Papaverine-induced Relaxation Research Articles

Intravesical hyaluronic acid treatment improves bacterial cystitis and reduces cystitis-induced hypercontractility in rats.

To investigate the effect of intravesical hyaluronic acid on Escherichia coli-induced cystitis and cystitis-induced hypercontractility in rats. Bacterial cystitis was induced in Wistar female rats by intravesical inoculation of E. coli. Isotonic saline was instilled in the control group (n = 6). The rats were either non-treated, treated with gentamycin (4 mg/kg, 5 days) or treated intravesically with hyaluronic acid (0.5 mL, 0.5%). On the eighth day, the bladder tissues were excised for histological examination, and the measurements of myeloperoxidase, superoxide dismutase and catalase activities. Contraction/relaxation responses to carbachol, isoprotrenol and papaverine were studied. Tissue myeloperoxidase activity was increased, but superoxide dismutase and catalase activities were decreased in bacterial cystitis, while hyaluronic acid treatment reversed these changes. In the hyaluronic acid-treated group, healing of the uroepithelium was observed, while decreased inflammatory cell infiltration was obvious in gentamycin-treated group. E. coli-induced cystitis in all rats resulted in increased contraction responses to carbachol compared with controls (P < 0.01). Treatment with hyaluronic acid, but not gentamycin, significantly (P < 0.05) depressed hypercontractility at maximum carbachol concentrations. In all rats with cystitis, papaverine-induced relaxation was increased, whereas isoproterenol-induced relaxation curves were not different between the studied groups. Gentamycin treatment, despite its ameliorative effect on inflammation, had no impact on the contractile dysfunction of the injured bladder. Intravesical hyaluronic acid, in addition to its supportive role in the healing of the epithelium, seems to lower the increased threshold for contraction and to reduce oxidative stress. These findings support a potential role for hyaluronic acid in the treatment of bacterial cystitis.

Effects of papaverine on carbachol- and high K+ -induced contraction in the bovine abomasum.

The effects of papaverine on carbachol (CCh) -and high K+- induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 µM) -and KCl (65 mM) -induced contractions in a concentration-dependent manner. Forskolin or sodium nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca2+ from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K+-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine (30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.

Abstract 21: Retinol-binding Protein 7 (RBP7), a Peroxisome Proliferator-Activated Receptor Gamma (PPARG) Target Gene, is a Novel Regulator of Endothelial Function

PPARG, a ligand-activated transcription factor, in the endothelium regulates vascular function and blood pressure, through mechanisms that remain poorly defined. Endothelial-specific expression of dominant negative (DN) PPARG (E-V290M) caused endothelial dysfunction when combined with a high fat diet (HFD). RBP7 is an intracellular retinoid binding protein belonging to the family of fatty acid-binding proteins and a PPARG target gene. RBP7 expression in aorta is induced by rosiglitazone and repressed by DN PPARG, and its level of expression is highly correlated with PPARG in other tissues. RBP7 is also expressed in endothelium. We hypothesize that RBP7 may mediate some vascular protective effects of PPARG during HFD. We examined vascular responses in basilar artery (BA, pressurized myograph) and carotid artery (CA, wire myograph) of RBP7-deficient (KO), heterozygous (HZ), and wild type (WT) mice fed normal chow diet (ND) or HFD for 8 or 20 wks. Weight gain was similar in all three HFD-fed groups and was higher than ND-fed mice. There was no difference in the endothelium-dependent, acetylcholine (ACh)-induced relaxation in either BA or CA of any group under ND. Following 8 wks HFD, BA from KO mice exhibited impaired relaxation to ACh compared to control mice (at 100μM: 33±7% KO vs 73±7% HZ vs 83±10% WT, p&lt;0.05). A normal response was observed to the nitric oxide donor, nitroprusside (SNP). An impaired ACh-, but not SNP- or papaverine-induced relaxation was observed in CA of KO mice after 20 wks of HFD (ACh 30μM: 49±6% KO vs 61±3% HZ vs 70±4% WT, p&lt;0.05). No differences were found in phenylephrine- and endothelin-1-induced contractions in CA in any group. Pre-incubation of BA for 30 minutes with the superoxide scavengers tempol (1mM) or PEG-superoxide dismutase (PEG-SOD, 100 U/ml) completely restored ACh-induced relaxation to normal (ACh at 100μM: 79±7% Tempol-KO; 75±12% PEG-SOD-KO; 81±11% WT). This phenotype was very similar to that observed in HFD fed E-V290M mice. We conclude that loss of RBP7 causes endothelial dysfunction in response to a HFD through a mechanism involving oxidative stress. These findings provide the first evidence that RBP7 plays a protective role in the endothelium, which might be related to the endothelial protective effects of PPARG.

The effect of etanercept on aortic nitric oxide-dependent vasorelaxation in an unpredictable chronic, mild stress model of depression in rats

Stress has been recognized as a risk factor for cardiovascular disease and depression, but the correlation is not well understood. However, inflammation is known to have a crucial role in both cardiovascular disease and depression. Tumor necrosis factor alpha (TNF-α) is a major cytokine for the activation of neuroendocrine, immune and behavioral responses. Therefore, we aimed to explore the effects of etanercept, an anti-TNF-α fusion protein, on endothelium-dependent vascular reactivity, blood pressure and endothelial nitric oxide synthase (eNOS) immunoreactivity in a model of unpredictable chronic mild stress (UCMS). Male rats were exposed to UCMS for 8 weeks, and etanercept (0.8mg/kg, weekly) was administered during UCMS induction. The systolic blood pressure was recorded by the tail cuff method, and the relaxant responses of the aorta induced by carbachol, sodium nitroprusside (SNP) and papaverine were evaluated in an isolated organ bath system. UCMS rats exhibited an impaired carbachol-induced relaxant response compared to control rats, but there were no significant differences in the SNP- and papaverine-induced relaxant responses between the control and stressed rats. Etanercept treatment improved the carbachol-induced endothelium dependent relaxations observed in rats that experienced UCMS. No significant change in the systemic blood pressure was observed, but decreased expression of eNOS was detected in the UCMS group. Moreover, there were no significant changes in the etanercept treatment group compared to the control rats. Our results suggest that TNF-α could be a mediator of vascular dysfunction associated with UCMS, which leads to decreased expression of eNOS.

Effects of chronic ethanol consumption on rat upper gastrointestinal system: Functional and histologic findings

The purpose of the present study was to determine the effect of chronic alcohol consumption on reactivity of esophageal tunica muscularis mucosae (TMM) and lower esophageal sphincter (LES) smooth muscle. Six male rats in alcohol-fed group received ethanol (7.2% v/v) in a modified liquid diet for 4 weeks. Two control groups were used; six rats in the standard diet-fed group received rat chow and water for 4 weeks. Six rats in sucrose-fed group were given sucrose and received a liquid diet. The smooth muscle reactivity of TMM and LES strips from ethanol-fed and control animals was evaluated in organ chambers. Also histologic study was undertaken to show effects of chronic alcohol consumption. Maximum contractile responses of TMM to KCl and carbachol were decreased in the ethanol-fed group compared to the control groups. Relaxant responses to serotonin were decreased in the ethanol-fed group compared to the control groups. In TMM, isoproterenol- and papaverine-induced relaxant responses were similar in the ethanol-fed and control groups. In LES smooth muscle, relaxant responses to papaverine or isoproterenol were similar in the control groups and the ethanol-fed group. There was no change in agonist potency among the groups. The relaxation response elicited by nicotine and sodium nitroprusside (SNP) or contractile response elicited by carbachol and 80 mM KCl was decreased with maximum responses and pD2 values, in the ethanol-fed group compared to that of the control groups in LES. Decreased nNOS immunoreactivity in myenteric plexus was found in alcohol-exposed group compared to control groups. Our findings suggest that chronic alcohol consumption impairs relaxant and contractile responses of both TMM and LES smooth muscle and it may contribute to gastroesophageal reflux commonly seen after alcohol binges.

Effects of chronic low- and high-dose ethanol intake on the nitrergic relaxations of corpus cavernosum and penile nitric oxide synthase in the rabbit

Epidemiological evidence showed that chronic ethanol consumption is a major risk factor in the development of impotence. The present study investigated the effects of carbachol-, electrical field stimulation (EFS)-, sodium nitroprusside (SNP)- and papaverine-induced relaxant responses in the isolated corpus cavernosum tissues from rabbits submitted to an 12-week course of chronic low (5% v/v) or high ethanol intake (30% v/v). Increased carbachol- and EFS-induced relaxant responses but not SNP and papaverine, were observed in low ethanol-fed rabbits compared with controls. However, impaired carbachol- and EFS-induced relaxant responses were observed in high ethanol-fed rabbits compared with control rabbits. There were no significant differences in SNP- and papaverine-induced relaxant responses between control and high ethanol-fed rabbits. In addition, decreased neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) immunoreactivity in penile tissue were found in high ethanol-fed rabbits, but increased the immunoreactivity in low ethanol-fed group, compared with control group. These results suggest that alterations in nitric oxide (NO) production within the cavernous tissue in the high ethanol-fed rabbits are, at least in part, responsible for the erectile dysfunction.

Effects of Resveratrol on Ileal Smooth Muscle Reactivity in Polymicrobial Sepsis Model

To determine the effects of resveratrol on the ileal smooth muscle reactivity in polymicrobial sepsis. Polimicrobial sepsis was induced by the cecal ligation and perforation (CLP) procedure. Sprague Dawley rats were divided into three groups. Rats in resveratrol group received resveratrol after CLP (100 mg/kg, i.p.). Rats received saline immediately after CLP in the sepsis group. Control group rats underwent sham operation. The rats were sacrificed and the ileum was excised 24 h after the operation. Contractile and relaxant responses in isolated smooth muscle strips (SMS) were determined using an in vitro muscle technique. TNFα and IL-6 levels were measured in blood samples. Contractile responses to carbachol and KCl and relaxant responses to transmural electrical field stimulation (EFS) were significantly decreased in the sepsis group compared with control and resveratrol groups. No significant changes were observed for smooth muscle reactivity in the resveratrol and control groups. Sodium nitroprusside (SNP) or papaverine-induced relaxations were similar in the all groups. Resveratrol treatment supressed increased TNFα and IL-6 levels in blood seen in sepsis group. Ileal smooth muscle reactivity was improved after resveratrol treatment in rats with sepsis. The results of the present study indicate that the beneficial effects of resveratrol might be, at least in part, attributed to its effects on non-adrenergic non-cholinergic pathway and/or anti-inflammatory and antioxidant activity.

Abstract 1287: TRPV4-Deficient Mice Exhibit Impaired Endothelium-Dependent Relaxation Induced by Acetylcholine in Vitro and in Vivo

Agonist-induced Ca 2+ entry in endothelial cells is important for the synthesis and release of vasoactive factors, although mechanisms of Ca 2+ entry remain largely unknown. Emerging evidence suggests that the transient receptor potential vanilloid 4 (TRPV4) channel, a Ca 2+ -permeant TRP channel, is expressed in endothelial cells and may be involved in the regulation of vascular tone. Here we investigated the potential role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout (TRPV4 −/− ) mice model. Carotid arteries were isolated and preconstricted with the thromboxane A2 mimetic U46619. Concentration-dependent relaxations to acetylcholine (10 −9 –10 −5 M) were markedly reduced in carotids of TRPV4 −/− vs. wild-type (WT) mice (maximal relaxations of 31±12% vs 53±4%, respectively; n=4 mice). There was no significant change in the ED50 for Ach. In both WT and TRPV4 −/− , acetylcholine-induced relaxations were blocked and converted to constrictions by the NO synthase inhibitor L-NAME (maximal relaxations of −25±6% and −24±7%, respectively). There was no difference in papaverine-induced relaxations between WT and TRPV4 −/− mice (maximal relaxations of 93±3% vs. 90±3%, respectively). U46619 caused similar contractions in carotid arteries from those mice. We also compared in vivo vasodilator effects of acetylcholine by measuring changes in blood pressure in those animals. Intravenous administration of acetylcholine (15 ng/gm bolus) decreased blood pressure by 32±6 mmHg in WT mice (from 90±15 to 57±10 mmHg; n=6), whereas blood pressure was reduced by only 10 mmHg in TRPV4 −/− mice (from 67±6 to 56±4 mmHg; n=12). Acetylcholine caused similar reductions in heart rate in WT and TRPV4 −/− mice, with mean changes of 365±57 and 292±40 beats/min, respectively. We conclude that the endothelium-dependent vasodilator response to acetylcholine is reduced both in vitro and in vivo in TRPV4 −/− mice, and these findings may provide novel insight into the mechanisms of Ca 2+ entry evoked by chemical agonists in endothelial cells. The paradoxically lower baseline blood pressure in TRPV4 −/− mice requires further investigation.

The effect of papaverine on ion channels in rat basilar smooth muscle cells

Objectives: Papaverine has been used in treating vasospasm following subarachnoid hemorrhage (SAH). However, its action mechanism for cerebral vascular relaxation is not clear. Potassium and calcium channels are closely related to the contraction and relaxation of cerebral smooth muscle. Therefore, to identify the role of potassium and calcium channels in papaverine-induced vascular relaxation, we examined the effect of papaverine on potassium and calcium channels in freshly isolated smooth muscle cells from rat basilar artery.Method: The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recorded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. Papaverine was added to the bath solution.Results: Papaverine of 100 μM into bath solution increased the amplitude of the outward K+ current which was completely blocked by BKCa blocker, IBX (iberiotoxin) and a calcium chelator, BAPTA (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) in whole cell mode. Papaverine (100 μM) also inhibited L type Ca2+ current recorded in isolated smooth muscle cells from rat basilar artery.Discussion: These results strongly suggest that Ca2+-activated potassium channels and L type Ca2+ channels may be involved in papaverine-induced vascular relaxation in rat basilar artery.

Effect of chronic renal failure on foregut smooth muscle reactivity: an experimental study

Purpose An association between chronic renal failure (CRF) and gastroesophageal reflux (GER) is well known. The aim of this study was to pharmacologically characterize and investigate the possible contribution of smooth muscle reactivity pathways involving GER on the CRF rat model. Material and Methods Chronic renal failure was created in Sprague-Dawley rats by 5 of 6 nephrectomy. The rats were divided into 2 groups: the CRF-induced group (CRF group) and the sham-operated group (control group). Esophageal smooth muscle strips were studied in vitro for their contractile (KCl, carbachol) and relaxant (isoproterenole, serotonin, and papaverine) response to receptor activation in the organ chambers set up. Subsequently, the in vitro lower esophageal sphincter (LES) smooth muscle study was generated by KCl, carbachol, isoproterenole, nicotine, sodium nitroprusside (SNP), and papaverine. Results Compared with controls, esophageal strips taken from CRF-induced rats associated with decreased smooth muscle responses to carbachol, serotonin, and increased response to KCl. Isoproterenole- and papaverine-induced relaxant responses were not affected. Contractility of the isolated LES strips were significantly increased to KCl and carbachol in the CRF group compared with the control group. Similar relaxant responses were obtained in LES strips stimulated by isoproterenole, SNP, and papaverine in the CRF and control group. Nicotine-induced relaxant responses were decreased in the CRF group compared with the control group. Conclusions Our study revealed alterations of receptor-dependent esophageal and LES smooth muscle reactivity in the CRF-induced rats. Impaired foregut smooth muscle reactivity may contribute to the development of GER-related functional abnormalities in patients with CRF.

Esophageal smooth muscle reactivity is impaired in chronic reflux esophagitis by both receptor- and nonreceptor-mediated mechanisms

Aim Esophagitis is associated with an impaired esophageal peristalsis. A few studies have been aimed at understanding the pathophysiology of abnormal peristaltic activity. The mechanism of impaired esophageal smooth muscle reactivity in the chronic gastroesophageal reflux (GER) model is investigated in vitro for the first time. Materials and Methods The chronic GER rat model was created by partial gastric outlet obstruction. The histopathological findings related to esophagitis were evaluated. Smooth muscle strips of the tunica muscularis mucosa of esophagus were studied in standard organ chambers. Carbachol- and KCl-induced contractile responses and serotonin- and papaverine- induced relaxant responses in both reflux and sham-operated control groups were determined. Results Histopathologically, chronic reflux esophagitis was observed in all specimens of the reflux group. Contractile (carbachol- and KCl-induced) smooth muscle responses were significantly decreased in the reflux group. When compared to control group, relaxant response of smooth muscle to serotonin was also significantly decreased in the reflux group. However, there was no difference in papaverine-induced relaxant responses between 2 groups. Conclusions Our study describes the effects of chronic GER on rat esophageal smooth muscle contractility in vitro. We found that both receptor- (carbachol, serotonin) and nonreceptor-mediated (KCl) esophageal smooth muscle reactivity were impaired in chronic reflux esophagitis. These changes may correspond to the functional motor abnormalities of the esophagus seen in patients with chronic reflux esophagitis.

Ceramide-Induced Impairment of Endothelial Function Is Prevented by CuZn Superoxide Dismutase Overexpression

Ceramide is an important intracellular second messenger that may also increase superoxide. The goal of this study was to determine whether overexpression of CuZn superoxide dismutase (SOD) protects against ceramide-induced increases in vascular superoxide and endothelial dysfunction. Carotid arteries from CuZnSOD-transgenic (CuZnSOD-Tg) and nontransgenic littermates were examined in vitro. Immunohistochemistry confirmed that CuZnSOD protein was greater in carotid artery from CuZnSOD-Tg compared with nontransgenic mice. Ceramide (N-acetyl-d-sphingosine; 1 and 10 micromol/L) produced concentration-dependent impairment (P<0.05) of vasorelaxation in response to the endothelium-dependent agonist acetylcholine (ACh) in nontransgenic mice. For example, 100 micromol/L ACh relaxed arteries from nontransgenic mice by 96+/-4% and 52+/-5% in the presence of vehicle and 10 micromol/L ceramide, respectively. In contrast, ceramide (1 or 10 micromol/L) had no effect (P>0.05) on responses of carotid artery to ACh in CuZnSOD-Tg mice. Ceramide had no effect on nitroprusside- or papaverine-induced relaxation in CuZnSOD-Tg or nontransgenic mice. Ceramide increased superoxide in arteries from nontransgenic vessels, and this effect was prevented by polyethyleneglycol-SOD (50 U/mL) or overexpression of CuZnSOD. These results suggest that ceramide-induced increases in superoxide impair endothelium-dependent relaxation, and that select overexpression of the CuZn isoform of SOD prevents ceramide-induced oxidative stress in vessels.

Characterization of capsaicin-induced, capsazepine-insensitive relaxation of ileal smooth muscle of rats

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC 20) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6±7.5%, n=10 and 97.1±0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca 2+ channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro- l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca 2+-free, EGTA solution. Phosphorylation of MLC 20 (a percentage of phosphorylated to total MLC 20) was increased 1 min after application of 10 μM acetylcholine (7.8±2.0%, n=6 vs. 22.6±3.2%, n=6) and of 65.9 mM KCl (2.2±0.3%, n=8 vs. 10.7±1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC 20 phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC 20 phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC 20 phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca 2+ influx via non-L-type Ca 2+ channels, possibly via store-operated Ca 2+ channels and Ca 2+ release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC 20 phosphorylation.

The effect of epigallocatechin gallate on intestinal motility in normal and endotoxemic mice

The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) and (−)-epigallocatechin-3-gallate (EGCg) on motility of jejunal segments isolated from mice. Endotoxemia, induced by intraperitoneal injection of 10 mg/kg LPS, evoked a significant decrease in the amplitude of spontaneous isometric contractions and an increase in the papaverine-induced relaxations. EGCg induced a dose-dependent relaxation and a decrease in the amplitude and the rate of spontaneous contractions. The effect was observed at relatively low concentration of EGCg (0.125 mM) and was not inhibited by naloxone, phentolamine, or yohimbine. The response to acetylcholine in the presence of EGCg was bigger in endotoxemic animals. Naloxone enhanced the acetylcholine-induced contractions in the presence of EGCg in saline-treated animals, while in endotoxemic animals it was not effective. In conclusion, endotoxemia induces an impairment of spontaneous jejunal contractions recorded in vitro. The EGCg-induced relaxations are smaller in endotoxemic tissues and do not depend on alpha adrenergic and opioid receptors. Additionally, an EGCg-induced increase in the acetylcholine-evoked contractions in endotoxemic animals suggests that EGCg may have a positive effect on LPS-induced impairment in intestinal motility.

