Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Si-Eun Lee; Duck-Sun Ahn; Young-Ho Lee

doi:10.4196/kjpp.2009.13.3.241

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Lee, Ahn, and Lee: Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Original Article

Korean J Physiol Pharmacol 2009;13(3):241-249.

Published online: 17 June 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.3.241

Role of T-type Ca²⁺ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Si-Eun Lee, Duck-Sun Ahn, Young-Ho Lee

Department of Physiology, BK 21 Project for Medical Sciences, College of Medicine, Yonsei University, Seoul 120-752, Korea

Corresponding to: Young-Ho Lee, Department of Physiology, College of Medicine, Yonsei University, C.P.O. Box 8044, Seoul 120-752, Korea. (Tel) 82-2-2228-1708, (Fax) 82-2-393-0203, (E-mail) yhlee@yumc.yonsei.ac.kr

Received 27 May 20094 June 2009 Accepted 19 June 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

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (< 1 μM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α 1G, α 1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.

Keywords: Calcium channels, Nickel, Mibefradil, NNC 55-0396, Spontaneous contractility

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Fig. 1.

Effect of removing external Ca²⁺ and nifedipine on spontaneous Ca²⁺ transients and contractions. (A, B) Effect of removing external Ca²⁺ (0-Ca²⁺) and 1 μM nifedipine on spontaneous Ca²⁺ transients and contractions. Representative recoding (C) and statistical evaluation (D) showing the concentration-response curve obtained by cumulative addition of low concentrations of nifedipine. Data are expressed as relative percentage of the control (amplitude before treatment of nifedipine). Results are expressed as mean±SEM of six experiments. ∗Control vs Nifedipine (p<0.05).

Fig. 2.

Expression of mRNAs and proteins for α subunits (α1G and α1H) of T-type Ca²⁺ channel in longitudinal muscle layer of pregnant rat myometrium. Representative data of RT/PCR (A) and western blot (B). Immunoblots are representative of four independent preparations. The PCR was performed with 35 cycles and PCR products were followed by electrophoresis on a 1% agarose gel.

Fig. 3.

Dose-response curve for the effect of T-type Ca²⁺ channel blockers on the spontaneous Ca²⁺ transients and contractions. (A₁, B₁, C₁) Concentration-related reduction of the amplitude and frequency of spontaneous Ca²⁺ transients. (A₂, B₂, C₂) concentration-related reduction of the amplitude and frequency of spontaneous contractions. Mibefradil (A), NNC 55-0396 (B), and nickel (C) were added cumulatively. Data are expressed as relative percentage of control (amplitude before treatment of blockers). Results are expressed as mean±SEM of seven experiments. ∗Control vs Blockers, ∗∗Amplitude vs Frequency (p<0.05).

Fig. 4.

Representative recording for the effect of T-type Ca²⁺ channel blockers on spontaneous Ca²⁺ transients and contractions. Spontaneous Ca²⁺ transients (top) and contractions (bottom) before and during 1 μM mibefradil (A), 5 μM NNC 55-0396 (B), and 100 μM nickel (C) application. Data are representative of ten independent preparations.

Fig. 5.

Effect of the T-type Ca²⁺ channel blockers on the frequency and slope of rising phase of spontaneous Ca²⁺ transients. Superimposed spontaneous Ca²⁺ transients under control conditions and in presence of 1 μM Mibefradil (A), 5 μM NNC 55-0396 (B), and 100 μM nickel (C), respectively. Each blocker was added in the bath solution after spontaneous Ca²⁺ transients were stable. Statistical evaluation (D) showing the Ca²⁺ ratio and frequency obtained by adding mibefradil, NNC 55-0396, and nifedipine. Data are expressed as relative percentage of control. Results are expressed as mean±SEM of six experiments.

Fig. 6.

Effect of (A) nifedipine (1 μM), (B) mibefradil (1 μM), (C) NNC 55-0396 (5 μM), and (D) nickel (100 μM) on the 70 mM KCl-induced increase in [Ca²⁺]_i and force. All drugs were added for 10 min before 70 mM KCl-induced contraction. Data are representative of seven~nine independent preparations.

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