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

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(3)R 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

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) and induces Ca²⁺release from endoplasmic reticulum (ER) stores.</p> <p>Methods</p> <p>In the present study, we monitored the cytosolic Ca²⁺transients using the UV light photolysis technique to uncage caged Ca²⁺or caged IP₃into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca²⁺receptors present in the ER, and measured their Ca²⁺dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).</p> <p>Results</p> <p>We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP₃R) dependent-Ca²⁺response following both correcting treatments compared to uncorrected cells in such a way that Ca²⁺responses (CF+treatment <it>vs </it>wild-type cells) were normalized. This normalization of the Ca²⁺rate does not affect the activity of Ca²⁺-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP₃R1, we observed a decrease of the implication of IP₃R1 in the Ca²⁺response in CF corrected cells. We observed a similar Ca²⁺mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP₃R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.</p> <p>Conclusion</p> <p>These results suggest reversal of the IP₃R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP₃R Ca²⁺release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.</p