Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries.

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01-10 µM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by L-NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane depolarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium inhibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. These findings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

The effect of sub-chronic systemic ethanol treatment on corpus cavernosal smooth muscle contraction: the contribution of RhoA/Rho-kinase.

The aim of this study was to evaluate whether the sub-chronic systemic ethanol exposure has direct effect on cavernosal smooth muscle contractions induced by KCl (depolarizing) and phenylephrine (α1-receptor agonist), and the possible involvement of RhoA/Rho-kinase pathway. Sub-chronic systemic ethanol was applied to mice with inhalation route for 14 days. The blood levels in ethanol-treated mice averaged 121.2 ± 9.1 mg/dl. KCl (80 mM) and phenylephrine (10 nM-100 µM) induced sustained contractions in corpus corporal strips from sham-treated mice. Sub-chronic ethanol treatment reduced the contractions to KCl. However, phenylephrine-induced contractions were not affected by ethanol treatment. Rho-kinase inhibitor fasudil (50 µM) and Y-27632 (50 µM) inhibited contractions to KCl and phenylephrine in sham-treated mice. Ethanol treatment increased the inhibitory effect of Rho-kinase inhibitors on contractions to phenylephrine. The relaxations induced by fasudil (100 µM) and Y-27632 (500 µM) did not change in ethanol treatment group. In ethanol-treated group, the expression of RhoA decreased compared to sham-treated group. Also, ROCK1 expression was reduced by ethanol but not statically significant to sham-treated group; however, the expression of ROCK2 increased in ethanol group. From these findings, it seems that phenylephrine and KCl-induced contractions depends on RhoA/Rho-kinase-mediated Ca(2+) sensitization. Also, these results suggest that the ethanol treatment decreased the expression of RhoA, and the inhibitory effect of ethanol on KCl-induced contractions may be due to, at least in part, the inhibition of a RhoA/Rho-kinase in mouse corpus cavernosum.

Evaluating the Teratogenicity of Ritodrine and Nifedipine using a Frog Embryo Teratogenesis assay (FETAX).

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was used to assess the teratogenic potential of two tocolytics. Embryos of the South African clawed frog, Xenopus laevis, were exposed to ritodrine or nifedipine. Exposure media were changed and monitored at 24-hour intervals. The 96-hour LC50 (Lethal concentration), the 96-hour EC50 (Malformation), and the No Observable Adverse Effect Concentrations (NOAEC) and the Lowest Observable Adverse Effect Concentration (LOAEC) for mortality, malformation and length were determined for each drug. Nifedipine was determined to be the more toxic and teratogenic than ritodrine, with a LC50 of 0.606 µg/L, an EC50 of 0.006 µg/L, and a teratogenicity Index (TI) value (LC50/EC50) of 101. On the other hand, the LC50 of ritodrine was 28.571 mg/L. In addition; the LC50, EC50 and TI values for nifedipine in the 5 mg/L ritodrine + nifedipine combination group were determined as 1.050 µg/L, 0.868 µg/L and 1.5 respectively. For ritodrine, the NOAEC and LOAEC values were determined as 2 mg/L and 4 mg/L, respectively. For the nifedipine and the ritodrine + nifedipine groups; while the LOAEC values of these groups were 0.0001 µg/L and 0.1 µg/L, respectively. NOAEC value couldn't be determined. Our results demonstrated that nifedipine administration was associated with higher levels of teratogenic and toxic effects. However, the ritodrine + nifedipine combination form reduced the toxic and teratogenic effects of nifedipine on Xenopus embryos. Further studies should be conducted in order to investigate the optimal combination concentrations of these substances for the treatment of preterm labor.

Effect of silymarin on bladder overactivity in cyclophosphamide-induced cystitis rat model.

The purpose of this study was to investigate the effects of silymarin, a phytotherapeutic agent, on bladder overactivity in a cyclophosphamide (CYP)-induced cystitis rat model. Female Wistar Albino rats received a single intraperitoneal injection of CYP (150 mg/kg) or saline and after 72 h, bladder function was evaluated by in vitro preparations of whole bladders and cystometry with continuous saline infusion under urethane anesthesia. Silymarin or a vehicle was orally given for 7 days in rats. CYP was injected on the 5th day of silymarin or vehicle treatment and then the animals were killed on the 8th day. CYP-treatment dramatically potentiated the basal spontaneous contractions of isolated whole bladders compared to control rats. In anesthetized rats, during continuous infusion cystometry, intercontraction interval (ICI) was significantly shorter, but bladder voiding pressure was not significantly changed in CYP-injected rats compared to control rats. In the CYP-injected group, silymarin treatment significantly decreased the amplitude, frequency (contractions/min) and area under the curve of spontaneous contractions, but failed to change carbachol-induced contraction in isolated whole bladder. Also, silymarin treatment significantly increased the ICI in comparison to the vehicle treatment. In the saline-injected group, no significant changes in the bladder function were observed between the silymarin and vehicle-treated groups. Histopathological examination showed that CYP-induced bladder inflammation tended to be lower in the silymarin+CYP-treated group. In conclusion, the oral administration of silymarin suppressed CYP-induced bladder overactivity. Silymarin may be considered as an attractive treatment for CYP-induced bladder overactivity.

