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Published as doi: 10.1096/fj.05-5292fje.
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(The FASEB Journal. 2006;20:1260-1262.)
© 2006 FASEB

Control of intestinal motility by the Cav1.2 L-type calcium channel in mice

Jörg W. Wegener1, Verena Schulla, Angela Koller, Norbert Klugbauer, Robert Feil and Franz Hofmann

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

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

ABSTRACT

The Cav1.2 L-type Ca2+ channel is the dominant voltage-activated Ca2+ channel in heart and smooth muscle. The functional significance of this channel was studied in intestinal smooth muscle from mice carrying a smooth muscle-specific, conditional inactivation of the Cav1.2 gene (Cav1.2SMACKO mice). Inactivation was complete within 4 wk after tamoxifen treatment and confirmed by RT-PCR, Western blot and functional analysis. Cav1.2SMACKO mice show reduced feces excretion, absence of rhythmic contractions in small and large intestinal muscle and signs of paralytic ileus. Extracellular field stimulation evoked smaller contractions in jejunum muscles from Cav1.2SMACKO than from CTR mice, whereas carbachol-induced contractions of similar magnitude in both muscles. The Ca2+ needed for contraction in jejunum was provided mainly by Cav1.2 channels and by store-operated channels in muscles from CTR and Cav1.2SMACKO mice, respectively. In conclusion, the Cav1.2 channel is essential for electromechanical coupling and important for pharmaco-mechanical coupling in intestinal smooth muscle and cannot be substituted functionally by other Ca2+ entry pathways.—Wegener, J. W., Schulla, V., Koller, A., Klugbauer, N., Feil, R., Hofmann, F. Control of intestinal motility by the Cav1.2 L-type calcium channel in mice.


Key Words: intestinal contraction • constipation • SKF96365 • store-operated channels

CONTRACTILE ACTIVITY OF intestinal smooth muscle generates the peristalsis that determines effective digestion and propulsion of digesta. This activity is controlled by neuronal reflexes and by pacemaker cells of smooth muscle precursor origin, that is, interstitial cells of Cajal (ICC), involving both electromechanical and pharmaco-mechanical coupling. Electromechanical coupling is mediated by the opening of voltage-dependent Ca2+ channels, which is initiated by electrical activity conducted from the ICC to smooth muscle cells via gap junctions (1) . The role of these Ca2+ channels in intestinal pharmaco-mechanical coupling, in which acetylcholine (Ach) is the major excitatory agonist (2) , is less clear.

Rhythmic contractile activity in the intestine is modulated by neuronal input. This modulation involves both intramuscular (i.m.) ICCs and smooth muscle cells. At present, it is unclear whether neuronal transmitters exclusively affect the activity of ICCs or whether these transmitters also act directly on smooth muscle. ICCs are essential for nitrergic and cholinergic signaling in the stomach (3) and lower esophagus sphincter (4) , as evidenced by studies using W/Wv mice. In support of this view, i.m. ICCs in the stomach (3) and colon (5) have synapse-like contacts with nerve varicosities and gap-junction contacts with smooth muscle cells. In contrast, small intestinal muscle from W/Wv mice showed no spontaneous activity, but responded normally to nerve stimulation (6) . This behavior of the small intestinal muscle suggested that neurotransmitter can bypass the ICCs in small intestine and directly activate the smooth muscle.

Ach released from nerve endings can increase smooth muscle contraction either directly or indirectly via ICCs (7) , because M3 receptors are expressed in intestinal smooth muscle (8) and ICCs (7) . Activation of muscarinic receptors induces IP3-dependent Ca2+ release from intracellular stores (9) , PKC translocation (10) and initiates an increase in [Ca2+] by activation of Ca2+ release and by opening of cationic ion channels (11) . Ca2+ released from intracellular stores is believed to be the main trigger for smooth muscle contraction. However, contractions evoked by cholinergic agonists were reduced by L-type Ca2+ channel blockers in intestinal muscle (12 , 13) . Thus, it is unclear to what extent muscarinic-mediated contractions depend on a L-type Ca2+ channel in intestinal smooth muscle.

