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 Freund's 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.

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

Blockade of mGluR1 receptor results in analgesia and disruption of motor and cognitive performances: effects of A-841720, a novel non-competitive mGluR1 receptor antagonist.

BACKGROUND AND PURPOSE: To further assess the clinical potential of the blockade of metabotropic glutamate receptors (mGluR1) for the treatment of pain. EXPERIMENTAL APPROACH: We characterized the effects of A-841720, a novel, potent and non-competitive mGluR1 antagonist in models of pain and of motor and cognitive function. KEY RESULTS: At recombinant human and native rat mGluR1 receptors, A-841720 inhibited agonist-induced calcium mobilization, with IC50 values of 10.7+/-3.9 and 1.0 +/- 0.2 nM, respectively, while showing selectivity over other mGluR receptors, in addition to other neurotransmitter receptors, ion channels, and transporters. Intraperitoneal injection of A-841720 potently reduced complete Freund's adjuvant-induced inflammatory pain (ED50 = 23 micromol kg(-1)) and monoiodoacetate-induced joint pain (ED50 = 43 micromol kg(-1)). A-841720 also decreased mechanical allodynia observed in both the sciatic nerve chronic constriction injury and L5-L6 spinal nerve ligation (SNL) models of neuropathic pain (ED50 = 28 and 27 micromol kg(-1), respectively). Electrophysiological studies demonstrated that systemic administration of A-841720 in SNL animals significantly reduced evoked firing in spinal wide dynamic range neurons. Significant motor side effects were observed at analgesic doses and A-841720 also impaired cognitive function in the Y-maze and the Water Maze tests. CONCLUSIONS AND IMPLICATIONS: The analgesic effects of a selective mGluR1 receptor antagonist are associated with motor and cognitive side effects. The lack of separation between efficacy and side effects in pre-clinical models indicates that mGluR1 antagonism may not provide an adequate therapeutic window for the development of such antagonists as novel analgesic agents in humans.

Effects of castration and androgen replacement on erectile function in a rabbit model.

We investigated, in a rabbit model, the effects of castration and testosterone replacement on: 1) the hemodynamics of the corpus cavernosum; 2) alpha-1 adrenergic receptor protein expression; 3) neural NO synthase protein expression and activity; 4) phosphodiesterase type 5 activity; and 5) trabecular smooth muscle/connective tissue balance. One week after bilateral orchiectomy, animals were treated for 7 days with vehicle alone, testosterone, or estradiol. Intact control animals received vehicle only. Systemic arterial blood and intracavernosal pressures (ICP) were measured in each animal before and after electrical stimulation of the cavernosal nerve. Alpha1-adrenergic receptor protein expression was determined by ligand binding studies. NO synthase expression and activity were determined by Western blot analyses and conversion of L-arginine to citrulline, respectively. Phosphodiesterase type 5 activity was determined by hydrolysis of guanosine 3',5'-cyclic monophosphate (cGMP) in tissue extracts in the absence or presence of 100 nM sildenafil. Smooth muscle content was assessed by Masson's trichrome staining and computer-assisted histomorphometry. Castration significantly reduced ICP, but it did not alter systemic arterial blood pressure during stimulation of the cavernosal nerve. Testosterone, but not estradiol, treatment prevented the effects of castration and restored ICP to values similar to those obtained in intact animals. Castration reduced expression of alpha1-adrenergic receptor, and this reduction was prevented or reversed by testosterone replacement. Neural NO synthase protein expression and total activity were not altered significantly by castration or after testosterone replacement. However, phosphodiesterase type 5 activity increased in castrated animals treated with testosterone. Castration significantly reduced trabecular smooth muscle content, and this reduction was restored by testosterone (but not estradiol) treatment. The results of this study demonstrate that androgen deprivation alters the functional responses and structure of erectile tissue.

Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis of RNA.

The Milli-Q PF Plus water-polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC)-treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue-cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver kidney and bladder as well as cultured human corpus cavernosum smooth muscle cells. RNA was prepared by guanidinium isothiocyanate solubilization, phenol/chloroform extraction and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (ca. 7.6 kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2 kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water for Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water and Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute for DEPC-treated water for the preparation of RNA and Northern blot analysis.

