Modulation of gap junction mediated intercellular communication in TM3 Leydig cells R C S Goldenberg 1 , F S A Fortes 1 , J M Cristancho 1,3 , M M Morales 1 , C R Franci 2 , W A Varanda 2 and A C Campos de Carvalho 1 1 Institute of Biophysics Carlos Chagas Filho, UFRJ, Brazil 2 Department of Physiology, Faculty of Medicine of Ribeirão Preto, USP, Av. Bandeirantes, 3900 14049-900 Ribeirão Preto, Brazil 3 Surcolombian University, Neiva, Colombia (Requests for offprints should be addressed to A C C de Carvalho, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowsky s/n, Cidade Universita´ria, Ilha do Fundão, Rio de Janeiro RJ 21941-900, Brasil; Email: acarlos@biof.ufrj.br) Abstract 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 con- fluence 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. Journal of Endocrinology (2003) 177, 327–335 Introduction Gap junctions are plasma membrane specializations responsible for the transfer of small molecules (up to 1 kDa) and ions between adjoining cells (Bennett et al. 1991). Gap junction mediated intercellular communi- cation has been implicated in fundamental cellular pro- cesses such as embryonic development, cell differentiation, proliferation and growth control. The proteins forming gap junctions, named connexins, are phosphoproteins (with the exception of connexin 26 (Cx26)) that can be phos- phorylated by protein kinases activated by neurotransmit- ters, growth factors, hormones, oncogenes and exogenous chemicals. In particular, activation of protein kinases A and C is known to modulate junctional communication between cells. PKC has been shown to phosphorylate Cx43 in serine and threonine residues of the carboxyl tail of the protein (Moreno et al. 1994, Oh et al. 1991), but to date the phosphorylation of Cx43 by PKA has not been unequivocally shown. Nevertheless, 12-O-tetradecanoyl- phorbol-13-acetate (TPA) and cAMP have been found to induce a variety of effects in cells expressing Cx43, leading both to inhibition and to enhancement of gap junction communication, depending on cell type and developmen- tal stage (Oh et al. 1991, Risley et al. 1992, Xie & Hu 1992, Hünster & Weingant 1993, Lampe 1994). In the testis, cellular interactions are essential for adequate functioning of many different cell types (Skinner 1982) and, to date, 11 types of connexin have been identified in its constituent cells (Kadle et al. 1991, Haefliger et al. 1992, Risley et al. 1992, Risley 2000). Freshly dissociated Leydig cells show both electrical and metabolic coupling, and Cx43 has been reported as the main functional component of their gap junctions (Pérez- Armendariz et al. 1994, Varanda & Campos de Carvalho 1994). Although electrical characteristics of the gap junction in these cells have been well characterized, modulation processes have not yet been extensively 327 Journal of Endocrinology (2003) 177, 327–335 0022–0795/03/0177–327  2003 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org