To clarify the molecular identity and the functional role of the L-type Ca2+ channels involved in intestinal smooth muscle motility, a mouse strain was generated by tamoxifen-mediated mutagenesis (14) , which selectively lacked an intact Cav1.2 gene in smooth muscle. The analysis of these mutants indicates that the lack of this channel leads to paralytic ileus, because the intestinal smooth muscle Cav1.2 L-type Ca2+ channel is essential for rhythmic contractile activity of small and large intestine.

MATERIALS AND METHODS

Generation of mice
All experiments conformed to the animal protection laws of Germany. The generation of smooth muscle {alpha}1c-subunit calcium channel knock out (Cav1.2SMACKO) mice has been described (15) . All mice had one "knock-in" allele with a tamoxifen-dependent Cre recombinase under the control of the smooth muscle-specific SM22 promoter (14) . Knockout mice carried a "loss-of-function" and a modified allele of the Cav1.2 gene (loxP-flanked exons 14 and 15) and were generated by injection of tamoxifen. The corresponding control (CTR) mice carried a wild-type and a modified allele of the Cav1.2 gene (loxP-flanked exons 14 and 15) and were also treated with tamoxifen.

Animals were kept under standard conditions with water and food ad libitum. 6 mice were kept in solitary confinement; the weight of the consumed food and of the excreted feces from these mice was measured daily. At an age of 3 to 12 mo, Cav1.2SMACKO and CTR mice were i.p. injected with tamoxifen (1 mg per day) for 5 consecutive days. After development of the phenotype (see Results), mice were killed by cervical dislocation, and the intestine was removed. Data obtained from wild-type and CTR animals were not statistically different, indicating that 1) tamoxifen treatment and/or 2) the presence of the knock-in allele and/or 3) the inactivation of the Cav1.2 gene in a single allele had no effect by itself on the parameters measured. Salts and drugs used were as pure as available and purchased from Sigma (Sigma-Aldrich, Munich, Germany) and Calbiochem (Merck, Darmstadt, Germany) unless otherwise indicated.

RT-PCR of intestinal tissue
Poly(A)-mRNA was isolated from the small intestine (10 mg) using Dynabeads Oligo (dT)25 (Hamburg, Germany), as described previously (16) . The following primers were used (a) for Cav1.2: VS9 5'- ACA CAG CCA ATA AAG CCC TCC TG-3' and VS18 5'- GGC CAG CTT CTT CCT CTC CTT-3'; and (b) for hypoxanthine-guanine-phosphoribosyltransferase (HGPRT): QG 197 5'-GTA ATG ATC AGT CAA CGG GGG AC-3' and QG 198 5'-CCA GCA TTG CAA CCT TAA CCA-3'.

Western blot
Microsomal membranes were prepared from isolated longitudinal smooth muscle from small and large intestine and solubilized as described (17) . Membrane fractions were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. Blots were treated with 5% nonfat milk powder in TRIS-buffered saline and labeled with anti-Cav1.2 or anti-Cav1.3 antibody (Ab) (Chemicon, Temecula, CA).

Tension recordings
Segments from jejunum and colon were mounted in longitudinal orientation to organ baths (Myograph 601, Danish Myotechnology, Aarhus, Denmark) as described (18) . Tension was recorded isometrically at 36 ± 1°C using segments of 5–7 mM length. Resting tension was set to 10–15 mN. Depolarization was achieved by exchanging equimolar Na+ with K+. Drugs were applied as single dose or cumulatively to achieve the concentrations as indicated. Inhibitors or activators (test substances) were applied 10–20 min before initiation of contraction.

Contractions in response to neuronal stimulations were elicited by electrical field stimulation (EFS). The responses were abolished by tetrodotoxin (1 µM) confirming that they were due to neuronal activity. EFS was performed with electrodes positioned either in parallel to the muscle (square wave pulses of 30 ms duration at 5 Hz, train duration of 30 s, 10 V) or across the muscle (square wave pulses of 1- to 2-ms duration at 10 Hz, train duration of 10 s, 10 V). Both EFS protocols were checked to produce maximal contraction amplitudes and were generated by a PowerLab 4/20 (ADinstruments, Spechbach, Germany). Results from both types of stimulation were statistically not different and, therefore, pooled.

Evaluation of results
Results are presented as images of gels and blots, original recordings, or expressed as means ± SEM. Force was normalized to the wet weight of the muscle preparation. Effects of drugs were analyzed in steady-state conditions. Changes in tension were determined with respect to the resting tension before stimulation. Phasic responses to stimulation were measured at maximum, whereas tonic responses were measured 5–10 min after stimulation. Statistical comparisons of data sets were performed by a Student’s t test using Prism 4 software (GraphPad, San Diego, CA). Differences were considered significant at P < 0.05.

RESULTS

Characterization of Cav1.2SMACKO mice
Cav1.2SMACKO mice showed a reduced motility and an enlarged lower abdomen at ~3–4 wk after start with the injection of tamoxifen. At this stage, excretion of feces was significantly reduced (Fig 1 A) and intake of food decreased during the following 1 to 2 days (Fig. 1B ). Animals died with symptoms of a paralytic ileus. Necropsy showed intestinal situs with grossly extended small and large intestine of the Cav1.2SMACKO mice (Fig 1C, D ) supporting the diagnosis of a paralytic ileus (15) . The disease was caused by the inactivation of the gene coding for Cav1.2 L-type Ca2+ channel (Fig. 2 ). RT-PCR analysis showed that the Cav1.2 gene was inactivated in jejunum smooth muscle 7 days after the start of the injections with tamoxifen (Fig. 2A ). Western blot and functional analysis demonstrated that the gene inactivation resulted in a time-dependent loss of the protein (Fig 2B ) and failure of the dihydropyridine isradipine (0.1 µM) to relax depolarization-induced contractions (Fig. 2C ). In wild type (CTR) muscle strips, isradipine relaxed K+-induced contractions with an EC50 of 1 nM. The disappearance of protein and isradipine-mediated relaxation were complete within 4 wk after start of the injections. At this time, Cav1.2 protein was also absent in colon muscle from Cav1.2SMACKO mice (Fig. 2D ). The different times needed for the inactivation of the gene and for the development of disease symptoms obviously reflect the slow turnover of the Cav1.2 protein. We estimated that the half-life of the Cav1.2 protein was ~14 days in this intestinal smooth muscle. The loss of the Cav1.2 protein was not compensated by an up-regulation of the expression of the dihydropyridine-sensitive Cav1.3 ({alpha}1D) Ca2+ channel (Fig. 2E, F ) as reported for embryonic hearts (19) . Together, these results demonstrate the successful inactivation of the Cav1.2 gene in intestinal smooth muscle.


Figure 1
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  		Figure 1. Time course of feces excretion (A) and food intake (B) after treatment with tamoxifen. The Cav1.2SMACKO mice showed reduced motility at day 25 (dotted vertical line). Days were numbered with the start of the injection with tamoxifen. Data points represent the mean ± SD (n=3 each group). Intestinal situs of a CTR (C) and Cav1.2SMACKO (D) mouse. Arrows point at the jejunum. Note the extended jejunum and caecum in (D).


Figure 2
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  		Figure 2. Inactivation of the Cav1.2 gene in intestinal smooth muscle. Stripped longitudinal smooth muscle was used for all experiments. Numbers above gel lines and on the abscissa correspond to days after the start of injection with tamoxifen. A) Time-dependent PCR-analysis of jejunum from tamoxifen-treated Cav1.2SMACKO mice (L1/L2T). L2 contains the "floxed" exons 14 and 15 and encodes a functional Cav1.2 gene. In L1, exons 14 and 15 were deleted; this deletion causes a loss-of-function allele, as described (16) . The signal for L2 disappeared within 7 days after injection with tamoxifen. HGPRT was used as an internal standard. M indicates marker line. B) Time-dependent Western blot analysis of jejunum membrane fractions from four tamoxifen-treated Cav1.2SMACKO (L1/L2T) mice and one CTR (+/L2T) mouse using an Ab against Cav1.2 Ca2+ channel (16) . + indicates the wild-type allele. The protein band for the Cav1.2 Ca2+ channel disappeared time-dependently after injection with tamoxifen. The loading control was ß-actin. C) Time-dependent change in isradipine sensitivity of K+-contracted jejunum smooth muscle strips from CTR and Cav1.2SMACKO mice. Muscles were contracted by 85 mM K+. Isradipine was used at 0.1 µM. Data points represent means ± SEM (n=8–12). D) Western blot of colon and brain membrane fractions from CTR (+/L2T) and Cav1.2SMACKO (L1/L2T) mice for the Cav1.2 protein. No protein was detected in colon preparations from Cav1.2SMACKO (L1/L2T) mice. E, F) Western blot of jejunum (E), colon (F), and brain membrane fractions from CTR (+/L2T) and Cav1.2SMACKO (L1/L2T) mice for the Cav1.3 protein. The Ab detected the Cav1.3 protein only in brain membranes.

