Effects of Nitric Oxide-Related Agents on Opioid Regulation of Rat Testicular Steroidogenesis"

Transcription

1 BIOLOGY OF REPRODUCTION 54, (1996) Effects of Nitric Oxide-Related Agents on Opioid Regulation of Rat Testicular Steroidogenesis" Michael L. Adams, 2 Edward R. Meyer, and Theodore J. Cicero Department of Psychiatry, Washington Ulniversity School of Medicine, St. Louis, Missouri ABSTRACT These studies examined whether nitric oxide (NO) mediates opioid suppression of testicular steroidogenesis. Adult male rats were treated with various combinations of a NO synthase (NOS) inhibitor (NG-nitro-L-arginine methyl ester; NAME), a NO donor (isosorbide dinitrate; ISDN), an opioid agonist (morphine), and an opioid antagonist (naltrexone). Serum LH and testosterone and testicular interstitial fluid (TIF) testosterone concentrations were then measured. Inhibition of NO production by NAME reversed morphine-suppressed testosterone secretion; treatment with the NO donor, ISDN, reversed naltrexone-stimulated testosterone secretion. NAME did not alter morphine's effects on LH secretion and attenuated morphine's suppression of hcg-stimulated testosterone secretion, indicating that these effects occur directly in the testes and are not dependent on LH secretion. Even though these effects suggested possible interactions between NO and opioid systems, no additive or synergistic effects were found with suppressive combinations of morphine and ISDN, or with stimulatory combinations of naltrexone and NAME at doses that had little effect on testosterone secretion when given alone. These results indicate that opioid and NO exert independent effects on testicular steroidogenesis through separate pathways or mechanisms and that NO does not mediate opioid-induced testicular suppression. INTRODUCTION Nitric oxide (NO) is an intercellular messenger [1-3] formed by NO synthase (NOS) from L.-arginine [4, 5]. It functions as a neurotransmitter in the central and peripheral nervous systems, but it is a small, gaseous, and reactive molecule. Its biosynthesis, biodisposition, and mechanisms of action differ from those of other neurotransmitters. Some major actions of NO result from binding to the iron atom in the heme group of guanylate cyclase, which stimulates the production of cyclic guanylic acid [4, 6]. The role of NO in a diverse range of physiological and pathological functions is studied with substrates and inhibitors of NOS [7, 8] and nitrovasodilators that produce or donate NO [9, 10]. The present studies examined whether NO produced in vivo in male rats interacts with opioids to suppress testicular function. NOS has been localized to the vascular endothelium of the male rat testis and to other reproductive organs [11], suggesting that NO might influence testicular secretory function. We have reported evidence that NO inhibits testosterone secretion in rats [12, 13] and that NOS inhibition reverses alcohol-induced suppression of testicular steroidogenesis [14]. We have also reported that morphine suppresses two major regulatory aspects of testicular function, i.e., testosterone secretion and testicular interstitial fluid (TIF) formation [15], as does alcohol [16, 17]. This evidence Accepted January 12, Received April This Aork wias supported by grants AA-09222, AA-071t4, and AA-07t66 from the National Institute on Alcohol Abuse and Alcoholism and DA from the National Institute on )rug Abuse. T I.C is the recipient of a Research Scientist Award (DA-0095) from the National Institute on )rug Abuse. 2 Correspondence: Michael Adaims. Ph.1), Washington tuniversity School of Medicine I)epartment of Psychiatry. Box 8134, 4940 Children s Place. St Loulis MO FAX: (31 ) suggests that NO could be a mediator of morphine's suppression of testicular steroidogenesis and that NOS inhibitors might specifically block the effects of morphine on testosterone secretion through decreasing NO production. If this suggestion is true, then it can be predicted that 1) blocking NOS would inhibit opioid effects; 2) increasing NO concentrations would block opioid antagonist effects; and 3) the suppressive effects of an opioid agonist would be additive or synergistic with the effects of a NO donor, and the stimulatory effects of an opioid antagonist would be additive or synergistic with the effects of a NOS inhibitor. The present studies were designed to examine these issues. Secretion of