Prejunctional facilitatory effect of a thiol-alkylating agent N-Ethylmaleimide on neurogenic contractions in rat prostate smooth muscle.

AIMS: The effect of a non-specific thiol-alkylating agent N-ethylmaleimide (NEM) was studied on neurogenic contractile mechanisms in rat ventral prostate gland. METHODS: Male Wistar albino rats were used. The rats were killed by cervical dislocation under sevoflurane anesthesia and ventral prostate gland was removed. Two preparations were obtained from each lobe. Neurally evoked isometric contractions were induced using trains of electrical field stimulation (EFS; 0.5, 1, 4, or 8 Hz). The effect of NEM on the contractions to EFS was examined in the absence or presence of adrenergic and/or purinergic antagonists. RESULTS: NEM enhanced the EFS-evoked contractions without altering the basal tone. These effects were significantly suppressed by an α(1) -adrenergic receptor antagonist (prazosin), a P2-purinergic antagonist (suramin), a specific P2X-receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), an ATP analog (α,ß-methylene ATP), or a calcium channel blocker (verapamil). This facilitating effect of NEM did not occur following the administration of L-cysteine or glutathione which saturated NEM with excess thiols. However, a thiol-oxidant diamide failed to affect the contractions to EFS. An adrenergic neuron blocker (guanethidine) completely suppressed the responses to NEM. On the other hand, an α(2) -adrenergic receptor blocker (yohimbine), a nitric oxide synthase inhibitor (N(ω) -nitro-L-arginine) or a cholinergic muscarinic receptor antagonist (atropine) did not significantly affect the facilitatory response of NEM. CONCLUSIONS: These findings suggest that NEM has a prejunctional facilitatory action on the adrenergic nerves in rat prostate tissue to enhance release of transmitters, noradrenaline, and ATP. NEM sensitive proteins involved in transmitter release mechanisms can play a role in this effect.

Differential effect of L-cysteine in isolated whole-bladder preparations from neonatal and adult rats.

The present study was undertaken to compare the effects of the thiol reagents L-cysteine and (diazene dicarboxylic acid bis 5N,N-dimethylamide) diamide on contractile activity of neonatal and adult rat bladders. In vitro whole-bladder preparations from Wistar rats were used to study the modulation of spontaneous bladder contractions by thiol reagents. After blocking cholinergic and adrenergic transmission with atropine and guanethidine, L-cysteine facilitated spontaneous bladder contractions in neonatal rat bladders. The effect of L-cysteine was suppressed by diamide. Diamide alone did not change basal activity of the neonatal rat bladder. The facilitatory effects of L-cysteine were reduced by the L-type Ca2+ channel-blocking agent nifedipine and the calcium-activated K+ channel opener NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one]. ATP or suramin, a purinergic receptor antagonist, significantly inhibited the effect of L-cysteine in neonatal bladders, whereas the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine was ineffective. L-cysteine did not elicit any detectable effects in the adult rat bladder; whereas diamide caused a large-amplitude sustained tonic contraction. The contraction induced by diamide in adult bladder did not occur when the preparation was pretreated with L-cysteine. Also, L-Cysteine administered during the diamide-evoked contraction completely inhibited the contraction to diamide. In conclusion, our results suggest that L-cysteine has markedly different effects in isolated whole-bladder preparations from neonatal and adult rats. Thus thiol-sensitive mechanisms may modulate contractility by regulation of Ca2+ and K+ channels and/or purinergic transmission in the neonatal bladder. The effects of L-cysteine and diamide were reversed in adult bladders, indicating that the regulation of bladder contractility by thiols is markedly altered during postnatal development.

Effects of vitamin E and sodium selenate on impaired contractile activity by bacterial lipopolysaccharide in the rat vas deferens.

