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Role of various kinases in muscarinic M3 receptor-mediated contraction oflongitudinal muscle of rat colon

View Article: PubMed Central - PubMed

ABSTRACT

The longitudinal muscle layer in gut is the functional opponent to the circular musclelayer during peristalsis. Differences in innervation of the layers allow for thecontraction of one layer concurrently with the relaxation of the other, enabling thepassage of gut contents in a controlled fashion. Differences in development have given thecells of the two layers differences in receptor populations, membrane lipid handling, andcalcium handling profiles/behaviors. The contractile activity of the longitudinal muscleis largely mediated by cholinergic neural input from myenteric plexus. Activation ofmuscarinic receptors leads to rapid activation of several kinases including MLC kinase,ERK1/2, CaMKII and Rho kinase. Phosphorylation of myosin light chain (MLC20) byMLC kinase (MLCK) is a prerequisite for contraction in both circular and longitudinalmuscle cells. In rat colonic longitudinal muscle strips, we measured muscarinicreceptor-mediated contraction following incubation with kinase inhibitors. Basal tensionwas differentially regulated by Rho kinase, ERK1/2, CaMKII and CaMKK. Selective inhibitorsof Rho kinase, ERK1/2, CaMKK/AMPK, and CaMKII each reduced carbachol-induced contractionin the innervated muscle strips. These inhibitors had no direct effect on MLCK activity.Thus unlike previously reported for isolated muscle cells where CaMKII and ERK1/2 are notinvolved in contraction, we conclude that the regulation of carbachol-induced contractionin innervated longitudinal muscle strips involves the interplay of Rho kinase, ERK1/2,CaMKK/AMPK, and CAMKII.

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Contractile response to carbachol is mediated via m3 receptors. Longitudinalmuscle strips of rat colon were placed in an organ bath and subjected to 1 g ofbasal tension. After 30 min of equilibration, strips were incubated for 15 min with1 µM of the m2 receptor antagonist methoctramine or 1 µM of the m3 receptorantagonist 4-DAMP and then with CCh (1 µM). Contractile response with maximum forcewas measured as peak contraction (A) and total response for first 2 min, measured asarea under curve (AUC), was considered as total contraction (B). Carbachol elicitedcontraction was selectively blocked by 4-DAMP. Values are means ± SEM of 4experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-induced contraction.The inset illustrates an original tracing showing abolition of carbachol-inducedcontraction by 1 µM 4-DAMP but not by 1 µM tetrodotoxin (TTX) or 1 µM methoctramine."W" indicates when the preparation was washed with fresh Kreb's buffer after peakcontraction. Horizontal bar indicates time in minutes and the vertical barillustrates force in grams.
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fig_002: Contractile response to carbachol is mediated via m3 receptors. Longitudinalmuscle strips of rat colon were placed in an organ bath and subjected to 1 g ofbasal tension. After 30 min of equilibration, strips were incubated for 15 min with1 µM of the m2 receptor antagonist methoctramine or 1 µM of the m3 receptorantagonist 4-DAMP and then with CCh (1 µM). Contractile response with maximum forcewas measured as peak contraction (A) and total response for first 2 min, measured asarea under curve (AUC), was considered as total contraction (B). Carbachol elicitedcontraction was selectively blocked by 4-DAMP. Values are means ± SEM of 4experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-induced contraction.The inset illustrates an original tracing showing abolition of carbachol-inducedcontraction by 1 µM 4-DAMP but not by 1 µM tetrodotoxin (TTX) or 1 µM methoctramine."W" indicates when the preparation was washed with fresh Kreb's buffer after peakcontraction. Horizontal bar indicates time in minutes and the vertical barillustrates force in grams.

Mentions: Repeated measurements of peak contraction and area under the curve in response to 1 µMCCh were conducted on strips following wash for 15 min in Krebs buffer and contractionswere calculated as percentage of initial contraction before wash. There were nosignificant differences in either peak contraction or AUC with repeated measurements.Following a 15 min incubation after initial contraction, peak contraction was 98.29% ±3.99% (n=13, 5 animals) and the AUC for 2 min was 102.1% ± 1.88%(n=13, 5 animals) of the initial contraction (Fig. 2A and 2BFig. 2.


Role of various kinases in muscarinic M3 receptor-mediated contraction oflongitudinal muscle of rat colon
Contractile response to carbachol is mediated via m3 receptors. Longitudinalmuscle strips of rat colon were placed in an organ bath and subjected to 1 g ofbasal tension. After 30 min of equilibration, strips were incubated for 15 min with1 µM of the m2 receptor antagonist methoctramine or 1 µM of the m3 receptorantagonist 4-DAMP and then with CCh (1 µM). Contractile response with maximum forcewas measured as peak contraction (A) and total response for first 2 min, measured asarea under curve (AUC), was considered as total contraction (B). Carbachol elicitedcontraction was selectively blocked by 4-DAMP. Values are means ± SEM of 4experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-induced contraction.The inset illustrates an original tracing showing abolition of carbachol-inducedcontraction by 1 µM 4-DAMP but not by 1 µM tetrodotoxin (TTX) or 1 µM methoctramine."W" indicates when the preparation was washed with fresh Kreb's buffer after peakcontraction. Horizontal bar indicates time in minutes and the vertical barillustrates force in grams.
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Related In: Results  -  Collection

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fig_002: Contractile response to carbachol is mediated via m3 receptors. Longitudinalmuscle strips of rat colon were placed in an organ bath and subjected to 1 g ofbasal tension. After 30 min of equilibration, strips were incubated for 15 min with1 µM of the m2 receptor antagonist methoctramine or 1 µM of the m3 receptorantagonist 4-DAMP and then with CCh (1 µM). Contractile response with maximum forcewas measured as peak contraction (A) and total response for first 2 min, measured asarea under curve (AUC), was considered as total contraction (B). Carbachol elicitedcontraction was selectively blocked by 4-DAMP. Values are means ± SEM of 4experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-induced contraction.The inset illustrates an original tracing showing abolition of carbachol-inducedcontraction by 1 µM 4-DAMP but not by 1 µM tetrodotoxin (TTX) or 1 µM methoctramine."W" indicates when the preparation was washed with fresh Kreb's buffer after peakcontraction. Horizontal bar indicates time in minutes and the vertical barillustrates force in grams.
Mentions: Repeated measurements of peak contraction and area under the curve in response to 1 µMCCh were conducted on strips following wash for 15 min in Krebs buffer and contractionswere calculated as percentage of initial contraction before wash. There were nosignificant differences in either peak contraction or AUC with repeated measurements.Following a 15 min incubation after initial contraction, peak contraction was 98.29% ±3.99% (n=13, 5 animals) and the AUC for 2 min was 102.1% ± 1.88%(n=13, 5 animals) of the initial contraction (Fig. 2A and 2BFig. 2.

View Article: PubMed Central - PubMed

ABSTRACT

The longitudinal muscle layer in gut is the functional opponent to the circular musclelayer during peristalsis. Differences in innervation of the layers allow for thecontraction of one layer concurrently with the relaxation of the other, enabling thepassage of gut contents in a controlled fashion. Differences in development have given thecells of the two layers differences in receptor populations, membrane lipid handling, andcalcium handling profiles/behaviors. The contractile activity of the longitudinal muscleis largely mediated by cholinergic neural input from myenteric plexus. Activation ofmuscarinic receptors leads to rapid activation of several kinases including MLC kinase,ERK1/2, CaMKII and Rho kinase. Phosphorylation of myosin light chain (MLC20) byMLC kinase (MLCK) is a prerequisite for contraction in both circular and longitudinalmuscle cells. In rat colonic longitudinal muscle strips, we measured muscarinicreceptor-mediated contraction following incubation with kinase inhibitors. Basal tensionwas differentially regulated by Rho kinase, ERK1/2, CaMKII and CaMKK. Selective inhibitorsof Rho kinase, ERK1/2, CaMKK/AMPK, and CaMKII each reduced carbachol-induced contractionin the innervated muscle strips. These inhibitors had no direct effect on MLCK activity.Thus unlike previously reported for isolated muscle cells where CaMKII and ERK1/2 are notinvolved in contraction, we conclude that the regulation of carbachol-induced contractionin innervated longitudinal muscle strips involves the interplay of Rho kinase, ERK1/2,CaMKK/AMPK, and CAMKII.

No MeSH data available.


Related in: MedlinePlus