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. 2001 Sep 15;535(Pt 3):867–878. doi: 10.1111/j.1469-7793.2001.00867.x

Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models

Koji Nobe *, Roy L Sutliff *, Evangelia G Kranias *,†, Richard J Paul *,†
PMCID: PMC2278809  PMID: 11559781

Abstract

  1. Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). Its presence and/or functional significance in contractility of bladder, a smooth muscle tissue particularly dependent on SR function, is unknown. We investigated this by measuring the effects of carbachol (CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was ablated (PLB-KO mice). In the PLB-KO bladder, the maximum increases in [Ca2+]i and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50 values increased.

  2. Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB-KO bladder, localizing the effects to the SR.

  3. To determine whether these effects were specific to PLB, we generated mice with smooth-muscle-specific expression of PLB (PLB-SMOE mice), using the SMP8 α-actin promoter. Western blot analysis of PLB-SMOE mice showed approximately an eightfold overexpression of PLB while SERCA was downregulated 12-fold.

  4. In PLB-SMOE bladders, in contrast, the response of [Ca2+]i and force to CCh was significantly increased and the EC50 values were decreased. CPA had little affect on the CCh-induced increases in [Ca2+]i and force in PLB-SMOE bladder.

  5. These results show that alteration of the PLB:SERCA ratio can significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that PLB can play a major role in modulation of bladder contractility.

Contraction of bladder smooth muscle is reported to be dependent on mobilization of Ca2+ from intracellular stores, the sarcoplasmic reticulum (SR) (Levin et al. 1993; Zderic et al. 1993; Yoshikawa et al. 1996; Chacko et al. 1997; Damaser et al. 1997; Zderic et al. 1999). Receptor-mediated contractions are reported to be particularly sensitive to agents, such as cyclopiazonic acid or thapsigargin, which disrupt SR function by inhibiting its Ca2+ ATPase (SERCA) (Munro & Wendt, 1994; Rohrmann et al. 1996). The SR may also play a role in buffering of Ca2+ influx (Yoshikawa et al. 1996) and in the phasic component of bladder contraction via Ca2+-induced Ca2+ release (Ganitkevich & Isenberg, 1992). SR function is known to change during development (Zderic et al. 1995), and in hypertrophy (Levin et al. 1997) and other disease models (Zderic et al. 1996).

Phospholamban (PLB) is a 30 kDa pentameric protein associated with SERCA (Kadambi & Kranias, 1997). It has been studied extensively in the heart, in which it functions as an inhibitor of SERCA (Kim et al. 1990) via a mechanism involving decreasing its affinity for Ca2+ (Kranias, 1985). Importantly, phosphorylation of PLB relieves this inhibition and has long been known to be involved in β-adrenergic modulation of cardiac contractility (Koss & Kranias, 1996). A PLB gene-targeted ‘knockout’ (PLB-KO) mouse was used to show that PLB was, in fact, the major player in the β-adrenergic augmentation of cardiac contractility (Luo et al. 1994). Using this PLB-KO mouse, PLB was also shown to be an important factor in regulating contractility in aorta, not only by modulating smooth muscle contractility (Lalli et al. 1997, 1999), but also by altering endothelial cell function (Sutliff et al. 1999a). To our knowledge, it is not known whether PLB is present in bladder and, if so, whether it can modulate bladder contractility.

In this study, we first investigated the role of PLB in bladder using the PLB-KO mouse, showing that the absence of PLB considerably altered intracellular Ca2+ ([Ca2+]i) homeostasis and contractility. To further demonstrate that these effects were specific to PLB and to quantify the effects of varying the PLB:SERCA ratio, we developed a new mouse model in which PLB is expressed using the smooth-muscle-specific α-actin promoter (Sutliff et al. 1999b). We showed that PLB is present in bladder from wild-type mice, and that its overexpression causes downregulation of SERCA. Using simultaneous measurements of force and [Ca2+]i, we delimit the range of function that can be associated with PLB:SERCA ratios.

METHODS

Generation of gene-altered mouse models

The development of the PLB-KO mice has been previously detailed (Luo et al. 1994); the background strain is SVJ129 × CF1. PLB-SMOE transgenic mice were generated using a background strain of FVB/n with the SMP8 α-actin promoter, described in detail elsewhere (Sutliff et al. 1999b). Two different lines with high levels of PLB expression were isolated and studied. The mechanical properties were similar, suggesting the results were not attributable to an insertional mutation. Data presented here are from one line. Genotypes of all animals were determined by PCR analysis of tail DNA biopsies.

Bladder smooth muscle preparation

Ten- to 16-week-old mice were killed in a precharged CO2 chamber. The urinary bladder was dissected and rinsed in physiological saline solution (PSS), and fat and connective tissue were removed from both sides. The bladder was folded into a loop with the smooth muscle on the outside and the free ends joined together with a surgical wound clip and incubated in PSS at 37 °C for 30 min.

simultaneous measurement of [ca2+]i and isometric force

The bladder loops were incubated in test tubes filled with a fura loading solution containing 0.3 ml of Mops-PSS containing 13.3 μm fura-PE3 AM dissolved in DMSO (final concentration was 0.67 %). The non-cytotoxic detergent pluronic F127 (final concentration 0.1 %) was added to increase the solubility of fura-PE3 AM.

Tissues were incubated at room temperature for 5-6 h, then rinsed in 37 °C PSS for 15 min to remove free dye. The bladder loops were mounted via the wound clip to a fixed stainless steel wire and to another wire placed through the loop which was connected to a Harvard Apparatus force transducer. Resting tension was set at 5 mN, where the tissue length was established to be in the optimal range for force generation. Isometric force values were represented as millinewtons per square millimetre, and cross-sectional areas were estimated by dividing the blotted tissue weight by the length.

The force measurement apparatus was attached to a Teflon mount with an inflow and outflow port and fitted into an acrylic cuvette; the final chamber volume was 2.5 ml. The cuvette was connected to a Cole-Palmer circulating pump via polyethylene tubing in which PSS at 37 °C was perfused (21 ml min−1). The cuvette was placed in a 37 °C water-jacketed holder of a PTI Delta Scan-1 (Photon Technology International, South Brunswick, NJ, USA) dual wavelength spectrofluorimeter, configured for front face measurements. The cuvette was aligned such that the sample was placed perpendicular to the path of the excitation light beam. Fluorescence was excited at 340 and 380 nm, and emission was measured at 510 nm. As previously described, the fluorescence ratio was formed by dividing the emission intensity generated at the 340 nm excitation wavelength by that at 380 nm (R340/380). The R340/380 was calculated and calibrated to absolute values of [Ca2+]i (nm) as reported by Grynkiewicz et al. (1985). 10 μm ionomycin and Ca2+-EGTA solutions were used to establish Rmin and Rmax, and Mn2+ was used to quench the fura-PE3 fluorescence to measure the background fluorescence of the bladder; the Kd was taken as 224 nm.

Western blot analysis

Bladders were excised, and powdered at liquid N2 temperatures using a dental amalgamator. The powdered samples were resuspended in a homogenization buffer containing 25 mm imidazole, 300 mm sucrose, 1 mm dithiothreitol, 20 mm sodium metabisulfite and 0.1 mm phenylmethylsulfonyl fluoride, and protein concentrations were determined (Bradford protein assay, Bio-Rad, Goleta, CA, USA). Protein samples were solubilized in SDS sample buffer, and protein concentrations in the linear range for antibody detection were loaded on a 10-20 % gradient SDS-polyacrylamide gel. Samples were transferred electrophoretically to a polyvinyldene difluoride membrane. After the membrane was blocked with 5 % dry milk, the membrane was incubated for 1 h at room temperature with a monoclonal antibody to PLB (1:1000 dilution). The antibody- antigen complex was detected after incubation of the blot with horseradish peroxidase-conjugated secondary antibody and was visualized using enhanced chemiluminescence Western blotting reagents (Amersham, Arlington Heights, IL, USA). Blots were quantified using NIH Image software.

Materials

Fura-PE3 AM was purchased from TEF Lab, Inc. (Austin, TX, USA); carbachol (CCh), U73122, nicardipine and cyclopiazonic acid (CPA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals and materials were of reagent grade.

Our physiological salt solution (PSS) contained (mm): 122 NaCl, 4.73 KCl, 15.0 NaHCO3, 1.19 MgCl2, 0.02 EDTA, 1.19 KH2PO4, 2.5 CaCl2 and 11.1glucose, aerated with 95 % O2-5 % CO2 to give a pH of 7.4 at 37 °C. Mops-buffered PSS contained (mm): 140 NaCl, 4.70 KCl, 1.20 NaH2PO4, 20.0 Mops, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2 and 11.1 glucose, adjusted with NaOH to give a pH of 7.4 at 37 °C.

Data analysis

All values are expressed as means ±s.e.m., and n represents the number of mice. Significance was determined using standard ANOVA, followed by Student’t test with Bonferroni's correction for multiple comparisons.