We investigated whether bacterial lipopolysaccharide (LPS) treatment causes any hyporeactivity in rat vas deferens tissue and also whether vitamin E or sodium selenate has any restorative effect on this possible hyporesponsiveness. LPS treatment attenuated contractions to electrical field stimulation (EFS), phenylephrine, or ATP at the prostatic and epididymal ends. Treatment with the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine or vitamin E could prevent the impairment in contractile responses of both ends to EFS and phenylephrine but sodium selenate could restore these impaired contractions at only the epididymal end. LPS treatment also caused a similar significantly impairment on purinergic or adrenergic component of nerve-evoked contractions in the presence of prazosin or suramin, respectively, and vitamin E or sodium selenate could restored this impairment at both ends. On the other hand, both antioxidant agents failed to restore the impaired ATP-induced contractions in LPS-treated rats at both ends. In conclusion, LPS-treatment caused a hyporeactivity in the rat vas deferens. A possible increased oxidative activity in the vas deferens may be a major reason for the impairment of contractile responses. The restorative effects of vitamin E and/or sodium selenate on this hypocontractility may depend on their antioxidant properties or their inhibitory action on the iNOS.

Differential effect of neocuproine, a copper(I) chelator, on contractile activity in isolated ovariectomized non-pregnant rat, pregnant rat and pregnant human uterus.

The study was conducted to examine effects of a selective copper(I) chelator, neocuproine on the spontaneous or oxytocin-induced contractions in isolated ovariectomized non-pregnant rat, pregnant rat and pregnant human uterus. Uterus activity was evaluated in tissues obtained from bilaterally ovariectomized non-pregnant rats on the 21st day of the operation (n = 24), pregnant rats on the 19-21st day of gestation (n = 24) and women undergoing caesarean section at 38-42 weeks of pregnancy (n = 15). Neocuproine (100 microM) significantly suppressed the amplitude and frequency of the spontaneous contractions in the ovariectomized non-pregnant rat uterus while this agent facilitated the frequency of the spontaneous or oxytocin-induced contractions in the pregnant rat and human uterus without altering the amplitude of these contractions. At high concentration of 200 microM, neocuproine could enhance the amplitude of the contractions in the pregnant uterus. These effects were blocked by a purinergic receptor antagonist, suramin (100 microM) and did not occur following the administration of neocuproine-copper(I) complex or copper(II) chelator cuprizone. alpha, beta-methylene ATP increased the amplitude and frequency of contractions in the pregnant uterus, but not affected the contractions in the ovariectomized non-pregnant rat uterus, and neocuproine potentiated this facilitation effect. However, the suppressive effect of neocuproine on the ovariectomized non-pregnant rat uterus increased in the presence of alpha,beta-methylene ATP. Beta-adrenoceptor blocker, propranolol or nitric oxide synthase inhibitor, L-nitroarginine did not affect the responses to neocuproine. These findings suggest that neocuproine can affect the uterus contractile activity by modulation purinergic excitatory responses and that copper(I)-sensitive mechanisms may play a role in this effect.

Effect of phosphodiesterase type 4 inhibitor rolipram on cyclophosphamide-induced cystitis in rats.

Cyclophosphamide induces a severe haemorrhagic cystitis characterized by bladder overactivity. The study was conducted to examine effects of a phosphodiesterase 4 (PDE4) inhibitor rolipram on bladder overactivity in rats with cyclophosphamide treatment. 42 female Wistar rats were used. 30 rats received a single i.p. injection of cyclophosphamide, and after 72 h, bladder function was evaluated by (1) in vitro preparations of whole bladders and (2) cystometry with continuous saline infusion under urethane anesthesia. Cyclophosphamide-treatment dramatically potentiated the basal spontaneous contractions of isolated whole bladders compared to control rats. Atropine, guanethidine or suramin was ineffective on the spontaneous contractions whereas nifedipine completely abolished. Rolipram (5-80 microM) induced a significant concentration-dependent decrease on the amplitude, frequency (contractions/min) and area under the curve of spontaneous contractions. Carbachol elicited phasic contractions superimposed on a tonic contraction. Rolipram caused a relaxation on the tonic contraction whereas it could not affect the phasic contractions induced by carbachol. In anesthetized rats, during continuous infusion cystometry, intercontraction interval was significantly shorter in cyclophosphamide-injected rats than in control rats. Rolipram at 5-40 microM has no significant effect on the intercontraction interval and contraction pressure while it significantly decreased pressure threshold. At 80 microM, it significantly decreased the intercontraction interval and contraction pressure. In conclusion, PDE4 inhibitor rolipram caused a significant decrease on the amplitude, frequency and area under the curve of basal spontaneous contractions in cyclophosphamide-treated rats, at doses that have no effect on the carbachol-induced phasic contractions and cystometric parameters. PDE4 inhibitors may be considered as an attractive strategy for the treatment of cyclophosphamide-induced bladder overactivity.

The relaxant activity of 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]-pyridazine 1,5,6-trioxide in the mouse corpus cavernosum.

