[Ca²⁺]_i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity

Abstract

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, 0.031 μM) caused a rapid and graded increase in [Ca²⁺]_i up to a net amplitude of 492±26 nM (n=19) which gradually declined. The EC₅₀ for ACh was 0.13 μM. This first [Ca²⁺]_i peak was followed, when the ACh concentration increased, in approximately 5060% 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 an ACh concentration of 100 μM.

3. Removal of extracellular calcium (in the presence of EGTA, 0.4 mM) or addition of the voltage-dependent Ca²⁺-channel blocker verapamil (10 μM) 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 μM), completely abolished the [Ca²⁺]_i response. Thapsigargin (1 μM) also blocked the caffeine (5 mM)-induced transient rise in [Ca²⁺]_i.

4. Atropine (a non-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP, a selective M₃ antagonist) inhibited the [Ca²⁺]_i response to muscarinic cholinoceptor activation with an IC₅₀ of 13 and 20 nM, respectively. Pirenzepine (a selective M₁ antagonist) also totally inhibited the [Ca²⁺]_i response to ACh but with a higher IC₅₀ of 2 μM. Methoctramine (a selective M₂ antagonist) up to a concentration of 10 μM 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, 1 and 3 μM, and methoctramine at 1, 3 and 10 μM. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA₂ 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 M₃ receptor subtype.

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