Voltage-dependent Ca2+ Current Identified in Freshly Isolated Interstitial Cells of Cajal (ICC) of Guinea-pig Stomach

Young Chul Kim; Hikaru Suzuki; Wen-Xie Xu; Hikaru Hashitani; Woong Choi; Hyo-Yung Yun; Seon-Mee Park; Sei Jin Youn; Sang-Jeon Lee; Sang Jin Lee

doi:10.4196/kjpp.2008.12.6.323

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Kim, Suzuki, Xu, Hashitani, Choi, Yun, Park, Youn, Lee, and Lee: Voltage-dependent Ca2+ Current Identified in Freshly Isolated Interstitial Cells of Cajal (ICC) of Guinea-pig Stomach

Original Article

Korean J Physiol Pharmacol 2008;12(6):323-330.

Published online: 17 December 2008

DOI: https://doi.org/10.4196/kjpp.2008.12.6.323

Voltage-dependent Ca²⁺ Current Identified in Freshly Isolated Interstitial Cells of Cajal (ICC) of Guinea-pig Stomach

Young Chul Kim¹, Hikaru Suzuki², Wen-Xie Xu³, Hikaru Hashitani², Woong Choi⁴, Hyo-Yung Yun⁵, Seon-Mee Park⁶, Sei Jin Youn⁶, Sang-Jeon Lee⁵, Sang Jin Lee¹

¹Department of Physiology, Chungbuk National University, College of Medicine, Cheongju 361-763, Korea

²Department of Physiology, Nagoya City University Medical School, Nagoya 467-8601, Japan

³Department of Physiology, College of Medicine, Shanghai Jiaotong University, Shanghai 200240, P.R. China

⁴Department of Pharmacology, Chungbuk National University, College of Medicine, Cheongju 361-763, Korea

⁵Department of Surgery, Chungbuk National University, College of Medicine, Cheongju 361-763, Korea

⁶Department of Internal Medicine, Chungbuk National University, College of Medicine, Cheongju 361-763, Korea

Corresponding to: Young Chul Kim, Department of Physiology Chungbuk National University College of Medicine, 12, Gaeshindong, Heungduk-gu, Cheongju 361-763, Korea. (Tel) 82-43-261−2859, (Fax) 82-43-261-2859, (E-mail) physiokyc@chungbuk.ac.kr

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

The properties of voltage dependent Ca²⁺ current (VDCC) were investigated in interstitial cells of Cajal (ICC) distributed in the myenteric layer (ICC-MY) of guinea-pig antrum. In tissue, ICC-MY showed c-Kit positive reactions and produced driving potentials with the amplitude and frequency of about 62 mV and 2 times min⁻¹, respectively, in the presence of 1 μM nifedipine. Single ICC-MY isolated by enzyme treatment also showed c-Kit immunohistochemical reactivity. These cells were also identified by generation of spontaneous inward current under K⁺-rich pipette solution. The voltage clamp experiments revealed the amplitude of – 329 pA inward current at irregular frequency. With Cs+-rich pipette solution at V_h=−80 mV, ICC-MY produced voltage-dependent inward currents (VDIC), and nifedipine (1 μM) blocked VDIC. Therefore, we successfully isolated c-Kit positive single ICC from guinea-pig stomach, and found that ICC-MY potently produced dihydropiridine sensitive L-type voltage-dependent Ca²⁺ currents (VDCC_L).

Keywords: Gastrointestinal (GI) tract, Stomach, Interstitial cells of Cajal (ICC), Voltage-dependent Ca²⁺ current (VDCC)

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Fig. 1.

Identification of ICC in the antral region of guinea-pig stomach by c-Kit immunohistochemical activity. (A) reveals the network of ICC-MY in myenteric border of guinea-pig gastric antrum. In (A), arrow head and arrow indicate cell body and processes from ICC-MY, respectively. (B) ICC-IM in circular muscle layers is shown. Scale bars in (A) and (B) are 40 μM.

Fig. 2.

Pacemaker potential of ICC-MY in myenteric area of guinea-pig gastric antrum. Spontaneous electrical activity of ICC-MY in guinea-pig antrum was recorded in the presence of 1 μM nifedipine. (A) and (B) Driving potential from ICC-MY was recorded in the presence of 1 μM nifedipine. (B) Expended event of pacemaker potential was displayed.

Fig. 3.

Identification of freshly isolated single ICC-MY. Single ICC was isolated by enzyme treatment from myenteric border of gastric antrum in guinea-pig. (A) ICC-MY with many processes from myenteric border of guinea-pig gastric antrum is shown. ICC expressed c-Kit activity (B). (B) shows phenotype (left; arrow head) and c-Kit immunohistochemical reactivity (right; arrow head) of ICC-MY. Lower figures of (B) represent magnification of the upper (B). However, gastric smooth muscle cells (arrows) did not express c-Kit activity (right upper panel). Scale bars in (A) and (B) indicate 40 μM. Note: The left side of panel (B) is phase contrast image from guinea-pig gastric ICC. The right side of panel (B) is c-Kit immunoreactivity image.

Fig. 4.

Spontaneous inward current recorded from freshly isolated single gastric ICC from guinea-pig antrum. Using the conventional whole cell patch-clamp technique, we observed spontaneous inward current in freshly isolated single ICC from guinea-pig gastric antrum. (A) Spontaneous inward current was recorded irregularly under K⁺-rich pipette solution. (B) and (C) ICC showed spontaneous inward current in the presence (C) and absence (B) of 1 μM nifedipine under Cs⁺-rich pipette solution. The data from (B) and (C) are summarized in (D).

Fig. 5.

Voltage-dependent Ca²⁺ currents (VDCC) in freshly isolated single ICC from guinea-pig antrum. At a holding potential of −80 mV, spontaneous inward current was recorded in freshly isolated single ICC as shown in Fig. 4B. From these cells, voltage-dependent inward current (VDIC) was recorded by step pulses. (A) Currents were elicited by 20 mV depolarizing step pulses from a holding potential of −80 mV to +40 mV (voltage protocol is shown in inset). (B) Currents were elicited by voltage steps from a holding potential of −80 mV to 0 mV. The peak amplitudes generated were compared before and after nifedipine (1 μM) treatment. (C) Current-voltage relationships of before (filled circles) and after (open circles) treatment of 1 μM nifedipine are shown. (D) The blocking effect of nifedipine on VDIC is summarized.

Fig. 6.

Voltage-dependent activation and inactivation kinetics of VDIC in freshly isolated single ICC from guinea-pig antrum. Voltage dependences of activation and inactivation curves of VDIC are shown as a plot of normalized peak currents. (A) Raw current traces recorded for the analysis of steady-state inactivation before (left pannel) and after (right pannel) treatment of 1 μM nifedipine are shown. Note that generation of spontaneous inward current during the recording of VDCC (see arrows in (A)). (B) The steady-state activation and inactivation curves in the absence of nifedipine are plotted and displayed. Filled circles and open circles represent the activation and the inactivation curve, respectively. In the absence of nifedipine, a half-activation and -inactivation voltage (V_0.5) were −1±2.9 mV and −48±11 mV, respectively. And slope factors (k) of them were 8±1.6 and 9±1.2, respectively.

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