Eginhard S. Waldkirch

Transient receptor potential A1 (TRPA1) activity in the human urethra--evidence for a functional role for TRPA1 in the outflow region.

BACKGROUNDnA role for the transient receptor potential (TRP) A1 ion channel in rat lower urinary tract (LUT) sensation and disease has been proposed, but in the human LUT no information on TRPA1 activity is available.nnnOBJECTIVESnTo investigate the distribution of TRPA1 in the human urethra and to study the effect of TRPA1 agonists on isolated urethral strip preparations.nnnDESIGN, SETTINGS, AND PARTICIPANTSnUrethral specimens were obtained preoperatively from 10 patients and were freshly prepared for Western blot, immunohistochemistry, and functional in vitro investigations.nnnMEASUREMENTSnThe expression patterns of TRPA1 were studied with Western blot and immunohistochemistry. The effects of allyl isothiocyanate (AI), cinnamaldehyde (CA), and NaHS (donor of H(2)S) on tension of urethral strips were investigated in tissue baths.nnnRESULTS AND LIMITATIONSnTRPA1 immunoreactivity (-IR) was found in nerve fibres in the suburothelial space and was also located to nerve fibres of the muscle layer. Single TRPA1-IR nerves extended into the urothelium. A majority, but not all TRPA1-IR nerves also expressed immunoreactivity for CGRP or TRPV1. In the urothelium, TRPV1 was located to the outer layers whereas TRPA1 was observed in basal urothelial cells. Interspersed between strands of smooth muscle cells of the urethral wall, TRPA1- and vimentin-IR cells containing central nuclei and slender cytoplasmatic extensions were observed. In functional experiments, TRPA1-agonists had no contractile effect in urethral preparations. After precontraction with phenylephrine, AI, CA, and NaHS caused concentration-dependent relaxations of urethral strip preparations.nnnCONCLUSIONSnThe localization of TRPA1 to nerves that also express TRPV1 and CGRP, and in urothelial cells and interstitial cells, as well as the findings that TRPA1 agonists can modify tone of urethral preparations, propose a role for TRPA1 in afferent and efferent sensory signaling of the human outflow region.

Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone

Nitric oxide (NO)/cyclic GMP (cGMP)-mediated mechanisms have a pivotal function in reducing the tone of the penile smooth musculature during normal erectile responses. The cyclic AMP (cAMP) signaling pathway is also involved in the adjustment of smooth muscle contractility, and suggestions for interactions between cGMP- and cAMP-mediated mechanisms have been presented. Using activators of the cGMP- or the cAMP-pathway, as well as inhibitors of protein kinase A (PKA; cAMP-dependent kinase) and protein kinase G (PKG; cGMP-dependent kinase), the present study was undertaken to further delineate the functional relation between these pathways in the penis. In addition, the distribution of PKA and some cAMP-binding phosphodiesterases (cAMP-PDEs) were investigated in human erectile tissue. Functional experiments were performed on isolated human corpus cavernosum (HCC). The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10xa0µM), or the PKG, Rp-8-pCPT-cGMPS (10xa0µM), on relaxation induced by the cumulative administration of sodium nitroprusside (SNP), forskolin, sildenafil or tadalafil (IC351) were studied in preparations of HCC precontracted with 1xa0µM norepinephrine (NE). Using immunohistochemical procedures, the presence of immunoreactivity for cAMP-PDEs PDE3, PDE4, and PDE4A, as well as for PKA was investigated in specimens of HCC from which preparations were also used in the functional experiments. Forskolin, SNP, sildenafil, and IC 351 dose-dependently reversed NE-induced tension of isolated HCC preparations. The relaxing effects of SNP were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. In contrast, relaxation induced by forskolin, sildenafil and tadalafil were significantly reversed by both Rp-8-pCPT-cGMPS and Rp-8CPT-cAMPS. Abundant immunoreactivity for PDE3 and PKA was observed in the corpus cavernosum smooth muscle cells. Immunoreactivity for PDE4 was also detected in the smooth musculature and in the cytoplasm of endothelial cells lining the cavernous sinusoids, as well as in nerve fibres interspersing the trabecular stroma. The present results support the hypothesis of interactions between cGMP- and cAMP-mediated signals in the HCC, and suggest that the effects of inhibitors of PDE5 on isolated erectile tissue may also partly or indirectly include actions of the cAMP second messenger system. The exact mechanism by which such an interaction occurs is not clear, but it may involve altered activity of the cGMP-inhibited PDE3 brought about by a change in the intracellular levels of cGMP by the inhibition of PDE5. This will in turn lead to increasing levels of cAMP, facilitating the interaction of cAMP with the PKA. The immunoreactivity specific for PDE3, PDE4, PDE4A and PKA registered in HCC section is also in support of an important role for the cAMP/PKA-system for penile smooth muscle function.

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

OBJECTIVESnThe 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.nnnMATERIALS AND METHODSnUsing 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.nnnRESULTSnRelaxation 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.nnnCONCLUSIONnOur 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.

Cyclic AMP-specific and cyclic GMP-specific phosphodiesterase isoenzymes in human cavernous arteries—immunohistochemical distribution and functional significance

Objectives: It has been well established that male erectile dysfunction is frequently associated with vascular diseases. The normal function of cavernous arteries is considered a prerequisite to maintain sufficient blood flow to the trabecular spaces in order to enable penile erection. Contractility of cavernous arteries is regulated by the peripheral autonomic nervous system and endogenous factors released from the endothelial cell layer. A significant increase of blood flow in the central cavernous arteries is the initial event leading to penile tumescence and rigidity. Besides the significance of the nitric oxide/cyclic guanosine monophosphate (cGMP)-mediated mechanisms, the cyclic AMP (cAMP) signalling pathway is also involved in the regulation of tone of the erectile tissue, and interactions between cGMP- and cAMP-mediated mechanisms have been demonstrated. The aim of the present study was to investigate by means of immunohistochemistry the presence of the phosphodiesterase (PDE) isoenzymes 3, 4 (cAMP-specific PDEs) and 5 (cGMP-specific PDE) in thin sections of human central cavernous arteries (HCA) and their functional significance in the mechanism of vessel tone regulation. Methods: Functional experiments were performed using circular segments of HCA and strip preparations of the human corpus cavernosum (HCC). Relaxant effects induced by the cumulative addition of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram (PDE4 inhibitor) and sildenafil (PDE5 inhibitor; 0.01, 0.1, 1 and 10xa0M) were studied in preparations of HCA and HCC challenged by 1xa0M norepinephrine (NE). Moreover, immunohistochemistry was carried out in order to evaluate the expression of PDE3, PDE4 and PDE5 in thin sections of HCA. Results: Milrinone, rolipram and sildenafil dose-dependently reversed the NE-induced tension of the isolated vascular segments and HCC strips with sildenafil being the most effective drug. Neither rolipram nor milrinone reached an EC50 value. Abundant immunoreactivities specific for PDE3, PDE4 and PDE5 were observed in the entire smooth musculature of the wall of HCA and resistance arteries. In addition, immunoreactivity for PDE4 was also detected in the cytoplasm of endothelial cells lining the cavernous arteries. Conclusions: The cGMP-dependent relaxation of cavernous arteries is not only dependent on the normal function of the peripheral autonomic nervous system but also on the functional integrity of the vascular endothelium. The expression of the cGMP-specific PDE5 and the ability of the PDE5 inhibitor sildenafil to reverse the adrenergic tension of isolated segments of HCA underline the important role of the NO/cGMP pathway in the control of smooth muscle tone of human trabecular smooth musculature and penile cavernous arteries. Our results also suggest a significance of the cAMP-dependent signaling mechanisms in the regulation of tension of central HCAs. The present findings are also in support of the hypothesis of interactions between the cGMP- and cAMP-mediated signaling pathways in HCAs. Further investigations are indicated in order to outline potential differences between central HCAs and helicine resistance arteries. This may help to understand better the relations between structural and functional changes in the penile erectile tissue in patients with cardiovascular diseases and endothelial dysfunction.

Expression of cAMP-dependent protein kinase isoforms in the human prostate : functional significance and relation to PDE4

OBJECTIVESnTo investigate the expression of isoforms of the cyclic AMP (cAMP)-dependent protein kinase (cAK) in the transition zone of the human prostate and the functional significance of the enzyme in the control of prostate smooth muscle.nnnMETHODSnUsing Western blot analysis and immunohistochemistry, the expression and distribution in the prostate of cAKIalpha, cAKIbeta, cAKIIalpha, and cAKIIbeta in relation to alpha-actin and the phosphodiesterase PDE4 (types A and B) were investigated. The effects of the cAK inhibitor Rp-8-CPT-cAMPS on the reversion of the adrenergic tension of isolated prostate tissue induced by forskolin, rolipram, sodium nitroprusside (SNP), and tadalafil were examined by means of the organ bath technique.nnnRESULTSnImmunosignals specific for cAKIalpha, cAKIIalpha, and cAKIIbeta were observed in the smooth musculature and glandular structures of the prostate. Double stainings revealed the colocalization of alpha-actin and PDE4 with the cAK isoforms. The expression of the cAK isoforms was confirmed by Western blot analysis. The relaxation of the tension induced by norepinephrine brought about by forskolin, rolipram, SNP, and tadalafil was significantly attenuated by Rp-8-CPT-cAMPS.nnnCONCLUSIONSnThe colocalization of smooth muscle alpha-actin and PDE4 with cAK, as well as the results from the organ bath experiments, provide further evidence for a pivotal role of the cAMP-dependent signaling in the regulation of prostate smooth muscle contractility. Compounds interacting with the cAMP/cAK pathway might represent a new therapeutic avenue to treat symptoms of benign prostatic hyperplasia and lower urinary tract symptomatology.

