[Ca 2+ ] i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity

Cytosolic calcium concentration ([Ca 2+ ] 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. In isolated myocytes, acetylcholine (ACh, 0.031 μ m ) caused a rapid and graded increase in [Ca 2+ ] i up to a net amplitude of 492±26 n m ( n =19) which gradually declined. The EC 50 for ACh was 0.13 μ m . This first [Ca 2+ ] i peak was followed, when the ACh concentration increased, in approximately 5060% of the cells, by successive peaks of decreased amplitude ([Ca 2+ ] i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca 2+ ] i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min −1 with an ACh concentration of 100 μ m . Removal of extracellular calcium (in the presence of EGTA, 0.4 m m ) or addition of the voltage‐dependent Ca 2+ ‐channel blocker verapamil (10 μ m ) did not alter the first [Ca 2+ ] i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca 2+ ‐ATPase, thapsigargin (1 μ m ), completely abolished the [Ca 2+ ] i response. Thapsigargin (1 μ m ) also blocked the caffeine (5 m m )‐induced transient rise in [Ca 2+ ] i . Atropine (a non‐selective muscarinic cholinoceptor antagonist) and 4‐diphenyl acetoxy N‐methyl piperidine (4‐DAMP, a selective M 3 antagonist) inhibited the [Ca 2+ ] i response to muscarinic cholinoceptor activation with an IC 50 of 13 and 20 n m , respectively. Pirenzepine (a selective M 1 antagonist) also totally inhibited the [Ca 2+ ] i response to ACh but with a higher IC 50 of 2 μ m . Methoctramine (a selective M 2 antagonist) up to a concentration of 10 μ 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 n m ; pirenzepine 0.3, 1 and 3 μ m , and methoctramine at 1, 3 and 10 μ 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. In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca 2+ ] i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca 2+ ] i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca 2+ ] i and the contractile responses are primarily dependent on activation of the M 3 receptor subtype.British Journal of Pharmacology (1997) 120 , 1294–1301; doi: 10.1038/sj.bjp.0701061