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 increase 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 microM) 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 microM SNP, respectively). Similarly, significant increases in ouabain-sensitive Rb-uptake were obtained with 1 and 10 microM S-NO-Glu. In contrast, incubation of HCCSMC with 8-bromo-cyclic GMP (100 microM) 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 microM, respectively) of HCC strips contracted with 15 mM K+. Ouabain (0.1 to 10 microM) 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

The expression of functional postsynaptic alpha2-adrenoceptors in the corpus cavernosum smooth muscle.

1. The purpose of this study was to determine if corpus cavernosum smooth muscle expresses functional postsynaptic alpha2-adrenoceptors (AR). 2. The alpha2-adrenoceptor agonist UK 14,304 elicited concentration-dependent contractions in rabbit corpus cavernosum smooth muscle (CCSM). The half-maximal response occurred at 0.32+/-0.03 microM and the maximum contraction at 10 microM UK 14,304. 3. Pretreatment of CCSM strips with selective alpha2-adrenoceptor antagonists, rauwolscine and RS-15385, produced rightward shifts in the dose-response curves to UK 14,304 (pA2 values 7.1 and 8.5, respectively). In contrast, these antagonists did not alter contraction induced by the alpha1-adrenoceptor agonist phenylephrine (PE) or oxymetazoline. UK 14,304-induced contractions were also inhibited by prazosin (pA2 = 9.08). 4. UK 14,304-induced contractions, unlike those to PE, were highly dependent on the presence of extracellular Ca2+. 5. [3H]-rauwolscine bound to CCSM membranes with high affinity (Kd = 1.5 nM). [3H]-rauwolscine binding was displaced by unlabelled rauwolscine, RS-15385, UK 14,304 and prazosin, but not by PE. 6. UK 14,304 inhibited forskolin and prostaglandin E1 (PGE1)-induced increases in intracellular cyclic AMP concentration in primary cultures of rabbit CCSM cells. 7. These results demonstrate that CCSM expresses Gi-coupled postsynaptic alpha2-adrenoceptors, and activation of these receptors causes contraction of trabecular smooth muscle.

Pharmacotherapeutic advances in the treatment of erectile dysfunction.

An estimated 20 million to 30 million American men have erectile dysfunction (ED). The past 2 decades of research defining erectile physiology and investigating the pathogenesis of ED have led to the recognition of a predominantly vascular basis for organic male sexual dysfunction. These scientific advances have laid the foundation for the advent of pharmacotherapies. The Food and Drug Administration approval of intracavernosal, intraurethral, and oral pharmacotherapeutics for ED has revolutionized non-surgical management of this condition. The primary care physician is faced with the challenges of diagnosis and treatment of ED, as well as referral of patients to urologists. In this article, erectile physiology and pathophysiology are reviewed, and pharmacotherapeutics are classified and discussed by their mechanisms of action and the means of administration. A thorough understanding of these new therapeutic options is key to the accurate diagnosis and successful treatment of ED and maximal patient satisfaction and care.

Immunodetection of nmt55/p54nrb isoforms in human breast cancer.

BACKGROUND: We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54nrb, whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. RESULTS: Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54nrb mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54nrb protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54nrb protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54nrb that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. CONCLUSIONS: Our study indicates that nmt55/p54nrb protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54nrb in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression.

Loss of expression of a 55 kDa nuclear protein (nmt55) in estrogen receptor-negative human breast cancer.