Impaired gastric motility in the gastroesophageal reflux rat model: an in vitro study

Purpose The present study investigated the effects of acid and mixed reflux on the responsiveness of gastric smooth muscle in the gastroesophageal reflux (GER) rat model. Material and methods Three groups of rat were studied encompassing acid reflux, mixed reflux and sham operation. Acid reflux was induced by pyloric ligation (AR group) and mixed reflux was induced by jejunal ligation 1 cm distal to Treitz ligament (MR group). Similar surgical manipulations were carried out in the sham operated rats (SO group). Carbachol-, serotonin-, KCl-induced contractile response and nicotine-, sodium nitroprusside-, papaverine-induced relaxant response in isolated gastric fundus smooth muscle strips were determined using in vitro muscle technique 24 h after surgery. Results Isolated gastric fundus smooth muscle contractility to serotonin, carbachol or KCl was significantly reduced in the AR and MR groups with decreased Emax and pD 2 values compared with the SO group. Relaxant responses to nicotine was significantly increased in the AR and MR groups with increased Emax and pD 2 values compared with the SO group. Sodium nitroprusside and papaverine-induced-relaxant responses were similar in all of the groups and there was no change in agonist potency. Conclusion The present study indicates that decreased contractile and increased nicotine-induced relaxant response of the gastric smooth muscle in the surgically created GER model. These findings suggest that impaired gastric smooth muscle reactivity at least in part may play a role in gastric dysmotility in GER.

Augmentation of NO-mediated vasodilation in metabolic acidosis

Reduction of perivascular pH in acidemia produces hyporesponsiveness of vascular bed to vasoconstrictors. In the present study, we examined the effects of modest acidification on dilatory responses of isolated rat thoracic aorta. Acetylcholine produced endothelium-dependent relaxation in phenylephrine-precontracted aorta, which was markedly enhanced by acidification of Krebs-Henseleit solution from pH 7.4 to 7.0. A similar augmentation was observed in the relaxing responses to NO donors (SNP, SIN-1, SNAP), 8-Br-cGMP and NS-1619 (a putative K Ca channel opener and/or Ca channel inhibitor) in endothelium-denuded, phenylephrine-contracted aorta. However, papaverine-induced relaxation was not affected by the change in pH. At pH 7.4, the relaxing responses to acetylcholine and SNP were partially inhibited by charybdotoxin (K Ca channel inhibitor) but not glibenclamide (K ATP channel inhibitor), while at pH 7.0 the relaxation induced by either drug was not affected by K + channel inhibitors. Relaxation induced by 8-Br-cGMP or NS-1619 was not inhibited by charybdotoxin or glibenclamide. Acidification to pH 7.0 increased the cGMP production in response to acetylcholine in endothelium-intact aorta and to SNP in endothelium-denuded aorta. These results show that modest acidification augments NO-mediated relaxation in rat aorta, probably due to an enhancement of cGMP-dependent but K + channel-unrelated relaxation mechanisms.

Aging Impairs Nitric Oxide-Mediated Relaxant Responses of Rabbit Corporal Smooth Muscle

Aging has been reported to cause impotence, the mechanism of which is unknown. Therefore, we investigated the effect of aging on electrical stimulation-induced neurogenic, carbachol-induced, endothelium-dependent, and sodium nitroprusside-induced cGMP-dependent relaxant responses of rabbit corporal smooth muscle. Male New Zealand white rabbits were divided into young (4 months), intermediate (8 months), and old (24 months) groups. Electrical stimulation-, carbachol-, sodium nitroprusside-, and papaverine-induced relaxant response in isolated corporal smooth muscle strips were determined using in vitro muscle technique. Although there was no significant difference in the relaxant response of corporal strips to papaverine among the groups, relaxant responses to carbachol and sodium nitroprusside were significantly lower in corporal strips of old group than both young and intermediate groups. However, in the old and intermediate groups, electrical field stimulation-induced neurogenic relaxation was significantly reduced compared with the young group. KCl-induced (124 mM) contractile responses were the same in all groups. Our data indicate that the presence of age-dependent differences in the NO/cGMP-mediated relaxant responses of corporal tissue in the male rabbits. This may contribute to the development of impotence.

