Airway smooth muscle relaxation results from a reduction in the frequency of Ca ²⁺ oscillations induced by a cAMP-mediated inhibition of the IP ₃ receptor

Abstract

Background

It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca ²⁺ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca ²⁺ oscillations.

Methods

The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca ²⁺ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.

Results

All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca ²⁺ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca ²⁺ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca ²⁺ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca ²⁺ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca ²⁺ available to stimulate contraction because an elevation of intracellular Ca ²⁺ concentration induced by exposure to a Ca ²⁺ ionophore (ionomycin) or by photolysis of caged-Ca ²⁺ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca ²⁺-activated K ⁺ channels, which would be expected to increase Ca ²⁺ influx and contraction. By contrast, the photolysis of caged-IP ₃ in the presence of agonist, to further elevate the intracellular IP ₃ concentration, reversed the slowing of the frequency of the Ca ²⁺ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP ₃R to IP ₃ was reduced by FSK and this was supported by the reduced ability of IP ₃ to release Ca ²⁺ in SMCs in the presence of FSK.

Conclusion

These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca ²⁺ oscillation frequency by reducing internal Ca ²⁺ release through IP ₃ receptors.