We previously showed that plasma membrane Ca2+-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca2+ ([Ca2+]i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1+/−, Pmca4+/−, Pmca4−/−, and Pmca1+/−Pmca4−/− mice. There were no differences in basal [Ca2+]i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca2+]i (130–180%) in smooth muscle from Pmca1+/− and Pmca1+/−Pmca4−/− bladders than those in WT or Pmca4−/−. The responses to carbachol (CCh: 10 μM) were also greater in Pmca1+/− (120–150%) than in WT bladders. In contrast, the responses in Pmca4−/− and Pmca1+/−Pmca4−/− bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca2+]i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4−/− (130–190%) and Pmca1+/−Pmca4−/− (120–250%) bladders, but not in Pmca1+/− bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca2+ clearance, while PMCA4 is essential for the [Ca2+]i increase and contractile response to the CCh receptor-mediated signal transduction pathway.
- bladder smooth muscle
- gene-altered mice
the plasma membrane Ca2+-ATPase (PMCA) is a P-type Ca2+-ATPase, with four different isoforms encoded by four separate genes (16–18). In addition, there are a number of variants for each isoform due to diverse mRNA splices (17). PMCA isoforms have different expression levels during development, with PMCA1 being the most critical isoform during embryogenesis (12) and with PMCA1 and PMCA4 being the major isoforms after birth (21). They also have tissue-specific distributions: PMCA1 and PMCA4 are ubiquitously expressed, while PMCA2 and PMCA3 are found predominantly in neuronal tissues (11). Although PMCA isoforms have ∼80–90% identity in their amino acid composition and all contain 10 transmembrane segments and a PDZ-binding site in the COOH terminus (14, 15), their functions are not redundant. PMCA1 homozygotes (Pmca1−/−) are embryonically lethal, indicating that PMCA1 is an essential housekeeping pump (11). PMCA2 homozygous mice are deaf and have balance deficiencies (7). PMCA4 null mutant males are infertile due to the reduced motility of sperm cells (11, 13, 20).
We previously studied contractile function of bladder smooth muscle of a PMCA4 null mutant (Pmca4−/−) and PMCA1/PMCA4 double gene-targeted mice (Pmca1+/−Pmca4−/−) (8). Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger (NCX), as well as gene-targeting of PMCA isoforms, all prolonged the relaxation half-time. The contribution to relaxation of PMCA was calculated to be 25–30%; that of SERCA2, 20%; and NCX, 70%. In this study, we address the roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle. We measured cytosolic Ca2+ ([Ca2+]i) using fura-PE3 and simultaneously measured contractility in the intact bladder smooth muscle of WT, Pmca1+/−, Pmca4−/−, and Pmca1+/−Pmca4−/− mice. Our evidence indicates distinct isoform functions, with the PMCA1 isoform involved in overall Ca2+ clearance, while PMCA4 is essential for the contractile response to the carbachol (CCh) receptor-mediated signal transduction pathway.
MATERIALS AND METHODS
Mice were 129/SvJ and Black Swiss mixed background, between 4 and 6 mo of age, with the majority being 5 mo. For each mouse type studied, only age-matched and sex-matched pairs of WT and gene-targeted mice were used.
Mice were euthanized in a precharged CO2 chamber. Urinary bladders were dissected from WT, Pmca1+/−, Pmca4+/−, Pmca4−/−, and Pmca1+/−Pmca4−/− mice, and were rinsed with physiological salt solution (PSS) of the following composition (in mM): 120 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4 1.1 NaH2PO4, 23.8 NaHCO3, 11.2 glucose, and bubbled with 95% O2-5% CO2, pH 7.4. The mucosal layer in the lumen was separated along natural tissue lines and removed. The bladder was cut open and the middle part of the mucosa-denuded bladder was cut into a longitudinal strip of 1.5-mm width. The bladder strip was folded into a loop; free ends were joined together with a surgical thread. For simultaneous Ca2+ and force measurements, bladder preparations were incubated 4 h or overnight at room temperature with stirring in 10 μM fura-PE3 AM loading solution dissolved in DMSO. The noncytotoxic detergent pluronic F127 (0.025%) was added to increase the solubility of fura-PE3 AM. Experimental details were as reported previously by Nobe et al. (10). Animal treatment and experimental protocols were approved by the Institutional Animal Care and Use Committee, University of Cincinnati.
Simultaneous Ca2+ and force measurements.
Bladder loops were mounted for measurements of isometric force between a Harvard Apparatus force transducer and a fixed stainless steel wire. The mounting assembly was fitted into a Teflon holder and placed in a cuvette, which was designed to place the tissue in the light path of a dual-wavelength spectrofluorimeter (PTI Delta Scan-1; Photon Technology International, South Brunswick, NJ). The muscle loop was continuously perfused with 95% O2-5% CO2 aerated PSS at 37°C. The muscle loop was stretched three times to a peak force of 15 mN for 15 min and reached nearly twice the initial length, setting the length in the range for optimal force generation. Fluorescence was excited at 340 nm and 380 nm, and emission was measured at 510 nm. The fluorescence ratio of 340/380 nm was taken as measure of [Ca2+]i.
Resting [Ca2+]i calibration.
To ensure equal optical conditions, fluorescence for each tissue was excited at 360 nm, the isobestic wavelength of fura-PE3, and emission intensity at 510 nm set to 106 counts/s (cps) by adjusting the excitation slits of the monochromator. Then the 340/380 ratio was measured. Background autofluorescence was measured by adding MnCl2 (5 mM) to quench the fura-PE3 fluorescence. The basal [Ca2+]i was calculated by subtracting the background and was calibrated to the standard calibration curve. After removal of the tissue from the cuvette, 340/380 ratios were measured for various calibrated [Ca2+] solutions using a commercial kit (Invitrogen). The [Ca2+] (nM) vs. 340/380 ratio was used to establish a standard curve. After subtraction of the background, [Ca2+]i was calibrated from the basal 340/380 ratios using the standard calibration curve.
Concentration-response relations were generated using cumulative additions of CCh (0.1–10 μM). In our previous study (8), we reported that KCl-induced contractility among genotypes was not affected by atropine (10 μM), so the innervations of bladder do not appear to be a major factor. Contraction kinetics were measured using 80 mM KCl or 10−5 M CCh stimulation. Relaxation kinetics were measured by replacing the PSS with a Ca2+-free PSS containing 0.1 mM EGTA. Half-times of the increase/decrease of tension and [Ca2+]i were measured as an index of rates. Ca2+ signals are expressed as the 340/380 ratio and isometric force as milli-Newtons per square millimeter, with the cross-sectional area estimated as the wet weight/length.
Statistical analyses were performed using ANOVA with the Holm-Sidak multiple comparison test. A value of P < 0.05 was taken as indicative of a statistically significant value. All values are expressed as means ± SE; n represents the number of mice.
PMCAs have a high affinity for Ca2+ and alterations of basal [Ca2+]i might account for the differences in contractility previously reported (9). However, our measurements indicated that there were no significant differences in the unstimulated basal [Ca2+]i between genotypes, when expressed as the ΔF340/380 ratio (Fig. 1A), or expressed as calibrated values, which ranged from 157 to 165 nM in bladder smooth muscle from the four mouse types studied (Fig. 1B).
CCh concentration-force and CCh concentration-[Ca2+]i relations.
A simultaneous recording of the Ca2+ signals and tension responses to cumulative addition of carbachol in WT are shown in Fig. 2. Beginning at 0.3 μM CCh, increases in Ca2+ were accompanied by increases in force. We compared the Ca2+ signal and tension responses to carbachol (Figs. 3 and 4) in bladders from WT, Pmca1+/−, Pmca4−/−, and Pmca1+/−Pmca4−/− mice. Figure 3A shows the averaged 340/380 ratio values. Pmca1+/−Pmca4−/− bladders have significantly decreased Ca2+ responses to CCh. In Fig. 3B, the values were normalized to their respective maximal responses; both Pmca4−/− and Pmca1+/−Pmca4−/− were moderately less sensitive than WT bladders to higher concentrations of CCh, but the Pmca1+/−Pmca4−/− preparations were somewhat more sensitive at lower concentrations.
Consistent with the altered Ca2+ signals, the isometric forces in the different genotypes showed similar tendencies (Fig. 4). Bladders from Pmca4−/− and Pmca1+/−Pmca4−/−, but not from Pmca1+/− mice, produced significantly less force than that of WT (Fig. 4A). The tension responses, normalized to their respective maximal developed force, show that the contractile response of bladders from Pmca4−/− and Pmca1+/−Pmca4−/− mice were less sensitive to CCh than WT (Fig. 4B).
KCl depolarization vs. receptor-mediated stimulation.
For KCl depolarization, bladders from Pmca1+/− and Pmca1+/−Pmca4−/− mice have higher responses than those of Pmca4−/−, which were similar to those of WT mice. This pattern was quite different than that for CCh stimulation; Pmca1+/− responses were greater than WT, which, in turn, was greater than Pmca4−/− or Pmca1+/−Pmca4−/−.
Another interesting difference between mouse types can be seen when comparing the magnitudes of the CCh forces to those of the corresponding KCl contracture. CCh-induced forces in bladder smooth muscle from Pmca4−/− and Pmca1+/−Pmca4−/− mice were similar in magnitude to those of their corresponding KCl contractures. On the other hand, CCh-induced forces from Pmca1+/− and WT mice were significantly greater than forces produced in response to KCl depolarization. Figure 5 shows the Ca2+ and force records from experiments comparing responses from bladders from Pmca1+/− (Fig. 5, middle) and Pmca4−/− (Fig. 5, bottom) mice. The most notable feature is that for the Pmca1+/− bladder, the magnitudes of both force and [Ca2+]i for CCh stimulation are much greater than those for KCl (Fig. 5, middle). In contrast, the responses to KCl or CCh are of similar but smaller magnitudes in Pmca4−/− bladders (Fig. 5, bottom). The averaged data for the peak magnitudes from this protocol for Ca2+ and force are presented in Fig. 6, A and B. The overall pattern amongst the genotypes is similar for Ca2+ and force.
The differences between genotypes for KCl and CCh stimulation are emphasized when presented as the CCh response divided by the KCl response as shown for [Ca2+]i in Fig. 7A and force in Fig. 7B. The ratios for Pmca4−/− and Pmca1+/−Pmca4−/−are considerably lower than those for Pmca1+/− and WT.
If the observed responses were simply due to higher [Ca2+]i, as a consequence of a generalized decrease in PMCA Ca2+ extrusion, then one might anticipate that the highest values in response to KCl should occur in the Pmca1+/−Pmca4−/− bladders. The Pmca1+/−Pmca4−/− values are larger than those of WT bladders, but similar to those of Pmca1+/− bladders. On the other hand, Pmca1+/−Pmca4−/− values for CCh stimulation are the lowest, while those for Pmca1+/− bladders are the highest.
Ca2+ and force kinetics.
To further investigate the differences in function in bladders from different genotypes, we measured the kinetics in terms of the half-times for the rise and washout of the responses to KCl or CCh (Fig. 8). Fig. 9 shows the summarized data for the Ca2+ and force increases. Bladders from Pmca1+/− mice have the shortest rise half-times, though only marginally different from the WT. Half-times for Pmca4−/− and Pmca1+/−Pmca4−/− bladders were two to three times longer. This pattern also held true for CCh stimulation. Again, if simple loss of Ca2+ clearance played the major role, Pmca1+/−Pmca4−/− preparations should exhibit the most rapid contraction rather than the slowest.
Figure 10 shows similar data for the half-times of Ca2+ decay and relaxation after the washout in Ca2+-free PSS. There were only small differences between the genotypes for relaxation following KCl stimulation. However, for relaxation following CCh stimulation, a similar pairing to those observed for contraction magnitudes was found. That is, the half-time for Pmca1+/− was similar to the half-time for WT bladders, but both were considerably shorter than half-times for Pmca4−/− and Pmca1+/−Pmca4−/− bladders.