Involvement of BK Ca channels in the relaxation of detrusor muscle via β-adrenoceptors

Detrusor muscle relaxes upon activation of β-adrenoceptors on smooth muscle cells. However, the mechanism of relaxation following the stimulation of β-adrenoceptors remains unclear. In order to clarify the mechanism, we investigated the involvement of ion channels in bladder relaxation. In guinea-pig isolated bladder strips precontracted by high-K +, isoproterenol caused concentration-dependent relaxation. The relaxation caused by isoproterenol (1 μM) was larger in 30 mM K + than in 120 mM K + (54.2±8.0% and 18.2±4.1% of papaverine-induced relaxation, respectively, n=4). Iberiotoxin (100 nM) inhibited the isoproterenol-induced relaxation (vehicle 69.5±8.0% vs. iberiotoxin 24.9±6.2%, respectively, n=5). Whole-cell patch-clamp recording revealed that isoproterenol as well as forskolin increased the iberiotoxin-sensitive K + currents, and this increase was abolished by protein kinase inhibitor. These results suggest that the isoproterenol-induced relaxation of guinea-pig bladder smooth muscle is mainly mediated by facilitation of BK Ca channels subsequent to the activation of the cAMP/protein kinase A pathway.

Inhibitory mechanism of papaverine on the smooth muscle of guinea pig urinary bladder.

In guinea pig urinary bladder, the hyperosmotic 65 mM KCI (H-65K+)- or carbachol (CCh)-induced contraction was inhibited by an addition of papaverine in a concentration-dependent manner. The cAMP content of the muscle in the presence of H-65K+ or CCh was increased by papaverine only at the higher concentration of 100 microM, but cGMP content was not affected by papaverine. Forskolin, compared with papaverine, increased cAMP content in a concentration-dependent manner, and nitroprusside did not significantly increase cGMP content. In a fura 2 loaded muscle, papaverine did not affect an increase of [Ca2+]i level by high K+ or CCh. The increase of oxidized flavoprotein (FPox) fluorescence and muscle contraction in the presence of H-65K+ or CCh was decreased by papaverine (1 - 100 microM), and the increase of pyridine nucleotide (PNred) fluorescence was not affected by papaverine. In summary, it was concluded that papaverine induced relaxation by inhibiting mitochondrial respiration in guinea pig urinary bladder as well as ileum. Moreover, it is proposed that the mechanism of papaverine-induced relaxation in the smooth muscle, which shows predominantly a metabolic dependency on its contraction, is an inhibition of mitochondrial respiration.

Parathyroid hormone-related protein in rat penis: expression, localization, and effect on cavernosal pressure.

Although PTH-related protein-(1-36) [PTHrP-(1-36)] is known to be expressed in smooth muscle and to exert potent myorelaxant effects, its tonic effects on cavernosal smooth muscle has not yet been explored. Using the RT-PCR technique, the present study establishes that PTHrP messenger RNA is present in microdissected corpus cavernosa in the rat. In immunohistochemical studies using affinity-purified antibodies to middle regions of PTHrP, immunostaining was localized throughout the penile structures, including vessels, cavernosal smooth muscle, and trabecular fibroblasts. Strong immunostaining for PTHrP was also detected in the dorsal nerve bundles. In anesthetized rats, intracavernosally injected boluses of increasing doses of PTHrP-(1-36) (0.3-30 pmol in 100 microl saline) had little effect on intracavernosal pressure. However, they markedly potentiated the dilatory response to papaverine (8-800 nmol), increasing the papaverine-induced intracavernous pressure by 2.5-fold, close to the mean arterial pressure. In conclusion, the cavernosal expression of PTHrP messenger RNA, the distribution of immunoreactive PTHrP throughout the structuro-functional components of the erectile apparatus and its strong potentiating action on papaverine-induced cavernosal relaxation, collectively suggest that PTHrP participates in the control of cavernosal tone.