RESULTS

characterization of cch-induced [ca2+]i and isometric force responses in wild-type mouse bladder

Resting levels of [Ca2+]i and isometric force averaged 181.9 ± 27.4 nm and 1.4 ± 0.2 mN mm−2 (n = 10), respectively. CCh (10 μm) induced significant increases in both [Ca2+]i and isometric force (Fig. 1); maximal responses occurred at 26.0 ± 2.3 s for [Ca2+]i, and 148 ± 13.8 s for force (n = 5).Figure 2 shows summary data for these experiments. [Ca2+]i increased nearly threefold to 774.9 ± 20.1 nm and active isometric force averaged 12.0 ± 0.6 mN mm−2 (n = 10). The responses to CCh were reproducible following a rinse in PSS for 15 min. The muscarinic receptor antagonist atropine (1 μm, 10 min pretreatment) or the phospholipase C inhibitor U73122 (10 μm, 10 min preincubation) abolished the CCh-induced increases in [Ca2+]i and force. In contrast, preincubation with the Ca2+ channel blocker nicardipine (3 μm) for 10 min had only moderate effects on the CCh-induced responses with 81.0 % and 71.4 % of CCh-induced [Ca2+]i and isometric force, respectively, remaining in the presence of nicardipine. These data indicate that the CCh responses are mediated by muscarinic receptor stimulation. The inhibition by U73122 or nicardipine indicates that an IP3-mediated Ca2+ release from intracellular stores plays a much larger role than Ca2+ influx in pharmacomechanical coupling.

Figure 1. Carbachol (CCh) induced changes in fluorescent intensities (A) and [Ca2+]i and force (B) in wild-type mouse bladder.

Figure 1

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Fura-PE3-loaded tissue was stimulated by 10 μm CCh at 37 °C for 3 min. Changes in the fluorescent emission intensities at 510 nm excited by 340 nm (•) and 380 nm (○) were recorded. [Ca2+]i (○) was calculated as described in Methods, and isometric force (•) were indicated. CCh induced an increase in F340 and a decrease in F380, indicating a true increase in [Ca2+]i. This change is correlated with the increase in isometric force.

Figure 2. Baseline characteristics of wild-type mouse bladder: effects of atropine (Atr), U73122 or nicardipine treatment on the CCh-induced [Ca2+]i and force responses.

Figure 2

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Bladders were preincubated in the presence or absence of the muscarinic receptor antagonist (1 μm Atr), a phospholipase C inhibitor (10 μm U73122) or a calcium channel blocker (3 μm nicardipine) for 10 min, and then 10 μm CCh was added. [Ca2+]i (Inline graphic) and isometric force (Inline graphic) were measured simultaneously as described in Methods. Each value represents the mean ±s.e.m. of at least three independent determinations. * and # values significantly different from resting and CCh responses, respectively, at P < 0.05.

PLB-KO mouse bladder

One expectation, based on previous studies on the aorta (Lalli et al. 1997) is that, in the absence of PLB, an uninhibited Ca2+ pump might lead to a lower [Ca2+]i for any given level of activation. Basal [Ca2+]i tended to be lower in the PLB-KO bladder, but did not achieve statistical significance. We studied the effects of PLB gene ablation by comparing the [Ca2+]i and force relations generated by cumulative additions of CCh; typical responses of individual WT and PLB-KO bladders are shown in Fig. 3A; the averaged concentration-response relations from these experiments for both bladder types are summarized in Fig. 3B. Maximal increases in WT [Ca2+]i and isometric force, attained with 10 μm CCh, were 809.1 ± 36.4 nm and 11.8 ± 0.5 mN mm−2 (n = 6), respectively. At higher levels of CCh both force and [Ca2+]i declined. Decreases at high concentrations of CCh (30 μm) have been reported (Lundbeck & Sjogren, 1992). The basis for this decline is not know with certainty but is suggested to be attributable to a G-protein-mediated inhibition attributed to high levels of muscarinic receptor activation.

Figure 3. Effects of CCh on the [Ca2+]i and isometric force in bladder from wild-type and PLB-KO mice.

Figure 3

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CCh-induced changes in [Ca2+]i (○) and isometric force (•) were measured simultaneously as described in Methods. A, typical changes seen in a single bladder preparation (left panel, wild-type; right panel, PLB-KO). B, concentration-response relations for CCh-induced [Ca2+]i and isometric force responses in bladder from WT (circles) and PLB-KO (squares) mice. Averaged [Ca2+]i (○) and isometric force (•) responses from experiments as shown in Fig. 3A. C, isometric force plotted as a function of [Ca2+]i. Each value represents the mean ±s.e.m. of at least four independent determinations. *P < 0.05; values significantly different from WT.

In the PLB-KO mouse, the maximal increases in [Ca2+]i and isometric force were considerably depressed: 402.6 nm and 5.8 mN mm−2, or 41.5 % and 47.4 %, respectively, of the WT responses. Significant decreases were also detected at 3.0 and 10.0 μm CCh. The EC50 values for [Ca2+]i and isometric force are presented in Table 1. The EC50 for PLB-KO [Ca2+]i was significantly rightward shifted compared to control. [Ca2+]ivs. isometric force relations are shown in Fig. 3C. They show strong correlations between [Ca2+]i and isometric force for both WT and PLB-KO mice: r = 0.997 and 0.965, respectively.

Table 1.

Carbachol EC50 values (μm) for [Ca2+]i and isometric force in mouse bladder

Bladder type [Ca2+]i Isometric force
WT (SVJ129 × CF1) 2.55 ± 0.26 0 2.25 ± 0.17
WT + CPA 0.43 ± 0.07 0.68 ± 0.08
KO 3.65 ± 0.43* 2.00 ± 0.30
KO +CPA 0.60 ± 0.08 0.76 ± 0.07
WT (FVB/n) 2.11 ± 0.20 2.51 ± 0.15
WT +CPA 0.49 ± 0.04 0.72 ± 0.05
SOME 0.72 ± 0.08* 0.79 ± 0.04*
SMOE +CPA 0.72 ± 0.13 0.94 ± 0.02*
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Values given as means ± S.E.M., n = 4–8. All +CPA values differ (P < 0.05) from corresponding values in the absence of CPA, except for SMOE.

*

Gene-altered bladders (KO, SMOE) differ (P < 0.05) from corresponding control.

effects of cyclopiazonic acid (cpa) on cch-induced increases in [ca2+]i and isometric force in WT and PLB-KO bladder

The depressed responses of the PLB-KO are consistent with an increased SERCA activity due to loss of PLB inhibition. If this were the case, then elimination of SR function should mitigate or abolish these differences. We used this approach with a pharmacological SERCA inhibitor, CPA (10 μm), and the summarized data are presented in Fig. 4. In the WT bladder (Fig. 4A), pretreatment with CPA for 10 min enhanced the increases in [Ca2+]i and isometric force in response to CCh compared to control (absence of CPA), in the first cumulative CCh challenge after CPA treatment. This is reflected in the significant decreases in EC50 values for both [Ca2+]i and isometric force in the presence of CPA (Table 1). The maximal [Ca2+]i and force responses in the initial series following CPA treatment were not different from control. After a washout of the initial CCh stimulation with a CPA-containing PSS for 15 min, a second concentration-response relation was attempted. In the second challenge, the responses were severely blunted, maximum responses of 9.3 % and 10.7 % of the control. This suggests that the SR Ca2+ content is depleted by generation of the first concentration-response series and is consistent with the data shown in Fig. 2 suggesting that intracellular Ca2+ stores are the major source of Ca2+ for contraction.

Figure 4. Effects of cyclopiazonic acid (CPA) on the relations between [CCh] and [Ca2+]i (open symbols) or isometric force (filled symbols) for (A) wild-type and (B) PLB-KO bladder.

Figure 4

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Bladders were preincubated in the presence (first CCh after CPA; diamonds) or absence (control; circles or squares) of 10 μm CPA for 10 min, and then the concentration of CCh was increased cumulatively. CPA and CCh treated tissues were rinsed with 10 μm CPA-containing PSS for 15 min, and then the cumulative stimulation was repeated (second CCh after CPA; triangles). Each value represents mean ±s.e.m. of at least four independent determinations. * and # values significantly different from control and first CCh responses after CPA treatments, respectively, at P < 0.05.

In the PLB-KO bladder (Fig. 4B), pretreatment with 10 μm CPA markedly increased the [Ca2+]i and isometric force responses to CCh (first CCh stimulation after CPA treatment). Significant increases compared to responses of PLB-KO in the absence of CPA (control) were detected at all concentrations > 0.1 μm CCh. Maximal responses were significantly increased to 748.8 ± 14.9 nm and 12.7 ± 0.7 mN mm−2 (n = 7), and were not different from either WT or WT + CPA. EC50 values for the first post-CPA challenge with CCh for [Ca2+]i and isometric force were again similar to those for WT or WT + CPA (Table 1). As for the WT, CPA treatment severely blunted the second challenge to CCh: maximal responses were 50.7 % and 21.9 % of control responses. The normalization of the [Ca2+]i and force responses to CCh of the PLB-KO to those of the WT bladder after CPA treatment is consistent with the SR being the locus of the differences with respect to the WT.

PLB-SMOE bladder

Using the PLB-SMOE mouse (Sutliff et al. 1999b), we tested whether overexpression of PLB would have the opposite effects to that of its ablation. That is, a significant increase in PLB inhibition of SERCA would be expected to lead to increased [Ca2+]i and force. Two different lines with high levels of PLB expression were isolated and studied. The mechanical properties were similar, suggesting the results were not attributable to an insertion mutation occurring during the incorporation of the added transgene. Resting [Ca2+]i levels in the PLB-SMOE bladder were higher than that in WT (183.7 vs. 73.3 μm; P < 0.05; see Fig. 5B). Typical cumulative CCh responses for individual bladders are shown in Fig. 5A and averaged concentration-response relations from these experiments in Fig. 5B. The PLB-SMOE responses (Fig. 5A, right panel) were greatly increased at lower CCh concentrations (0.3-1.0 μm) but maximal responses were not different from the WT (770.4 ± 21.8 nm and 13.4 ± 0.8 mN mm−2vs. 784.8 ± 54.2 nm and 12.8 ± 0.6 mN mm−2). EC50 values of [Ca2+]i and isometric force PLB-SMOE bladders were significantly reduced by about threefold compared to the WT (Table 1). Isometric force as a function of [Ca2+]i is shown in Fig. 5C. This relation was near linear in the WT with a correlation coefficient of 0.995. In the PLB-SMOE bladder the relation was more curvilinear; the force vs.[Ca2+]i relation was rightward shifted for CCh concentrations between 0.1 and 1.0 μm, but at higher concentrations coincided with the relation for the WT.

Figure 5. Effects of CCh on the [Ca2+]i and isometric force in bladder from wild-type and PLB-SMOE mice.

Figure 5

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CCh-induced changes in [Ca2+]i and isometric force were measured simultaneously as described in Methods. A, typical responses for [Ca2+]i (○) and isometric force (•) of an individual bladder (left panel, wild-type; right panel, PLB-SMOE). B, concentration-response relations for CCh-induced [Ca2+]i and isometric force responses in bladder from WT (circles) and PLB-SMOE (triangles) mice. Averaged values for [Ca2+]i (open symbols) and isometric force (filled symbols) responses from experiments as shown in Fig. 5A. C, isometric force plotted as a function of [Ca2+]i. Each value represents the mean ±s.e.m. of at least four independent determinations. *P < 0.05; values significantly different from WT.

effects of cpa on cch-induced increases in [ca2+]i and isometric force in WT and PLB-SMOE bladder

Using a similar strategy to that for the PLB-KO, we investigated the SR dependence of these differences with CPA inhibition of SERCA (Fig. 6). In the WT mouse, pretreatment with CPA enhanced the sensitivity of [Ca2+]i and isometric force to CCh as indicated by the leftward shifts in the concentration-force relations (Fig. 6A). This enhancement by CPA was similar to that previously shown for the WT on the PLB-KO genetic background (Fig. 4A). Maximal responses in the presence of CPA were measured at 3.0 μm CCh and these values were not different from the untreated control responses. Similar to the previous results (Fig. 4), little response to a second CCh challenge was noted.

Figure 6. Effects of CPA on the relations between [CCh] and [Ca2+]i (open symbols) or isometric force (filled symbols) for (A) wild-type and (B) PLB-SMOE bladder.

Figure 6

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Bladders were preincubated in the presence (first CCh after CPA; diamonds) or absence (control; circles or inverted triangles) of 10 μm CPA for 10 min, then the concentration of CCh was increased cumulatively. Subsequently, tissues were rinsed with 10 μm CPA containing PSS for 15 min, and the cumulative stimulation repeated (second CCh after CPA; triangles). Each value represents mean ± s.e.m. of at least four independent determinations. * and # values significantly different from control and first CCh responses after CPA treatments, respectively at P < 0.05.

In contrast, in the PLB-SMOE bladder (Fig. 6B) pretreatment with 10 μm CPA had little effect on the CCh concentration-[Ca2+]i or -isometric force relations (first CCh stimulation after CPA treatment). The EC50 for [Ca2+]i was unchanged in the presence of CPA, whereas force was slightly but significantly increased (Table 1). These results are consistent with significant inhibition of SERCA in the PLB-SMOE bladder, such that further inhibition with CPA has little further effect.

ca2+ loading and refilling of the sarcoplasmic reticulum

One might also anticipate that not only steady state relations, but also the Ca2+ recycling by the SR would be altered by the genetic manipulation that changes the PLB/SERCA levels. Direct measures in situ of SR Ca2+ loading are not available, but indirect estimates based on the ability to develop force and increase [Ca2+]i in protocols involving multiple contractions have been reported (Devine et al. 1972; Bond et al. 1984; Taggart & Wray, 1998). We employed a similar experimental design measuring the ability to respond to repeated CCh challenges in a series of isometric contractions of 2 min duration at 2.5 min intervals as shown in the upper panels of Fig. 7 and Fig. 8. The maximum levels of force and [Ca2+]i can be related to both the initial [Ca2+]i loading and refilling capacity of the SR. In WT aorta, both the peak [Ca2+]i and force decreased with each stimulation throughout the series, with the fifth contracture being about 50 % of that observed in the initial contraction. In the PLB-KO bladder, force was maintained at the initial levels throughout; peak [Ca2+]i decreased by about 30 %. In contrast, in the PLB-SMOE bladder, [Ca2+]i and force decreased sharply throughout the series and were nearly unresponsive to the fifth challenge with CCh. These results suggest that the refilling of SR Ca2+ stores is essential for maintenance of the peak responses and that this is clearly affected by PLB inhibition of SERCA. Since the peak force and [Ca2+]i were in the order PLB-SMOE > WT > PLB-KO, SR Ca2+, uptake rather than SR Ca2+ content and release appears to be the dominant factor in the determination of the magnitude of the peak responses.

Figure 7. Effects of multiple challenges with CCh on the [Ca2+]i and isometric force in bladder from wild-type and PLB-KO mice.

Figure 7

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Bladders were subjected to five cycles of a 2 min exposure to CCh (10 μm) then rinsed at 2.5 min intervals. A, typical responses for [Ca2+]i (○) and isometric force (•) of an individual bladder (left panel, wild-type; right panel, PLB-KO). Responses were averaged. B, [Ca2+]i (open symbols) and isometric force (filled symbols) as a function of the cycle number in bladder from WT (circles) and PLB-KO (squares) mice. Each value represents the mean ±s.e.m. of at least four independent determinations. *P < 0.05; values significantly different from WT.

Figure 8. Effects of multiple challenges with CCh on the [Ca2+]i and isometric force in bladder from wild-type and PLB-SMOE mice.

Figure 8

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Bladders were subjected to five cycles of a 2 min exposure to CCh (10 μm) then rinsed at 2.5 min intervals. A, typical responses for [Ca2+]i (○) and isometric force (•) of an individual bladder (left panel, wild-type; right panel, PLB-SMOE). Responses were averaged. B, [Ca2+]i (open symbols) and isometric force (filled symbols) as a function of the cycle number in bladder from WT (circles) and PLB-SMOE (triangles) mice. Each value represents mean ±s.e.m. of at least four independent determinations. *P < 0.05; values significantly different from WT.

These data also can be analysed with respect to the transient behaviour in terms of the half-times (t1/2) for the rise and fall of [Ca2+]i and force, which are presented in Table 2. The t1/2 for the increase in [Ca2+]i upon addition of CCh was greater (P = 0.02) in the PLB-KO bladder than WT. The t1/2 for relaxation upon washout was not different. In the first contraction of the series, the force in the PLB-KO bladder was only about 50 % of that in the WT bladder. Because this difference might play a role in the time courses, responses were also compared in the fifth contraction in which the forces were nearly the same (see Fig. 3 and Fig. 8). In this case, there were no significant differences. There were no significant differences in the t1/2 values for the rise or fall of [Ca2+]i or force.

Table 2.

Half-times for the increase (t1/2,rise) and decrease (t1/2,relax) of [Ca2+]i and isometric force for the first contraction–relaxation cycle

[Ca2+]i Isometric force
Bladder type n t1/2,rise t1/2,relax t1/2,rise t1/2,relax
WT 5 10.4 ± 0.7 20.0 ± 2.4 17.4 ± 1.8 47.0 ± 5.0
PLB-KO 4 22.0 ± 4.5 24.0 ± 1.8 21.8 ± 2.4 32.8 ± 7.1
FVB/n 6 14.7 ± 2.7 22.7 ± 3.9 30.0 ± 4.4 54.8 ± 6.7
PLB-SMOE 6 21.5 ± 4.8 38.8 ± 6.1* 45.3 ± 9.2* 57.7 ± 11.2
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Asterisks indicate P < 0.05 from appropriate WT or FVB/n.

For the PLB-SMOE bladder, t1/2 values for both the rise and fall of [Ca2+]i were slower than the WT, but only the latter achieved statistical significance (P = 0.05).t1/2 for the rise of force was similarly slower than the WT, but did not achieve statistical significance. There were no differences noted in the relaxation of force upon washout, despite the difference in [Ca2+]i.

Western blot analysis of PLB and SERCA in PLB-KO and SMOE mouse bladder smooth muscle

Critical to any interpretation of these data is knowledge not only of the relative amounts of PLB but also of SERCA in these gene-altered mice. It is possible, for example, that changes in PLB are matched by compensatory changes in SERCA, to preserve the WT Ca2+-ATPase activity. Figure 9 shows Western blots of PLB and SERCA in these models. In the PLB-SMOE bladder, clear evidence of the expression of the PLB transgene can be seen relative to the level of PLB observed in FVB/n wild-type. Surprisingly, SERCA expression in the PLB-SMOE is significantly reduced. This gel was probed for both PLB and SERCA from the same loading to visually emphasize the large increase in the PLB/SERCA ratio in the PLB-SMOE bladder, due to the increase in PLB, as well as the decrease in SERCA. In separate gels, in which the loading could be controlled to insure the PLB and SERCA were both in their respective linear ranges, PLB was increased eightfold and SERCA decreased 12-fold. For the PLB-KO, the SERCA content of the KO was not different from that of the wild-type, within the limits of Western blot analysis. Interestingly, when comparing the wild-type mice, the FVB/n mice showed approximately a fourfold greater content for both PLB and SERCA than the SVJ129 × CF1 strain used for the gene-targeted (KO) mice.

Figure 9. Western blot analysis of PLB and SERCA in bladder smooth muscle from PLB-KO, PLB-SMOE and WT mice.

Figure 9

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A, Western blot using anti-PLB (upper panel) and anti-SERCA (lower panel) antibodies for bladder, and heart as a reference. WT refers to the background for the PLB-KO mice (SVJ129 × CF1) and WT (FVB/n) to that for the PLB-SOME mice. B, relative density vs. protein for PLB (open symbols) and SERCA (filled symbols). Left panel: PLB-KO (squares) and its WT (circles); right panel: PLB-SMOE (triangles) and its WT (circles). Relative density analysis indicates that PLB is eightfold greater in the PLB-SMOE than its FVB/n wild-type. SERCA is significantly downregulated (12-fold) in the PLB-SMOE mouse. In the PLB-KO, SERCA was not different from its wild-type (SVJ129 × CF1). The different wild-type backgrounds were associated with considerably different PLB and SERCA levels (FVB/n > fourfold that of SVJ129 × CF1).

DISCUSSION

To study the role of PLB in bladder smooth muscle contractility, we used both gene-targeted PLB-KO and transgenic PLB-SMOE mice models. Our hypothesis is that PLB inhibition of SERCA can significantly modulate [Ca2+]i and consequently contractility. Data from each model strongly support a major role for PLB in the regulation of bladder contractility. We focused on the receptor-mediated contractions elicited by CCh, which our data indicate are largely dependent on intracellular Ca2+ stores. This inference agrees with similar conclusions of other reports on mouse bladder (Schaufele et al. 1995; Sugita et al. 1998).

The PLB-KO mice form one limit on the possible modulation of function, in which SERCA is not inhibited. [Ca2+]i in the steady state is a balance of Ca2+ release and uptake. With greater Ca2+ uptake, one might anticipate a lower steady state [Ca2+]i for any given level of Ca2+ release. Alternatively, an uninhibited SERCA might lead to greater SR Ca2+ loading, leading to a greater Ca2+ release per any given stimulus level. Since the Ca2+ response to CCh in the PLB-KO bladder was significantly suppressed, it appears in this smooth muscle that SR Ca2+ uptake is the dominant factor in the steady state.

Our results with the PLB-SMOE model also support this hypothesis. These provide the opposite extreme, with PLB increased and SERCA decreased. At the protein level, PLB is substantially expressed, attributable to the robustness of the α-actin promoter. The downregulation of SERCA was unexpected if one anticipates the Ca2+ uptake capacity to be maintained. One the other hand, upregulation of ligand often leads to downregulation of receptor. The basis of this coupling of PLB and SERCA expression is unknown. Our prediction is that with significant overexpression of PLB, SR Ca2+ uptake would be blunted leading to higher [Ca2+]i per stimulus relative to the WT. This is indeed what was observed (Fig. 5), the opposite of that observed for the PLB-KO.

For both models, the changes in isometric force, measured simultaneously in fura-PE3 AM-loaded bladder, were parallel to those of [Ca2+]i. This suggests that the decreased contractility was related to the altered Ca2+ signalling, as Ca2+-calmodulin activation of myosin regulatory light chain kinase is a major route of activation of smooth muscle (de Lanerolle & Paul, 1991).

To validate our hypothesis, it is necessary to demonstrate that the site of the altered Ca2+ homeostasis was the SR. Using CPA to inhibit SERCA, the differences in both [Ca2+]i and contractility between WT and PLB-KO in response to the initial challenges with CCh were abolished. As may be expected from the loss of a major [Ca2+]i clearing mechanism upon inhibition of SERCA, both WT and PLB-KO bladder were more sensitive to CCh, but their responses were identical. It was particularly striking that the 50 % suppression of both maximum force and [Ca2+]i seen in the PLB-KO relative to the WT bladder was relieved with CPA. The near identical concentration-response relations for the WT and PLB-KO bladder after CPA inhibition of SERCA further suggests that no compensatory responses at either the mechanical or Ca2+ homeostatic levels (excluding the SR) occurred.

