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

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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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Effect of CaMKK inhibitor, STO-609 on carbachol-induced contraction. 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 withthe selective CaMKK inhibitor STO-609 (10 µM) and then with different concentrationsof CCh (10 nM to 100 µM). Contractile response with maximum force was measured aspeak contraction (A) and total response for first 2 min, measured as area undercurve (AUC), was considered as total contraction (B). Values are means ± SEM of 4–6experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-inducedcontraction.
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fig_006: Effect of CaMKK inhibitor, STO-609 on carbachol-induced contraction. 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 withthe selective CaMKK inhibitor STO-609 (10 µM) and then with different concentrationsof CCh (10 nM to 100 µM). Contractile response with maximum force was measured aspeak contraction (A) and total response for first 2 min, measured as area undercurve (AUC), was considered as total contraction (B). Values are means ± SEM of 4–6experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-inducedcontraction.

Mentions: Treatment of muscle strips with CaMKK inhibitor STO-609 (10 µM) for 10 min had nosignificant effect on basal tone (0.76 ± 0.10 grams versus 0.74 ± 0.10 grams withSTO-609). STO-609, however, significantly reduced contraction in response to differentconcentrations of CCh. Peak contractions (in grams) for control versus with STO-609 were0.51 ± 0.16 versus 0.32 ± 0.14 (P<0.05, n=6) at 10 nMCCh; 1.31 ± 0.20 versus 0.81 ± 0.15 (P<0.01, n=23)for 1 μM CCh; and 1.96 ± 0.37 versus 1.22 ± 0.20 (P<0.05,n=16) for 100 µM (Fig. 6AFig. 6.


Role of various kinases in muscarinic M3 receptor-mediated contraction oflongitudinal muscle of rat colon
Effect of CaMKK inhibitor, STO-609 on carbachol-induced contraction. 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 withthe selective CaMKK inhibitor STO-609 (10 µM) and then with different concentrationsof CCh (10 nM to 100 µM). Contractile response with maximum force was measured aspeak contraction (A) and total response for first 2 min, measured as area undercurve (AUC), was considered as total contraction (B). Values are means ± SEM of 4–6experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-inducedcontraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862207&req=5

fig_006: Effect of CaMKK inhibitor, STO-609 on carbachol-induced contraction. 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 withthe selective CaMKK inhibitor STO-609 (10 µM) and then with different concentrationsof CCh (10 nM to 100 µM). Contractile response with maximum force was measured aspeak contraction (A) and total response for first 2 min, measured as area undercurve (AUC), was considered as total contraction (B). Values are means ± SEM of 4–6experiments and each experimental value derived from several strips.**P<0.05 significant inhibition of CCh-inducedcontraction.
Mentions: Treatment of muscle strips with CaMKK inhibitor STO-609 (10 µM) for 10 min had nosignificant effect on basal tone (0.76 ± 0.10 grams versus 0.74 ± 0.10 grams withSTO-609). STO-609, however, significantly reduced contraction in response to differentconcentrations of CCh. Peak contractions (in grams) for control versus with STO-609 were0.51 ± 0.16 versus 0.32 ± 0.14 (P<0.05, n=6) at 10 nMCCh; 1.31 ± 0.20 versus 0.81 ± 0.15 (P<0.01, n=23)for 1 μM CCh; and 1.96 ± 0.37 versus 1.22 ± 0.20 (P<0.05,n=16) for 100 µM (Fig. 6AFig. 6.

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