Dopamine receptor repertoire of human granulosa cells

<p>Abstract</p> <p>Background</p> <p>High levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.</p> <p>Methods</p> <p>Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4) were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system.</p> <p>Results</p> <p>We found members of the two DA receptor families (D₁- and D₂ -like) associated with different signaling pathways in human GCs, namely D₁ (as expected) and D₅ (both are Gs coupled and linked to cAMP increase) and D₂, D₄ (Gi/Gq coupled and linked to IP3/DAG). D₃ was not found. The presence of the trophic hormone hCG (10 IU/ml) in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR) or protein levels (immunocytochemistry/Western blotting) of D_1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D₁ and D₂, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D₂ expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D₂L, D₂S). Irrespective of these variants, D₂ proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed in the absence of extracellular calcium and was abolished by a D₂ blocker (L-741,626). DA treatment (48 h) of human GCs resulted in slightly, but significantly enlarged, viable cells.</p> <p>Conclusion</p> <p>A previous study showed D₂ in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D₂-like receptors, linked to calcium. Ovarian DA can act thus via D_1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.</p

Prostaglandin E 2 (PGE 2 ) is a testicular peritubular cell-derived factor involved in human testicular homeostasis

In man, blockage of prostaglandin (PG)-production e.g. by non-steroidal anti-inflammatory drug (NSAIDs) may have negative testicular side effects, implying beneficial actions of PGs in the testis. We examined human testicular samples and isolated human testicular peritubular cells (HTPCs) to explore sites of PG-synthesis and targets. HTPCs express cyclooxygenase 1 (COX1) and secrete PGE 2 . Receptors (EP1, 2, 4) were specifically identified in peritubular cells. In HTPCs PGE 2 significantly increased mRNA levels of the contractility protein calponin, but did not induce contractions. PGE 2 , as well as EP1 and EP4 receptor agonists, significantly increased glia cell line derived neurotrophic factor (GDNF) mRNA and/or protein levels. Importantly, the NSAID ibuprofen reduced PGE 2 and this action also lowered SMA and calponin mRNA levels and levels of secreted GDNF protein. The results reveal an unknown PGE 2 system in the human testis, in involving peritubular cells, which may be prone to interference by NSAIDs.Fil: Rey Ares, Veronica. Ludwig Maximilians Universitat; AlemaniaFil: Rossi, Soledad Paola. Ludwig Maximilians Universitat; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Dietrich, Kim Gwendolyn. Ludwig Maximilians Universitat; AlemaniaFil: Köhn, Frank Michael. Ludwig Maximilians Universitat; AlemaniaFil: Ullrich Schwarzer, J. Andrologicum; Múnich, Alemania; AlemaniaFil: Welter, Hared. Ludwig Maximilians Universitat; AlemaniaFil: Frungieri, Monica Beatriz. Ludwig Maximilians Universitat; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mayerhofer, Artur. Ludwig Maximilians Universitat; Alemani

Alpha 1 adrenergic receptor-mediated inflammatory responses in human testicular peritubular cells

Stress activates the sympathetic nervous system and is linked to impaired fertility in man. We hypothesized that catecholamines by acting on testicular cells have a role in these events, possibly by fostering an inflammatory environment. The cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), express adrenergic receptors (ADRs) α1B, α1D, β1 and β2. A selective α1-ADR agonist, phenylephrine, increased intracellular Ca 2+ -levels in cultured HTPCs and induced COX-2, IL-6 and MCP-1 mRNA expression without affecting IL-1β mRNA. These changes were paralleled by a significant increase in the secretion of IL-6 and MCP-1. Epinephrine was also effective, but salbutamol, a selective β2-ADR agonist was not. Our results suggest that stress-associated elevation of catecholamines may be able to promote inflammatory events by targeting peritubular cells in the human testis. Blockage of α1-ADRs may therefore be a novel way to interfere with stress-related impairment of male reproductive functions.Fil: Rossi, Soledad Paola. Ludwig Maximilians Universitat; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Walenta, Lena. Ludwig Maximilians Universitat; AlemaniaFil: Rey Ares, Veronica. Ludwig Maximilians Universitat; AlemaniaFil: Köhn, Frank-Michael. Andrologicum; AlemaniaFil: Ulrich Schwarzer, J.. Andrology Center Munich; AlemaniaFil: Welter, Harald. Ludwig Maximilians Universitat; AlemaniaFil: Calandra, Ricardo Saul. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Frungieri, Monica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mayerhofer, Artur. Ludwig Maximilians Universitat; Alemani

Human Umbilical vein: Involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor sensitized responses

In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-senzitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 μmol/l) or NS-398 (10, 30 μmol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.Fil: Errasti, Andrea Emilse. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rey Ares, Veronica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Daray, Federico Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Rogines Velo, M. P.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Sardi, Sergio Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Paz, Cristina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Podesta, Ernesto Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rothlin, Rodolfo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin