Prof Dr Y. Sarioglu, Cumhuriyet Universitesi, Tip Fakultesi, Farmakoloji Anbilim Dali, 58140 Sivas, Turkey. e-mail: email@example.com
Objective To investigate the changes in relaxation responses to omeprazole in corpus cavernosal strips from rabbits with diabetes induced by alloxan.
Materials and methods Diabetes was induced in 10 New Zealand white rabbits. After 8 weeks, the reactivity to electrical-field stimulation, carbachol and omeprazole of corporal strips from the penises of the diabetic animals and from 10 untreated controls was assessed in organ chambers.
Results In the diabetic group, the relaxation responses of corpus cavernosal strips to omeprazole were comparable with those of the control group, whereas the relaxation responses to electrical field stimulation and carbachol were impaired.
Conclusion The relaxant effect of omeprazole in corpus cavernosum was not affected by diabetes, in which neurogenic and endothelium-mediated relaxations were impaired.
The physiology of penile erection involves the haemodynamic mechanisms of arterial vasodilatation and the restriction of venous return . Over the last decade there have been significant advances in the understanding of the physiology of penile erection. Neurogenically mediated relaxation responses that persist after adrenergic and cholinergic blockade are thought to be mediated by the nonadrenergic-noncholinergic (NANC) neurotransmitters. Nitric oxide (NO) generated in response to NANC transmission is the main event leading to vascular smooth muscle relaxation, through the activation of soluble guanylyl cyclase [2,3].
Diabetes mellitus is one of the most frequent causes of erectile impotence and the prevalence of impotence in the diabetic males has been estimated to be 35–75% [4,5]. Studies in diabetic men and animal models of diabetes show that diabetic-based impotence is associated with the impaired neurogenic and endothelium-dependent relaxation of trabecular smooth muscle. It has also been suggested this impairment in relaxation responses caused by diabetes appears to involve an alteration in NO/cGMP pathway [5–8].
The H+-K+ ATPase mediates K+ uptake into cells and operates an active transport mechanism exchanging K+ for H+ efflux . Recent studies suggest that non-epithelial cells may also possess H+-K+ ATPase activity and inhibition caused a reduction in K+ uptake, K+ content and intracellular pH in cultured vascular smooth muscle cells, as well as in intact vascular tissue . The change in the activity of many key enzymes like Na-K ATPase has been shown in neural or vascular tissue and vascular cells in cultures obtained from diabetic animals . We reported that the H+-K+ ATPase inhibitor omeprazole caused relaxation of corpus cavernosum smooth muscle in vitro. The aim of the present study was to investigate the relaxant effect of omeprazole in the corpus cavernosum of a diabetic rabbit model.
Materials and methods
Diabetes was induced in 14 litter-mate and weight-matched, mature New Zealand white rabbits, as described previously . At the end of first week 10 rabbits with blood glucose levels of 2.5 g/L were accepted as diabetic and maintained for 8 weeks; a further 10 normal rabbits were maintained as litter-mate controls for 8 weeks.
After 8 weeks the rabbits were killed and their penises excised. The corporal tissues were carefully dissected free from tunica albuginea and mounted in organ baths containing Krebs solution maintained at 37 °C. One end of each corporal strip was attached to the bottom of the organ bath and the other tied to a force transducer (FT 03, Grass Instruments, Quincy, MA) connected to a pen polygraph (Grass 79E). Each strip was allowed to equilibrate with a basal tension of 2 g for 1 h; this basal tension was chosen because it provided reproducible relaxation responses to omeprazole. Corporal tissues had an intact endothelium, as assessed by the capacity of acetylcholine (1 µmol/L) to produce relaxation. The physiological saline was of the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3 25, MgSO4 1.2, KH2PO4 1.2, glucose 11. The pH of this solution was 7.4, after being bubbled with a 95% O2−5% CO2 gas mixture.
Corpus cavernosum tissue was prepared and used in organ chamber experiments as described previously , with relaxation assessed against a background of precontraction by phenylephrine (10 µmol/L), adjusted to 70–80% maximal contraction. At the plateau of contraction, relaxation responses to cumulative concentrations of carbachol or sodium nitroprusside (SNP), to test endothelium-dependent and independent relaxations, respectively, were assessed, with only one drug for each preparation. Relaxation responses to electrical-field stimulation (EFS) were determined after submaximal contraction with phenylephrine (10 µmol/L). The technical procedure used for obtaining the responses to EFS was described previously . One strip from each rabbit was contracted with phenylephrine (10 µmol/L) and the second strip maintained unstimulated. In some experiments, atropine (1 µmol/L), Nw-nitro l-arginine methyl ester (L-NAME, an NO synthase inhibitor; 30 µmol/L) or indomethacin (a prostaglandin synthase inhibitor, 10 µmol/L) was added to the tissue bath during contractile activity. After active muscle tone was exerted by phenylephrine, omeprazole (10–1000 µmol/L) was cumulatively applied to the bath. In some experiments, tetraethylammonium (TEA) (0.5 mmol/L), an inhibitor of ATP-sensitive potassium channels, ammonium chloride (7.5 mmol/L) or sodium acetate (7.5 mmol/L) was added to the bath after the corpus cavernosal contraction induced by phenylephrine. In some experiments, L-NAME (30 µmol/L) or indomethacin (10 µmol/L) were added to the organ bath 15 min before the precontraction to assess the effects of NO and prostaglandins, which might have contributed to corporal smooth muscle relaxation induced by omeprazole. Nimodipine was also cumulatively (0.1–10 µmol/L) applied to the bath after precontraction with KCl (80 mmol/L).
The following drugs were used in the experiments: omeprazole (Astra, Södertalje, Sweden), glibenclamide (Sigma Chemicals, St Louis, MO), indomethacin (Sigma), atropine sulphate (Sigma), L-NAME (Sigma), TEA (ICN, UK), KCl, acetylcholine chloride, SNP, atropine, guanethidine, carbachol, nimodipine (Sigma), phenylephrine hydrochloride (Sigma). All drugs were dissolved in distilled water except for indomethacin, omeprazole and glibenclamide, which were dissolved in 1% Na2CO3, polyethylene glycol 400 and DMSO, respectively. All drugs were freshly prepared on the day of the experiments. In the high K+ solution NaCl was exchanged for equimolar amounts of KCl.
All data were expressed as the mean (sem). In tissues contracted with phenylephrine or KCl, the relaxant responses were expressed as a percentage of the active muscle tone induced by phenylephrine or KCl. To evaluate the effects of an agonist, the maximum response (Emax), the concentration for a half-maximal response (EC50) and pD2 values were calculated from the concentration-response curve (CRC) obtained in each experiment, as predicted from the Scatchard equation for drug–receptor interaction, where:
– 1/EC50× response + maximum response/EC50
The pD2 value was expressed as the negative logarithm of the EC50. Groups were compared using general linear models of anova followed by Scheffe's F-test, with P < 0.05 considered to be indicate statistical significance.
The blood glucose concentrations and body weights of control and diabetic groups at the end of 8 weeks are shown in Table 1; compared with the control group, the differences in these variables were significant (P < 0.05) in the diabetic group.
Table 1. Characteristics of the animals, and the pD2 and Emax (µmol/L) of the omeprazole-induced relaxation and those with other agents
Similar tensions were obtained in both group when strips were contracted with 124 mmol/L KCl. In corpus cavernosal strips precontracted with phenylephrine at submaximal concentration, cumulative concentrations of carbachol (0.01–10 µmol/L) caused concentration-dependent relaxation. The impairment of relaxation responses in the diabetic group was statistically significant (P < 0.05). EFS gave frequency-dependent relaxation. The Emax value was significantly lower in the diabetic group (P < 0.05) than in the controls. In corpus cavernosal strips precontracted with phenylephrine, SNP (0.01–30 µmol/L) and papaverine caused concentration-dependent relaxation responses, but there were no significant differences in Emax and pD2 values between the groups (data not shown).
Omeprazole caused a concentration-dependent relaxation of cavernosal strips precontracted with phenylephrine in both groups (Fig. 1). Omeprazole also decreased and finally abolished all spontaneous contractile activity which developed in some strips of corpus cavernosum after they were mounted in the organ bath. There was no statistically significant difference in the relaxation responses to omeprazole between the groups (P < 0.05; Table 1). The relaxation responses induced by omeprazole did not change in the tissues pretreated with indomethacin (10 µmol/L) or L-NAME (30 µmol/L). Additionally, neither alkalinization with ammonium chloride (7.5 mmol/L) nor acidification with sodium acetate (7.5 mmol/L) had a significant effect on the relaxant effect of omeprazole either group. Responses to omeprazole were similar before and after a 15-min application of 1 µmol/L glibenclamide, an inhibitor of ATP-sensitive potassium channels, and 0.5 mmol/L TEA, an inhibitor of calcium-activated potassium channels, in both groups (P > 0.05; Table 1). Nimodipine, an l-type calcium-channel blocker, relaxed corporal strips precontracted with KCl in both groups.
The present results show that omeprazole induces relaxation in diabetic rabbit corpus cavernosum strips similar to that in controls animals. In the corpus cavernosum strips from diabetic rabbits, the endothelium-mediated and EFS-induced relaxation responses were impaired, whereas the relaxation responses induced by omeprazole were no different from those in controls. The relaxation responses of corpus cavernosum strips, obtained from both diabetic and normal rabbits, were not mediated by cyclo-oxygenase products and/or NO synthesized and released from NANC nerves and corporal endothelium.
All spontaneous contractile activity in the corpus cavernosum strips were blocked by adding omeprazole, but not by indomethacin, L-NAME or atropine. Rhoden et al. suggested that NO, cyclo-oxygenase products and acetylcholine have no role in normal spontaneous contractile activity of corpus cavernosum, and that spontaneous activity involves H+-K+ ATPase or a similar pathway. Holmquist et al. showed that spontaneous activity was not affected by tetrodotoxin, atropine and phentolamine, suggesting a myogenic origin.
The mechanism underlying the relaxant effect of omeprazole is unclear. Although, several other smooth muscles are relaxed by a decrease in intracellular pH , the corporal relaxation induced by omeprazole was not changed by alkalinization and acidification . Likewise the dose-dependent relaxation of omeprazole was not affected by glibenclamide (an inhibitor of ATP sensitive potassium channels) or by TEA (an inhibitor of calcium-activated potassium channels). As pretreatment of corporal tissue with L-NAME, indomethacin, ammonium chloride or glibenclamide had no effect on the omeprazole-induced relaxation responses, they are not mediated by cyclo-oxygenase products and NO synthesized and released from NANC nerves and endothelium; the relaxation induced by omeprazole is not related to its ability to inhibit the H+-K+ ATPase inhibition. Additionally, a decrease in corporal smooth muscle tone by decreased intracellular pH cannot be responsible because relaxation responses induced by omeprazole were unchanged by alkalinazation or acidification. The possibility of an increase in tone caused by the depolarization which resulted from a decrease in K+ uptake into cells, and stimulation of the opening of voltage-dependent calcium channels (which may increase tension) is also unlikely to explain the effect of omeprazole, because it caused no such contraction.
The high concentration of proton-pump inhibitors affect other ion-motive ATPases like Na+-K+ ATPase and vacuolar H+-ATPase . Omeprazole inhibits responses to phenylephrine and suggests that it may inhibit Ca+ influx through the receptor-operated channels. The ability of omeprazole to inhibit KCl responses indicates that omeprazole may inhibit voltage-operated calcium channels, because the l-type calcium-channel blocker nimodipine also inhibited responses to KCl in rabbit corporal smooth muscle.
In conclusion, theoretically omeprazole may have a beneficial effect in the treatment of erectile dysfunction. There was no evidence that the relaxant effect was produced by the mechanisms damaged in diabetes; relaxation responses to omeprazole were unchanged in this animal model of impaired neurogenic and endothelium-mediated relaxation responses. Thus omeprazole may have some advantages in treating erectile dysfunction caused by diabetes.
S. Ayan, MD, Assistant Professor of Urology.
Y. Sarioglu, PhD, Professor of Pharmacology.
S. Yildirim, MD, PhD, Assistant Professor of Pharmacology.