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

BACKGROUND: Hyperpolarization-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. METHODS: We performed whole-cell patch clamp, RT-PCR, and Ca(2+)-imaging in cultured ICCs from mouse mid colon. RESULTS: SQ-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 Ca(2+) [Ca(2+)](i) oscillations, rolipram and 8-bromo-cAMP increased [Ca(2+)](i) oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca(2+)](i) oscillations. CONCLUSIONS: HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity

Effects of Ca2+-Activated Cl- Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal

Background/Aims: Anoctamin1 (Ca2+-activated Cl- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca2+-activated Cl- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. Methods: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca2+ ([Ca2+]i) imaging were performed in cultured ICC obtained from mice. Results: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca2+-activated Cl- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca2+]i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca2+ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl2 (30 µM) (T-type Ca2+ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. Conclusion: These results indicate that though ANO1 and T-type Ca2+ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon

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

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2–MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70

Effects of prostaglandin F2α on small intestinal interstitial cells of Cajal

AIM: To explore the role of prostaglandin F2α (PGF2α)) on pacemaker activity in interstitial cells of Cajal (ICC) from mouse small intestine

Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression ▿ ‡

Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κB complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival

Phentolamine Inhibits the Pacemaker Activity of Mouse Interstitial Cells of Cajal by Activating ATP-Sensitive K(+) Channels

The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATP-sensitive K(+) channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30 degrees C in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous [Ca(2+)](i) oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive K(+) channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive K(+) channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular Ca(2+) signaling.Epperson A, 2000, AM J PHYSIOL-CELL PH, V279, pC529Deka DK, 2004, EUR J PHARMACOL, V492, P13, DOI 10.1016/j.ejphar.2004.03.057Silva LFG, 2005, INT J IMPOT RES, V17, P27, DOI 10.1038/sj.ijir.3901269Rodrigo GC, 2005, CURR PHARM DESIGN, V11, P1915Jun JY, 2005, BRIT J PHARMACOL, V144, P242, DOI 10.1038/sj.bjp.0706074Kito Y, 2005, AM J PHYSIOL-CELL PH, V288, pC710, DOI 10.1152/ajpcell.00361.2004Choi S, 2006, CELL PHYSIOL BIOCHEM, V18, P187Sanders KM, 2006, ANNU REV PHYSIOL, V68, P307, DOI 10.1146/annurev.physiol.68.040504.094718Ward SM, 2006, J PHYSIOL-LONDON, V576, P675, DOI 10.1113/jphysiol.2006.117390Park CG, 2007, N-S ARCH PHARMACOL, V376, P175, DOI 10.1007/s00210-007-0187-1Hoy M, 2001, J BIOL CHEM, V276, P924Vemulapalli S, 2001, FUNDAM CLIN PHARM, V15, P1Jun JY, 2001, AM J PHYSIOL-CELL PH, V281, pC857Brayden JE, 2002, CLIN EXP PHARMACOL P, V29, P312Jun JY, 2004, BRIT J PHARMACOL, V141, P670, DOI 10.1038/sj.bjp.0705665Mannhold R, 2004, MED RES REV, V24, P213, DOI 10.1002/med.10060Suzuki H, 2000, J PHYSIOL-LONDON, V525, P105Proks P, 1997, P NATL ACAD SCI USA, V94, P11716Kubo M, 1997, J PHYSIOL-LONDON, V503, P489Shepherd RM, 1996, BRIT J PHARMACOL, V119, P911MURPHY ME, 1995, J PHYSIOL-LONDON, V486, P47HUIZINGA JD, 1995, NATURE, V373, P347Rustenbeck I, 1995, EXP CLIN ENDOCR DIAB, V103, P42WARD SM, 1994, J PHYSIOL-LONDON, V480, P91ZHANG L, 1994, AM J PHYSIOL, V267, pG494WILDE AAM, 1994, CARDIOVASC RES, V28, P847QUAYLE JM, 1994, J PHYSIOL-LONDON, V475, P9JONAS JC, 1992, BRIT J PHARMACOL, V107, P8SCHWIETERT R, 1992, EUR J PHARMACOL, V211, P87SMALL RC, 1992, BRAZ J MED BIOL RES, V25, P983PLANT TD, 1991, BRIT J PHARMACOL, V104, P385DUNNE MJ, 1991, BRIT J PHARMACOL, V103, P1847HOFFMANN BB, 1991, PHARMACOL BASIS THER, pCH10PLANT TD, 1990, BRIT J PHARMACOL, V101, P115