Activation of muscarinic cholinergic receptors enhances the volume‐sensitive efflux of myo‐inositol from SH‐SY5Y neuroblastoma cells

Abstract

A mechanism used by cells to regulate their volume under hypo-osmotic conditions is the release of organic osmolytes, one of which is myo-inositol. The possibility that activation of phospholipase-C-linked receptors can regulate this process has been examined for SH-SY5Y neuroblastoma cells. Incubation of cells with hypo-osmolar buffers (160-250 mOsm) led to a biphasic release of inositol which persisted for up to 4 h and could be inhibited by inclusion of anion channel blockersresults which indicate the involvement of a volume-sensitive organic anion channel. Inclusion of oxotremorine-M, a muscarinic cholinergic agonist, resulted in a marked increase (80-100%) in inositol efflux under hypo-osmotic, but not isotonic, conditions. This enhanced release, which was observed under all conditions of hypo-osmolarity tested, could be prevented by inclusion of atropine. Incubation of the cells with either the calcium ionophore, ionomycin, or the phorbol ester, phorbol 12-myristate 13-acetate, partially mimicked the stimulatory effect of muscarinic receptor activation when added singly, and fully when added together. The ability of oxotremorine-M to facilitate inositol release was inhibited by removal of extracellular calcium, depletion of intracellular calcium or down-regulation of protein kinase C. These results indicate that activation of muscarinic cholinergic receptors can regulate osmolyte release in this cell line. Keywords: calcium, muscarinic cholinergic receptors, myoinositol, osmolyte, protein kinase C, volume regulation. Regulation of cell volume is essential for many physiological processes and is of prime importance to the CNS, because of the restricted volume of the skull. Brain cells can swell in response to either changes in plasma osmolarity (hypoosmotic swelling) or from changes in intracellular ion and water distribution (isotonic swelling). The latter is also referred to as cellular or cytotoxic edema (Kimelberg 2000; Pasantes-Morales et al. 2000). Hypo-osmotic swelling frequently occurs as a result of hyponatremia, which is associated with clinical conditions such as congestive heart failure, nephrotic syndrome and hepatic cirrhosis. Water overload may also occur in some psychiatric disorders, such as schizophrenia, or in athletes and in instances of the inappropriate secretion of anti-diuretic hormone. The majority of symptoms observed are neurological and include disorientation, mental confusion and seizures.In response to hypo-osmotic stress, neural cells swell, and to restore osmotic balance a loss of K + and Cl -ions is initially observed. However, as large changes in ion concentrations can adversely impact cell excitability, cells subsequently utilize 'compatible' or 'non-perturbing' osmolytes which are specifically designed to counter changes in osmolarity without compromising cell function. Three distinct classes of osmolytes can be identified, namely (i) amino acids, such as glutamate or taurine, (ii) methylamines, such as betaine and glycerophosphorylcholine, and (iii) polyols, such...

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

“…It has become increasingly clear that agonists of these receptors, and ATP in particular, may accelerate cell-volume recovery under anisosmotic conditions and potently modulate volume-sensitive channels as well as transporters in cells, even in the absence of cell-volume changes (15,28,30,54). It has also been established that signaling linked to G protein-coupled receptors may impact cell volume, as, for example, in mouse Means Ϯ SE values (in nmol/mg of protein) from experiments performed in triplicate are shown.…”

Hydrogen Peroxide Potentiates Volume-sensitive Excitatory Amino Acid Release via a Mechanism Involving Ca2+/Calmodulin-dependent Protein Kinase II

“…Two Ca 2ϩ /calmodulin-dependent enzymes known to be activated by H 2 O 2 , the myosin light chain kinase (MLCK), and CaMKII, have also been found to regulate VRACs (20,27,49,50). We therefore explored whether changes in intracellular Ca 2ϩ and the activation of Ca 2ϩ /calmodulin-dependent enzymes are involved in the H 2 O 2 effect.…”

Two conventional protein kinase C isoforms, α and βI, are involved in the ATP‐induced activation of volume‐regulated anion channel and glutamate release in cultured astrocytes

“…However, a number of laboratories have demonstrated that stimulation of GPCRs, including several metabotropic P2Y receptors for ATP and ADP, produces limited activation of a VRAC-like pathway in non-swollen cells and greatly increases its activity in cells exposed to hypoosmotic medium (Jeremic et al 2001;Mongin and Kimelberg 2002;Mongin and Kimelberg 2003;Loveday et al 2003;Darby et al 2003;Franco et al 2004). Depending on the experimental system and the GPCR involved, PKCs have been found to play major or minor roles in the regulation of VRAC activity by GPCR agonists (Loveday et al 2003;Falktoft and Lambert 2004;Mongin and Kimelberg 2005;Heacock et al 2006;Cheema et al 2007;Ramos-Mandujano et al 2007). In HeLa cells, HTC rat hepatoma cells, and Mz-ChA-1 human cholangiocarcinoma cells, PKC activity was found to be critical for hypotonic (i.e.…”

“…Several groups discovered that ATP and agonists of several other G-protein coupled receptors (GPCR) strongly increase VRAC activity in swollen cells. GPCR agonists may also produce limited VRAC activation in non-swollen cells of both neural and non-neural origin (Tilly et al 1994;Mongin and Kimelberg 2002;Loveday et al 2003;Darby et al 2003;Franco et al 2004;Falktoft and Lambert 2004;Heacock et al 2006;Cheema et al 2007). Regulation of VRAC activity by several purinergic (ATP) receptors may be especially important in the brain.…”

“…In our previous work, we have found that although some of the PKC inhibitors (such as chelerythrine and bisindolylmaleimide) potently blocked ATP-dependent VRAC activation, others (such as Go6983 and Ro-32-0432) showed little efficacy Haskew-Layton et al 2005). In other cell types, PKC has been reported to play either major or minor role in the GPCR-dependent regulation of organic osmolyte release via VRAC (Loveday et al 2003;Falktoft and Lambert 2004;Cheema et al 2007). In the present study we used isoform-specific pharmacological inhibitors and siRNAs to explore the functional significance of individual PKC isozymes.…”