Non‐genomic effects of androgens on isolated human vascular and nonvascular penile erectile tissue

To evaluate non‐genomic effects of testosterone and dihydrotestosterone (DHT) on isolated human cavernosal arteries (HCA) and corpus cavernosum (HCC) using organ‐bath studies and radio‐immunoassays (RIA), as non‐genomic effects of androgens are reported for vascular smooth musculature and there is evidence that the relaxant response involves a modulation of cyclic nucleotide tissue levels.

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

INTRODUCTIONnBesides 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.nnnAIMnTo 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).nnnMETHODSnCryostat 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.nnnMAIN OUTCOME MEASURESnExpression of cGKI alpha and beta isoforms in relation to smooth muscle alpha-actin, cGMP, and eNOS in human penile erectile tissue.nnnRESULTSnImmunoreactivities 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.nnnCONCLUSIONSnOur 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.

Phosphodiesterase type 5 (PDE5) is co-localized with key proteins of the nitric oxide/cyclic GMP signaling in the human prostate

PurposeExperimental studies have provided the basis for the evaluation of inhibitors of the phosphodiesterase type 5 (PDE5) in the treatment of lower urinary tract symptomatology (LUTS) secondary to benign prostatic hyperplasia (BPH). It has been speculated that the clinical efficacy of PDE5 inhibitors in patients with LUTS/BPH can be explained by their effects on the urinary bladder rather than on the prostate. Hence, the significance of the nitric oxide (NO)/cyclic GMP signaling in the control of the human prostate requires further clarification.MethodsThe present study aimed to investigate by means of immunohistochemistry in the human prostate the expression and distribution of key mediators of the NO pathway, namely cyclic GMP, the neuronal nitric oxide synthase (nNOS), and cyclic GMP-binding protein kinases type I (cGKIα, cGKIß), in relation to PDE5, protein kinase A (cAK), and the vasoactive intestinal polypeptide (VIP).ResultsIn the smooth muscle portion of the transition zone, immunosignals specific for the PDE5 were found co-localized with cyclic GMP, cGKIα, and cGKIß, as well as with the cyclic cAMP-binding protein kinase A. Smooth muscle bundles were seen innervated by slender varicose nerves characterized by the expression of nNOS. Some of these nerves also presented staining related to the neuropeptide VIP.ConclusionsThe findings give hints that the cyclic GMP- and cyclic AMP-dependent signal transduction may synergistically work together in regulating muscle tension in the transition zone. This might be of significance for the identification of new pharmacological avenues to treat patients with symptomatic BPH.

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

INTRODUCTIONnRho 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.nnnAIMnTo investigate by means of immunohistochemistry and organ bath studies the significance of the Rho pathway in human cavernous arteries.nnnMAIN OUTCOME MEASURESnThe 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.nnnMETHODSnSpecimens 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.nnnRESULTSnAlpha-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.nnnCONCLUSIONnThe findings are in support for a role of the Rho/ROK-mediated signaling in the regulation of muscle tone of human cavernous arteries.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Seminal Vesicles.

INTRODUCTIONnPhosphodiesterase (PDE) isoenzymes have been shown to play a role in the control of human male genital tissues. There are hints from basic research and clinical studies that PDE5 inhibitors may have the ability to retard the male ejaculatory response. While the expression of PDE isoenzymes in the human seminal vesicles (SVs) has been described, the distribution of cyclic adenosine monophosphate (AMP)- and cyclic guanosine monophosphate (GMP)-PDEs has not yet been investigated.nnnAIMnThe aim of this study was to elucidate the expression and distribution of PDE isoenzymes PDE3A, PDE4 (isoforms A and B), PDE5A, and PDE11A in human SV tissue.nnnMETHODSnUsing immunohistochemistry (double-labeling techniques, laser fluorescence microscopy), the occurrence of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A, the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and protein gene product 9.5 (PGP 9.5) was examined in sections of SV. Cytosolic supernatants prepared from isolated human SV tissue were subjected to Western blot analysis using specific anti-PDE antibodies.nnnMAIN OUTCOME MEASUREnThe expression and distribution by of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A in the human SV were investigated by means of immunohistochemistry and Western blot analysis.nnnRESULTSnImmunosignals specific for PDE3A were seen in both the smooth muscle and the glandular epithelium, whereas staining for PDE4A, PDE5A, and PDE11A was mainly limited to epithelial cells. Varicose nerve fibers transversing the sections also presented staining for PDE3A. In nerve fibers and nerve endings, PDE4A and PDE4B were found co-localized with VIP; PDE5A-positive nerves also presented immunosignals specific for CGRP. The expression of said PDE isoenzymes was confirmed by Western blotting.nnnCONCLUSIONSnThe results indicate that cyclic AMP- and cyclic GMP-PDE isoenzymes are involved in the control of secretory activity and efferent neurotransmission in the SV. These findings might be of importance with regard to the identification of new therapeutic avenues to treat premature ejaculation.