Effects of endopeptidase inhibition on the contraction-relaxation response of isolated human vaginal tissue.

INTRODUCTION.: Vasoactive peptides, such as bradykinin, C-type natriuretic peptide (CNP), vasoactive intestinal polypeptide (VIP), and endothelin 1 (ET-1), are assumed to be involved in the control of female genital vascular and nonvascular smooth muscle. Tissue levels of said peptides are controlled by the activity of endopeptidase enzymes. Theoretically, in female genital tissues, inhibiting the degradation of bradykinin, CNP, and VIP, or the conversion of Big ET-1 into ET-1 should result in an enhancement in smooth muscle relaxation and, thus, an improvement in sexual response. AIM.: Elucidate the effects of the endopeptidase inhibitor KC 12615 on the contraction/relaxation response of isolated human vaginal smooth muscle to Big ET-1, bradykinin, CNP, or VIP. METHODS.: Tissue bath experiments were carried out to ascertain the responses of human vaginal tissue challenged by ET-1 (0.1 µM) to increasing concentrations of bradykinin, CNP, and VIP (0.01 µM, 0.1 µM, and 1 µM, respectively). The effects were also evaluated following preexposure to KC 12615 (10 µM, for 20 minutes). MAIN OUTCOME MEASURES.: Measure the effects of KC 12615 on the relaxation of isolated human vaginal smooth muscle brought about by bradykinin, CNP, or VIP and the contraction mediated by Big ET-1. RESULTS.: The tension induced by ET-1 was reversed by bradykinin, CNP, or VIP (-25 ± 6.6%, -13.3 ± 2.2%, and -17.6 ± 10%, respectively). Big ET-1 induced contraction of the vaginal tissue. Preexposure of the tissue to KC 12615 increased the relaxation exerted by bradykinin, CNP, or VIP (to -39.2 ± 5.8%, -40.7 ± 7.3%, and -44.6 ± 19%, respectively). The contraction induced by Big ET-1 was attenuated in the presence of KC 12615 (to approximately 25% of the initial response). CONCLUSION.: Inhibition of endopeptidase activity can antagonize the contraction of human vaginal tissue induced by Big ET-1 and increase the relaxation induced by vasoactive endogenous peptides.

Rho kinase-related proteins in human vaginal arteries: an immunohistochemical and functional study.

INTRODUCTION: The calcium-sensitizing Rho A/Rho kinase pathway has been suggested to play a role in the control of nongenital vascular smooth muscle. Rho-associated kinases (ROKs) cause calcium-independent modulation of smooth muscle contraction, and have been demonstrated in the bladder, prostate, and corpus cavernosum. Until now, it is not known whether ROKs and related proteins play a role in the control of vaginal blood flow. AIM: To investigate by means of functional studies and immunohistochemistry the significance of the Rho pathway in human vaginal arteries. METHODS: Vaginal tissue was obtained from five postmenopausal women. Specimens were processed for immunohistochemistry for ROK1, ROK2, RhoA, and RhoGDI. Segments of sub-epithelial vaginal arteries were mounted in a tissue bath. Effects of Y27632 on the concentration-response curves to phenylephrine (Phe) or Phe-precontracted preparations were investigated. MAIN OUTCOME MEASURE: The expression of Rho kinases ROK1, ROK2, and the Rho-associated protein RhoGDI in human vaginal arteries was investigated by means of immunohistochemistry. Tissue bath studies were conducted in order to characterize the effects of the ROK inhibitor Y27632 on isolated vaginal arteries. RESULTS: A meshwork of α-actin immunoreactive arterioles was located in the sub-epithelium of human vaginal specimens. Immunoreactivities for ROK1, ROK2, RhoA, and RhoGDI were expressed in the smooth musculature of these arteries. At 0.1 and 1 µM Y27632, the contraction to Phe (10 µM) was 99 ± 17% and 28 ± 12% that of 124 mM K(+) . In Phe-contracted preparations, Y27632 produced relaxant responses. CONCLUSIONS: The activation of alpha(1) -adrenoceptors contracts sub-epithelial human vaginal arteries via ROK-sensitive mechanisms. A role for these signals in the regulation of vaginal blood flow might be considered.

Endogenous vasoactive peptides and the human vagina--a molecular biology and functional study.

