Korean J Physiol Pharmacol.  2011 Jun;15(3):129-135. 10.4196/kjpp.2011.15.3.129.

5-Hydroxytryptamine Generates Tonic Inward Currents on Pacemaker Activity of Interstitial Cells of Cajal from Mouse Small Intestine

• Shahi PK
• Choi S
• Zuo DC
• Yeum CH
• Yoon PJ
• Lee J
• Kim YD
• Park CG
• Kim MY
• Shin HR
• Oh HJ
• Jun JY

• KMID: 1448915
• DOI: http://doi.org/10.4196/kjpp.2011.15.3.129

Abstract

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular Ca2+ ([Ca2+]i) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-HT2B, 3, 4, and 7 receptors in ICC. However, SDZ 205557 (a 5-HT4 receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT3 antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT2B receptor antagonist). Based on [Ca2+]i analysis, we found that 5-HT increased the intensity of [Ca2+]i. The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-HT3, 4, and 7 receptors via [Ca2+]i mobilization and regulation of mitogen-activated protein kinases.

Keyword

Interstitial cells of Cajal (ICC); Intestinal motility; Pacemaker currents; 5-hydroxytryptamine (5-HT)

MeSH Terms

Animals
Flavonoids
Gastrointestinal Motility
Imidazoles
Interstitial Cells of Cajal
Intestine, Small
Mice
Mitogen-Activated Protein Kinases
para-Aminobenzoates
Patch-Clamp Techniques
Phenols
Pyridines
Receptor, Serotonin, 5-HT2B
Receptors, Serotonin
Receptors, Serotonin, 5-HT4
Serotonin
Sulfonamides
Flavonoids
Imidazoles
Mitogen-Activated Protein Kinases
Phenols
Pyridines
Receptor, Serotonin, 5-HT2B
Receptors, Serotonin
Receptors, Serotonin, 5-HT4
Serotonin
Sulfonamides
para-Aminobenzoates

Figure

• 

  Fig. 1. Effects of 5-HT on pacemaker potentials recorded in ICC from mouse small intestine. (A) shows the pacemaker potentials of ICC exposed to 5-HT (10 μM) in the current clamping mode (I=0). 5-HT induced membrane depolarization and inhibited the amplitude and frequency of pacemaker potential in ICC. The dot lines indicate the control resting membrane potentials levels. Responses to 5-HT are summarized in (B) and (C). Bars represent the means±SE. ∗Asterisks indicate significantly different from controls (p<0.05). RMP, resting membrane potentials.

• 

  Fig. 2. Effects of 5-HT on pacemaker currents recorded in cultured ICC from mouse small intestine. (A), (B) and (C) show pacemaker currents of ICC exposed to 5-HT (1, 10, or 100 μM) at a holding potential of –70 mV. Vertical solid line represents amplitude of pacemaker current and horizontal solid line represents duration of recording(s) pacemaker currents. The dot lines indicate the control resting current levels. (D) and (E) summarize the effects of 5-HT on pacemaker currents in ICC. Bars represent the means±SE. ∗Asterisks indicate significantly different from controls (p<0.05). Con, control.

• 

  Fig. 3. Agarose gels of the RT-PCR products of 5-HT receptor subtypes using separated ICC. Amplified cDNA prepared from pure ICC separated by magnetic cell separation and visualized in 2% gel. c-Kit – ICC marker, myosin – smooth muscle cells marker, PGP9.5 – neuronal cell marker.

• 

  Fig. 4. Effects of 5-HT receptor subtype antagonists on 5-HT-induced responses on pacemaker currents in cultured ICC of the mouse small intestine. (A) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of 5-HT4 receptor antagonist (SDZ 205557; 10 μM). (B) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of 5-HT7 receptor antagonist (SB 269970; 10 μM). (C) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of 5-HT3 receptor antagonist 3-TCM (10 μM). (D) Pacemaker currents of ICC exposed to the selective 5-HT2B receptor antagonist SB 204741 (10 μM). Responses to 5-HT in presence of different receptor antagonists are summarized in (E). Bars represent the mean values±S.E. The dotted lines indicate the zero current levels. ∗Asterisks indicate significantly different from the control (p<0.05). Con, control.

• 

  Fig. 5. Effects of 5-HT on [Ca2+]i oscillation in cultured ICC from mouse small intestine. (A) shows the basal and peak point of ICC image and (B) Ca2+ oscillation in the normal condition. (C) shows the basal and peak point of ICC image and (D) Ca2+ oscillation in the presence of 5-HT (10 μM). The interval of representative frame was 1 second and the exposure time of each frame was 500 ms.

• 

  Fig. 6. Effects of various MAPK inhibitors on 5-HT induced responses on pacemaker currents in cultured ICC of the mouse small intestine. (A) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of PD 98059, a p42/44 MAPK inhibitor (10 μM). (B) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of SB 203580, a p38 MAPK inhibitor (10 μM). (C) Pacemaker currents of ICC exposed to 5-HT (10 μM) in the presence of JNK II inhibitor (10 μM). Responses to 5-HT in presence of different MAPK inhibitors are summarized in (D). Bars represent mean values±S.E. The dotted lines indicate the zero current levels. ∗Asterisks indicate significantly different from control (p<0.05). Con, control.

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