The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

Jun Hong Park; Hyun Sik Kim; Sun Young Park; Chaeuk Im; Ji Hoon Jeong; In Kyeom Kim; Uy Dong Sohn

doi:10.4196/kjpp.2009.13.5.393

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Park, Kim, Park, Im, Jeong, Kim, and Sohn: The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

Original Article

Korean J Physiol Pharmacol 2009;13(5):393-400.

Published online: 18 February 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.5.393

The Influences of G Proteins, Ca²⁺, and K⁺ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

Jun Hong Park^1,^∗, Hyun Sik Kim^1,^∗, Sun Young Park¹, Chaeuk Im¹, Ji Hoon Jeong², In Kyeom Kim³, Uy Dong Sohn^1,

¹College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea

²School of Medicine, Chung-Ang University, Seoul 156-756, Korea

³School of Medicine, Kyungpook National University, Daegu 700-422, Korea

Corresponding to: Uy Dong Sohn, Department of Pharmacology, College of Pharmacy, Chung-Ang University, 221, Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea. (Tel) 82-2-820-5614, (Fax) 82-2-826-8752, (E-mail) udsohn@cau.ac.kr

^∗ The first two authors contributed equally to this article.

Received 15 September 2009 Revised 24 September 2009 Accepted 12 October 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 μM) and atropine (1 μM). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of ‘on’ and ‘off’ contraction and the effects of G-proteins, phospholipase, and K⁺ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a G_i inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, G_s inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K⁺ channel opener) decreased these contractions, and tetraethylammonium (TEA, K⁺_Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca²⁺ channel may be activated by G-protein α subunits. Furthermore, K⁺_Ca– channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca²⁺ channel and to investigate the effects of other K⁺ channels on EFS-induced on and off contractions.

Keywords: EFS, Cat esophageal, Circular smooth muscle, NO, L-type Ca²⁺ channel

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Fig. 1.

Identification of ‘on’ and ‘off’ contraction. (A) ‘Off’ contractions were evoked by electrical field stimulation (EFS) in esophageal circular smooth muscle. (B) L-NAME (100 μM, a NOS inhibitor) was pretreated for 10 min, thus the contractions was provoked during EFS (‘On’ contractions).

Fig. 2.

Effects of atropine and tetrodotoxin on EFS-induced contractions. ‘On’ and ‘off’ contractions were abolished by tetrodotoxin (1 μM, administered 15 min before EFS) and atropine (1 μM, 15 min before EFS). Values are expressed as means± SEM. ^∗∗p<0.01 versus control value.

Fig. 3.

Effect of Ca²⁺ blocker on EFS-induced contractions. Nimodipine (an L-type Ca²⁺ channel blocker) inhibited EFS-induced ‘off’ and ‘on’ contractions in a concentration-dependent manner, which suggested that Ca²⁺ channel contributes to the activation of muscle contraction in response to EFS. Values are expressed as means± SEM. ^∗p<0.05, ^∗∗p<0.01 versus untreated controls.

Fig. 4.

Effects of PTX (A) and CTX (B) on response to EFS. EFS-induced contractions were significantly inhibited by PTX (800 ng/ml, a Gi inactivator, n=5) after 2 hr of pretreatment, which indicated that Gi protein mediated EFS-induced muscle contraction. Furthermore, EFS-induced contractions were also significantly inhibited by CTX (2 μg/ml, Gs inactivator, n=5), which indicated that Gs protein mediated EFS-induced muscle contraction. Values are expressed as means± SEM. ^∗∗p<0.01 versus untreated controls.

Fig. 5.

Effects of phospholipases inhibitors on EFS-induced contractions. D609 (10 μM, a phosphatidylcholine-PLC inhibitor), pCMB (10 μM, a PLD inhibitor), U73122 (10 μM, a phosphatidylinositol-PLC inhibitor), and DEDA (10 μM, a phospholipase A₂) had no effect on EFS-induced (A) ‘off’ or (B) ‘on’ contractions. Values are expressed as means± SEMs.

Fig. 6.

Effect of a K⁺ Channel opener on EFS-induced contractions. Pinacidil (a K⁺ Channel opener) decreased the amplitude of both ‘on’ and ‘off’ EFS-induced contractions, which suggested that K⁺ efflux through K⁺ channel reduced response to EFS. Values are expressed as means±SEMs. ^∗p<0.05, ^∗∗p<0.01 versus untreated controls.

Fig. 7.

Effect of K⁺_ca- Channel Blocker on response to EFS. EFS-induced ‘off’ and ‘on’ contractions were found to be augmented by TEA (a Ca²⁺-dependent K⁺ channel blocker). Values are expressed as means±SEMs. ^∗p<0.05, ^∗∗p<0.01 versus untreated controls.

Fig. 8.

The signaling diagram induced by EFS in cat esophageal muscle. The ‘off’ contraction (A) is probably mediated via NO-mediated G kinase dependent relaxation. Interestingly, our findings suggest that ‘on’ and ‘off’ (B) contractions are mediated by Gi/s proteins via the opening of L-type Ca²⁺ channel, and that these contractions are augmented by the blocking of Ca²⁺-dependent K⁺ channel. However, it remains to be determined which muscarinic receptor and whether contractile proteins and intracellular Ca²⁺ stores are involved in EFS-induced ‘on’ and ‘off’ contraction.

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