Robert B. Moreland

Investigative Urology: PGE sub 1 Suppresses the Induction of Collagen Synthesis by Transforming Growth Factor-beta sub 1 in Human Corpus Cavernosum Smooth Muscle

Collagen synthesis has been examined in primary cultures of human corpus cavernosum smooth muscle cells (HCCSMC), the major mesenchymal cell type of the corpus cavernosum. These cultures were grown from human surgical specimens and characterized by morphological and biochemical characteristics. These cells express mRNA for transforming growth factor-beta 1 (TGF-beta 1), a major regulator of extracellular matrix synthesis, as well as all three subtypes of TGF-beta receptors. Human corpus cavernosum smooth muscle cells primarily synthesize types I and III fibrillar collagen. Treatment of HCCSMC with exogenous TGF-beta 1 (80 pM.) induced a 2.5- to 4.5-fold increase in the synthesis of types I and III collagen and resulted in detectable levels of type V/XI collagen. Treatment of HCCSMC with the eicosanoid PGE1 in combination with TGF-beta 1 suppressed the induction of collagen synthesis by TGF-beta 1 in a dose-dependent manner with concomitant decreases in types I, III and V/XI collagen. The expression of TGF-beta 1 mRNA as well as types I and II TGF-beta receptors was induced by exogenous TGF-beta 1. Transforming growth factor-beta 1 mRNA induction was suppressed by PGE1. These data suggest that prostaglandins and TGF-beta 1 may play a key role in modulation of collagen synthesis in the corpus cavernosum, and in the regulation of fibrosis of the corpus cavernosum.

TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists.

Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N′-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freunds adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.

Mechanisms of Venous Leakage: A Prospective Clinicopathological Correlation of Corporeal Function and Structure

PURPOSE We investigated the pathophysiology of structurally based corporeal veno-occlusive dysfunction. MATERIALS AND METHODS We prospectively evaluated 24 impotent patients (mean age plus or minus standard error 46 +/- 3 years) who had exposure to vascular risk factors and/or disorders inducing diffuse trabecular structure alterations and who underwent penile prosthesis insertion. Preoperative indexes of veno-occlusive function (flow to maintain, venous outflow resistance and pressure decay measurements using repeat dosing pharmacocavernosometry) were correlated with postoperative erectile tissue computer assisted color histomorphometry (percent trabecular smooth muscle to total erectile tissue area). To develop further study findings and correlate histomorphometric findings with molecular biological properties molecular biological studies (ribonuclease protection analysis, reverse transcription-polymerase chain reaction assay for expression of transforming growth factor-beta 1 messenger [m] ribonucleic acid [RNA] and protein affinity labeling techniques for specific transforming growth factor-beta receptors) were performed in representative patients with high (39 to 43%), intermediate (30 to 37%) and low (13 to 29%) trabecular smooth muscle content (normal 42 to 50%). RESULTS Flow to maintain, venous outflow resistance and pressure decay values significantly correlated with trabecular smooth muscle cell content (r = -0.89, 0.82 and -0.85, respectively). In the high, intermediate and low smooth muscle content subgroups flow to maintain, venous outflow resistance and pressure decay values were 1 to 5, 9 to 30 and 50 to 120 ml. per minute, 17 to 84, 3 to 9 and 1 to 2 mm. Hg/ml. per minute, and 40 to 60, 48 to 80 and 110 to 120 mm. Hg decrease in 30 seconds from 150 mm. Hg, respectively. There were no significant differences in patient age or prevalence of risk factors among the 3 subgroups. Patients representative of all 3 subgroups had transforming growth factor-beta 1 mRNA, auto-induction of transforming growth factor-beta 1 mRNA and induction and/or increased availability of all 3 types of transforming growth factor-beta receptors. CONCLUSIONS The pathophysiology of structurally based corporeal veno-occlusive dysfunction is related to elevated corporeal connective tissue content. Based on our data and those in the literature corporeal fibrosis is hypothesized to develop secondary to abnormalities in the regulation of normal collagen synthesis and degradation, most likely associated with adverse influences of chronic ischemia.

Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels.

