Am J Physiol Cell Physiol Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 292: C423-C431, 2007. First published September 6, 2006; doi:10.1152/ajpcell.00313.2006
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/C423    most recent
00313.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, L.
Right arrow Articles by Paul, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, L.
Right arrow Articles by Paul, R. J.

MUSCLE CELL BIOLOGY AND CELL MOTILITY

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

Li Liu,1 Yukisato Ishida,1 Gbolahan Okunade,2 Gail J. Pyne-Geithman,1 Gary E. Shull,2 and Richard J. Paul1

1Department of Molecular and Cellular Physiology and 2Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 5 June 2006 ; accepted in final form 30 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

PMCA; 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. 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.

Tissue preparation. 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.

Experimental protocols. 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.

Data analysis. 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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal [Ca2+]i. 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 {Delta}F340/380 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).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Bar graphs showing basal [Ca2+]i in bladder smooth muscle preparations from wild-type (WT) mice and the plasma membrane Ca2+ ATPase (PMCA) gene-altered mice, Pmca1+/–, Pmca4–/–, and Pmca1+/–Pmca4–/–. A: ratio of {Delta}F340/380. B: calibrated Ca2+ concentration (nM). Data are means ± SE. There were no statistically significant differences.

 
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.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Typical experimental record showing simultaneous recording of the time course of the Ca2+ signal and tension responses to carbachol (CCh) in WT. Typical experimental record of the simultaneous measurement of Ca2+ signal and tension in response to CCh ({blacktriangleup}). Upper curve: the 340/380 ratio of fluorescence intensities (right axis); Lower curve: isometric force in mN (left axis).

 

Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. Relations between [Ca2+]i and CCh concentration in bladder smooth muscle from PMCA gene-altered mice ({circ} wild-type, {square} Pmca1+/–, {blacktriangleup} Pmca4–/–, bullet Pmca1+/–Pmca4–/–). A: values were expressed as the change in the 340/380 ratio of the fluorescence intensities and presented as means ± SE. B: values were normalized to the maximal response and presented as means ± SE.

 

Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. Relations between force and carbachol concentration in bladder smooth muscle from PMCA gene-altered mice ({circ} wild-type, {square} Pmca1+/–, {blacktriangleup} Pmca4–/–, bullet Pmca1+/–Pmca4–/–). A: developed force normalized to cross-sectional area given as means ± SE. B: the tension responses normalized to the maximal developed force and presented as means ± SE.

 
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.


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 5. Typical experimental records of the time course of the Ca2+ signal (upper curves, right axis) and contraction (lower curves, left axis) for responses to KCl and CCh for bladders from WT (top), Pmca1+/– (middle), and Pmca4–/– (bottom). Event marks ({blacktriangleup}) show where bladder smooth muscle preparations were first exposed to 80 mM KCl for 5 min, which elicited increases of both [Ca2+]i and tension; after washout of KCl with Ca2+-free physiological salt solution (PSS) containing 0.1 mM EGTA, which leads to force relaxation and Ca2+ decay; readmission of Ca2+, returning cytosolic [Ca2+]i to the basal level. The muscles were then treated with 10 µM CCh, which elicited increases of both [Ca2+]i and tension. Finally, CCh was washed out with Ca2+-free PSS containing 0.1 mM EGTA.

 

Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 6. KCl- and CCh-induced Ca2+ increases (A) and developed force (B) in bladder smooth muscle preparations from WT, Pmca1+/–, Pmca4–/–, and Pmca1+/–Pmca4–/– mice. Bars represent means (also given as numbers), and error bars represent ± SE. *Significant difference (P < 0.05) from WT. {ddagger}Significant difference (P < 0.05) from group indicated.

 
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.


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 7. CCh-induced Ca2+ increase (A) and tension (B) normalized to the respective KCl-induced responses in bladder smooth muscle preparations from WT, Pmca1+/–, Pmca4–/–, and Pmca1+/–Pmca4–/– mice. Bars represent means (also given as numbers), and error bars represent ± SE. *Significant difference (P < 0.05) from WT. {ddagger}Significant difference (P < 0.05) from group indicated.

 
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 8
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 8. Experimental records demonstrating the protocol for the measurement of the half-times (tau) of Ca2+ increase and decay (A) and contraction-relaxation (B) in response to KCl and CCh, as well as washout of KCl and CCh. For further details, see experimental protocol section in MATERIALS AND METHODS.

 

Figure 9
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 9. Half-times of Ca2+ increases (A) and developed forces (B) in response to KCl and CCh in bladder smooth muscle preparations from WT, Pmca1+/–, Pmca4–/–, and Pmca1+/–Pmca4–/– mice. Bars represent means (also given as numbers), and error bars represent ± SE. *Significant difference (P < 0.05) from WT. {ddagger}Significant difference (P < 0.05) from group indicated.

 
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.


Figure 10
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 10. Half-times for Ca2+ decay (A) and for relaxation of force (B) after washout of KCl and CCh with Ca2+-free PSS containing 0.1 mM EGTA in bladder smooth muscle preparations from WT, Pmca1+/–, Pmca4+/–, and Pmca4–/– mice. Bars represent means (also given as numbers), and error bars represent ± SE. *Significant difference (P < 0.05) from WT. {ddagger}Significant difference (P < 0.05) from group indicated.