On the other hand, in the PLB-SMOE bladder, inhibition of SERCA with CPA had little effect while showing the expected sensitization in the WT (Fig. 6; Table 1). Our interpretation is that the high levels of PLB relative to SERCA lead to an inhibition of SR Ca2+ uptake and further inhibition with CPA does not initially lead to additional effects. In the continued presence of CPA, a second CCh concentration-response series could not be generated. This is consistent with a Ca2+-depleted SR and the data in Fig. 2, suggesting that SR Ca2+ release is the primary source of Ca2+ for contraction. Since this was observed for the PLB-SMOE bladder as well, the SR is still its main player in activation of contraction, despite the downregulation of SERCA. It is worth noting that the inhibition by CPA differs significantly from PLB, in that PLB shifts the affinity of SERCA for Ca2+, rather than decreasing Vmax (Brittsan et al. 2000). Thus at high [Ca2+]i levels SERCA activity is not affected by PLB, consistent with the fact that differences were seen in the PLB-SMOE bladder only in the lower stimulus and hence [Ca2+]i ranges. After CPA treatment, WT, PLB-KO and PLB-SMOE bladder all showed similar [Ca2+]i and force responses to CCh. This is consistent with the hypothesis that Ca2+ release is similar in all models and the differences are related to altered SERCA Ca2+ uptake.

The essential role of SR Ca2+ for contraction can also be seen in the ability of the smooth muscle to maintain force when challenged to multiple contraction-relaxation cycles. The ability to generate force and increase [Ca2+]i continuously decreased and was nearly abolish by the fifth cycle in the PLB-SMOE bladder, whereas the PLB-KO bladder was able to maintain the responses. We attribute the former to an increased PLB inhibition of SR Ca2+ loading and the opposite for the latter.

Interestingly, despite the combination of increased PLB and decreased SERCA, the PLB-SMOE bladders are able to relax and maintain [Ca2+]i at levels below the threshold for activation of contraction. Thus other Ca2+ removal systems, such as the plasma membrane Ca2+ pumps and Na+-Ca2+ exchanger, must play significant roles. If other Ca2+ extrusion systems were significantly upregulated in the PLB-SMOE bladder relative to the WT, one might anticipate that after CPA treatment, the CCh concentration-response relations would differ. The differences post-CPA were moderate (Fig. 6), suggesting that these systems are not significantly upregulated in the PLB-SMOE bladder.

Interpretation of the transient data for the increases and relaxations of [Ca2+]i and force is less straightforward than those of the steady state. While Ca2+ release and Ca2+ uptake are the end determinants of [Ca2+]i, there are many other potential rate limiting steps. At first glance for the de-inhibited SERCA in the PLB-KO, one might anticipate, based on cardiac muscle (Luo et al. 1994), a more rapid Ca2+ release upon stimulation due to increased Ca2+ loading of the SR, and a more rapid Ca2+ removal upon washout. Neither was observed, with the rise in [Ca2+]i actually being slower in the PLB-KO bladder, though not statistically significant (Table 1). This would suggest that Ca2+ uptake and/or extrusion, not release, is dominant. On the other hand, the decrease in [Ca2+]i upon washout was not different and, if anything, slower in the PLB-KO bladder. This suggests that other rate limiting steps may be involved. If, for example, receptor inactivation were a rate-limiting step, then a difference in SR Ca2+ pump activity between the PLB-KO and WT bladder would not necessarily lead to a more rapid decline in [Ca2+]i.

The results for the PLB-SMOE bladder are consistent with our hypothesis of PLB inhibition of SERCA. Both the rise and fall times (t1/2 values) of [Ca2+]i were slower, the latter significantly so (Table 2). While the slower rise time did not achieve statistical significance, this was mirrored by the slower increase in force, which was significant. In contrast to an increase in SR Ca2+ pump activity in the uninhibited PLB-KO bladder, a decrease in activity with overexpression of PLB might supplant a faster rate-limiting step. The t1/2 for the decrease in force with washout of CCh was not different despite the difference in [Ca2+]i, again suggesting that other processes, such as dephosphorylation of the myosin regulatory light chain, are likely to be rate limiting for mechanical relaxation.

In summary, our findings indicate that PLB via its inhibition of SERCA can be a significant modulator of [Ca2+]i and thus contractility in mouse bladder smooth muscle. Though the physiological role of PLB remains to be clarified, our results indicate that therapeutic targeting of phospholamban can be a new approach to ameliorating bladder dysfunction.

Acknowledgments

This work was supported by NIH HL09781 (R.L.S.) HL26057 (E.G.K.), HL25318 (E.G.K.), P40RR12358 (E.G.K.), HL64018 (E.G.K.) and HL54829 (R.J.P.).

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