Michael Sohn

In vitro effects of a novel class of nitric oxide (NO) donating compounds on isolated human erectile tissue.

OBJECTIVES The discovery of nitric oxide (NO) as one of the major effectors in penile erectile function has led to the development of various drugs which are able to elevate intracellular levels of cGMP. Recently, a novel class of NO donors have been developed, exemplified by S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine-ethylester (SNACET), as well as compounds combining both phosphodiesterase inhibitory and NO donating activity, such as NCX 911 (sildenafil nitrate). In our study, we assessed the effects of GSNO, SNACET and NCX 911 on adrenergic tension and electrically induced relaxation of isolated human corpus cavernosum (HCC) and the in vitro formation of cGMP. Effects were compared to those of sodium nitroprusside (SNP) and sildenafil citrate. MATERIALS AND METHODS Using the organ bath technique, drug effects were investigated on norepinephrine-induced tension and electrically induced relaxation of HCC. HCC strips were exposed to increasing concentrations of the compounds (0.01/0.1-10/100 microM) and the accumulation of cGMP was determined by means of a radioimmunoassay. RESULTS Relaxation of HCC induced by means of electrical field stimulation (EFS) was abolished by tetrotodoxin, guanylyl cyclase inhibitor ODQ and nitric oxide synthase inhibitor L-NNA. Adrenergic tension of HCC strips was dose-dependently reversed by the drugs. The rank order of potency was: SNP > GSNO > NCX911 > sildenafil > SNACET. Compounds also dose-dependently increased EFS-induced amplitudes of relaxation (SNP > NCX911 > sildenafil > SNACET/GSNO). Relaxing effects of the drugs were paralleled by an increase in tissue levels of cGMP. CONCLUSION Our results provide a rationale for future use of NCX 911 and S-nitrosothiols in the pharmacotherapy of erectile dysfunction (ED). Since the compounds are assumed to exert no considerable hemodynamic effects, they might be developed into new oral treatments for ED.

Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue

INTRODUCTION Besides the bioavailability of nitric oxide (NO), downstream guanine monophosphate (cGMP) effector proteins are also considered to play a significant role in penile vascular disease. In animal studies, a downregulation of the cGMP-dependent protein kinase-1 (cGKI) alpha isoform has been linked to erectile dysfunction and diabetes mellitus. So far, the expression of cGKI alpha and beta isoforms has not been evaluated in human penile erectile tissue. AIM To evaluate the expression of cGKI alpha and beta isoforms in relation to smooth muscle alpha-actin, cGMP, and endothelial NO synthase (eNOS) in human cavernous arteries (HCAs) and human corpus cavernosum (HCC). METHODS Cryostat sections of HCA and HCC were incubated with primary antibodies directed against alpha-actin, cGMP, eNOS, cGKI, cGKI alpha, and cGKI beta. Visualization of double-labeled immunofluorescent stainings was achieved by laser microscopy. Western blot analysis was performed in order to confirm the expression of cGKI isoforms. MAIN OUTCOME MEASURES Expression of cGKI alpha and beta isoforms in relation to smooth muscle alpha-actin, cGMP, and eNOS in human penile erectile tissue. RESULTS Immunoreactivities specific for cGKI, cGKI alpha, and cGKI beta were observed within the smooth musculature and the endothelium of cavernous arteries and sinusoids. Double stainings revealed the colocalization of alpha-actin, cGMP, eNOS, and cGKI isoforms. The expression of cGKI isoforms was confirmed by Western blot analysis. CONCLUSIONS Our results demonstrate, for the first time, the expression of both cGKI alpha and beta isoforms in the smooth musculature of HCA and HCC. Corresponding to recent findings from animal studies, the presence of cGKI alpha and beta provides further evidence for a significant role of these enzymes in the control of smooth muscle function in human penile erectile tissue.

Rho Kinase (ROK)-Related Proteins in Human Cavernous Arteries: An Immunohistochemical and Functional Approach.