testosterone into the general circulation and into TIF, and the formation of TIF, were used as measures of testicular function [18]. Testosterone concentrations in TIF directly measure the biosynthesis, release, and bioavailability of testosterone inside the testicular capsule around the seminiferous tubules where testosterone exerts paracrine and autocrine effects on steroidogenesis and spermatogenesis [19, 201. TIF testosterone concentrations parallel testosterone concentrations in the general circulation, so serum testosterone levels reflect TIF testosterone levels [15, 17]. TIF volumes are a measure of extracellular and extratubular fluid formation inside the testis and vascular perfusion of the testis [21-23]. TIF formation within the testes controls the access of nutrients, hormones, anti other substances to intratesticular cells and structures [21, TIF paracrine control systems play a major role in idiopathic infertility in men [191. Thus, these measures provide functionally important information on the role of drugs in gonadal activity. Serum LH was also measured because LH is a major physiological stimulus of testosterone secretion and a major factor in the regulation of TIF formation [19-21]. 1128

2 NO, OPIOIDS, AND RAT TESTOSTERONE 1129 The role of NO in opioid-induced testicular suppression was assessed in male rats through the injection of morphine after pretreatment with a NOS inhibitor (NG-nitro-L-arginine methyl ester; NAME), and by the effects of NO donor (isosorbide dinitrate; ISDN) pretreatment on opioid antagonist (naltrexone) stimulation of testosterone secretion. We examined the effects of NAME on morphine-induced testicular suppression in hcg-treated rats to assess whether morphine-induced decreases in gonadotropins [15] are involved in the interaction between morphine and NO in the regulation of gonadal steroidogenesis. We also combined low doses of morphine and ISDN or naltrexone and NAME to find possible additive or synergistic effects to support a role for NO in mediating the testicular effects of opioids. Materials and Animals MATERIALS AND METHODS All experiments used adult (60 day old) male Sprague- Dawley-derived rats that were bred in our laboratories from a stock obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). NAME, ISDN, naltrexone, and hcg were obtained from Sigma Chemical Company (St. Louis, MO). Morphine sulfate was obtained from the National Institute on Drug Abuse (Rockville, MD). The testosterone antiserum (R- 15P) was obtained from Radioimmunoassay Systems Laboratories, Inc. (Carson, CA) and had approximately 18% cross-reactivity with 5-dihydrotestosterone and less than 1% cross-reactivity with all other steroids. The National Hormone Pituitary Program (Dr. Salvatore Raiti, NIDDK, Bethesda, MD) supplied the rat double-antibody RIA kits used for all serum LH measurements. Treatments In all experiments, rats in groups of 9-15 of the same age were randomized. Morphine sulfate ( mg/kg), naltrexone ( mg/kg), NAME (1-300 mg/kg), and hcg (20 IU/kg) or saline were injected s.c. in volumes of 1 ml/kg. ISDN ( mg/kg) suspensions in water with constant stirring were given orally by gavage in volumes of 10 ml /kg. Because the ISDN powder contained 60% lactose, the amounts of lactose in all control solutions and ISDN treatment suspensions were adjusted so that all animals in each ISDN experiment were treated with equal amounts of lactose in water. NAME was injected 30 min before morphine; hcg was mixed with and injected simultaneously with morphine; ISDN was given 2 h before naltrexone; combined treatments of morphine and ISDN were given within 15 sec; and combined injections of naltrexone and NAME were administered 11/2 inches apart within 10 sec. Morphine/NAME Dose Response To examine the effect of NOS inhibition on opioid-suppressed testosterone secretion, NAME (30 mg/kg) was injected before various doses of morphine, and serum and TIF samples were collected 2 h after the morphine injection. The dose dependency of NAME's inhibition of morphine's testicular effects was tested with NAME (1-300 mg/kg) pretreatment before administration of morphine (0.5 or 1 mg/ kg); serum and TIF samples were collected 2 h after morphine injection. Morphine/NAME with hcg Rats were treated with hcg (20 IU/kg) simultaneously with morphine (10 mg/kg) or saline, after NAME (30 mg/ kg) or saline pretreatment, to examine whether or not the effects of NAME on morphine-induced decreases in testosterone secretion were dependent on prior morphine-induced changes in circulating gonadotropins [15]. Samples were collected 2 h after morphine and hcg treatments. Naltrexone/ISDN Dose Response To further examine the role of NO in opioid suppression of testosterone secretion, rats were treated with a large dose of the opioid antagonist naltrexone (5 mg/kg) after pretreatment with several doses of ISDN, an exogenous NO donor. This dose of naltrexone stimulates LH secretion and testosterone secretion. In our laboratory, the peak effect of naltrexone on LH secretion occurred 1 h postinjection, and the peak effect on testosterone secretion occurred 2 h postinjection (unpublished results). Samples were collected 2 h after naltrexone injection, when stimulation of testosterone secretion was at its peak. Morphine/ISDN and Naltrexone/NAME Combinations The role of NO in opioid regulation of testosterone secretion was also tested by combining doses of morphine (0.1 or 0.2 mg/kg) and ISDN (20 or 40 mg/kg) that were near the threshold of doses needed to suppress testosterone secretion. These groups were compared to control groups given saline and lactose vehicle. In addition, animals received doses of naltrexone (0.03 mg/kg) and NAME (1 mg/ kg) that were not quite effective in stimulating testosterone secretion when administered alone, and the results were compared to those obtained with control injections with saline. In these experiments, samples were collected 2 h after treatments. TIF Collection TIF was collected as previously described [16, 17]. Immediately after serum collection from trunk blood, both testes were removed, small holes were cut in the caudal end of each testis, and each testis was then suspended in a tube to allow TIF drainage overnight. TIF volumes were then measured with a pipette, and TIF testosterone was measured by RIA.

3 1130 ADAMS ET AL. * * 4 ~ 1,,&, 5 c po W 0 E- U, o Ga C, 0 mg/kg a c ;:. - c 0 rn To ne 0.5 mg/kg Morphine (mg/kg) NAME (mg/kg) FIG. 1. Dose-response curves of the effects of morphine (0-1 mg/kg) on serum testosterone with and without pretreatment with NAME (30 mg/kg). Values are means + SEM of 9-10 animals per group. *Significantly (p < 0.05, ANOVA) different from the respective saline control groups. FIG. 3. Dose-response curves of the effects of NAME ( mg/kg) pretreatment before administration of saline or morphine (0.5 mg/kg) on serum testosterone levels. Values are means + SEM of 9-10 animals per group. *Significantly (p < 0.05, ANOVA) different from the respective control groups (morphine-treated). Testosterone and LH RIAs Testosterone was extracted from serum with ethanol as previously described [14, 15]. The dried aliquots were reconstituted in RIA buffer and then assayed by RIA; TIF aliquots were diluted with RIA buffer and then subjected to testosterone RIA without extraction as previously described [16, 17]. LH was measured by RIA in unextracted serum aliquots, as previously described, with use of instructions and materials from the National Hormone Pituitary Program [16]. Statistics ANOVA followed by post hoc analysis with Fisher's protected least significant difference tests was used to determine significant differences between groups as described previously [17]. The level of significance was set atp < All experimental groups contained 9-15 rats, and the results were reproducible in separate experiments. RESULTS Morphine Dose Response with NAME Pretreatment Morphine at mg/kg doses after saline pretreatment decreased serum testosterone (Fig. 1). NAME, as expected, was a testosterone secretion stimulant, increasing testosterone levels when saline instead of morphine was injected. NAME (30 mg/kg) pretreatment also completely reversed the effect of mg/kg doses of morphine on testosterone secretion (Fig. 1). Because TIF testosterone levels paralleled changes in serum testosterone levels in all of these studies, only serum testosterone levels are reported. Serum LH concentrations were not altered by morphine at this 2- h time point because peak effects on LH secretion occur earlier (data not shown). Since none of the treatments used in these studies altered the effects of morphine or naltrexone on serum LH or TIF volumes, serum LH and TIF volumes are not reported. In a separate experiment we examined whether the NAME-induced reversal of morphine's suppression of testosterone secretion could be overcome by large doses of morphine. A dose of NAME (30 mg/kg) given 30 min before large doses of morphine (up to 15 mg/kg) completely inhibited morphine's effects on both serum and TIF testosterone (data not shown). FIG. 2. Dose-response curve of the effect of NAME (0-30 mg/kg) pretreatment on morphine (1 mg/kg)-induced suppression of serum testosterone levels. Values are means + SEM of 9-10 animals per group. *Significantly (p < 0.05, ANOVA) different from the control group (saline injections only). Morphine with NAME Pretreatment Dose Response As shown in Figure 2, the inhibition of morphine-induced suppression of testosterone secretion by NAME pretreatment was dose-dependent. Doses of NAME of 10 mg/ kg or higher significantly reversed the effect of 1 mg/kg morphine. We also found that morphine (0.5 mg/kg)-induced inhibition of NAME-stimulated testosterone secretion

4 NO, OPIOIDS, AND RAT TESTOSTERONE 1131 FIG. 4. Effects of saline or NAME (30 mg/kg) pretreatment followed by saline or morphine (10 mg/kg) treatment on serum testosterone levels in hcg-stimulated rats (20 IU hcg/kg). Values are means + SEM of 9-10 animals per group. Significantly (p < 0.05, ANOVA) different from the control group (saline and hcg only). was not overcome even by mg/kg doses of NAME pretreatment (Fig. 3). Morphine and NAMIE in hcg-stimulated Rats Morphine, as expected, inhibited hcg (20 IU/kg)-stimulated testosterone secretion (Fig. 4). NAME (30 mg/kg) completely reversed morphine's inhibition of testosterone secretion in hcg-stimulated rats, suggesting that these changes are not mediated by changes in LH secretion. Naltrexone with ISDNPretreatment The possible involvement of NO in opioid-induced testosterone suppression was further examined by testing whether a NO donor, ISDN, could reverse the effects of the opioid antagonist naltrexone. ISDN has been found to decrease testosterone secretion by itself [131. As shown in Figure 5, naltrexone (5 mg/kg), as expected, increased testosterone secretion in saline-pretreated rats. The NO donor ISDN ( mg/kg), given 2 h before naltrexone, dosedependently inhibited naltrexone-induced increases in testosterone secretion. The higher doses of ISDN (600 and 1000 mg/kg) completely attenuated the stimulation of testosterone secretion by naltrexone; testosterone levels were decreased to levels similar to those seen after administration of ISDN alone [13]. Morphine with ISDN Treatment To help determine whether or not NO mediates morphine's suppression of testosterone secretion, combinations of low closes of morphine and ISDN were administered to ascertain whether the suppressive effects were additive or synergistic. Morphine at a dose of 0.1 mg/kg combined with ISDN at a dose of 20 mg/kg, neither of which produced any effect on testosterone secretion by itself, did not alter testosterone secretion (Fig. 6). This experiment was repeated several times with different dosages of morphine and ISDN that were near the threshold of dosages needed to exert FIG. 5. Dose-response curve of the effect of ISDN ( mg/kg) pretreatment on naltrexone (5 mg/kg)-induced stimulation of serum testosterone levels. Values are means SEM of 9-10 animals per group. *Significantly (p < 0.05, ANOVA) different from the control group (lactose vehicle and saline only). significant effects on testosterone secretion; in no case did combined morphine and ISDN alter testosterone secretion to a greater extent than the same doses of each agent given alone, supporting the hypothesis that morphine and ISDN do not exert additive or synergistic effects on testosterone secretion. A higher dose of morphine (0.2 mg/kg) and a higher dose of ISDN (40 mg/kg)-doses that were effective in decreasing testosterone concentrations-were also not additive or synergistic when combined, as combined treatment decreased serum testosterone to the same extent as treatment with either agent alone (Fig. 7). Naltrexone with NAME Treatment We also gave low doses of naltrexone and NAME to help determine whether NO is involved in mediating opioid reg- FIG. 6. Effects of treatment with the combination of saline and lactose vehicle, saline and ISDN (20 mg/kg), morphine (0.1 mg/kg) and lactose vehicle, or morphine (0.1 mg/kg) and ISDN (20 mg/kg) on serum testosterone. Values are the means + SEM of 15 animals per group. No significant differences were found.

5 1132 ADAMS ET AL. FIG. 7. Effects of treatment with the combination of saline and lactose vehicle, saline and ISDN (40 mg/kg), morphine (0.2 mg/kg) and lactose vehicle, or morphine (0.2 mg/kg) and ISDN (40 mg/kg) on serum testosterone. Values are the means + SEM of 15 animals per group. *Significantly (p < 0.05, ANOVA) different from the saline/lactose vehicle group values. ulation of testosterone secretion. The combination of naltrexone at 0.03 mg/kg and NAME at 1 mg/kg, doses that exerted slightly stimulatory but nonsignificant effects on testosterone secretion by themselves, did not significantly increase serum testosterone (Fig. 8). DISCUSSION The results of these studies support the hypothesis that endogenous arginine-nos-no and opioid pathways both suppress testicular steroidogenesis and, consequently, male FIG. 8. Effects of treatment with the combination of saline and saline, saline and NAME (1 mg/kg), naltrexone (0.03 mg/kg) and saline, or naltrexone (0.03 mg/kg) and NAME (1 mg/kg) on serum testosterone. Values are the means ± SEM of 15 animals per group. No significant differences were found. fertility. This conclusion is based on several observations. I)ecreases in NO formation, induced by NOS inhibition, markedly increased testosterone secretion and decreased morphine's inhibitory effects on testosterone secretion (Figs. 1-3). During conditions in which NAME could not stimulate testosterone secretion (because of maximal stimulation of testosterone production by hcg), morphine suppressed testosterone levels and NAME totally reversed the effect of morphine (Fig. 4). Our results show that NAME reverses the effect of morphine on testosterone secretion and that morphine attenuates the effect of NAME on testosterone secretion. In addition, the NO donor, ISDN, suppressed testosterone secretion by itself 13] and also inhibited stimulation of testosterone secretion by the opioid antagonist naltrexone (Fig. 5). At first glance, these results suggest that NO might mediate a major portion of opioid-induced suppression of testosterone secretion. However, a careful examination of these data, combined with observations from other experiments in which low doses of morphine and ISDN or naltrexone and NAME were given (Figs. 6-8), indicates that the interaction between the NO and opioid systems regulating testosterone secretion involves separate pathways or mechanisms that happen to lead to the same effect on testosterone secretion. The interaction does not appear to involve common or closely linked pathways in which NO mediates opioid-induced inhibition of testosterone secretion. The data represent a summation of stimulatory effects of NAME or naltrexone and inhibitory effects of morphine or ISDN acting through independent NO and opioid systems. Certain aspects of our results contain evidence for the lack of a common mechanism or pathway in the NO and opioid systems that regulate testicular steroidogenesis. First, NAME elevates testosterone secretion by itself and ISDN suppresses testosterone secretion by itself, as seen in this report (Figs. 1, 3, and 7) and previous reports [ This suggests the possibility that the effects of NO-related agents on the action of opioid agents are a summation of opposite, independent effects. Second, morphine and NAME are structurally dissimilar, and the effect of NAME on morphine suppression of testosterone secretion was not overcome by very high doses of morphine (reported in Results). In addition, the effect of morphine on NAME stimulation of testosterone secretion was not overcome by very high doses of NAME (Fig. 3). This suggests that interactions between morphine and NAME are noncompetitive and could arise from effects on independent pathways that both affect testosterone secretion. The third, most important piece of evidence for a lack of mediation of opioid testicular effects by NO is found in the results of experiments combining low doses of morphine and ISDN or low doses of naltrexone and NAME. The NO donor ISDN was neither additive nor synergistic with morphine when both were given at doses that were not effective

