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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5292fjev1 20/8/1260    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 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.

Control of intestinal motility by the Ca_v1.2 L-type calcium channel in mice

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

SPECIFIC AIMS

The contractility of the intestinal smooth muscle is controlled by different mechanisms. We investigated the functional consequences of smooth muscle-specific disruption of the gene encoding the L-type Cav1.2 Ca²⁺ channel.

PRINCIPAL FINDINGS

1. Disruption of the gene encoding the L-type Ca_v1.2 Ca²⁺ channel leads to paralytic ileus
Mice (Ca_v1.2^SMACKO mice) were studied in which the Ca_v1.2 gene was disrupted by a smooth muscle-specific, tamoxifen-triggered approach using the Cre/loxP system. Ca_v1.2^SMACKO mice showed an enlarged lower abdomen and a reduced excretion of feces at 3–4 wk after injection with tamoxifen. Thereafter, animals died with symptoms of a paralytic ileus.

2. The Cav1.2 channel protein disappears 4 wk after gene inactivation
RT-PCR analysis showed that the Ca_v1.2 gene was inactivated in jejunum smooth muscle 7 days after treatment with tamoxifen. Western blot and functional analysis demonstrated that the gene inactivation resulted in a time-dependent 1) loss of the protein and 2) failure of the dihydropyridine isradipine (0.1 µM) to relax depolarization-induced contractions. The disappearance of protein and isradipine-mediated relaxation was complete within 4 wk. The different times needed for the inactivation of the gene and for the disappearance of the protein obviously reflect the slow turnover of the Ca_v1.2 protein; half-life was estimated to be 14 days. The loss of the Ca_v1.2 protein was not compensated by an up-regulation of the expression of the dihydropyridine-sensitive Ca_v1.3 (_1D) Ca²⁺ channel.

3. The Ca_v1.2 channel controls spontaneous contractile activity
All intact segments of jejunum and colon from control (CTR) mice showed spontaneous contractile activity; the magnitude but not the frequency was concentration-dependently blocked by isradipine (EC₅₀ 13 nM, Fig. 1 A, B). Segments from nearly all Ca_v1.2^SMACKO mice exhibited no contractile activity (Fig. 1D ). In preparations from those Ca_v1.2^SMACKO mice that showed a tiny activity (jejunum: 8 out of 66; colon: 4 out of 20), the frequency of contractions was not different to those from CTR mice. This result indicates that the Ca_v1.2 channel determines the contraction magnitude but does not modulate the frequency of the rhythmic contractions.

View larger version (18K): [in this window] [in a new window]   Figure 1. 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).

4. The Cav1.2 channel participates in electrically evoked contractions
Electrical field stimulation (EFS) of jejunum strips induced significantly less contraction in Ca_v1.2^SMACKO mice than in CTR mice, whereas direct stimulation of muscarinic receptors by carbachol induced similar tension levels (Fig. 1E, F ). The muscarinic antagonist atropine abolished contractions elicited by EFS or carbachol (Fig. 1E, F ) and slightly depressed spontaneous contractions. The NO synthase inhibitor L-NAME (100 µM) enhanced similarly spontaneous contractions and the response to EFS in both mice strains.

5. The Cav1.2 channel is substituted during cholinergic-induced contractions in jejunum
Carbachol-induced contractions were significantly reduced in colon from Ca_v1.2^SMACKO as compared to CTR mice (Fig. 2 A–C). Surprisingly, carbachol-induced contractions were not different in magnitude in jejunum from CTR and from Ca_v1.2^SMACKO mice (Fig. 2D-F ). However, carbachol-induced contraction was reduced by isradipine (0.1 µM) in muscles from CTR but not from Ca_v1.2^SMACKO mice (Fig. 2G-I ). Addition of the bona fide inhibitor of store-operated channels (SOC), SKF96365, completely abolished the carbachol-induced contraction (Fig. 2G-I ). Further analysis revealed that contractions in response to capacitative Ca²⁺ entry (induced either by reintroduction of Ca²⁺ or by treatment with thapsigargin) were much larger in jejunum muscles from Ca_v1.2^SMACKO than from CTR mice (Fig. 2L ). These contractions were concentration-dependently blocked by SKF96365 in muscles from both CTR and Ca_v1.2^SMACKO mice (Fig. 2J, K ) with IC₅₀’s of 12 and 14 µM, respectively.

View larger version (30K): [in this window] [in a new window]   Figure 2. 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.

CONCLUSION AND SIGNIFICANCE

The successful inactivation of the Ca_v1.2 L-type Ca²⁺ channel in intestinal smooth muscle was confirmed by the lack of the Ca_v1.2-mRNA and -protein and the missing effect of the dihydropyridine isradipine on K⁺-induced contraction. The absence of the Ca_v1.2 protein in the intestinal smooth muscle caused a severe dysfunction resulting in an extended small and large bowel, the missing/reduction of rhythmic contractions, difficulties to excrete feces, and, finally, death most likely by a paralytic ileus. A similar phenotype was described after inactivation of the interstitial cells of Cajal (ICC) by anti-c-kit antibodies in neonate mice, whereas mutant mice exhibiting a defect in neuronal signaling pathways, that is, mice lacking neuronal NO synthase or M2/M3-type muscarinic receptors, did not show such a prominent intestinal phenotype.

Rhythmic contractions of intestinal smooth muscle are the result of electromechanical coupling between smooth muscle cells (SMC) and ICC. The present study shows that this coupling depends critically on the presence of the Ca_v1.2 Ca²⁺ channel. The magnitude of the contractile spontaneous activity was almost absent in the Ca_v1.2^SMACKO mice. No change in the frequency was observed in muscles from Ca_v1.2^SMACKO mice or from CTR mice treated with isradipine. It is suggested that the Ca_v1.2 Ca²⁺ channel acts as both voltage-sensor and -executor that initiates and determines contraction in smooth muscle, leaving the pacemaker activity of the ICC untouched (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Proposed role of the Cav1.2 Ca2+ channel in smooth muscle from jejunum. Pacemaker activity of a ICC initiates contraction in smooth muscle cells (SMC) via electrical coupling followed by Cav1.2 channel activation. Cholinergic input from neurons stimulates subtypes of muscarinic receptors (m2R, m3R) in interstitial cells of Cajal and SMC and induces contraction indirectly via electrical coupling and directly via activation of Cav1.2 channels and store-operated channels (SOC).

Nerve stimulation still induced an atropine-sensitive response in longitudinal muscle from jejunum of Ca_v1.2^SMACKO mice, although the coupling of ICC to smooth muscle is functionally defective as evidenced by the lack of spontaneous contractions. However, this response was 50% smaller than that observed in CTR mice, indicating that part of the response in CTR mice is due to electrically coupling mediated by ICC. Therefore, we suggested that, in longitudinal jejunum, response to nerve stimulation is mediated by i.m. ICC and SMC (Fig. 3) .

Carbachol induced smaller contractions in colon smooth muscle from Ca_v1.2^SMACKO than from CTR mice, in agreement with previous reports that contractions elicited by muscarinic receptor stimulation required opening of Ca_v1.2 channels. Surprisingly, bath-applied carbachol-contracted jejunum strips from both mouse lines to the same extent. Contraction was inhibited by the dihydropyridine isradipine in strips from CTR mice but not from Ca_v1.2^SMACKO mice. Thus, the Ca_v1.2 channel was replaced by an alternative Ca²⁺ entry pathway in the Ca_v1.2^SMACKO animals. Several lines of evidence suggest that this pathway involves Ca²⁺ entry via store-operated channels (SOC): (a) the magnitude of contraction induced by either Ca²⁺ re-entry or by thapsigargin was 2 times larger in muscles from Ca_v1.2^SMACKO than from CTR mice. Both contractions are thought to be mediated by SOC (b). Contractions induced by either Ca²⁺ re-entry or by thapsigargin in muscles from Ca_v1.2^SMACKO mice were inhibited by the SOC inhibitor SKF96365. SOC are supposed to belong to the family of transient receptor channels (TrpC). Interestingly, such a channel type (TrpC3) is up-regulated in vascular smooth muscle from TrpC6-deficient mice. 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 Ca_v1.2^SMACKO mice, obviously because it did not support spontaneous contractility.

In summary, the present study shows that 1) the Ca_v1.2 channel is essential for electromechanical coupling and important for pharmaco-mechanical coupling in intestinal smooth muscle; 2) the Ca_v1.2 channel can not be substituted adequately by other Ca²⁺ entry pathways; and 3) Ach or NO released from nerve terminals are insufficient to regulate intestinal rhythmic contraction in the absence of the smooth muscle Ca_v1.2 Ca²⁺ channel.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5292fje;

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5292fjev1 20/8/1260    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 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.