Developmental changes in Ca2+homeostasis and contractility in gallbladder smooth muscle

Abstract

Relatively little is known about the contribution of Ca2+-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca2+homeostasis and Ca2+sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca2+concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca2+mobilization, suggesting immaturity of Ca2+sensitization mechanisms. The enhanced Ca2+release in the newborn gallbladder was the result of the increase in the size of the releasable Ca2+pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca2+pump nor the Na+/Ca2+exchanger collaborate in the extrusion of Ca2+. In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca2+-ATPase pump. The reduced Ca2+sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.