 
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.


Figure 11
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 11. KCl- and CCh-induced contractions in bladder smooth muscle preparations from WT, Pmca1+/–, Pmca4+/–, and Pmca4–/– mice. A: KCl- and CCh-induced peak tensions; values were normalized to cross-sectional area. B: ratio of CCh-induced peak tension divided by KCl-induced peak tension. Bars represent means (also given as numbers), and error bars represent means ± SE. *Significant difference (P < 0.05) from WT. {ddagger}Significant difference (P < 0.05) from group indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Paul, Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (e-mail: paulrj{at}ucmail.uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Brandt R, Oddoy A, Bohnke J, Schilling W. A model of the isolated blood perfused lung of rats in testing hypoxic pulmonary vasoconstriction. Z Erkr Atmungsorgane 173: 145–150, 1989.[Medline]

2. Darby PJ, Kwan CY, Daniel EE. Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+ handling. Am J Physiol Lung Cell Mol Physiol 279: L1226–L1235, 2000.[Abstract/Free Full Text]

3. Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol 120: 1147–1157, 1993.[Abstract/Free Full Text]

4. Gomez-Viquez L, Guerrero-Serna G, Garcia U, Guerrero-Hernandez A. SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells. Biophys J 85: 370–380, 2003.

5. Greeb J, Shull GE. Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca2+-transporting ATPase that is expressed predominantly in brain and skeletal muscle. J Biol Chem 264: 18569–18576, 1989.[Abstract/Free Full Text]

6. Hilfiker H, Strehler-Page MA, Stauffer TP, Carafoli E, Strehler EE. Structure of the gene encoding the human plasma membrane calcium pump isoform 1. J Biol Chem 268: 19717–19725, 1993.[Abstract/Free Full Text]

7. Kozel PJ, Friedman RA, Erway LC, Yamoah EN, Liu LH, Riddle T, Duffy JJ, Doetschman T, Miller ML, Cardell EL, Shull GE. Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2. J Biol Chem 273: 18693–18696, 1998.[Abstract/Free Full Text]

8. Liu L, Ishida Y, Okunade G, Shull GE, Paul RJ. Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice. Am J Physiol Cell Physiol 290: C1239–C1247, 2006.[Abstract/Free Full Text]

9. Liu L, Okunade G, Ishida Y, Wachterman J, Shull GE, Paul RJ. PMCA isoforms and bladder contractility: evidence from PMCA gene- targeted mice. FASEB J 18: A1083–A1083, 2004.

10. Nobe K, Sutliff RL, Kranias EG, Paul RJ. Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models. J Physiol 535: 867–878, 2001.[Abstract/Free Full Text]

11. Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O'Connor KT, Neumann JC, Andringa A, Miller DA, Prasad V, Doetschman T, Paul RJ, Shull GE. Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 279: 33742–33750, 2004.[Abstract/Free Full Text]

12. Prasad V, Okunade GW, Miller ML, Shull GE. Phenotypes of SERCA and PMCA knockout mice. Biochem Biophys Res Commun 322: 1192–1203, 2004.[CrossRef][ISI][Medline]

13. Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Liebermann J, Williams JC, Armesilla AL, Emerson M, Oceandy D, Knobeloch KP, Neyses L. Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility. J Biol Chem 279: 28220–28226, 2004.[Abstract/Free Full Text]

14. Schuh K, Uldrijan S, Gambaryan S, Roethlein N, Neyses L. Interaction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK. J Biol Chem 278: 9778–9783, 2003.[Abstract/Free Full Text]

15. Schuh K, Uldrijan S, Telkamp M, Rothlein N, Neyses L. The plasma membrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I. J Cell Biol 155: 201–205, 2001.[Abstract/Free Full Text]

16. Shull G, Greeb J. Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases. J Biol Chem 263: 8646–8657, 1988.[Abstract/Free Full Text]

17. Shull GE. Gene knockout studies of Ca2+-transporting ATPases. Eur J Biochem 267: 5284–5290, 2000.[ISI][Medline]

18. Strehler EE, Zacharias DA. Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81: 21–50, 2001.[Abstract/Free Full Text]

19. Usachev YM, DeMarco SJ, Campbell C, Strehler EE, Thayer SA. Bradykinin and ATP accelerate Ca2+ efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca2+ pump isoform 4. Neuron 33: 113–122, 2002.[CrossRef][ISI][Medline]

20. Withers S, Cartwright EJ, Neyses L. Sperm phenotype of mice carrying a gene deletion for the plasma membrane calcium/calmodulin dependent ATPase 4. Mol Cell Endocrinol 250: 93–97, 2006.[CrossRef][ISI][Medline]

21. Zacharias DA, Kappen C. Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse. Biochim Biophys Acta 1428: 397–405, 1999.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/C423    most recent
00313.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, L.
Right arrow Articles by Paul, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, L.
Right arrow Articles by Paul, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.