We have studied the effect of an activator of soluble guanylate cyclase 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO) on the tone and nitrergic relaxation responses of mouse cavernous strips and compared FPTO to a known nitric oxide donor sodium nitroprusside. FPTO thiol-dependently generated nitric oxide measured by polarography and activated purified human soluble guanylate cyclase. FPTO and sodium nitroprusside relaxed the cavernous tissue in a concentration-dependent manner. A nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine did not alter the relaxations to FPTO or sodium nitroprusside, whereas soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) suppressed relaxation to FPTO and sodium nitroprusside. Exogenously added thiols L-cysteine or dithiothreitol inhibited the relaxant responses to FPTO but not to sodium nitroprusside, whereas glutathione did not influence the responses to both agents. Thiol alkylation agent N-ethylmaleimide significantly enhanced FPTO-induced relaxation, and thiol-modifying agent diamide inhibited relaxation to FPTO. The potentiating effect of N-ethylmaleimide was neutralized by coadministration of N-ethylmaleimide with glutathione, L-cysteine, dithiothreitol, or ODQ. N-Ethylmaleimide but not diamide significantly inhibited relaxation induced by sodium nitroprusside. FPTO potently suppressed contraction to electrical field stimulation, which was prevented by glutathione or L-cysteine. In addition, FPTO did not affect relaxation produced by electrical field stimulation in phenylephrine-precontracted tissue. Our results show that FPTO can relax mouse corpus cavernosum and that the relaxant activity of this agent is thiol- and soluble guanylate cyclase-dependent. This effect could be potentiated by N-ethylmaleimide. FPTO does not potentiate nitrergic relaxation induced by electrical field stimulation.

Neocuproine, a copper (I) chelator, potentiates purinergic component of vas deferens contractions elicited by electrical field stimulation.

Effects of the specific copper (I) chelator, neocuproine, on the purinergic and adrenergic components of nerve-evoked contractions were investigated in the prostatic rat vas deferens. Electrical field stimulation (EFS; 4 Hz) induced bimodal contractions of vas deferens tissue in the presence of alpha1-adrenoceptor antagonist prazosin (to isolate the purinergic component) or purinoceptor antagonist suramin (to isolate the adrenergic component). Neocuproine significantly potentiated the purinergic component of the contractile responses to EFS. However, the same agent failed to elicit any significant effect on the adrenergic component of nerve-evoked contractions. The copper (II) chelator cuprizone could not affect the purinergic component of contractions. The potentiating effect of neocuproine which was reversible after washout of the drug, did not occur following the application of the pre-prepared neocuproine-copper (I) complex. A nitric oxide synthase inhibitor, L-nitroarginine; a cyclooxygenase inhibitor, indomethacin or an alpha2-adrenoceptor antagonist, yohimbine, failed to alter the responses to neocuproine on the purinergic component of the contraction to EFS. Neocuproine did not elicit any significant effect on preparations in which the purinergic receptors were desensitized with alpha,beta-methylene ATP. In conclusion, our results suggest that neocuproine potentiates the purinergic component of rat vas deferens contractions elicited by EFS, presumably by facilitating purinergic neurotransmission and that copper (I)-sensitive mechanisms can modulate purinergic transmission in this tissue.

Effects of melatonin on impaired neurogenic and endothelial relaxations by bacterial lipopolysaccharide in the mouse corpus cavernosum.

We investigated whether bacterial lipopolysaccharide treatment causes any neuronal and vascular hyporeactivity in mouse cavernous tissue and also whether melatonin has any restorative effect on this possible neuronal and vascular hyporesponsiveness. Lipopolysaccharide treatment attenuated contractions in response to phenylephrine. Treatment with the inducible nitric oxide synthase inhibitor aminoguanidine or melatonin restored the hypocontractility of the cavernous smooth muscle to phenylephrine. Relaxant responses of corpus cavernosum precontracted by phenylephrine to acetylcholine or electrical field stimulation were significantly impaired in mice treated with bacterial lipopolysaccharide. Treatment with aminoguanidine or melatonin could prevent the impairment of the neuronal and endothelial relaxations. There was no significant difference between control and lipopolysaccharide-treated groups in the contractile response to high-dose KCl and in the relaxant response to papaverine. In conclusion, bacterial lipopolysaccharide treatment caused a neuronal and endothelial dysfunction in the mouse corpus cavernosum. A possible increased oxidative activity in the cavernous tissue may be a major reason for the impairment of relaxant responses and hypocontracility of tissue. The restorative effects of melatonin on this hyporeactivity may depend on its antioxidant properties and partly on its inhibitory action on the inducible nitric oxide synthase production.