Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices

To investigate the phenomenon of Ca2+ sensitization, we developed a new intact airway and arteriole smooth muscle cell (SMC) "model" by treating murine lung slices with ryanodine-receptor antagonist, ryanodine (50 mu M), and caffeine (20 mM). A sustained elevation in intracellular Ca2+ concentration ([Ca2+](i)) was induced in both SMC types by the ryanodine-caffeine treatment due to the depletion of internal Ca2+ stores and the stimulation of a persistent influx of Ca2+. Arterioles responded to this sustained increase in [Ca2+](i) with a sustained contraction. By contrast, airways responded to sustained high [Ca2+](i) with a transient contraction followed by relaxation. Subsequent exposure to methacholine (MCh) induced a sustained concentration-dependent contraction of the airway without a change in the [Ca2+](i). During sustained MCh-induced contraction, Y-27632 (a Rho-kinase inhibitor) and GF-109203X (a protein kinase C inhibitor) induced a concentration-dependent relaxation without changing the [Ca2+](i). The cAMP-elevating agents, forskolin (an adenylyl cyclase activator), IBMX ( a phosphodiesterase inhibitor), and caffeine (also acting as a phosphodiesterase inhibitor), exerted similar relaxing effects. These results indicate that 1) ryanodine-caffeine treatment is a valuable tool for investigating the contractile mechanisms of SMCs while avoiding nonspecific effects due to cell permeabilization, 2) in the absence of agonist, sustained high [Ca2+](i) has a differential time-dependent effect on the Ca2+ sensitivity of airway and arteriole SMCs, 3) MCh facilitates the contraction of airway SMCs by inducing Ca2+ sensitization via the activation of Rho-kinase and protein kinase C, and 4) cAMP-elevating agents contribute to the relaxation of airway SMCs through Ca2+ desensitization.