&NA; Acid Sensing Ion Channels (ASICs) are a group of sodium‐selective ion channels that are activated by low extracellular pH. The role of ASIC in disease states remains unclear partly due to the lack of selective pharmacological agents. In this report, we describe the effects of A‐317567, a novel non‐amiloride blocker, on three distinct types of native ASIC currents evoked in acutely dissociated adult rat dorsal root ganglion (DRG) neurons. A‐317567 produced concentration‐dependent inhibition of all pH 4.5‐evoked ASIC currents with an IC50 ranging between 2 and 30 μM, depending upon the type of ASIC current activated. Unlike amiloride, A‐317567 equipotently blocked the sustained phase of ASIC3‐like current, a biphasic current akin to cloned ASIC3, which is predominant in DRG. When evaluated in the rat Complete Freuds Adjuvant (CFA)‐induced inflammatory thermal hyperalgesia model, A‐317567 was fully efficacious at a dose 10‐fold lower than amiloride. A‐317567 was also potent and fully efficacious when tested in the skin incision model of post‐operative pain. A‐317567 was entirely devoid of any diuresis or natriuresis activity and showed minimal brain penetration. In summary, A‐317567 is the first reported small molecule non‐amiloride blocker of ASIC that is peripherally active and is more potent than amiloride in vitro and in vivo pain models. The discovery of A‐317567 will greatly help to enhance our understanding of the physiological and pathophysiological role of ASICs.

Sildenafil Citrate, a Selective Phosphodiesterase Type 5 Inhibitor:: Research and Clinical Implications in Erectile Dysfunction

Under normal physiological conditions, following sexual stimulation, release of nitric oxide (NO) from penile non-adrenergic, non-cholinergic nerves and the endothelium activates guanylyl cyclase and induces intracellular cGMP synthesis in erectile tissue trabecular smooth muscle cells. Increased cGMP levels reduce intracellular Ca2+ concentrations, inhibiting smooth muscle contractility and thereby initiating the erectile response. Phosphodiesterase type 5 (PDE type 5) is the predominant enzyme responsible for cGMP hydrolysis in trabecular smooth muscle. Activation of PDE type 5 terminates NO-induced, cGMP-mediated smooth muscle relaxation, resulting ultimately in restoration of basal smooth muscle contractility and penile flaccidity. Sildenafil citrate is a potent PDE type 5 reversible and selective inhibitor that blocks cGMP hydrolysis effectively (Ki approximately 3 nM). Under conditions of excessive adrenergic tone or impaired neurovascular status, following sexual stimulation, sildenafil acts to enhance NO-mediated smooth muscle relaxation, resulting in improved penile erection in men with erectile dysfunction. In this review, we summarize the current state of knowledge of the physiology of penile erection and the pharmacology, metabolism and clinical experience with sildenafil citrate in the management of erectile dysfunction.

Cavernosal expandability is an erectile tissue mechanical property which predicts trabecular histology in an animal model of vasculogenic erectile dysfunction

PURPOSE Reliable, clinically available, non-invasive measurements able to predict trabecular histology without the need for erectile tissue biopsy would improve impotence management, since the percentage of trabecular smooth muscle content has been shown to be associated with corporal veno-occlusive dysfunction. The purpose was to identify whether the erectile tissue mechanical property, cavernosal expandability, correlated with the percentage of trabecular smooth muscle content in an animal model of hypercholesterolemia and ischemic-induced corporal fibrosis. MATERIALS AND METHODS New Zealand White rabbits (6 to 7 months old, 3 to 3.5 kg.), were divided into control (n = 7), hypercholesterolemic (n = 5, 0.5% cholesterol diet) and atherosclerotic groups (n = 8, 0.5% cholesterol diet with balloon de-endothelialization). At 16 weeks, the corpora cavernosa were removed en bloc and submerged in physiologic salt solution, and volume-pressure data were plotted at 20 mm. Hg pressure intervals under trabecular smooth muscle relaxation. Cavernosal expandability, X, (the measure of the ability to achieve high corporal expansion at relatively low intracavernosal pressure) and tunical distensibility, V(E)/V(F), (relative volume of fully erect to flaccid penis) were calculated. Erectile tissue was assessed by computer-assisted color histomorphometry with Massons trichrome stained sections (30 to 45 high power fields/animal) to assess percentage of trabecular smooth muscle content. RESULTS The overall mean percentage of trabecular smooth muscle content and mean cavernosal expandability values were 45.4 +/- 1.6, 39.2 +/- 0.9, 33.9 +/- 0.6 and 0.0165 +/- 3.04 x 10(-3), 0.0116 +/- 1.63 x 10(-3), 0.0118 +/- 1.26 x 10(-3) mm. Hg(-1) for the control, hypercholesterolemic and atherosclerotic groups, respectively (r = 0.87). Significant differences in trabecular smooth muscle content were observed among all 3 groups, and in cavernosal expandability, between control and atherosclerotic groups, as well as between control and hypercholesterolemic groups but not between atherosclerotic and hypercholesterolemic groups. CONCLUSIONS The erectile tissue mechanical property, cavernosal expandability, correlated with erectile tissue structural quality. Since cavernosal histology has been shown to predict corporal veno-occlusive function, it is hypothesized that the measurement of cavernosal expandability may become a valuable functional clinical parameter in the diagnosis and treatment of men with erectile dysfunction.

