Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells.
We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i, rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. -60 to -10 mV. As expected, we found that nicotine (10 microM) depolarized the membrane (from ca. -60 to -20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 microM) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.
Luxoro
M
M
Laboratory of Cellular Physiology, Faculty of Sciences, University of Chile, Viña del Mar.
Nassar-Gentina
V
V
Rojas
E
E
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009538:Nicotine / Q000494:pharmacology; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000494:pharmacology; D015398:Signal Transduction; D012964:Sodium / Q000494:pharmacology*; D014403:Tubocurarine / Q000494:pharmacology
D010095:Oxotremorine; C042743:oxotremorine M; D009538:Nicotine; D015544:Inositol 1,4,5-Trisphosphate; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D014403:Tubocurarine