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This Article Full Text (PDF) All Versions of this Article: 18/10/1159 04-1516fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WEGENER, J. W. Articles by HOFMANN, F. Search for Related Content PubMed PubMed Citation Articles by WEGENER, J. W. Articles by HOFMANN, F.

An essential role of Ca_v1.2 L-type calcium channel for urinary bladder function

Institut für Pharmakologie und Toxikologie, Technische Universität München, München, Germany

¹Correspondence: Institut für Pharmakologie und Toxikologie, Technische Universität München, Biedersteiner Str. 29, D-80802 München, Germany. E-mail: Wegener{at}ipt.med.tu-muenchen.de

SPECIFIC AIMS

To clarify the contribution of L-type Ca²⁺ channels to smooth muscle function, we inactivated the murine smooth muscle L-type Ca_v1.2 Ca²⁺ channel by a conditional and time-controlled knockout approach.

PRINCIPAL FINDINGS

1. Intact L-type Ca²⁺ Ca_v1.2 channel protein is absent in urinary bladder from SMACKO mice
Bladder smooth muscle from control (CTR) mice expressed mRNA for the cardiac type Ca_v1.2a and the smooth muscle type Ca_v1.2b channel (Fig. 1 B). We created a mouse line (SMACKO mice, smooth muscle _1c subunit calcium channel knockout mice) in which both isochannel were inactivated by our gene targeting strategy (Fig. 1A ). In bladder muscle from SMACKO mice, intact mRNA and the protein of the Ca_v1.2 channels were not detected by RT-PCR and Western blot analysis, respectively (Fig. 1C, D ). L-type Ba²⁺ current was missing in bladder myocytes from SMACKO mice (Fig. 1E-H ). The L-type Ca²⁺ channel blocker isradipine (1 µM) reduced the L-type Ba²⁺ current in myocytes from CTR but was without effect in myocytes from SMACKO mice. These results confirm the successful inactivation of the Ca_v1.2 gene in bladder muscle from SMACKO mice.

View larger version (30K): [in this window] [in a new window]   Figure 1. Phenotype of murine SMACKO bladder. A) Diagram of the modified Cav1.2 (L2) allele (I) and inactivated Cav1.2 (L1) allele (II) obtained after Cre-mediated recombination of the L2 allele. Filled boxes denote exons 13 to 17 of the Cav1.2 gene. Filled triangles indicate loxP sequences. Excision of exons 14 and 15 introduced a frame shift resulting in a premature stop codon. VI8, VI4, and VI10 indicate location of the respective primers used for genotype analysis. B, E, C, and A = restriction sites for the restriction enzymes BamHI, EcoRI, ClaI, and Acc65I. B) RT-PCR analysis of mRNA isolated from the urinary bladder (UB) of CTR mice for the cardiac (Cav1.2a) and smooth muscle (Cav1.2b) calcium channel expression. C) RT-PCR analysis of mRNA isolated from the urinary bladder (UB) of CTR (+/L2T) and SMACKO (L1/L2T) mice and from the heart (H) of SMACKO (L1/L2T) mice, all treated with tamoxifen (T). Note that the wild-type and L2 allele generate the same transcripts and therefore cannot be distinguished by RT-PCR analysis. +/L2 and L1 represent wild-type/L2 and L1 transcripts, respectively. The L2 signal vanished in bladder but not in heart from SMACKO (L1/L2T) mice confirming the tissue specificity of Cre recombination. The L1 signal appeared in bladder from CTR (+/L2T) mice. The second band close to the L1 transcript has been sequenced. It contains the authentic Cav1.2 sequence with a 80 bp deletion. HGPRT was used as internal standard. D) Western blot analysis of Cav1.2 protein expression. Arrows indicate the positions of the Cav1.2 protein and ß-actin, respectively. The samples were prepared from urinary bladder of CTR (+/L2T) and SMACKO (L1/L2T) mice after treatment with tamoxifen. Western blots of the membrane (pellet) and cytosolic fraction are shown. E–H) IBa of detrusor myocytes of CTR (E, G) and SMACKO (F, H) mice. E, F). Original recordings in the absence (control) and presence of 1 µM isradipine (ISR). Currents were activated by depolarization from –80 mV to 0 mV. G, H) Voltage current relation. Data points represent means ± SE (n=4–12). I) Urine spots of freely moving male mice. **P <0.01. J) Wet weight of the bladder, heart, and body from CTR and SMACKO mice. n = 9–15 animals; n.s., nonsignificant; ***P <0.001.

2. The Ca_v1.2 channel controls spontaneous contractile activity
As a functional consequence of Ca_v1.2 gene inactivation, SMACKO mice developed bladder dysfunction characterized by diminished micturition (Fig. 1I ). Isolated bladder muscle (i.e., detrusor) showed spontaneous contractile activity if slightly depolarized by increasing extracellular [K⁺] from 5 to 30 mM. Spontaneous activity was also induced in CTR muscles by 10 µM carbachol (CCh). In contrast, detrusor from SMACKO mice lacked spontaneous activity. These results indicate that Ca_v1.2 channel is crucial for rhythmic muscle activity of the bladder.

3. CCh-induced contraction depends on functional Ca_v1.2 channels
In detrusor muscle strips, depolarization by high extracellular K⁺ (85 mM) or stimulation with CCh (10 µM) induced contraction consisting of a phasic, followed by a tonic, contractile component (Fig. 2 A, B). Both components were significantly smaller in smooth muscles from SMACKO mice than in those from CTR mice (Fig. 2A, B ). The L-type Ca²⁺ channel blockers, isradipine (1 µM; Fig. 2A, B ) and cadmium (300 µM) reduced both components in muscles from CTR but not in those from SMACKO mice. CCh-induced contractions were reduced in muscles from CTR but not in those from SMACKO mice if extracellular Ca²⁺ was scavenged by BAPTA (10 mM) without emptying intracellular stores (Fig. 2B ). These findings suggest that CCh-induced contraction depends on calcium influx through the Ca_v1.2 channel.

View larger version (31K): [in this window] [in a new window]   Figure 2. Depolarization- and carbachol-induced contraction. Original recordings of tension of CTR (a) and SMACKO (b) detrusor muscle response to depolarization by 85 mM K+ (A) or 10 µM CCh (B, C). Bars indicate the presence of 85 mM K+ or 10 µM CCh. Traces after 5 min preincubation with 1 µM isradipine (ISR) (A, B) 20 s after bath application of 10 mM BAPTA (B), 30 min after addition of 1 µM thapsigargin (TG) (C), and 15 min after incubation in Ca2+ free solution containing 1 mM EGTA (C) are graphically superimposed. Statistics of peak (c) and tonic (d) contractile responses in control conditions (control) and in the presence of ISR, BAPTA, TG and EGTA are shown. Tonic responses were obtained 5 min after addition of K+ or CCh. Open and filled bars indicate experiments with muscles from CTR and SMACKO mice, respectively. Data represent means ± SE. Numbers indicate number of experiments. *P <0.05; **P <0.01; ***P <0.001; n.s., nonsignificant.

4. Ca²⁺ release from intracellular stores is not crucial for CCh-induced phasic contractions
Emptying of intracellular stores by long-time treatment with thapsigargin (1 µM, 30 min) did not change CCh-induced phasic contractions of bladder muscle from CTR and SMACKO (Fig. 2C ). Inhibition of Ca²⁺ release by the PLC inhibitors U73122 (10 µM) and 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate (100 µM) did not affect CCh-induced phasic contractions. However, if the muscles were stressed by conditions of increased intracellular [Ca²⁺], which probably leads to a maximal filling of the intracellular Ca²⁺ stores, treatment with thapsigargin or U72122 attenuated CCh-induced phasic contractions by 15%. These results argue against a significant contribution of Ca²⁺ release from intracellular stores, even in forced conditions, to CCh-induced phasic contractions.

5. Contraction via Rho-kinase signaling cannot compensate loss of Ca_v1.2 channel function
Incubation of CTR and SMACKO muscle strips with the Rho-kinase inhibitor Y27632 (20 µM) reduced CCh-stimulated contraction by 40 and 80 %, respectively. Thus, the CCh-induced contractions of SMACKO muscle, but not of CTR muscle, are mostly caused by Rho-kinase-dependent inhibition of myosin phosphatase. However, bladder contractions in SMACKO mice reached only 25% of those observed in CTR mice (Fig. 2B ). Obviously, the Rho-kinase pathway is insufficient to compensate the loss of Ca_v1.2 function.

CONCLUSIONS AND SIGNIFICANCE

In the present study, the Ca_v1.2 gene was inactivated in urinary bladder from mice by using a smooth muscle-specific, tamoxifen-activated Cre/lox system. Inactivation of the Ca_v1.2 gene in bladder muscle was confirmed by the lack of protein and L-type calcium channel current. The absence of the Ca_v1.2 protein resulted in the lack of rhythmic contractions and a reduction of contractile responses to external stimuli. SMACKO mice show severe difficulties in urinating shown by reduced micturition. These results show that the Ca_v1.2 channel is critically involved in bladder function.

A major finding of this study is that stimulation of bladder contraction by muscarinic acetylcholine receptors depended mostly on the presence of the Ca_v1.2 channel. The decisive experiments for this conclusion are that 1) stimulus-induced tension in muscle strips from SMACKO mice is almost 4-fold smaller than in CTR mice, 2) short term removal of extracellular Ca²⁺ by BAPTA prevented CCh-induced contraction, and 3) preincubation with thapsigargin had no effect on CCh-induced phasic contraction. The small residual contraction induced by CCh in muscles from SMACKO mice is mostly due to activation of the Ca²⁺-independent Rho/Rho-kinase pathway and party to an unidentified Ca²⁺ influx pathway.

The coupling mechanism between muscarinic receptors and Ca_v1.2 channels is unclear. Although a recent study postulated that coupling of Ca_v1.2 and muscarinic receptors involves PI₃-kinase and PKC in vascular myocytes, inhibitors of PLC, PI₃-kinase, and PKC either had no effect or blocked Ca²⁺ channels (J. W. Wegener and A. Welling, unpublished results). We can rule out the possibility that Ca²⁺ release from intracellular stores by activation of the muscarinic receptors activates directly myosin light chain kinase leading to contraction. However, Ca²⁺ from intracellular stores may activate the Ca_v1.2 channel via activation of Ca²⁺-dependent Cl^– channels.

In conclusion, this study clearly identified the Ca_v1.2 channel as an essential part of the regulation of bladder contractility. Release of Ca²⁺ from intracellular stores, Ca²⁺ entry via non-Ca_v1.2 channels, and inhibition of myosin phosphatase via Rho/Rho-kinase signaling cannot compensate the lack of the Ca_v1.2 channel. Thus, these results challenge the generality of the hypothesis that hormone triggered smooth muscle contraction depends mainly on intracellular calcium release via IP₃ and/or on a calcium-independent signaling pathway.

View larger version (18K): [in this window] [in a new window]   Figure 3. Simplified scheme of agonist-induced contraction in urinary bladder smooth muscle. Upon stimulation of muscarinergic receptors (mR) Cav1.2 channel and Rho/Rho-kinase are activated. Ca2+ entry via Cav1.2 channels induces contraction via calmodulin-dependent activation of myosin light chain kinase (MLCK). In parallel, inhibition of myosin light chain phosphatase (MLCP) via phosphorylation of myosin phosphatase inhibitor (MYPT1) through Rho-kinase potentiates the effects of Cav1.2 channel activation. Thickness of arrows corresponds to the contribution of each signaling pathway.

FOOTNOTES

² Present address: Institut für Experimentelle und klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Albertstr. 25, 79104 Freiburg, Germany.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1516fje; doi: 10.1096/fj.04-1516fje

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