We have identified and characterized a 55 kDa nuclear protein (referred to as nmt55) from human breast tumors and MCF-7, human adenocarcinoma breast cell line, using site-directed monoclonal antibodies. Measurements of estrogen receptors (ER) and progesterone receptors (PR), by ligand binding assays, in cytosols of 63 human breast tumors permitted classifications of these tumors into four phenotypes (ER+/PR+, ER+/ PR-, ER-/PR-, ER-/PR+). Nuclear protein (nmt55) expression in these tumors, as determined from Western blot analyses, showed a statistically significant association (p = 0.001) with tumor hormonal phenotype. Review of the pathologic characteristics of tumors analyzed suggested that lack of nmt55 expression was significantly associated with mean tumor size (p < 0.03), mean ER (p = 0.001) and mean PR (p < 0.002), but was not associated with tumor stage, grade, or type. To further study this protein, we cloned and sequenced a 2.5 kb cDNA using a monoclonal antibody to nmt55. The complete predicted open reading frame encodes a protein with 471 amino acids and a calculated molecular mass of 54,169 Da. The deduced amino acid sequence exhibited unique regions rich in glutamine, histidine, arginine, and glutamic acid. Northern blot analysis of RNA from MCF-7 cells and ER+/PR+ human breast tumors showed a 2.6 kb mRNA. Southern blot analysis suggested the presence of a single copy of this gene. Chromosomal mapping, using fluorescent in situ hybridization (FISH), located nmt55 gene to the X chromosome, region q13. The extensive homology between nmt55 and RNA binding proteins suggested that nmt55 may be involved in hnRNA splicing. The strong association observed between expression of nmt55, tumor hormonal phenotype, mean tumor size, mean ER, and mean PR content suggests that loss of nmt55 expression may be related to events involved in hormone insensitivity, tumor differentiation, and unregulated tumor cell growth and metastases.

A rapid and simple method for the detection of prostate-specific antigen mRNA in archival tissue specimens using a reverse transcription-polymerase chain reaction assay.

OBJECTIVES: Polymerase chain reaction (PCR) amplification of DNA from archival, fixed tissue sources is now performed routinely. In this report, we describe a reproducible technique for mRNA amplification from archival tissues. METHODS: Archival, fixed tissue was treated with a modification of a rapid, acid guanidinium technique. The RNA yielded was reverse transcribed and subjected to 40 cycles of two-step PCR, using a panel of primers designed to encompass relatively short (less than 250 bp) cDNA fragments. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. RESULTS: Using fixed, paraffin-embedded specimens of human prostate, we have demonstrated a reproducible pattern of reverse transcription (RT)-PCR products using several different primer sets. Our data suggest that RNA degradation proceeds to fragments approximately 250 bp or shorter in length but that many of these fragments survive intact over extended periods of time and are of suitable quality to serve as a template for RT and subsequent PCR amplification. CONCLUSIONS: We describe a technique that will allow the retrospective analysis of archival tissues for gene expression. These methods are generally applicable to a wide variety of systems. Such a method will make it possible to perform retrospective studies of gene expression in the archival tissues of patients whose eventual clinical course is already known, greatly shortening the time needed for genetic outcome studies in slowly growing tumors, such as prostate cancer.

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.

Pathophysiology of erectile dysfunction: the contributions of trabecular structure to function and the role of functional antagonism.

Erectile dysfunction (ED) is estimated to impact more than 150 million men this year worldwide. An understanding of the pathophysiology of ED both furthers the basic scientific knowledge of disease processes and provides a rational design of pharmacotherapy. At present, there are two major views regarding the pathophysiology of ED. In the first hypothesis, the oxygen tension-dependent changes in the penis during erection are proposed to impact corpus cavernosum structure by inducing various cytokines, vasoactive factors and growth factors at the two different oxygen tensions (flaccidity and erection) which, in turn, alter smooth muscle metabolism and connective tissue synthesis. Decreases in the corpus cavernosum smooth muscle/connective tissue ratio have been correlated with an increased likelihood of diffuse venous leak and a failure of the veno-occlusive mechanism in prospective patient studies. Evidence for such a hypothesis incorporates nocturnal penile tumescence and circadian changes in oxygenation as important in maintaining erectile health. The alternate hypothesis proposes that ED is the result of a metabolic imbalance between relaxatory and contractile processes within the trabecular smooth muscle such that contractile processes predominate. Based on this hypothesis, therapy can be accomplished via drugs which shift this balance towards vasodilatation, or by gene therapy approaches to supplement the deficient components favoring smooth muscle relaxation. Both of these hypotheses predict a management strategy for ED that impacts pharmacotherapeutics. In this review of the pathophysiology of ED, each hypothesis will be examined and a synthesis devised incorporating both views. The future of research in this area as well as pharmacotherapy in ED in terms of pathophysiology is discussed including the merits and drawbacks of prophylaxis and prevention of ED. International Journal of Impotence Research (2000) 12, Suppl 4, S39-S46.

Is there a role of hypoxemia in penile fibrosis: a viewpoint presented to the Society for the Study of Impotence.

During erection, oxygen tension changes in the corpus cavernosum penis from 25-40 mm Hg in the flaccid state to 90-100 mm Hg in the erect state. The relationship between corpus cavernosum trabecular structure and erectile function is dependent on a critical balance of smooth muscle to connective tissue for successful veno-occlusion. In this article, the potential role for transforming growth factor beta(1) (TGF-beta(1)) and prostaglandin E (PGE) in maintaining a functional smooth muscle/connective tissue balance are discussed as well as the importance of oxygen tension in the synthesis of these factors. Correlations between animal models of disease as well as clinical reports are presented in support of a role for hypoxemia in penile fibrosis. A case is presented for a biological basis of nocturnal penile tumescence in the preservation of potency and an overall hypothesis for the molecular pathology of erectile dysfunction is proposed.

Pathophysiology of Peyronie's disease.

Peyronie's disease is an inflammatory condition characterized by the formation of fibrous, noncompliant nodules in the tunica albuginea which can impede tunical expansion during penile erection, leading to deformity and bending. While the cause of this disease is thought to be due to microvascular trauma and abnormal wound healing, other hypotheses include genetic predisposition. In this review the pathophysiology of Peyronie's disease is discussed as well as current hypotheses regarding its origin.

Development of an organ culture experimental system to investigate collagen synthesis in corpora cavernosa.

An organ culture model of rabbit corpus cavernosum has been developed to investigate fibrillar collagen synthesis in intact organ. Rabbit peni were removed en bloc, the corpora cavernosa were dissected, sliced into 5 mm pieces and incubated in culture media up to 24 h. Tissue viability and metabolic and functional integrity were assessed by (a) lactate dehydrogenase (LDH) release, (b) protein synthesis, and (c) ability to respond to exogenously added cytokines. Explants, maintained for 24 h in organ culture, exhibited minimal LDH release and maintained functional integrity determined from protein synthesis and ability to synthesize collagen in response to TGF-beta(1). These data suggest that explants of rabbit corpus cavernosum in organ culture represent a viable model to investigate connective tissue protein biosynthesis in vitro.

Alpha-adrenergic receptor blockade by phentolamine increases the efficacy of vasodilators in penile corpus cavernosum.

Penile trabecular smooth muscle tone, a major determinant of erectile function, is highly regulated by numerous inter- and intracellular pathways. The interaction between pathways mediating contraction and relaxation has not been studied in detail. To this end, we investigated the functional effects of alpha adrenergic receptor blockade with phentolamine and its interaction with vasodilators (sildenafil, vasoactive intestinal polypeptide (VIP) and PGE1) that elevate cyclic nucleotides on penile cavernosal smooth muscle contractility. In organ bath preparations of cavernosal tissue strips contracted with phenylephrine, phentolamine significantly enhanced relaxation induced by sildenafil, VIP and PGE1. Sildenafil, VIP or PGE1 also significantly enhanced relaxation induced by phentolamine in cavernosal tissue strips contracted with phenylephrine. To study the effects of alpha adrenergic receptor blockade and modification of cyclic nucleotide metabolism during active neurogenic input, cavernosal tissue strips in organ bath preparations were contracted with the non-adrenergic agonist endothelin-1 and subjected to electrical field stimulation (EFS) in the absence or presence of phentolamine and/or sildenafil. EFS (5-40Hz) typically caused biphasic relaxation and contraction responses. Phentolamine alone enhanced relaxation and reduced or prevented contraction to EFS. Sildenafil enhanced relaxation to EFS at lower frequencies (< or = 5 Hz). The combination of phentolamine and sildenafil enhanced EFS-induced relaxation at all frequencies tested. EFS, in the presence of 10 nM phentolamine and 30 nM sildenafil, produced enhanced relaxation responses which were quantitatively similar to those obtained in the presence of 50 nM sildenafil alone. Thus, blockade of alpha-adrenergic receptors with phentolamine increases the efficacy of cyclic nucleotide-dependent vasodilators. Furthermore, phentolamine potentiates relaxation and attenuates contraction in response to endogenous neurotransmitters which are released during EFS. These findings suggest that antagonism of alpha-adrenergic signaling enables other independent relaxatory pathways to predominate within penile trabecular smooth muscle.

Role of alpha adrenergic receptors in erectile function.

Penile erection is a complex physiological process in which the regulation of penile hemodynamics depends on signal input from central and peripheral nervous systems, and on the balance and interplay between several local physiological mediators (neurotransmitters, vasoactive agents and endocrine factors). A role for the sympathetic nervous system in attaining or maintaining penile flaccidity and detumescence is well recognized. However, the exact mechanisms of alpha-adrenergic receptor mediated erectile function remain poorly defined. The objective of this review is to summarize data presented in the literature and from our laboratory on alpha-adrenergic receptors, and to discuss the conceptual framework by which the alpha-adrenergic receptor pathway modulates penile corpus cavernosum smooth muscle contractility. We will integrate the current state of knowledge of penile erection into one framework encompassing the biochemical and physiological pathways responsible for trabecular smooth muscle relaxation (erection) and contraction (detumescence). We will focus our discussion on local control mechanisms of alpha-adrenergic receptors and explore the following topics: (1) functional activity of alpha-1 and alpha-2 adrenergic receptors in the physiology of human penile erection; (2) identification, classification and characterization of alpha-1 and alpha-2 adrenergic receptor subtypes in human penile erectile tissue; (3) molecular mechanism of action of alpha-1 and alpha-2 adrenergic receptors in human penile erectile tissue; (4) blockade of alpha-1 and alpha-2 adrenergic receptor action by selective and non-selective alpha-1 and alpha-2 adrenergic receptor antagonists (blockers); (5) physiologic (functional) antagonism of alpha-1 and alpha-2 adrenergic receptor activity by vasodilators mediating smooth muscle relaxation; and (6) key areas of consensus, as well as critical gaps in the existing scientific knowledge concerning the rationale and the potential use of alpha-blockers in erectile function.

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 E(1) (PGE(1)) (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 microg 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 microg of MUSE intraurethrally immediately before intercourse. Independently, either 100 mg of sildenafil citrate or 1000 microg 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.

Cyclic AMP modulates TGF-beta 1-induced fibrillar collagen synthesis in cultured human corpus cavernosum smooth muscle cells.

INTRODUCTION AND OBJECTIVES: The mechanisms by which PGE1 suppresses transforming growth factor beta 1 (TGF-beta 1) induced fibrillar collagen synthesis in human corpus cavernosum smooth muscle cells (HCC SMC) remain undefined. Since PGE1 induces cyclic AMP (cAMP) synthesis in HCC SMC, the aim of this research is to investigate the role of cAMP in the regulation of connective tissue biosynthesis in human corporal smooth muscle cells in culture. MATERIALS AND METHODS: HCC SMC were incubated for 24 h in media containing [3H] proline with and without TGF-beta 1 in the presence or absence of agents which modulate cAMP or cGMP synthesis, or hydrolysis. Fibrillar collagen synthesis was determined by [3H]-proline incorporation into pepsin-resistant, trichloroacetic acid and precipitable protein. RESULTS: TGF-beta 1-induced collagen synthesis in HCC SMC was inhibited by receptor-mediated (PGE1) and non-receptor-mediated (forskolin) increases in cAMP synthesis, as well as the cell-permeable analog, dibutyryl cAMP. Sodium nitroprusside, a nitric oxide donor, and the cell-permeable analog, dibutyryl cGMP had no effect on TGF-beta 1-induced collagen synthesis. CONCLUSIONS: Cyclic AMP synthesis in HCC SMC inhibits TGF-beta 1-induced collagen synthesis. Agents which increase cAMP through specific G-protein-coupled receptors, direct stimulation of adenylate cyclase, or inhibition of phosphodiesterase activity could have beneficial effects as modulators of corpus cavernosum connective tissue biosynthesis.