Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca2+ requirement for phospholipase C activation

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Abstract
1 The relationship between muscarinic receptor-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown and the increase of intracellular Ca2+ ([Ca2+])i has been examined in canine cultured tracheal smooth muscle cells (TSMCs). 2 Addition of acetylcholine (ACh) and carbachol led to a 2–3 fold increase in [Ca2+]i over the resting level as determined by fura-2, with half-maximal stimulation (EC50) obtained at concentrations of 97 and 340 nm, respectively. Addition of the partial agonist, bethanechol, showed a smaller increase in PIP2 turnover and [Ca2+]i than did ACh or carbachol. 3 Addition of ACh or carbachol to TSMCs that had been prelabelled with [3H]-inositol led to the rapid (5–15 s) release of inositol mono, bis and trisphosphates IP1, IP2 and IP3. The time course of IP3 accumulation is correlated with the time course of the peak rise in [Ca2+]i 4 Inclusion of EGTA lowered the resting [Ca2+]i and markedly reduced the extent of the agonist-induced rise in [Ca2+]i. When assayed under conditions similar to those used for the [Ca2+]i measurements, EGTA reduced the muscarinic agonist-stimulated inositol phosphates (IPs) accumulation. Conversely, ionomycin could stimulate IPs accumulation and elevate [Ca2+]i. The addition of Ca2+ (2.7–617 nm) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. 5 Both Ca2+ and guanosine- 5′-O-(3-thiotriphosphate) (GTPγS) stimulated the formation of IPs in digitonin-permeabilized TSMCs prelabelled with [3H]-inositol. A further calcium-dependent increase in IPs accumulation was obtained by inclusion of either GTPγS or carbachol. The combined presence of carbachol and GTPγS elicited a synergistic effect on IPs accumulation, with half-maximal stimulation observed at approximately 8 nm free Ca2+. 6 These results indicate that (i) the magnitude of the initial rise in [Ca2+]i is directly related to the production of IPs and (ii) the phospholipase C-mediated PIP2 breakdown in TSMCs is sensitive to regulation by physiologically relevant concentrations of free Ca2+ ([Ca2+]f).