Muscarinic Receptors in Human Luteinized Granulosa Cells: Activation Blocks Gap Junctions and Induces the Transcription Factor Early Growth Response Factor-1

Abstract

Acetylcholine (ACh) was recently described to be produced by and act on human luteinizing granulosa cells (GCs). Cholinergic agents increase intracellular calcium levels and stimulate GC proliferation via muscarinic receptors. Based on this observation and because endocrine cells of the forming human corpus luteum (CL), which are the in vivo counterparts of GCs, also proliferate in vivo, we hypothesized that ACh may be a factor involved in the regulation of the complex cellular events occurring during ovulation and formation of the CL. We addressed this possibility by investigating ACh/muscarinic receptor-mediated events in GCs. Normally, cultured GCs and their in vivo counterparts are coupled via gap junctions (GJ) consisting of connexin 43. Treatment with carbachol impaired GJ coupling of GCs within seconds, as shown in single cell, whole cell, patch-clamp studies. The cholinergic antagonist atropine and the muscarinic receptor antagonist pirenzepine specifically blocked this effect. Disruption of GJ communication of GCs is probably due to increased phosphorylation of connexin 43 at serine residues, as shown in immunoprecipitation experiments with carbachol-challenged GCs. Ovulation/formation of the CL include reprogramming of luteinizing cells, and in the rat this involves gonadotropin- induced expression of the transcription factor early growth response factor-1 (egr-1). In human GCs we found that carbachol as well as hCG can mimic this effect, as shown by cDNA arrays and semiquantitative RT-PCR. In conclusion, our results obtained in GCs suggest that endogenous, locally produced ACh may contribute to the cellular remodeling of the forming CL via muscarinic receptor/egr-1, thereby affecting proliferation, GJ communication, and regulation of gene expression in luteinizing granulosa cells.

Two Types of Calcium Channels in Human Ovarian Endocrine Cells: Involvement in Steroidogenesis

“…There is evidence that the expression of voltage-activated Ca 2ϩ channels undergoes dynamic changes during cell proliferation and differentiation (22)(23)(24). Because human GCs in culture are dynamic cells, which also proliferate (45) and differentiate, it is possible that the expression of isoforms of Ca 2ϩ channels is confined to certain phases of the cell cycle and/or differential stages. Our study, in which GCs were treated with hCG, further suggested this possibility and indicated regulation of Ca 2ϩ channel expression.…”

Acetylcholine beyond neurons: the non‐neuronal cholinergic system in humans

“…In the pancreas, ACh increases paracellular permeability by interfering with tight junctions ( Jansen et al ., 1980 ). mAChRs affect the activity of gap junctions in gastric epithelial cells (rat) and in granulosa cells of the ovary ( Ueda et al ., 1995 ; Fritz et al ., 2002 ), most likely an extremely old regulatory target on the evolutionary timescale. Very recently it has been demonstrated that ACh is the transmitter responsible for regulating gap‐junction activity of the central synapse of Drosophila ( Allen and Murphey, 2007 ).…”

Effects of carbachol on rat Sertoli cell proliferation and muscarinic acetylcholine receptors regulation: an in vitro study

“…Acetylcholine has been reported to be a mitogen in various cell types (Ashkenazi et al, 1989;Guizzetti et al, 1996;Luthin et al, 1997;Rayford et al, 1997;Guizzetti et al, 1998;Metzen et al, 2003). Furthermore, stimulation of M 3 and M 5 mAChRs in human luteinized granulosa cells increased intracellular Ca + + concentrations and resulted in an increase of cell proliferation (Fritz et al, 1999(Fritz et al, , 2001, gap junctions communication and regulation of gene expression were also affected (Fritz et al, 2002). Thus, the effect of carbachol on Sertoli cells proliferation points out for physiological significance and opens questions that remain to be answered such as the effect of carbachol in the pattern of protein expression secreted by Sertoli cells and/or in the modulation of gap junctions communication.…”