Implications of prostate micrometastases in pelvic lymph nodes: An archival tissue study

OBJECTIVES In the United States, radical retropubic prostatectomy for adenocarcinoma usually includes a staging pelvic lymphadenectomy. If frozen section analysis of the lymph nodes fails to reveal any evidence of metastases, the prostate is removed. We have previously noted that as many as 56% of patients undergoing radical prostatectomy demonstrate rising serum prostate-specific antigen (PSA) levels by 4 years postoperatively. This report was designed to determine whether micrometastases undetectable by conventional pathologic methods: could have accounted for these biochemical failures. METHODS A retrospective analysis of formalin-fixed paraffin-embedded pelvic lymph node material was undertaken using a reverse transcription-polymerase chain reaction (RT-PCR)-based assay designed to amplify messenger RNA from PSA. All specimens were obtained from a group of 57 patients with prostate cancer who had undergone staging pelvic lymphadenectomy at the time of radical prostatectomy, and whose long-term follow-up was known. RESULTS Although all of these nodes appeared to be free of tumor by conventional pathologic methods, a RT-PCR assay was used to identify evidence of prostate metastases in 44% of evaluable samples. Of these, 14 of 16 went on to manifest rising serum PSA values by 5 years postoperatively. CONCLUSIONS These results suggest that molecular staging of pelvic lymph nodes prior to planned therapy for clinically organ-confined prostate cancer may better distinguish between patients with local disease and those for whom local therapy alone will not be curative. To our knowledge, this is the first large-scale retrospective gene expression study published.

Rationale for combination therapy of intraurethral prostaglandin E(1) and sildenafil in the salvage of erectile dysfunction patients desiring noninvasive therapy.

Corpus cavernosum smooth muscle relaxation and hence penile erection are regulated in part by increases in smooth muscle synthesis of the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). The object of this study was to determine 30-month follow-up results in motivated patients desiring noninvasive medical therapy using sildenafil citrate (Viagra) in combination with intraurethral prostaglandin E1 (PGE1) (Medicated Urethral System for Erection [MUSE]). Twenty-eight patients (mean±s.d. age, 59±7.3 y; 17 who had undergone radical prostatectomy and 11 who had a diagnosis of organic erectile dysfunction) were included in this study. Detailed history taking and physical examinations were performed and vascular risk factors noted. In these patients, treatment with either 100 mg of sildenafil citrate and/or 1000 μg of MUSE had failed. None of these patients desired intracavernosal injection. Duplex Doppler ultrasonography after redosing was carried out on all patients. Dynamic infusion corpus cavernosography/cavernosometry was obtained in 17 of 28 patients, and combination therapy was initiated using 100 mg of sildenafil citrate orally 60 min before intercourse and 500 μg of MUSE intraurethrally immediately before intercourse. Independently, either 100 mg of sildenafil citrate or 1000 μg of MUSE was not efficacious in inducing an erection sufficient for vaginal penetration in any of the 28 patients. After initiating a combination therapy, at 30 months, all 28 patients were reporting erections sufficient for vaginal penetration, with 3.6 intercourse episodes per month. None of the patients crossed over to intracavernosal therapy or penile prosthesis. During therapy, eight of 28 patients reduced the dose of sildenafil citrate to 50 mg. Combination therapy with MUSE and sildenafil may be more efficacious in the salvage of patients who desire noninvasive therapy but in whom single-treatment modalities fail. Although both cAMP- and cGMP-mediated vasodilation can lead to penile erection, combining therapies that incorporate both pathways may succeed when single therapies fail.

