Pawan Kumar Shahi

The possible roles of hyperpolarization-activated cyclic nucleotide channels in regulating pacemaker activity in colonic interstitial cells of Cajal

BackgroundHyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs.MethodsWe performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon.ResultsSQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations.ConclusionsHCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

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

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 Ca(2+) ([Ca(2+)](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-HT(2B, 3, 4, and 7) receptors in ICC. However, SDZ 205557 (a 5-HT(4) receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT(3) antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT(2B) receptor antagonist). Based on [Ca(2+)](i) analysis, we found that 5-HT increased the intensity of [Ca(2+)](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-HT(3, 4, and 7) receptors via [Ca(2+)](i) mobilization and regulation of mitogen-activated protein kinases.

Interplay of hydrogen sulfide and nitric oxide on the pacemaker activity of interstitial cells of cajal from mouse small intestine.

We studied whether nitric oxide (NO) and hydrogen sulfide (H2S) have an interaction on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of NO and H2S on pacemaker activities were investigated by using the whole-cell patch-clamp technique and intracellular Ca2+ analysis at 30℃ in cultured mouse ICC. Exogenously applied (±)-S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, or sodium hydrogen sulfide (NaHS), a donor of H2S, showed no influence on pacemaker activity (potentials and currents) in ICC at low concentrations (10 µM SNAP and 100 µM NaHS), but SNAP or NaHS completely inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction at high concentrations (SNAP 100 µM and NaHS 1 mM). Co-treatment with 10 µM SNAP plus 100 µM NaHS also inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction. ODQ, a guanylate cyclase inhibitor, or glibenclamide, an ATP-sensitive K+ channel inhibitor, blocked the SNAP+NaHS-induced inhibition of pacemaker currents in ICC. Also, we found that SNAP+NaHS inhibited the spontaneous intracellular Ca2+ ([Ca2+]i) oscillations in cultured ICC. In conclusion, this study describes the enhanced inhibitory effects of NO plus H2S on ICC in the mouse small intestine. NO+H2S inhibited the pacemaker activity of ICC by modulating intracellular Ca2+. These results may be evidence of a physiological interaction of NO and H2S in ICC for modulating gastrointestinal motility.

Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase

AIM To investigate lipopolysaccharide (LPS) related signal transduction in interstitial cells of Cajal (ICCs) from mouse small intestine. METHODS For this study, primary culture of ICCs was prepared from the small intestine of the mouse. LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique. Immunocytochemistry was used to examine the expression of the proteins in ICCs. RESULTS LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809, a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-L-arginine Methyl Ester, an inhibitor of nitric oxide (NO) synthase. Toll-like receptor 4, inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs. Catalase (antioxidant agent) had no action on LPS-induced action in ICCs. LPS actions were blocked by nuclear factor κB (NF-κB) inhibitor, actinomycin D (a gene transcription inhibitor), PD 98059 (a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases (MAPK) inhibitor]. SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO. CONCLUSION LPS inhibit the pacemaker currents in ICCs via prostaglandin E2- and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-κB are implicated in these actions.

Action of Lipopolysaccharide on Interstitial Cells of Cajal from Mouse Small Intestine

Background and Purpose: Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs. Methods: We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine. Results: LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4+/+ mice was not present in TLR4–/– mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K+ channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E2 (PGE2)-EP2 receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs. Conclusions: LPS can activate ICCs to release NO and PGE2 through TLR4 activation. The released NO and PGE2 inhibit pacemaker currents by activating ATP-sensitive K+ channels. The LPS actions are mediated by tyrosine kinase signaling pathways.

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na+-free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca2+-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca2+ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca2+-release through neurotensin receptor1.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3′,5′-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca2+ imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K+ channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca2+ ([Ca2+]i) oscillations revealed that ODQ and L-NAME increased [Ca2+]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca2+]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca2+ release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P1,3,4,5 agonist) and SEW 2871 (an S1P1 agonist) had no effects on pacemaker activity. Suramin (an S1P3 antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P2 antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P2 in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH2-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca2+-free solution or thapsigargin (a Ca2+-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca2+ ([Ca2+]i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca2+]i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P2 via regulation of external and internal Ca2+ and mitogenactivated protein kinase activation.

Capsaicin Inhibits the Spontaneous Pacemaker Activity in Interstitial Cells of Cajal From the Small Intestine of Mouse

Background/Aims Capsaicin (8-methyl-N-vanillyl-6-ninenamide), a compound found in hot peppers, has been reported to have different physiological actions on different cell types. Not much work has been done about the effect of capsaicin on the function of interstitial cells of Cajal (ICC). In the present study, we examined the action of external application of capsaicin on pacemaker activity in the cultured ICC from the small intestine of mouse. Methods We investigated the effect of capsaicin on pacemaker currents in cultured ICC from the small intestine of mouse using a whole cell patch-clamp technique and Ca2+-imaging analysis. Results When capsaicin was applied externally to the pacemaker generating ICC, it completely inhibited the pacemaker potential under current-clamp mode (I = 0) and the pacemaker current under voltage-clamp mode at a -70 mV of holding potentials. The effect of capsaicin on pacemaker activity in ICC was shown dose dependently. The effect of capsaicin was not through the transient receptor potential of the vanilloid type 1 (TRPV1) channel as capsazepine did not block the effect of capsaicin. L-NAME, an inhibitor of nitric oxide synthase, also did not block the capsaicin-induced effects. When the action of capsaicin was examined in the intracellular calcium oscillation, it completely abolished the calcium oscillation. Conclusions These results prove that the capsaicin has the inhibitory effects on the ICC which is carried out neither through TRPV channel nor the nitric oxide production. Intracellular Ca2+ was also an important target for actions of capsaicin on ICC.

epigallocatechin gallate inhibits the pacemaker activity of interstitial cells of cajal of mouse small intestine.

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at 30 and Ca(2+) image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive K(+) channel blocker and TEA, a Ca(2+)-activated K(+) channel blocker. Also, we found that EGCG inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced [Ca(2+)](i) oscillations by cAMP-, cGMP-, ATP-sensitive K+ channel-independent manner.