Role of plasma membrane Ca²⁺-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice

Abstract

We investigated the roles and relationships of plasma membrane Ca(2+)-ATPase (PMCA), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2, and Na(+)/Ca(2+) exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4(-/-)) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1(+/-)Pmca4(-/-)) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca(2+) transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca(2+)-free physiological salt solution containing EGTA. We measured the contributions of Ca(2+) clearance components by inhibiting SERCA2 (with 10 microM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 microM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be approximately 25-30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca(2+) extrusion-relaxation relationship, i.e., Ca(2+) homeostasis, of bladder smooth muscle.

Mice Lacking Hypertension Candidate Gene ATP2B1 in Vascular Smooth Muscle Cells Show Significant Blood Pressure Elevation

“…Using bladder smooth muscle cells, contractility measurements have documented the important role of ATP2B1 in the extrusion of Ca 2ϩ after carbachol stimulation or depolarization with potassium chloride. 9 Although bladder smooth muscle expresses both ATP2B4 and ATP2B1, ATP2B1 inhibition caused 3-time increments in intracellular calcium concentration and contraction of bladder smooth muscle compared with ATP2B4 blockade. Thus, ATP2B1 rather than ATP2B4 may have an important role in calcium handling and regulation in contraction of smooth muscle cells.…”

“…The ATP2B1-null mutant mouse has been reported to be embryolethal 8 ; thus, we need to make a conditional knockout (KO) mouse model of ATP2B1 using the Cre-loxP system to reveal the function of the gene. Because the ATP2B1 gene encodes one of the calcium pumps and plays an important role in contraction of bladder smooth muscle, 9 we selected vascular smooth muscle cells (VSMCs) as target tissue of KO. Because we have already demonstrated that ATP2B1 mRNA expression in human umbilical artery smooth muscle cells was significantly lower in those having the risk allele for hypertension than in those with no risk allele, 10 we hypothesized that VSMC ATP2B1 KO mice would exhibit high blood pressure.…”

The Plasma Membrane Calcium ATPasesand Their Role as Major New Players in Human Disease

“…With the use of inhibitors to SERCA and NCX in PMCA gene-ablated mice, the relative contribution of the PMCA to relaxation of bladder smooth muscle rings was estimated to be~25-30%, slightly greater than that of SERCA and with the NCX responsible for up to 70% of relaxation (216). This is largely compatible with data from isolated guinea pig detrusor smooth muscle cells which, using inhibitors to each system, estimated the relative contributions of PMCA, NCX, and SERCA to Ca 2ϩ clearance to be 27, 55, and 31%, respectively (138).…”

“…This is largely compatible with data from isolated guinea pig detrusor smooth muscle cells which, using inhibitors to each system, estimated the relative contributions of PMCA, NCX, and SERCA to Ca 2ϩ clearance to be 27, 55, and 31%, respectively (138). Interestingly, Liu et al (216) also found PMCA ablation to impair the rate of contraction, and in a further study in which contraction and [Ca 2ϩ ] i were measured simultaneously, it was determined that the loss of PMCA4 significantly inhibited the force and rate of contraction, as well as the rates of increase and decrease of [Ca 2ϩ ] i following cholinergic stimulation (215). In contrast, the authors found heterozygous PMCA1 deletion to elicit larger increases in [Ca 2ϩ ] i and rate and force of contraction following KCl stimulation, suggesting a greater role for this isoform in overall Ca 2ϩ extrusion.…”

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension

“…Indeed smooth muscle-specific genetic ablation of NCX1 lowers BP (Zhang et al, 2010;Zhao et al, 2011), whilst, conversely, mice overexpressing NCX1 in smooth muscle display increased BP (Blaustein & Lederer, 1999;Iwamoto et al, 2004); effects not consistent with a predominant role in Ca 2+ extrusion. Currently there is little known about the specific contribution of PMCAs to Ca 2+ efflux in VSM, but in bladder and myometrial smooth muscle extrusion by this route has been reported to contribute approximately 30% and 70% of total calcium efflux respectively (Matthew et al, 2004;Liu et al, 2006). Therefore, a greater understanding of the role of PMCAs in the microcirculation is important for further understanding of the regulation of BP and how it might be pharmacologically modulated.…”