Roles of PMCA Isoforms in Ca^2+-Homeostasis and Contractility of Bladder Smooth Muscle: Evidence from PMCA Gene-Ablated Mice
Plasma membrane Ca^2+ ATPase (PMCA) has four main isoforms encoded by different genes, but only PMCA1 and 4 are expressed in smooth muscle. We investigated the role and relation of the PMCA, sarco-(endo)plasmic reticulum Ca^2+ ATPase (SERCA2) and Na^+/Ca^2+ exchanger (NCX) in urinary bladder smooth muscle (UBSM) contractility and Ca^2+ homeostasis, by using Pmca1 heterozygous (Pmca1+/-), Pmca4 heterozygous (Pmca4+/-), Pmca4 null mutant mice (Pmca4-/-) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1+/-Pmca4-/-) mice. The gene manipulation did not alter the amounts of SERCA2 or NCX. Based on the half-time of relaxation rate after KCl stimulation, the contribution of PMCA to relaxation was calculated to be at least 25%, SERCA2 20% and NCX 70% (Liu et al., 2006). When [Ca^2+]i and contractility were simultaneously measured, KCl elicited both larger forces and Ca^2+ in Pmca1+/- and Pmca1+/-Pmca4-/-. The responses to carbachol (CCh) were also larger in Pmca1+/-. In contrast, the responses in Pmca4-/- and Pmca1+/-Pmca4-/- to CCh were significantly smaller. Our evidence indicates distinct isoform functions with PMCA1 involved in overall Ca^2+-clearance, while PMCA4 is essential for the [Ca^2+]i and contractile responses to CCh. We hypothesized that a localized increase of [Ca^2+]i in the absence of PMCA4 may inhibit the CCh-receptor-mediated function. CCh-induced contractility after treatments with iberotoxin (IBTX, Ca^2+ sensitive K^+ channels (BK^+Ca) blocker) and 2-aminoethoxydiphenyl borate (2-APB, capacitative Ca^2+ entry (CCE) blocker) did not eliminate the difference between WT and Pmca4-/-, indicating that neither BK+Ca channels nor CCE were altered. However, the L-type Ca^2+-channel (Cav1.2) blocker nifedipine exhibited significantly less inhibition in Pmca4-/- than in WT and Cav1.2 activators Bay K 8644 and KCl significantly increased the force in Pmca4-/- and restored the force to the same level as in WT. Western blot indicated the expression of Cav1.2 was not changed. Our data indicates the activities of Cav1.2 and ACh-receptor mediated signal transduction may be disrupted by localized increases in [Ca^2+]i due to the absence of PMCA4 in Pmca4-/-. Immunocytochemistry of PMCA and Cav1.2 indicates that PMCA1 and PMCA4 isoform have different distribution pattern, with PMCA1 uniformly distributed while PMCA4 punctated, yet, PMCA4 always associated with Cav1.2.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:pmca human gene symbols atp2b serca2 atp2a2 ncx bladder smooth muscle ca 2 homeostasis altered mice waves sparks fura pe3 fluo 4 indo 1 multi photon microscopy
Date of Publication:01/01/2007