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 potency 4-diphenylacetoxy-N-methylpiperidine methiodide > pirenzepine > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one, a pharmacological profile identical to that obtained for M3 mAChR-stimulated 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 α-cyano-(3,4-dihydroxy)cinnamonitrile. The results indicate that the activation of M3 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.
Footnotes
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The work was supported by National Institutes of Health Grant NS23831 (to S.K.F.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.072553.
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ABBREVIATIONS: CNS, central nervous system; VSOAC, volume-sensitive organic anion channel; mAChR, muscarinic cholinergic receptor; DIDS, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid; Oxo-M, oxotremorine-M; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; SITS, 4-acetamido-4′-isothiocyanatostilbene 2,2′-disulfonic acid; AG-9, α-cyano-(4-methoxyl)cinnamonitrile; AG-18, α-cyano-(3,4-dihydroxy)cinnamonitrile; AG-112, 2-amino-4-(4′-hydroxyphenyl)-1,1,3-tricyanobuta-1,3-diene; mpV, monoperoxo vanadium (picolinato); PD 98059, 2′-amino-3′-methoxyflavone; AF-DX 116, 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance.
- Received June 10, 2004.
- Accepted July 30, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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