[CA2-MUSCLE CELLS - RECEPTOR SUBTYPES AND CORRELATION WITH THE MECHANICAL-ACTIVITY(](I) OSCILLATIONS INDUCED BY MUSCARINIC STIMULATION IN AIRWAY SMOOTH)

1 Cytosolic calcium concentration ([Ca2+](i)) by indo 1 microspectrofl uorimetry in freshly isolated cells and isometric contraction of isola ted rings were measured in response to muscarinic cholinoceptor stimul ation in rat tracheal smooth muscle. 2 In isolated myocytes, acetylcho line: (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 dec lined, The EC(50) fur ACh was 0.13 mu M. This first [Ca2+](i) peak was followed. when the ACh concentration increased. in approximately 50-6 0% of tile cells. by successive peaks of decreased amplitude ([Ca2+](i ) oscillations) superimposed on the plateau phase, Whereas the percent age of cells exhibiting [Ca2+](i) oscillations remained consistent, th e frequency of these oscillations increased to up to 10 min(-1) with a ll 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, tha psigargin (1 mu M), completely abolished the [Ca2+](i) response. Thaps igargin (1 mu M) also blocked the caffeine (5 mM)-induced transient ri se in [Ca2+](i). 4 Atropine (a nea-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP. a sele ctive M(3) antagonist) inhibited the [Ca2+](i) response to muscarinic cholinoceptor activation with an IC50 of 13 and 20 nM, respectively. P irenzepine (a selective M(1) antagonist) also totally inhibited the [C a2+](i) response to ACh but with a higher IC50 of 2 mu M. Methoctramin e (a selective M(2) antagonist) up to a concentration of 10 mu M cause d only a 40% inhibition. The effect of muscarinic antagonists on cumul ative 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, 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 a nd 6.3, respectively. 5 In conclusion, the present study indicates tha t muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca2+](i) oscillations. The occurrence of these oscillations d epends on the graded amplitude of the first [Ca2+](i) rise and their f requency may play a role in the amplitude of the mechanical activity i n response to muscarinic cholinoceptor activation. Both the [Ca2+](i) and the contractile responses are primarily dependent on activation of the M(2) receptor subtype.