Possible role of Na+‐K+‐ATPase in the regulation of human corpus cavernosum smooth muscle contractility by nitric oxide

1 This study was designed to determine the role of sodium‐potassium adenosine triphosphatase (Na+‐K+‐ATPase) in the regulation of human corpus cavernosum smooth muscle contractility by nitric oxide (NO). In addition, we determined if the modulation of Na+‐K+‐ATPase activity by NO is dependent on the increases in intracellular cyclic GMP concentration. 2 The effect of NO donors, sodium‐nitroprusside (SNP) and S‐nitroso‐glutathione (S‐NO‐Glu), and a permeable cyclic GMP analogue, 8‐bromo‐cyclic GMP, on Na+‐K+‐ATPase activity (measured as ouabain‐sensitive 86Rb‐uptake) was studied in human cultured corpus cavernosum smooth muscle cells (HCCSMC). In addition, the effect of the cyclic GMP lowering agent, methylene blue, on NO‐induced increase in Na+‐K+‐ATPase activity was studied. 3 SNP (1 μm) caused time‐dependent increases in ouabain‐sensitive Rb‐uptake (33–72%) over 2–20 min in HCCSMC. The stimulation of ouabain‐sensitive Rb‐uptake by SNP was concentration‐dependent (30 and 102% with 0.1 and 1 μm SNP, respectively). Similarly, significant increases in ouabain‐sensitive Rb‐uptake were obtained with 1 and 10 μm S‐NO‐Glu. In contrast, incubation of HCCSMC with 8‐bromo‐cyclic GMP (100 μm) did not increase ouabain‐sensitive Rb‐uptake. 4 S‐NO‐Glu induced‐increase in intracellular cyclic GMP synthesis, but not the increase in ouabain‐sensitive Rb‐uptake, was completely inhibited by methylene blue in HCCSMC. 5 The Na+‐K+‐ATPase inhibitor, ouabain, caused a concentration‐dependent increase in tension (0.5 to 2 fold) in tissues contracted with 15 mM KCL. SNP and S‐NO‐Glu caused a concentration‐dependent relaxation (concentration required to cause half maximal relaxation (ED50) = 0.04 and 0.2 μm, respectively) of HCC strips contracted with 15 mM K+. Ouabain (0.1 to 10 μm) inhibited the response to SNP and S‐NO‐Glu by shifting the concentration‐response curves to the right and preventing full smooth muscle relaxation. 6 These results indicate that the activity of Na+‐K+‐ATPase modulates the contractility of HCC smooth muscle, and that NO stimulates Na+‐K+‐ATPase activity in HCCSMC independently of its ability to increase the intracellular cyclic GMP concentration. They also suggest that stimulation of Na+‐K+‐ATPase activity plays an important role in NO‐induced relaxation of HCC smooth muscle

Formation of corporal tissue architecture in vivo using human cavernosal muscle and endothelial cells seeded on collagen matrices.

We explored the feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle and endothelial cells in vivo, using three-dimensional acellular collagen matrices, which are similar in architecture to native corpora. Acellular collagen matrices were derived from processed donor rabbit corpora, using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were seeded on the acellular matrices. A total of 80 matrices, 20 without cells and 60 with cells, were implanted subcutaneously in athymic mice. An additional 36 matrices seeded with cells were maintained in culture for up to 4 weeks. Hydroxyproline quantification, Western blot analysis, RT-PCR, and scanning electron microscopy of the matrices, with and without cells, were performed at various time points. Animals were killed 3 days and 1, 2, 3, 4, 6, and 8 weeks after implantation. Immunocytochemical and histological analyses were performed to confirm the muscle and endothelial phenotype. Organ bath studies were performed in order to determine the degree of tissue contraction. Western blot analysis detected alpha-actin, myosin, and tropomyosin proteins from human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor (mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells before and 8 weeks after seeding. The implanted matrices showed neovascularity into the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and continued with time. The matrices were covered with the appropriate cell architecture 4 weeks after implantation. The matrices showed a stable collagen concentration over 8 weeks, as determined by hydroxyproline quantification. Immunocytochemical studies using alpha-actin and factor VIII antibodies confirmed the presence of corporal smooth muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no evidence of cellular organization in the control matrices. Organ bath studies showed that the cell-seeded corporal tissue matrices responded to electrical field stimulation, whereas the unseeded implants failed to respond. This study demonstrates that human cavernosal smooth muscle and endothelial cells seeded on three-dimensional acellular collagen matrices derived from donor corpora are able to form well-vascularized corporal tissues in vivo.