INTRODUCTION Rho kinases (ROKs) cause calcium-independent modulation of smooth muscle contraction. A significant role for the RhoA/ROK pathway in mediating the contraction of the penile erectile tissue has been suggested. Moreover, it has been postulated that ROK activity might represent a key factor in the pathophysiology of erectile dysfunction. Up until today, little is known on the significance of ROK and related proteins in the control of blood flow in the corpus cavernosum. AIM To investigate by means of immunohistochemistry and organ bath studies the significance of the Rho pathway in human cavernous arteries. MAIN OUTCOME MEASURES The expression of ROK1, ROK2, RhoA, and RhoGDI in human cavernous arteries was investigated by means of immunohistochemistry; myographic studies were conducted in order to characterize the effects of the ROK inhibitor Y27632 on isolated cavernous arteries. METHODS Specimens of human cavernous arteries were processed for immunohistochemistry for ROK1, ROK2, RhoA, and RhoGDI. Circular penile vascular segments were mounted in a tissue bath and the effects of increasing concentrations of the ROK inhibitor Y27632 on the tension induced by norepinephrine (NE, 1 µM) were investigated. RESULTS Alpha-actin immunoreactive cavernous arterioles also presented abundant staining specific for ROK1, ROK2, RhoA, and RhoGDI in the smooth musculature of the vascular wall. Cumulative addition of Y27632 dose-dependently reversed the tension induced by NE of isolated arterial segments. Y27632 produced relaxant responses with a reversion of tension of 34.3 ± 11.8% at a concentration of 1 µM. CONCLUSION The findings are in support for a role of the Rho/ROK-mediated signaling in the regulation of muscle tone of human cavernous arteries.

Systemic and cavernous plasma levels of endothelin 1 in healthy males during different functional conditions of the penis.

Abstract The role of the sympathetic adrenergic nerves in mediating the constant tone of penile flaccidity and returning the erect penis to its flaccid state is fairly well established. However, it is not yet known whether additional nonadrenergic transmitters might be involved in this process. Endothelin 1 (ET-1), a 21-amino-acid peptide with potent and long-lasting vasoconstrictor activity, may be one of the factors contributing to such control. The present study was undertaken to determine whether plasma levels of ET-1 changed during flaccidity, tumescence, rigidity, and detumescence. We determined plasma levels of ET-1 in the peripheral and cavernosal blood of 32 potent adult male volunteers, in whom penile tumescence and erection were elicited by exposure to visual and tactile erotic stimuli. Whole blood was aspirated from the corpus cavernosum and the cubital vein, and ET-1 was quantified in plasma aliquots obtained from the blood samples. Using the organ bath technique, we evaluated the contractile effects of ET-1 and norepinephrine (NE) on isolated human corpus cavernosum musculature. No significant change in ET-1 levels was observed in the peripheral or cavernosal blood in the process of developing erection, rigidity, or detumescence. The mean plasma level of ET-1 was 0.2–0.7 fmol/ml. In the organ bath, ET-1 elicited concentration-dependent contractions of isolated human corpus cavernosum, which were much more pronounced than those evoked by the adrenergic agonist NE. Our data indicate that despite the in vitro efficacy of ET-1 in stimulating contractile activity in isolated human cavernous smooth muscle, the peptide may not be of ultimate importance for the mechanism of flaccidity and detumescence in healthy males. Nevertheless, the exact role of ETs in the control of penile smooth muscle tone remains to be established.

Potential Mechanism of Action of Human Growth Hormone on Isolated Human Penile Erectile Tissue

OBJECTIVES To evaluate the mechanisms of growth hormone (GH) action on isolated human penile erectile tissue. Human GH (hGH) has been suggested to play a role in male reproductive function, including penile erection. Nevertheless, it still remains unclear which intracellular pathways mediate the physiological effects of GH on the human corpus cavernosum (HCC). METHODS Using the organ bath technique, the effects of GH were investigated on electrical field stimulation (EFS)-induced relaxation of isolated HCC in the absence and presence of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine (l-NOARG, 10 microm). Effects of GH on the production of tissue cyclic guanosine monophosphate (cGMP) in the absence and presence of ODQ and l-NOARG were also elucidated using radioimmunoassay. RESULTS ODQ and l-NOARG abolished the relaxation of the tissue induced by EFS, whereas amplitudes were increased by physiological concentrations of GH (1-100 nm). The attenuation of EFS-induced amplitudes by l-NOARG but not ODQ was, in part, reversed by GH. The production of cGMP (pmol cGMP/mg protein) induced by 10 nm GH was abolished in the presence of 10 microm ODQ. In contrast, the combination of GH (10 nm) and l-NOARG (10 microm) maintained cGMP production significantly greater than baseline (0.68 +/- 0.36 vs 1.07 +/- 0.48 pmol cGMP/mg protein). CONCLUSIONS Our data provide evidence that GH may act on human HCC by an NO-independent effect on guanylyl cyclase activity and may thus explain how growth factors, such as hGH, regulate male erectile function.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Male Urethra

OBJECTIVE To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. METHODS Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. RESULTS Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. CONCLUSION The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.

Phosphodiesterase isoenzymes in the human urethra: A molecular biology and functional study

Experimental and clinical studies have suggested a role for phosphodiesterase (PDE) isoenzymes in the control of the human lower urinary tract. This study aimed to investigate the expression of PDE isoenzymes and the effects of PDE inhibitors (PDE-Is) in isolated human urethral smooth muscle (USM). The expression of messenger ribonucleic acid (mRNA) specifically encoding for PDE isoenzymes and isoforms (1A, 1B, 1C, 2A, 4A, 4B, 4C, 4D, 5A and 11A) was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR). Using a tissue bath technique, the effects of vinpocetine (PDE1-I), erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA-HCl=MEP1) (PDE2-I), rolipram (PDE4-I), sildenafil, vardenafil and tadalafil (PDE5-Is) (0.01-10µM) on the tension of USM induced by norepinephrine were investigated. The production of cyclic guanosine monophosphate (cyclic GMP) and cyclic adenosine monophosphate (cyclic AMP) was measured by means of radioimmunoassays. RT-PCR analysis revealed the expression of PDE1B, PDE1C, PDE4A, PDE4C, PDE4D, PDE5A and PDE11A. The tension induced by norepinephrine (NE) was reversed by the PDE inhibitors with the following rank order of efficacy: rolipram (mean: -39%)≥sildenafil (-35%)>vardenafil (-26%)>tadalafil (-20%)>vinpocetine (-16%)>MEP1 (-2%). The relaxing effects of the drugs were paralleled by an elevation in tissue levels of cyclic AMP and cyclic GMP. Selective inhibitors of PDE4 and PDE5 can antagonize the tension induced by alpha-adrenergic stimulation of USM. PDE inhibition might represent an interesting option to facilitate the relaxation of the human outflow region.

Pharmacologic Characterization of Human Male Urethral Smooth Muscle: An In Vitro Approach

OBJECTIVE To elucidate the functional responses of isolated human urethral smooth muscle to various agents known to exert smooth muscle contraction or relaxation. METHODS Specimens of penile urethra were obtained from male patients who had undergone male-to-female gender reassignment surgery. Using the tissue bath technique, the contraction induced by increasing concentrations (1 nM-10 μM) of norepinephrine, phenylephrine, acetylcholine, carbachol, prostaglandin F2α, endothelin 1, angiotensin II, and oxytocin was measured. In another set-up, the effects of C-type natriuretic peptide (0.1 nM-1 μM), sodium nitroprusside, sildenafil, forskolin, alpha2-antagonist delquamine, and acetylcholine (1 nM/10 nM-10 μM) on the tension induced by norepinephrine were investigated. The production of cyclic guanosine monophosphate (GMP) and cyclic adenosine monophosphate (AMP) was measured by means of specific radioimmunoassays. RESULTS Endothelin 1, oxytocin, prostaglandin F2α, norepinephrine, and phenylephrine induced dose-dependent contraction of the isolated urethral tissue, whereas acetylcholine, carbachol, and angiotensin II had no or only minor contractile effects. The contraction induced by norepinephrine was reversed by the drugs with the following rank order of efficacy: sodium nitroprusside > delquamine > sildenafil > C-type natriuretic peptide > forskolin > acetylcholine. The maximal reversion of tension ranged from 68% (sodium nitroprusside) to 22% (acetylcholine). The relaxing effects of the drugs were paralleled by a several-fold increase in tissue levels of cyclic GMP and cyclic adenosine monophosphate. CONCLUSION The results provide evidence that urethral smooth muscle is under the control of endogenous compounds, such as adrenergic agonists (norepinephrine and phenylephrine), vasoactive peptides, prostagladins, NO/cyclic GMP, and acetylcholine, assumed to influence micturition at the peripheral level.

Expression and distribution of the transient receptor potential cationic channel A1 (TRPA1) in the human clitoris-comparison to male penile erectile tissue

The transient receptor potential cationic channel ankyrin 1 (TRPA1) is a channel protein assumed to act in various human tissues as mechano- and pain sensor and play a role in neurotransmission. The expression of TRPA has already been investigated in the human prostate and urethra, however, only very few studies have addressed the expression and distribution in the male and female genital tract. The present study aimed to investigate by means of immunohistochemistry (double-labeling technique, laser fluorescence microscopy) in the human clitoris and penile erectile tissue the localization of TRPA1 in relation to nNOS, the vasoactive intestinal polypeptide (VIP) and vesicular acetylcholine transporter (VAChT). In the clitoral tissue, TRPA1 was observed in basal epithelial cells and slender nNOS-positive nerve fibers transversing the subepithelial space. To a certain degree, in the clitoral epithelial cells, TRPA1 was found co-localized with vimentin. In human corpus cavernosum, immunoreactivity for TRPA1 was seen in nerves transversing the cavernous sinusoidal space and running alongside small arteries, these nerves also displayed expression of the vesicular acetylcholine transporter protein (VAChT). Varicose nerves containing nNOS or VIP were not immunoreactive for TRPA1. It seems likely that TRPA1 is involved in nitric oxide-mediated afferent sensory transmission in the clitoris while, in penile erectile tissue, a role for TRPA1 in cholinergic signaling might be assumed.