6 NO, OPIOIDS, AND RAT TESTOSTERONE 1133 alone (Fig. 6) or were slightly effective alone (Fig. 7). In addition, the nearly significant stimulatory effect of the NOS inhibitor NAME was not additive or synergistic with the nearly significant stimulatory effect of naltrexone (Fig. 8), because these two agents combined did not increase testosterone secretion above that observed after administration of either agent alone. If opioid-induced suppression of testosterone secretion depended on NO production, then some kind of additive effect would be seen between these combinations of morphine and ISDN or naltrexone, and NAME would have some kind of additive effect. The mechanisms involved in NO-induced suppression of testosterone secretion are unknown. However, some evidence indicates that NO might inhibit testosterone secretion by binding to heme groups of testicular steroidogenic enzymes to directly inhibit testicular steroidogenesis. NO is produced in the rat testis [11]. NO receptors include iron in heme groups of guanylate cyclase and other enzymes including steroidogenic enzymes [27-30]. In addition, carbon monoxide, which also inhibits testicular steroidogenesis [31, 32], binds heme iron as does NO [28]. An alternative hypothesis that resulted from our studies on NO-related agents and testicular steroidogenesis [13] is that the hemodynamic effects of NO-related agents are involved in the regulation of testosterone secretion. Some studies have suggested that testicular blood flow regulates testosterone secretion [33, 34] so that decreased blood flow is associated with decreased testosterone secretion. NO is a vasodilator [1-3] that is produced by testicular vascular endothelium [11]. NO decreases TIF formation [13], and NAME is a vasoconstrictor [7, 8]. It is known that NO decreases blood pressure and that NOS inhibitors increase blood pressure, but it is not known how ISDN or NAME influences testicular blood flow, testicular microvasculature, or testicular vascular permeability. We have found that two vasodilators without prominent NO-related effects, nicardipine and hydralazine, also decrease TIF formation and testosterone secretion and block NAME-stimulated testosterone secretion [13]. Opioids have some vasodilatory effects [35, 36] and suppress TIF formation [15]. In addition, TIF formation is directly linked to testicular blood flow [21, 37-39]. This evidence suggests that the effects of opioids and NO-related agents on vascular smooth muscle and blood flow, which have been linked to both TIF formation and testosterone secretion, may be involved in their effects on testicular steroidogenesis even though evidence for direct interactions between the two systems is absent. We found no evidence for an interaction between NO and opioid systems in regulating TIF formation, since NAME did not alter TIF volumes by itself or alter morphine's suppression of TIF formation. In addition, naltrexone did not alter TIF volumes by itself or alter ISDN's suppression of TIF formation. Our results confirm that the effects of NO-related agents on TIF formation are complex and require further study [12-14]. Our results confirm that NAME has no effects on basal [12, 13] or morphine-induced suppression of LH secretion. The findings indicate that the effect of NAME on morphineinduced suppression of testicular steroidogenesis was gonadotropin-independent or not related to prior inhibition of LH secretion by morphine, because NAME reversed morphine's suppression of testosterone secretion in hcg-stimulated rats (Fig. 4). This confirms previous evidence suggesting direct effects of both opioids and NO on testicular steroidogenesis [13, 15]. Recent studies have suggested both stimulatory [40-42] and inhibitory [43] effects of NO on LHRH release. We have reported that a NO donor and a NOS substrate can decrease LH secretion in vivo in rats [13], so further study is needed to characterize the roles of NO in LHRH/LH secretion. In summary, the present results indicate that inhibition of NOS can inhibit opioid-induced suppression of testosterone secretion and that exogenous NO can decrease opioid antagonist-stimulated testosterone secretion. But our results also suggest that the interaction between the NO and opioid testosterone secretion regulatory systems is independent, without common mechanisms or pathways, and that NO does not mediate opioid suppression of testosterone secretion. ACKNOWLEDGMENTS We thank Myrtle Barrett, Jingling Chen, and William Johnson for their technical assistance. REFERENCES 1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. 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