[Ca2+](i) oscillations induced by muscarinic stimulation in airway smooth muscle cells: Receptor subtypes and correlation with the mechanical activity

1 Cytosolic calcium concentration ([Ca2+](i)) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle. 2 In isolated myocytes, acetylcholine: (ACh, 0.03-1 mu M) caused a rapid and graded increase in [Ca2+](i) up ro a net amplitude of 492 +/- 26 nM (n = 19) which gradually declined, The EC(50) fur ACh was 0.13 mu M. This first [Ca2+](i) peak was followed when the ACh concentration increased in approximately 50-60% of the cells by successive peaks of decreased amplitude ([Ca2+](i) oscillations) superimposed on the plateau phase, Whereas the percentage of cells exhibiting [Ca2+](i) oscillations remained consistent, the frequency of these oscillations increased to up to 10 min(-1) with all ACh concentration of 100 mu. 3 Removal of extracellular calcium (in the presence of EGTA, 0.4 mM) or addition of the voltage-dependent Ca2+-channel blocker verapamil (10 mu M) did not alter the first [Ca2+](i) peak the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca2+-ATPase, thapsigargin (1 mu M), completely abolished the [Ca2+](i) response. Thapsigargin (1 mu M) also blocked the caffeine (5 mM)-induced transient rise in [Ca2+](i). 4 Atropine (a nea-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP, a selective M(3) antagonist) inhibited the [Ca2+](i) response to muscarinic cholinoceptor activation with an IC50 of 13 and 20 nM, respectively. Pirenzepine (a selective M(1) antagonist) also totally inhibited the [Ca2+](i) response to ACh but with a higher IC50 of 2 mu M. Methoctramine (a selective M(2) antagonist) up to a concentration of 10 mu M caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration-response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4-DAMP at 3, 10 and 30 nM: pirenzepine 0.3, and 3 mu M: and methoctramine at 1, 3 and 10 mu M. For these concentrations all of the antagonists produced a rightward shift of the CCRC for carbachol and pA(2) values were 9.2, 8.8, 6.7 and 6.3, respectively. 5 In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca2+](i) oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca2+](i) rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca2+](i) and the contractile responses are primarily dependent on activation of the M(2) receptor subtype.