MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle

Abstract

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca(2+)-free buffer but reappeared in normal Ca(2+)-containing buffer indicating that the contraction was Ca(2+) dependent. 4-aminopyridine (4-AP), voltage-dependent K(+) channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a G(i) inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca(2+) and K(+) channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca(2+), and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.

Keywords: Ca2+; Electrical field stimulation; Esophagus; G protein; K+; On contraction; Smooth muscle.

Fig. 1

Effect of external [Ca²⁺] on the on-contraction in the presence of L-NAME by EFS. (A) Representative trace of the effects of various external [Ca²⁺] on the on-contraction in the presence of 100 µM L-NAME (left). (B) The changes in amplitudes of the contraction affected by various external [Ca²⁺] were expressed as gram (g) (right). Results are expressed as means±S.E. of 4 experiments. ^*p<0.05, ^**p<0.01 compared with 0 mM external [Ca²⁺] for on-contraction in the presence of 100 µM L-NAME (ANOVA).

Fig. 2

Effect of 4-AP on the on-contraction in the presence of L-NAME by EFS. The changes in amplitudes of the on-contractions affected by 4-AP were expressed as % of control. The contraction was significantly augmented by 4-AP. Results are expressed as means±S.E. of 6 experiments ^*p<0.05, ^**p<0.01 compared with control of on-contraction in the presence of L-NAME (ANOVA).

Fig. 3

Effect of AlF the on-contraction in the presence of L-NAME. Muscle strips were incubated with AlF for 10 min or 30 min in various concentrations and then changes of EFS-induced contractile response in presence of L-NAME were measured. AlF increased the on-contraction in the presence of L-NAME in a concentration-dependent manner. Results are expressed as means±S.E. of 5 experiments. ^*p<0.05, ^**p<0.01, compared with control of on-contraction in the presence of L-NAME for set of 2 mM AlF, ^##p<0.01 compared with control of on-contraction in the presence of L-NAME for set of 10 mM AlF(ANOVA), ^$p<0.05 compared with on-contraction at 30 min pretreatment with 2 mM of AlF (Student t-test).

Fig. 4

Effect of PTX, C3 exoenzyme and Y27632 on the on-contraction in the presence of L-NAME. (A) Muscle strips were pretreated with PTX or C3 exoenzyme for 3 hr, and then the contraction was measured. PTX significantly inhibited the contraction; in particular, co-treatment with PTX and C3 toxin additively inhibited the contraction. (B) Muscle strips were incubated with various concentration of Y27632 for 30 min before EFS, and then the contraction was measured Results are expressed as means±S.E. of 3 experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with control of the on-contraction in the presence of L-NAME (ANOVA), ^ap<0.01 compared with PTX alone, ^bp<0.01 compared with C3 exoenzyme alone (ANOVA).

Fig. 5

Effect of ML-9 or chelerythrine on the on-contraction in the presence of L-NAME. (A) Representative trace of the effect of various ML-9 on the on-contraction in the presence of L-NAME. (B) The changes in amplitudes of the contraction affected by ML-9 (10, 30, 100 µM) were expressed as % of control. (C) Chelerythrine (1, 10 µM) was added in the muscle bath before 20 min and then the contraction in response to EFS was measured. Results are expressed as means±S.E. of 3 experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with control of on-contraction in the presence of L-NAME (ANOVA).