Potentiation of the Osmosensitive Release of Taurine and d-Aspartate from SH-SY5Y Neuroblastoma Cells after Activation of M₃Muscarinic Cholinergic Receptors

Abstract

The ability of muscarinic cholinergic receptors (mAChRs) to regulate the volume-sensitive efflux of two organic osmolytes, namely, taurine and D-aspartate, from human SH-SY5Y neuroblastoma cells has been examined. Incubation of the cells with hypoosmolar buffers resulted in an efflux of both osmolytes, with the threshold for release occurring at approximately 225 mOsM for taurine and D-aspartate. Inclusion of oxotremorine-M (Oxo-M), a muscarinic agonist, resulted in a marked enhancement of the volume-dependent efflux of both osmolytes and increased the threshold osmolarity for taurine and D-aspartate release to 340 (isotonic) and 320 mOsM, respectively. Maximum agonist stimulation of osmolyte release (350% of basal) was observed in the range of 225 to 250 mOsM. Oxo-M-stimulated osmolyte efflux was inhibited by muscarinic antagonists with a rank order of ,4]benzodiazepin-6-one, a pharmacological profile identical to that obtained for M 3 mAChRstimulated phosphoinositide hydrolysis. Agonist-stimulated efflux of both osmolytes could be inhibited by inclusion of either anion channel blockers known to inhibit the volume-sensitive organic anion channel (VSOAC) or by a tyrosine kinase inhibitor ␣-cyano-(3,4-dihydroxy)cinnamonitrile. The results indicate that the activation of M 3 mAChRs on SH-SY5Y neuroblastoma facilitates the ability of these cells to respond to very limited reductions in osmolarity via a release of osmolytes. mAChR-stimulated osmolyte efflux is mediated via a VSOAC and seems to require the activity of a tyrosine kinase.Although most cells possess homeostatic mechanisms for the maintenance of cell volume, these are particularly important to cells in the central nervous system (CNS) because of restrictions of the skull. Even modest alterations in brain volume can have profound effects on cell-cell signaling because the spatial relationship between neurons, astrocytes, and the extracellular space becomes compromised. Brain swelling, which can occur in response to conditions such as hyponatremia, inappropriate secretion of antidiuretic hormone, or after polydypsia, can lead to the compression of small blood vessels, and subsequently, cerebral anoxia and ischemia. Death can result from the displacement of brain parenchyma through the foramen magnum and the ensuing cardiac and respiratory arrest (Pasantes-Morales et al., 2000). To counter these deleterious changes, neural cells initially restore their osmotic balance via a loss of K ϩ and Cl Ϫ ions. However, because large changes in ion concentrations can adversely impact excitability, cells subsequently use "compatible" or nonperturbing organic osmolytes to counter changes in osmolarity without compromising cell function. In the CNS, the three quantitatively major organic osmolytes are taurine, glutamate, and myo-inositol. Organic osmolytes are released from neural cells via a volume-sensitive organic anion channel (VSOAC), a channel that has been extensively characterized both electrophysiologically and pharmacologically, although its molecular s...

Cholesterol Regulates Volume-Sensitive Osmolyte Efflux from Human SH-SY5Y Neuroblastoma Cells following Receptor Activation

“…Moreover, the increase in receptor-mediated efflux of taurine from cholesterol-depleted cells was greater than that due to the increase in basal release alone, i.e., a synergistic, rather than additive, enhancement of receptor-mediated osmolyte efflux was observed for all three receptors (p Ͻ 0.01; paired Student's t test). Because previous studies of SH-SY5Y cells have extensively characterized the coupling of PAR-1 and mAChRs to volume-dependent taurine efflux (Heacock et al, 2004(Heacock et al, , 2006Cheema et al, 2007), in the current study, emphasis has been placed on monitoring the effects of cholesterol depletion and repletion on the activity of these receptor subtypes. In addition, the effects (if any) of CD on signal transduction events can readily be assessed by monitoring mAChRstimulated phosphoinositide turnover in these cells.…”

“…Previously, we have demonstrated that receptor-mediated enhancement of osmolyte release is dependent on the degree of hypo-osmotic stress in SH-SY5Y cells (Heacock et al, 2004(Heacock et al, , 2006Cheema et al, 2007). Thus, the ability of cholesterol depletion to potentiate taurine release at different osmolarities was examined.…”

“…In this context, we and others Kimelberg, 2002, 2005;Heacock et al, 2004Heacock et al, , 2006Cheema et al, 2005Cheema et al, , 2007Ramos-Mandujano et al, 2007) have recently demonstrated that the volume-dependent release of osmolytes from a variety of neural cells can be dramatically enhanced following activation of certain G-protein-coupled receptors. Receptor activation not only increases the extent of osmolyte release, but it also lowers the threshold osmolarity ("set-point") at which osmolytes are released, thereby permitting cells to respond to small, physiologically relevant, reductions in osmolarity.…”

“…Osmolyte efflux from SH-SY5Y neuroblastoma cells was monitored essentially as previously described (Heacock et al, 2004;Cheema et al, 2007). In brief, cells were prelabeled to isotopic equilibrium with 18.5 kBq/ml [ 3 H]taurine at 37°C for 24 h. After prelabeling, the cells were washed twice with 2 ml of isotonic buffer A (142 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 3.6 mM NaHCO 3 , 1 mM MgCl 2 , and 30 mM HEPES, pH 7.4, 1 mg/ml D-glucose; approximately 340 mOsM).…”

“…We have previously demonstrated that these cells possess several pharmacologically distinct receptors that couple to osmolyte efflux, including proteinase-activated receptor (PAR) 1, muscarinic cholinergic receptor (mAChR), and sphingosine 1-phosphate (S1P; Heacock et al, 2004Heacock et al, , 2006Cheema et al, 2007). The results indicate that cholesterol depletion, induced by methyl-␤-cyclodextrin (CD), synergistically enhances the ability of all three receptors to increase taurine efflux above basal levels and that this potentiation can be reversed by administration of cholesterol or its stereoisomer, 5-cholesten-3␣-ol (epicholesterol).…”