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Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca 2+ requirement for phospholipase C activation
Author(s) -
Yang Chuen Mao,
Chou ShengPing,
Wang YenYi,
Hsieh JenTsung,
Ong Richard
Publication year - 1993
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1993.tb13948.x
Subject(s) - egta , ionomycin , carbachol , inositol , muscarinic acetylcholine receptor , muscarinic agonist , medicine , endocrinology , gtp' , calcium , chemistry , ryanodine receptor , phospholipase c , agonist , digitonin , biology , stimulation , biochemistry , receptor , enzyme
1 The relationship between muscarinic receptor‐mediated phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) breakdown and the increase of intracellular Ca 2+ ([Ca 2+ ]) 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 [Ca 2+ ] i over the resting level as determined by fura‐2, with half‐maximal stimulation (EC 50 ) obtained at concentrations of 97 and 340 n m , respectively. Addition of the partial agonist, bethanechol, showed a smaller increase in PIP 2 turnover and [Ca 2+ ] i than did ACh or carbachol. 3 Addition of ACh or carbachol to TSMCs that had been prelabelled with [ 3 H]‐inositol led to the rapid (5–15 s) release of inositol mono, bis and trisphosphates IP 1 , IP 2 and IP 3 . The time course of IP 3 accumulation is correlated with the time course of the peak rise in [Ca 2+ ] i4 Inclusion of EGTA lowered the resting [Ca 2+ ] i and markedly reduced the extent of the agonist‐induced rise in [Ca 2+ ] i . When assayed under conditions similar to those used for the [Ca 2+ ] i measurements, EGTA reduced the muscarinic agonist‐stimulated inositol phosphates (IPs) accumulation. Conversely, ionomycin could stimulate IPs accumulation and elevate [Ca 2+ ] i . The addition of Ca 2+ (2.7–617 n m ) to digitonin‐permeabilized TSMCs directly stimulated IPs accumulation. 5 Both Ca 2+ and guanosine‐ 5′‐O‐(3‐thiotriphosphate) (GTPγS) stimulated the formation of IPs in digitonin‐permeabilized TSMCs prelabelled with [ 3 H]‐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 n m free Ca 2+ . 6 These results indicate that (i) the magnitude of the initial rise in [Ca 2+ ] i is directly related to the production of IPs and (ii) the phospholipase C‐mediated PIP 2 breakdown in TSMCs is sensitive to regulation by physiologically relevant concentrations of free Ca 2+ ([Ca 2+ ] f ).