Comparisons of the contractile responses to KCl and CCh in bladder smooth muscle Pmca1+/−, Pmca4+/−, and Pmca4−/− mice.
In mouse bladder, we reported that the mRNA ratio for PMCA1 and PMCA4 was 4:5 (8). It is possible that the differences observed are related to the comparison of the heterozygous Pmca1+/− to the null Pmca4−/− bladders, which likely has a greater reduction in PMCA. In this context, the fact that Pmca1+/−Pmca4−/− bladder was similar to Pmca4−/− and distinctly different from Pmca1+/− argues against simple reduction of total PMCA as an underlying mechanism. Figure 11 shows peak forces for Pmca1+/− and Pmca4+/− bladders and WT and Pmca4−/− for comparison. Forces for Pmca1+/− for both KCl and CCh are significantly greater than those for Pmca4+/− bladders, which were similar to those for the WT bladder. Thus the effects in bladders in Pmca1+/− and Pmca4+/− mice are unlikely to be due to a simple reduction of total PMCA.
Measurements of bladder contractility and [Ca2+]i demonstrated different phenotypes depending on which PMCA isoforms were affected. Bladders from Pmca1+/− mice exhibited higher [Ca2+]i and force responses to both KCl and CCh stimulation than WT bladders. Pmca4−/− bladders, like WT bladders, responded similarly to KCl, but the responses to CCh were significantly suppressed. There are many potential explanations: for example, changes in the geometry of the smooth muscle layers. Gene manipulation did not cause significant cell death or damage (L. Liu, M. L. Miller, and R. J. Paul, unpublished observations). We have functional data that indicate that changes in the morphology of the muscle layers could not explain the results. A key parameter, the ratio of CCh to KCl force (Fig. 7), clearly distinguished between WT, Pmca1+/−, and Pmca4−/−. This parameter was identical in longitudinal and circular layers in studies on paired bladder preparations (n = 4; data not shown). Thus even if relative size or geometry of the layers changed, we wouldn't anticipate any differences in this ratio. We also used Western blot techniques to show that SERCA and NCX expression levels did not change after PMCA knockout. We also reported that the difference between WT and Pmca4−/− bladders remained after blocking SERCA and NCX by using cyclopiazonic acid and KB-R7943 plus Na+-free solution, respectively. Thus the differences are mainly attributable to the loss of PMCA (8).
Based on our previous studies (8), which showed that mRNA levels for each isoform were similar, it is unlikely that our observations relate simply to the loss of Ca2+ clearance due to the anticipated reduction in total PMCA. We would expect a greater reduction of PMCA in Pmca4−/− (∼50%) than Pmca1+/− bladders, leading to higher [Ca2+]i and force in the Pmca4−/− bladder. Force increases were greater in Pmca1+/− than Pmca4+/− bladders, which likely should have similar reductions in total PMCA. Moreover, Pmca1+/−Pmca4−/− bladders would have a predicted 75% reduction in total PMCA. Nevertheless, its responses to KCl were elevated, thus behaving like Pmca1+/− bladders, but its responses to CCh were suppressed similar to Pmca4−/−. Clearly a mechanism involving an increased [Ca2+]i simply proportional to the extent of the reduction of total PMCA Ca2+ clearance cannot accommodate our results.
Our results obtained here suggest that PMCA function in urinary bladder smooth muscle is dependent on the isoform involved. They are consistent with PMCA1 being a major housekeeping pump (1, 5, 6) and its reduction leading to higher [Ca2+]i and force for both KCl and CCh stimulation. PMCA4 on the other hand is associated with the suppressed responses to receptor-mediated, CCh stimulation. We would argue that the Pmca1+/−Pmca4−/− bladders might have greater responses to KCl due to their significantly reduced PMCA levels. On the other hand, we might predict depressed responses to CCh, due to their loss of PMCA4. However, the response to CCh was depressed, indicating that the loss of PMCA4 was dominant.
