO activation of soluble guanylate cyclase, increase in cGMP and cGMP-mediated relaxation of the penile erectile tissue (Nakane, 20 03) The peripheral effects of sex hormones on these functions of the penile smooth muscle have not been fully understood Erectile. .. relaxations in the treatment groups With the evidence for decreased NANC-nerve fibre density and neurotransmitter staining, generalized changes in autonomic reactivity and higher basal catecholamine concentration in the CC of the elderly (Wespes, 2002), age-associated changes of erectile function may consist of increased dominance of α1-adrenergic activity (increased corporal smooth muscle tone) with declines... 6 .3. 3.1 Response to Noradrenaline There was a trend towards increasing sensitivity and contractility (leftward shift) with the higher concentrations of NA used This increase in potency was accompanied by a 5.9±0.2% and 29.7±0.2% increase in maximal g tension generated in the groups of oestradiol and daidzein treated rabbits compared with the control groups (Figure 49) 6 .3. 3.2 Responses to Acetylcholine... accordance also with increased vascular smooth muscle sensitivity to catecholamine and α-adrenergic affinity in oestrogen treated male rats (Colucci et al., 1982) and the impaired coronary flow response to ACh in human males in the presence of 17-β oestradiol (Collins et al., 1995) There was a significant potentiation of ACh mediated relaxation during preincubation with daidzein in this investigation Oestradiol... 20 03) and nNOS (Fadel et al., 20 03) activities while inhibiting iNOS in bones (Cuzzocrea et al., 20 03) Furthermore, while testosterone upregulated both eNOS and nNOS isoenzymes in the CC (Marin et al., 1999), oestrogen down-regulated nNOS activity in the female genital tract (Min et al., 2001) In the present investigation, oestrogenic effect (oestradiol and daidzein) impaired the NO-mediated proerectile... 1988) indicating the involvement of neuronal response NA 9.46µM 1 minute 2Hz 5Hz 10Hz 20Hz 40Hz wash Figure 42: Typical Tracing of Rabbit Cavernosal Response to Electrical Field Stimulation Pro -erectile nitrergic (NANC) relaxation response of noradrenaline precontracted cavernosal strips from control rabbit during electrical field stimulation with increasing frequencies 6 .3. 2 Acute Incubation Study 6 .3. 2.1 . experiments. In case of noradrenaline, the drug contact time consisted of 2-5 minutes and the cycle time, 45-60 minutes. For acetylcholine and nitroglycerine, they were 1-2 minutes and 30 minutes. were incubated with 3. 5µM of atropine and 5.1µM of guanethidine for 30 minutes in order to block the contractile cholinergic and adrenergic responses and to unmask the relaxant response. 6.2 .3. 4 1 minute 0.22 0.88 3 10 32 97 220 659 Figure 40: Typical Tracing of Rabbit Cavernosal Response to Acetylcholine Sample tracing of the relaxant (pro -erectile) response o f noradrenaline