RESUMO
Long-term modulation of intercellular communication via gap junctions was investigated in TM3 Leydig cells, under low and high confluence states, and upon treatment of the cells for different times with activators of protein kinase A (PKA) and protein kinase C (PKC). Cells in low confluence were readily coupled, as determined by transfer of the dye Lucifer Yellow; on reaching confluence, the cells uncoupled. Western blots and RT-PCR revealed that connexin 43 (Cx43) was abundantly expressed in TM3 Leydig cells and its expression was decreased after the cells achieved confluence. Stimulation of PKA or PKC induced a decrease in cell-cell communication. Staurosporin, an inhibitor of protein kinases, increased coupling and was able to prevent and reverse the uncoupling actions of dibutyryl cAMP and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Under modulation by confluence, Cx43 was localized to the appositional membranes when cells were coupled and was mainly in the cytoplasm when they were uncoupled. In addition, cAMP and TPA reduced the surface membrane labeling for Cx43, whereas staurosporin increased it. These data show a strong correlation between functional coupling and the membrane distribution of Cx43, implying that this connexin has an important role in intercellular communication between TM3 cells. Furthermore, increased testosterone secretion in response to luteinizing hormone was accompanied by a decrease in intercellular communication, suggesting that gap junction mediated coupling may be a modulator of hormone secretion in TM3 cells.
Assuntos
Junções Comunicantes/efeitos dos fármacos , Células Intersticiais do Testículo/ultraestrutura , Animais , Transporte Biológico , Bucladesina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/química , Corantes , Conexina 43/análise , Conexina 43/genética , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Gliceraldeído-3-Fosfato Desidrogenases/genética , Isoquinolinas , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estaurosporina/farmacologia , Testosterona/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
We report results obtained with sera from 58 chronic chagasic patients that were evaluated for effects on heart rate and atrioventricular (AV) conduction in isolated rabbit hearts and screened for the presence of muscarinic and beta-adrenergic activity. We show that sera from 26 patients decreased heart rate, while 10 increased it and 22 had no effect. Additionally, sera from 20 of the 58 patients blocked AV conduction. Muscarinic activation seems to be involved in both effects, but is not the only mechanism, since atropine did not antagonize the decrease in heart rate in 23 percent of sera or AV block in 40 percent. Sera from patients with complex arrhythmias were significantly more effective in depressing both heart rate and AV conduction. Sera that induce increases in heart rate seem to operate exclusively through beta-adrenergic activation. Two of these sera, evaluated with respect to intercellular communication in primary cultures of embryonic cardiomyocytes were able to block gap junction conductance evaluated by a dye injection technique after 24-h exposure. The mechanisms underlying this uncoupling effect are currently being investigated