Expression and function of G-protein-coupled receptorsin the male reproductive tract

This review focuses on the expression and function of muscarinic acetylcholine receptors (mAChRs), α1-adrenoceptors and relaxin receptors in the male reproductive tract. The localization and differential expression of mAChR and α1-adrenoceptor subtypes in specific compartments of the efferent ductules, epididymis, vas deferens, seminal vesicle and prostate of various species indicate a role for these receptors in the modulation of luminal fluid composition and smooth muscle contraction, including effects on male fertility. Furthermore, the activation of mAChRs induces transactivation of the epidermal growth factor receptor (EGFR) and the Sertoli cell proliferation. The relaxin receptors are present in the testis, RXFP1 in elongated spermatids and Sertoli cells from rat, and RXFP2 in Leydig and germ cells from rat and human, suggesting a role for these receptors in the spermatogenic process. The localization of both receptors in the apical portion of epithelial cells and smooth muscle layers of the vas deferens suggests an involvement of these receptors in the contraction and regulation of secretion.Esta revisão enfatiza a expressão e a função dos receptores muscarínicos, adrenoceptores α1 e receptores para relaxina no sistema reprodutor masculino. A expressão dos receptores muscarínicos e adrenoceptores α1 em compartimentos específicos de dúctulos eferentes, epidídimo, ductos deferentes, vesícula seminal e próstata de várias espécies indica o envolvimento destes receptores na modulação da composição do fluido luminal e na contração do músculo liso, incluindo efeitos na fertilidade masculina. Além disso, a ativação dos receptores muscarínicos leva à transativação do receptor para o fator crescimento epidermal e proliferação das células de Sertoli. Os receptores para relaxina estão presentes no testículo, RXFP1 nas espermátides alongadas e células de Sertoli de rato e RXFP2 nas células de Leydig e germinativas de ratos e humano, sugerindo o envolvimento destes receptores no processo espermatogênico. A localização de ambos os receptores na porção apical das células epiteliais e no músculo liso dos ductos deferentes de rato sugere um papel na contração e na regulação da secreção.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de FarmacologiaUNIFESP, EPM, Depto. de FarmacologiaSciEL

APPROACH TO THE DISCOVERY OF NOVEL, SELECTIVE INHIBITORS OF P56(LCK) TYROSINE KINASE - IDENTIFICATION OF NON-HYDROXYLATED CHROMONES AS P56(LCK) INHIBITORS

The protein tyrosine kinase p56(lck), which is expressed predominantly in lymphocytes, plays a critical role in optimal T cell activation through the T cell antigen receptor. An approach is presented for the discovery of selective p56(lck) inhibitors, which are potential immunosuppressants. A non-radioactive assay for p56(lck) tyrosine kinase activity has been developed and adapted for high volume screening. This assay does not require purified enzyme. p56(lck) in the plasma membranes of a human T cell line is purified in situ by immobilization onto the wells of a microtiter plate using an antibody specific for p56(lck). Following the kinase reaction in the presence of test compound, autophosphorylated p56(lck) is detected with a biotinylated monoclonal antibody to phosphotyrosine. Using the approach described in this report, three simple chromones have been identified that inhibit p56(lck) autophosphorylation with low micromolar potencies and exhibit some selectivity fdr p56(lck) over the serine/threonine and other tyrosine kinases tested. These compounds constitute a novel group of p56(lck) tyrosine kinase inhibitors. (C) 1995 Wiley-Liss, Inc

Flow cytometric analysis with a fluorescent formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2-receptor

The histamine H2-receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMPdependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca2+]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression and potencies and efficacies of fMLP-induced increases in [Ca2+]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression