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The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine.

Daniel EE, Eteraf T, Sommer B, Cho WJ, Elyazbi A - J. Cell. Mol. Med. (2008)

Bottom Line: We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR).We found evidence that these channels were associated with Cav-1.These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Alberta, Edmonton, AB, Canada. edaniel@ualberta.ca

ABSTRACT
In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca (+) channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca(2+) channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

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(A) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. (B) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.
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fig06: (A) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. (B) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.

Mentions: Effects on frequencies in these experiments are summarized in Fig. 6A. In both Cav+/+ and in Cav−/− segments the frequencies decreased with each successive exposure to Ca2+-free medium and in all conditions; control, BayK 8644 and nicardipine. In Cav++ segments, as expected the addition of BayK 8644 enhanced frequencies significantly during all three exposures in Ca2+-free solutions compared to controls without BayK 8644. However, nicardipine had no significant effect to reduce frequencies significantly compared to controls or to BayK 8644. Thus exposure to CCH made the segments apparently less susceptible to block of L-type Ca2+ channels (compare Figs 2A and 6A). In Cav−/− segments, BayK 8644 increased frequencies significantly compared to controls during the second and third exposures to Ca2+ medium, while nicardipine reduced frequencies during the third exposure compared to controls and during the second and third exposures compared to BayK 8644. The only conditions in which Cav+/+ segments behaved significantly differently from Cav−/− segments were during the third exposure in BayK 8644 and during the second and third exposures in nicardipine. In all cases responses of Cav+/+ were larger. These results imply that Cav+/+ segments recycle the Ca2+ released by CCH from SR on each wash more efficiently than CavB- segments. This was true whether L-type Ca2+ channels were either open or closed, especially when closed. Also, the exposures to CCH facilitated recycling notably when L-type Ca 2+ channels were closed by nicardipine. This suggests that there is an alternate path for recycling calcium other than the L-type Ca + channels when caveolae are present and enhanced by exposure to CCH, presumably a store operated channel (Fig. 3). This recycling appears more effective when these channels are open.


The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine.

Daniel EE, Eteraf T, Sommer B, Cho WJ, Elyazbi A - J. Cell. Mol. Med. (2008)

(A) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. (B) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.
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Related In: Results  -  Collection

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fig06: (A) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. (B) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav+/+) (top panel) and Cav-1 knockout (Cav−/−) (bottom panel) tissues to Ca2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca2+-free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca2+-free medium were higher in Cav+/+ tissues than in Cav−/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.
Mentions: Effects on frequencies in these experiments are summarized in Fig. 6A. In both Cav+/+ and in Cav−/− segments the frequencies decreased with each successive exposure to Ca2+-free medium and in all conditions; control, BayK 8644 and nicardipine. In Cav++ segments, as expected the addition of BayK 8644 enhanced frequencies significantly during all three exposures in Ca2+-free solutions compared to controls without BayK 8644. However, nicardipine had no significant effect to reduce frequencies significantly compared to controls or to BayK 8644. Thus exposure to CCH made the segments apparently less susceptible to block of L-type Ca2+ channels (compare Figs 2A and 6A). In Cav−/− segments, BayK 8644 increased frequencies significantly compared to controls during the second and third exposures to Ca2+ medium, while nicardipine reduced frequencies during the third exposure compared to controls and during the second and third exposures compared to BayK 8644. The only conditions in which Cav+/+ segments behaved significantly differently from Cav−/− segments were during the third exposure in BayK 8644 and during the second and third exposures in nicardipine. In all cases responses of Cav+/+ were larger. These results imply that Cav+/+ segments recycle the Ca2+ released by CCH from SR on each wash more efficiently than CavB- segments. This was true whether L-type Ca2+ channels were either open or closed, especially when closed. Also, the exposures to CCH facilitated recycling notably when L-type Ca 2+ channels were closed by nicardipine. This suggests that there is an alternate path for recycling calcium other than the L-type Ca + channels when caveolae are present and enhanced by exposure to CCH, presumably a store operated channel (Fig. 3). This recycling appears more effective when these channels are open.

Bottom Line: We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR).We found evidence that these channels were associated with Cav-1.These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Alberta, Edmonton, AB, Canada. edaniel@ualberta.ca

ABSTRACT
In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca (+) channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca(2+) channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.

Show MeSH
Related in: MedlinePlus