Spontaneous and induced contractile activity
All intact segments of jejunum and colon from CTR mice showed spontaneous contractile activity; the magnitude but not the frequency was concentration-dependently blocked by isradipine (EC50 13 nM), as shown for jejunum muscle (Fig. 3 A, B). Almost no contractile activity was observed in segments of jejunum and colon from Cav1.2SMACKO mice (Fig. 3D ; Fig. 4 B). In those preparations that showed a tiny activity (jejunum: 0.04 ± 0.01 N/g in 8 out of 66 muscles; colon: 0.05 ± 0.01 N/g in 4 out of 20 muscles), the frequency of contractions was not different to those from CTR mice (Fig. 4D ). This result indicates that the Cav1.2 channel determines the contraction magnitude, but does not modulate the frequency of the spontaneous rhythmic contractions.


Figure 3
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  		Figure 3. Rhythmic and electrically evoked contractile activity in jejunum smooth muscle. A) Original recording from a jejunum muscle of a CTR mouse. Lines indicate application of isradipine; numbers correspond to log [isradipine] in M. B) Concentration-dependent effects of isradipine on amplitude and frequency of spontaneous contractile activity in muscles from CTR mice. Data points represent means ± SEM for both parts of the figure (n=4–12). At 1 µM isradipine, the amplitude of contraction was 1.5 ± 0.6% of control, and the frequency was determined to 0.38 ± 0.05 beats/s (n=4). C–D) Expanded original recordings from a jejunum muscle of a CTR (C) and a Cav1.2SMACKO mouse (D). E, F) EFS-induced contractions in jejunum smooth muscle from a CTR (E) and Cav1.2SMACKO (F) mouse. Bars indicate the application of EFS and 10 µM carbachol (CCh) in control conditions (left) and in the presence of 1 µM atropine (right).


Figure 4
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  		Figure 4. Rhythmic contractile activity in colon smooth muscle. A, B) Expanded original recordings from a colon muscle of a CTR (A) and a Cav1.2SMACKO mouse (B). C, D) Amplitude (C) and frequency (D) of rhythmic contractile activity in colon and jejunum muscles from CTR and Cav1.2SMACKO mice. Bars represent means ± SEM with the number of experiments. Note that the y axis for the frequency in colon is on the right-hand side.

The rhythmic contractile activity of intestinal smooth muscle is regulated by the intestinal nervous system (20) . Electrical field stimulation (EFS) of intestinal segments facilitates release of stimulatory and inhibitory neurotransmitters that modulate contractility. EFS of jejunum strips induced significantly less contraction in Cav1.2SMACKO mice than in CTR mice (Fig. 3 and Fig. 5 ), whereas direct stimulation of muscarinic receptors by carbachol induced similar tension levels. The muscarinic antagonist atropine reduced almost to zero contractions elicited by EFS or carbachol (Fig. 3 , 5) . The minor effect of EFS in muscles from Cav1.2SMACKO mice was not caused by an increased release of the inhibitory neurotransmitter NO. EFS-induced tension did increase slightly in the presence of the NO synthase inhibitor L-NAME (Fig. 5B ) to a similar extent in muscles from both mouse lines (CTR: 0.28±0.05 vs. 0.34±0.05 N/g; n=12; P=0.04. Cav1.2SMACKO: 0.07±0.02 vs. 0.09±0.02 N/g; n=12; P=0.04), suggesting that NO mediated inhibition of contraction was small but unaltered by the deletion of the Cav1.2 channel.


Figure 5
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  		Figure 5. Magnitude of EFS-induced contractions in jejunum smooth muscle. A) Magnitude of EFS-induced and carbachol-induced tension in steady state conditions in the absence and presence of atropine (1 µM). B) Magnitude of EFS-induced contraction in jejunum strips from CTR and SMACKO mice in the absence and presence of L-NAME (100 µM). Bars represent means ± SEM *, P < 0.05; n.s., not statistically significant.

In contrast to EFS- or carbachol-stimulated activities, spontaneous contractile activity was decreased by 35% in CTR muscle strips in the presence of atropine (Fig. 3E ; 1 µM; 100 vs. 66±4%; n=16), supporting the notion that this type of intestinal contractile activity is not exclusively determined by muscarinic receptors. Furthermore, the magnitude of spontaneous contractions was increased only by ~25% in the presence of the inhibitor of NO synthesis L-NAME (100 µM) (Fig 6 A, B), whereas contraction frequency was slightly reduced in muscles from CTR mice (31±1 vs. 28±1 contractions/min; n=12; P=0.01; paired Student’s t test; Fig. 6C ). No effect of L-NAME and/or atropine was observed in muscles from Cav1.2SMACKO mice (Fig. 3F , 6B ). These results show that the absence of rhythmic contractions in Cav1.2SMACKO mice is not due to an increased neuronal inhibition of smooth muscle activity nor to an inability of Ach to elicit contraction.


Figure 6
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  		Figure 6. Effect of L-NAME on rhythmic contractile activity. A) Original recordings of a jejunum muscle from a CTR mouse. Lines indicate the presence of 1 µM atropine or 1 µM atropine and 100 µM L-NAME. B,C) Amplitude (B) and frequency (C) of spontaneous contractile activity in jejunum of CTR and Cav1.2SMACKO mice. Bars represent means ± SEM *P < 0.05.

Cholinergic-induced contractions
The above shown experiments resulted in an unexpected discrepancy. In both jejunum and colon smooth muscle, spontaneous intestinal contractility was absent in the Cav1.2SMACKO. However, carbachol-induced contraction was normal in jejunum smooth muscle but clearly smaller in colon smooth muscle from Cav1.2SMACKO, as compared to the muscles from CTR mice (Fig. 7 A–C). This latter finding is in agreement with a previous report that contractions elicited by muscarinic receptor stimulation required opening of Cav1.2 channels in detrusor smooth muscle (18) . Cholinergic signaling has been reported to be the most prominent stimulatory pathway for contraction in the intestine (2) . This report and our findings are contrasted by the almost unchanged intestinal function after deletion of the muscarinic receptor M2 or M3 (21) . Therefore, we tried to unravel the cause of carbachol-induced contractions in jejunum.


Figure 7
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  		Figure 7. Contraction and relaxation in intestinal smooth muscle. Original recordings of a colon (A–C) and jejunum (D--F) smooth muscle from a CTR (A, D) and Cav1.2SMACKO mouse (B, E). Lines indicate the presence of 10 µM carbachol (CCh). C, F) Magnitudes of carbachol-induced tension in colon (C) and jejunum (F). In colon (A--C), the carbachol-induced contraction consisted of a phasic and a tonic response. In jejunum, the carbachol-induced contraction consisted of at least 3 components (D--F): a fast component at 5–10 s, a slow response at 10–120 s, and a tonic response within 10 min. G–I) Relaxation of carbachol-induced tonus by isradipine and SKF96365. G, H) Original recordings of muscles from a CTR (G) and a Cav1.2SMACKO (H) mouse. Lines indicate the presence of 10 µM carbachol (CCh), 1 µM isradipine (ISR), and 10 µM SKF96365 (SKF). I) Inhibition of CCh-induced contraction by 1 µM isradipine and 10 µM SKF96365. Bars represent means ± SEM (n=4, each). J–L) Relaxation by SKF96365 of Ca2+-re-entry-induced contraction in jejunum smooth muscle from CTR and Cav1.2SMACKO mice. J, K) Original recordings of a jejunum muscle strip from a CTR (J) and a Cav1.2SMACKO (K) mouse. Muscle strips were preincubated for 30–40 min in Ca2+-free buffer containing 1 mM EGTA to which 1 µM thapsigargin was added. Lines indicate the addition of 4 mM Ca2+, SKF96365, and Gd3+, respectively. Numbers indicate log[SKF96365] in M. 1 mM Gd3+ was used to induce maximal relaxation. L) Magnitude of contraction induced by Ca2+-re-entry. Bars represent means ± SEM. **P < 0.01.

Carbachol-induced contractions of the longitudinal smooth muscle from jejunum of CTR and Cav1.2SMACKO mice consisted of at least 3 components (Fig. 7D-F ): a fast component which had its maximum at ~5–10 s after stimulation; a slow response which had a maximum at ~10–120 s; and, finally, a tonic response which reached a steady state within 10 min. Interestingly, the amplitudes of these responses were not different between the CTR and Cav1.2SMACKO muscles (Fig. 7F ).

So far, these findings implied that the Cav1.2 channel is involved in cholinergic-induced contractions in colon but not in jejunum muscle. However, further analysis of jejunum muscle showed that the carbachol-induced contraction was reduced by isradipine (0.1 µM) in muscles from CTR but not from Cav1.2SMACKO mice (Fig. 7G--I ). Addition of the bona fide inhibitor of store-operated channels (SOC), SKF96365 (22) , completely abolished the carbachol-induced contraction (Fig. 7G--I ), suggesting that the muscarinic receptors couple in jejunum smooth muscle to the Cav1.2 channel and a member of the SOC family.

To further prove this possibility, contractions were induced by capacitative Ca2+ re-entry, which is supposed to be carried mainly by store-operated channels (23) . Surprisingly, the responses to capacitative Ca2+ entry were much larger in jejunum muscles from Cav1.2SMACKO than from CTR mice (Fig. 7J-L ). In addition, activation of Ca2+-entry by thapsigargin induced also larger contractions in muscles from Cav1.2SMACKO than from CTR mice (0.40±0.04 vs. 0.22±0.03 N/g; n=35–44; P<0.001), which were abolished by the putative SOC inhibitor Gd3+ (not shown). The contractions induced by capacitative Ca2+ re-entry were concentration-dependently blocked by SKF96365 in both muscles from CTR and Cav1.2SMACKO mice (Fig. 7J, K ) with IC50’s of 12 and 14 µM, respectively. These results indicate that, in jejunum muscle, muscarinic receptor activation can maintain a "normal" contraction in the Cav1.2SMACKO mice by switching from L-type calcium channel to a SOC-like channel (24) .

DISCUSSION

The selective inactivation of the L-type Cav1.2 Ca2+ channel in smooth muscle caused the death of the mice most likely by a combination of altered blood pressure regulation, bladder dysfunction, and a paralytic ileus (15 , 18) . The successful inactivation of the Cav1.2 L-type Ca2+ channel in intestinal smooth muscle was confirmed by the lack of the Cav1.2-mRNA and -protein and the missing effect of nanomolar concentrations of the dihydropyridine isradipine on K+-induced contraction. The absence of the Cav1.2 protein in the intestinal smooth muscle caused a severe dysfunction resulting in an extended small and large bowel, the absence of rhythmic contractile activity in 80 to 90% of the smooth muscle preparations and difficulties to excrete feces. The deletion also reduced electrically evoked contractions but apparently did not affect carbachol-induced contractions in the small intestine, whereas carbachol-induced contraction of the colon was reduced. A similar phenotype was described after inactivation of ICC by anti-c-kit antibodies in neonate mice (25) , whereas mutant mice exhibiting a defect in neuronal signaling pathways, i.e., mice lacking neuronal NO synthase (26) or M2/M3-type muscarinic receptors (21) , did not show such a prominent intestinal phenotype.

