• 대한의생명과학회지
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• 제18권3호
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• pp.218-226
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• 2012

Dopamine is an enteric neurotransmitter that regulates gastrointestinal motility. This study was done to investigate whether dopamine modulates spontaneous pacemaker activity in cultured interstitial cells of Cajal (ICCs) from mouse using whole cell patch clamp technique, RT-PCR and live $Ca^{2+}$ imaging analysis. ICCs generate pacemaker inward currents at a holding potential of -70 mV and generate pacemaker potentials in current-clamp mode. Dopamine did not change the frequency and amplitude of pacemaker activity in small intestinal ICCs. On the contrary dopamine reduced the frequency and amplitude of pacemaker activity in large intestinal ICCs. RT-PCR analysis revealed that Dopamine2 and 4-receptors are expressed in c-Kit positive ICCs. Dopamine2 and 4 receptor agonists inhibited pacemaker activity in large intestinal ICCs mimicked those of dopamine. Domperidone, dopamine2 receptor antagonist, increased the frequency of pacemaker activity of large intestinal ICCs. In $Ca^{2+}$-imaging, dopamine inhibited spontaneous intracellular $Ca^{2+}$ oscillations of ICCs. These results suggest that dopamine can regulate gastrointestinal motility through modulating pacemaker activity of large intestinal ICCs and dopamine effects on ICCs are mediated by dopamine2 receptor and intracellular $Ca^{2+}$ modulation.

• The Korean Journal of Physiology and Pharmacology
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• 제10권1호
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• pp.7-11
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• 2006

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, $23{\pm}1.5$ mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.

• The Korean Journal of Physiology and Pharmacology
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• 제9권4호
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• pp.203-207
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• 2005

Electrical rhythmicity in the gastrointestinal (GI) muscles is generated by pacemaker cells, known as interstitial cells of Cajal (ICC). In the present study, we investigated the effect of external divalent cations on pacemaking activity in cultured ICC from murine small intestine by using whole-cell patch clamp techniques. ICC generated pacemaker currents under a voltage clamp or electrical pacemaker potentials under a current clamp, and showed a mean amplitude of $-500{\pm}50$ pA or $30{\pm}1$ mV and the frequency of $18{\pm}2$ cycles/min. Treatments of the cells with external 0 mM $Ca^{2+}$ stopped pacemaking activity of ICC. In the presence of 2 mM $Ca^{2+}$, 0 mM external $Mg^{2+}$ depolarized the resting membrane potential, and there was no change in the frequency of pacemaking activity. However, 10 mM external $Mg^{2+}$ decreased the frequency of pacemaking activity ($6.75{\pm}1$ cycles/min, n=5). We replaced external 2 mM $Ca^{2+}$ with equimolar $Ba^{2+}$, $Mn^{2+}$ and $Sr^{2+}$, and they all developed inward current in the sequence of $Ba^{2+}$>$Mn^{2+}$>$Sr^{2+}$. Also the frequency of the pacemaking activity was stopped or irregulated. We investigated the effect of 10 mM $Ba^{2+}$, $Mn^{2+}$ and $Sr^{2+}$ on pacemaking activity of ICC in the presence of external 0 mM $Mg^{2+}$, and found that 10 mM $Ba^{2+}$ and $Mn^{2+}$ induced large inward current and stopped the pacemaking activity of ICC (n=5). Interestingly, 10 mM $Sr^{2+}$ induced small inward current and potentiated the amplitude of pacemaking activity of ICC (n=5). These results indicate that extracellular $Ca^{2+}$ and $Mg^{2+}$ are requisite for the pacemaking activity of ICC.

• Animal cells and systems
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• 제16권5호
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• pp.376-384
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• 2012

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of transient receptor potential melastatin 7 (TRPM7) channels. Here, we showed evidence suggesting that TRPM7 channels play an important role in ginseng total saponin (GTS)-mediated cellular injury. The combination techniques of electrophysiology, pharmacological analysis, small interfering RNA (siRNA) method and cell death assays were used. GTS depolarized the resting membrane potentials and decreased the amplitude of pacemaker potentials in cultured interstitial cells of Cajal (ICCs) in gastrointestinal (GI) tract. The TRPM7-like currents in single ICCs and the overexpressing TRPM7 in HEK293 cells were inhibited by GTS. However, GTS had no effect on $Ca^{2+}$-activated $Cl^-$ conductance. GTS inhibited the survival of human gastric (AGS) and brea (MCF-7) adenocarcinoma cells. Also, GTS inhibited the TRPM7-like currents in AGS and MCF-7 cells. The GTS-mediated cytotoxicity was inhibited by TRPM7-specific siRNA. In addition, we showed that overexpression of TRPM7 channels in HEK293 cells was inhibited by GTS. Thus, TRPM7 channels are involved in GTS-mediated cell death in AGS and MCF-7 cells, and these channels may represent a novel target for physiological disorders where GTS plays an important role.

• Journal of Korean Neurosurgical Society
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• 제42권3호
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• pp.205-210
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• 2007

Objective : Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. Methods : HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using software-searching method. Results : HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. Conclusion : Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.

• Molecules and Cells
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• 제20권3호
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• pp.435-441
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• 2005

Classical transient receptor potential channels (TRPCs) are thought to be candidates for the nonselective cation channels (NSCCs) involved in pacemaker activity and its neuromodulation in murine stomach smooth muscle. We aimed to determine the role of TRPC4 in the formation of NSCCs and in the generation of slow waves. At a holding potential of -60 mV, $50{\mu}M$ carbachol (CCh) induced $I_{NSCC}$ of amplitude [$500.8{\pm}161.8pA$ (n = 8)] at -60 mV in mouse gastric smooth muscle cells. We investigated the effects of commercially available antibodies to TRPC4 on recombinant TRPC4 expressed in HEK cells and CCh-induced NSCCs in gastric smooth muscle cells. TRPC4 currents in HEK cells were reduced from $1525.6{\pm}414.4pA$ (n = 8) to $146.4{\pm}83.3pA$ (n = 10) by anti-TRPC4 antibody and $I_{NSCC}$ amplitudes were reduced from $230.9{\pm}36.3pA$ (n = 15) to $49.8{\pm}11.8pA$ (n = 9). Furthermore, $I_{NSCC}$ in the gastric smooth muscle cells of TRPC4 knockout mice was only $34.4{\pm}10.4pA$ (n = 8) at -60 mV. However, slow waves were still present in the knockout mice. Our data suggest that TRPC4 is an essential component of the NSCC activated by muscarinic stimulation in the murine stomach.