INTRODUCTION: Endogenous peptides, such as vasoactive intestinal polypeptide (VIP), C-type natriuretic peptide (CNP), and bradykinin (BK), have been proposed to play a role in the female sexual arousal response by exerting relaxation of clitoral, labial, and vaginal smooth muscle. While the effects of endogenous peptides on the human male erectile tissue have already been described, only very few studies have been conducted to investigate the peptidergic control of female genital tissues, including the vagina. AIMS: To elucidate the expression of mRNA specifically encoding for peptide receptors in the human vagina and the effects of VIP, CNP, and BK on the tension induced by endothelin-1 (ET-1) of isolated human vaginal wall smooth muscle. The production of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in response to exposure of the tissue to the peptides was also measured. METHODS: The expression of mRNA encoding for receptor proteins specific for VIP, CNP, and BK were investigated by means of molecular biology (reverse transcriptase polymerase chain reaction [RT-PCR] analysis). Using the organ bath technique, the effects of VIP, CNP, and BK (0.1 nM to 1 µM) on the tension induced by 0.1 µM ET-1 of human vaginal strips were investigated. The tissue was also exposed to three different concentrations of VIP, CNP, and BK (0.01 µM, 0.1 µM, 1 µM) and the production of cAMP and cGMP determined by means of radioimmunoassays. MAIN OUTCOME MEASURES: Characterize the expression of peptide receptors in the human vagina and measure the relaxation exerted by BK, CNP, and VIP on the contraction induced by ET-1 of isolated human vaginal tissue. In addition, the effects of the peptides on the production of cAMP and cGMP were also elucidated. RESULTS: RT-PCR analysis revealed the expression of mRNA transcripts encoding for the VIP receptors VIP1R/vasoactive intestinal polypeptide receptor type 1 (VPAC1) and VIP2R/VPAC2, CNP receptors natriuretic peptide receptor type A (NPRA), natriuretic peptide receptor type B (NPRB) and natriuretic peptide receptor type C (NPRC), and BK receptor B2R. The tension induced by ET-1 was reversed by the peptides with the following rank order of efficacy: BK (21.7%) > VIP (20.9%) > CNP (13.3%). The relaxing effects of VIP and BK were paralleled by a 4.8-fold and fivefold increase in cAMP, while the production of cGMP was stimulated 38-fold and 119-fold in the presence of CNP or BK, respectively. CONCLUSION: Our results are in support of the hypothesis that endogenous peptides may contribute to the control of human vaginal smooth muscle tone through the involvement of the cyclic nucleotide-dependent pathways.

In vitro functional responses of isolated human vaginal tissue to selective phosphodiesterase inhibitors.

Only little is known as to the significance of the cyclic nucleotide-mediated signal transduction in the control of the function of human vaginal smooth musculature. Recently, the presence of the phosphodiesterase (PDE) isoenzymes 4 (cAMP-PDE) and 5 (cGMP-PDE) in the human vagina was reported. Thus, it was the aim of the study to elucidate the effects of some PDE inhibitors on the tension induced by endothelin 1 (ET-1), as well as on levels of cGMP and cAMP in isolated human vaginal wall tissue. Using the organ bath technique, the ability of norepinephrine (NE), carbachol, serotonin (5-HT), oxytocin and ET-1 to contract isolated vaginal wall muscle strips was evaluated. In another set-up, the effects of the PDE4 inhibitor rolipram and PDE5 inhibitors sildenafil and vardenafil (1 nM-10 microM) on the tension induced by 0.1 microM ET-1 of human vaginal wall tissue strips were investigated. In order to measure drug effects on tissue levels of cGMP and cAMP, vaginal tissue was exposed to different concentrations (0.1, 1 and 10 microM) of the compounds and the accumulation of cyclic nucleotides was determined. The adenylyl cyclase stimulating agents forskolin and nitric oxide donor sodium nitroprusside (SNP) (0.01, 0.1 and 1 microM) were used as reference compounds. While NE, carbachol and oxytocin failed to contract the vaginal tissue, ET-1 and, to a certain degree, 5-HT elicited contractile responses of the isolated strip preparations. The tension induced by 0.1 microM ET-1 was dose-dependently reversed by the drugs. The rank order of efficacy was sildenafil > forskolin > rolipram >or= vardenafil > SNP. Rmax values ranged from 24% (SNP) to 50% (sildenafil). With sildenafil being the only exception, none of the compounds reached an EC50 value. The relaxing effects of the drugs were paralleled by a fourfold to tenfold increase in tissue levels of cGMP and/or cAMP. Our results demonstrate that PDE inhibitors can relax human vaginal tissue and increase levels of cyclic nucleoside monophosphates. The findings with regard to the PDE5 inhibitors may indicate that the NO-cGMP pathway is, to a certain degree, involved in the control of vaginal smooth muscle tone. This might be of significance with regard to the pharmacological treatment of disorders connected with female sexual arousal and the ability to achieve orgasm.