Rhythmic contractions of intestinal smooth muscle are regarded as the result of electromechanical coupling between smooth muscle cells and the interstitial cells of Cajal (ICC) (27 , 28) . The present study shows that this coupling depends critically on the presence of the Cav1.2 Ca2+ channel. The magnitude of the contractile spontaneous activity was almost absent in the Cav1.2SMACKO mice. No change in the frequency was observed in muscles from Cav1.2SMACKO mice or from CTR mice treated with isradipine, indicating that the pacemaker activity controlling the spontaneous activity depends not on the Cav1.2 channel. In line with this finding, only a minor reduction was observed with 1 µM nicardipine (29) . This view is supported by a report showing that pacemaker activity of ICCs involved a voltage-dependent Ca2+ current, which was not influenced by dihydropyridines (30) . Thus, it is suggested that the Cav1.2 Ca2+ channel acts as both voltage-sensor and -executor that initiates and determines contraction in smooth muscle.

The loss of the smooth muscle Cav1.2 Ca2+ channel could not be compensated by transmitters released from the intestinal nerve system as evidenced by the reduced EFS stimulated contraction and the small effects of atropine and L-NAME on EFS-stimulated contractions. Ach released from the nerve terminals can induce contraction in the gastrointestinal tract by two routes. Indirectly via activation of ICCs coupled to smooth muscle cells and directly by activating smooth muscle muscarinic receptors (3 , 6 , 31) . In gastric fundus muscle, ICC mediate exclusively this transduction process, because muscles from mice lacking i.m. ICC (c-kit mutant W/Wv mice) did not respond to nerve stimulation but to externally applied cholinergic agonists (3) . In longitudinal muscle from jejunum of Cav1.2SMACKO, nerve stimulation still induced an atropine-sensitive response. However, this response was ~50% smaller than that observed in CTR mice, although the response to externally applied carbachol was identical. This finding indicates that part of the response in CTR mice is due to electrically coupling mediated by ICC. Therefore, it is suggested that, in longitudinal jejunum, cholinergic signaling is mediated by both i.m. ICC and muscarinic smooth muscle receptors. Indeed, in this tissue, nerves have been found that do not connect to i.m. ICC (32) .

In contrast to detrusor muscle (18) and colon muscle (Fig. 7A-C ), muscarinic receptor stimulation by bath-applied carbachol contracted jejunum smooth muscle strips from both CTR and Cav1.2SMACKO mice to the same extent. However, whereas contraction of CTR muscle was inhibited by the dihydropyridine isradipine at low concentration, isradipine had no effect at these concentration on contraction of Cav1.2SMACKO strips. Thus, the Cav1.2 channel was probably replaced by an alternative Ca2+ entry pathway in the Cav1.2SMACKO animals. Several pathways of Ca2+ entry have been proposed in cholinergic signaling in ileum, that is, m2AChR-mediated voltage-dependent Ca2+ entry and m3AChR-mediated voltage-independent Ca2+ entry (33) . Several lines of evidence suggest that Ca2+ entry via store-operated channels (SOC) almost completely substituted the loss of Ca2+ entry via Cav1.2 Ca2+ channels in the muscles from Cav1.2SMACKO mice: 1) the magnitude of contraction induced by either Ca2+ re-entry or by thapsigargin was 2 times larger in muscles from Cav1.2SMACKO than from CTR mice. Both contractions are thought to be mediated by SOC (34) . 2) Contractions induced by either Ca2+ re-entry or by thapsigargin in muscles from Cav1.2SMACKO mice were inhibited by the putative SOC inhibitor SKF96365. SOCs are supposed to belong to the family of transient receptor channels (TrpC, (35) ). Interestingly, up-regulation of a Ca2+ entry pathway carried by such a channel type (TrpC3) has been observed in vascular smooth muscle from TrpC6-deficient mice (36) . However, the increased coupling of the muscarinic receptors to such a Ca2+ entry pathway did not prevent the development of the lethal phenotype in the Cav1.2SMACKO mice, obviously because it did not support spontaneous contractility.

In summary, the present study shows that 1) the Cav1.2 channel is essential for electromechanical coupling and important for pharmaco-mechanical coupling in intestinal smooth muscle; 2) the Cav1.2 channel cannot be substituted adequately by other Ca2+ entry pathways; 3) Ach or NO released from nerve terminals are insufficient to regulate intestinal rhythmic contraction in the absence of the smooth muscle Cav1.2 Ca2+ channel.

ACKNOWLEDGMENTS

We thank Mrs. Susanne Paparisto for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie.

Received for publication October 26, 2005. Accepted for publication January 17, 2006.

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