The responses of Pmca4−/− and Pmca1+/−Pmca4−/− bladders were similar (Figs. 6 and 7). One might have anticipated that the reduction in PMCA1 would lead to somewhat higher [Ca2+]i and contractile response in Pmca1+/−Pmca4−/− bladders compared with Pmca4−/−. This may suggest that the Ca2+ released by CCh is less accessible for clearance by PMCA1. These results indicate that the bladder smooth muscle cell possesses distinct isoforms of PMCA for different functions, PMCA1 for generalized Ca2+ extrusion and PMCA4 for modulation of acetylcholine receptor signaling.
An isoform-dependent cellular function for PMCA is also suggested in other kinds of cells and tissues. Ca2+ extrusion through PMCA1 is suggested by the reported embryonic death of the null mutation of Pmca1 (11), presumably due to the high level of cellular Ca2+. Since PMCA1 is expressed in most cells and tissues, PMCA1 generally serves as the main Ca2+ extrusion pump (11, 17). In contrast, mutation of other PMCAs was not lethal. In sperm cells, the deletion of Pmca4 is reported to reduce motility, resulting in infertility (11). This reduced motility could be explained by a defect in cellular signaling induced by Pmca4 mutation. PMCA2 is expressed specifically in neuronal cells (20), and the mutation of Pmca2 leads to deafness and balance abnormalities (7), suggesting a signal-regulated role for PMCA in neuronal cells. The attenuation of CCh-induced responses occurred selectively in the bladder smooth muscle from Pmca4-mutated mice, suggesting that PMCA4 also acts to modulate receptor signaling. Therefore, PMCA1 may serve as the main Ca2+ extrusion pump and other isoforms of PMCA may function to modulate cellular signal transduction in various cells and tissues.
These conjectures raise the question of what is the mechanism, whereby PMCA4 deficiency influences the responses to CCh stimulation. It would appear reasonable that reduction of PMCA4 would lead to increases in [Ca2+]i in some cellular region. PMCA4 has been reported to be localized in caveolae (2, 3). So the question is, how can a local increase in Ca2+, presumably in caveolae, affect CCh contractures?
The suppressed Ca2+ and force responses in Pmca4−/− and Pmca1+/−Pmca4−/− bladders were accompanied by a moderate decrease in sensitivity of the [CCh]-response relations (Figs. 3 and 4). However, the predominant effect was a reduction in the magnitude of the responses to CCh. It is possible that the acetylcholine receptor number or function is altered in these mice, but this would have to be completely different in the Pmca1+/− bladder. The half-times of the responses to CCh and their relaxation upon washout were also significantly prolonged, suggesting that altered Ca2+ dynamics rather than receptor function underlies the changes in the Pmca4−/− and Pmca1+/−Pmca4−/− bladders.
PMCA4b in sensory neurons is reported to modulate the Ca2+ increase in response to external ATP. The associated phosphorylation of PMCA4 by PKC increases Ca2+ efflux, which, in turn, modified Ca2+-activated K+ channel activity (19). High [Ca2+] in the PMCA4-associated compartment would lead to increased activation of Ca2+-activated K+ channels, which, in turn, might lead to suppression of the responses to CCh in Pmca4−/− and Pmca1+/−Pmca4−/− bladders. Loss of PMCA4 could also affect the initial part of signal transduction between receptor activation and [Ca2+]i elevation in the CCh response. Another potential mechanism may involve a decrease in store-operated Ca2+ entry (SOCE). A high Ca2+ concentration localized in a region associated with the internal Ca2+ stores (sarcoplasmic reticulum, SR) could lead to a hyperloaded SR (4). SOCE may be suppressed in this case, as the SR never is sufficiently unloaded in response to CCh to trigger capacitative Ca2+ entry.
At this point, the mechanism(s) for the different functions will require further experimentation to resolve. Importantly, the data clearly indicate that the PMCA1 and PMCA4 isoforms subserve very different functions in Ca2+ homeostasis and consequently, contractility of the bladder.
This work was supported by National Institutes of Health Grant HL-66044 (to R. J. Paul) and HL-61974 (to G. E. Shull and R. J. Paul).
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- Copyright © 2007 the American Physiological Society