Regulation of intracellular Ca2+ concentration ([Ca2+]i) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca2+ release and reuptake. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is key to replenishment of SR Ca2+ stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca2+ indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca2+]i responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca2+]i transients to 1 μM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca2+]i oscillations (which were previously shown to result from repetitive SR Ca2+ release/reuptake). However, when ACh-induced [Ca2+]i oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2+]i transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca2+]i oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca2+ release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca2+ reuptake, potentially through altered PLN phosphorylation.

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca2+ stores is an important determinant of [Ca2+]i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca2+]i oscillations representing repetitive SR Ca2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca2+]i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca2+]i responses, and slowed or inhibited ongoing [Ca2+]i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca2+]i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca2+ release and reuptake, likely through PLB.Supported by NIH grant HL74309 (GCS)