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

Shahi PK, Choi S, Zuo DC, Kim MY, Park CG, Kim YD, Lee J, Park KJ, So I, Jun JY - J. Gastroenterol. (2013)

Bottom Line: CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity.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.HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, College of Medicine, Chosun University, Sesuk-dong, Dong-gu, Gwangju, 501-759, Korea.

ABSTRACT

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.

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Effects of protein kinase A (PKA) inhibitor, cAMP-regulated guanine nucleotide exchange factor (Epac) agonist, and cyclic nucleotide gated (CNG) channel blocker on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. KT-5720, a PKA inhibitor (10 μM) (a), 8-pCPT-2′-O-Me-cAMP, an Epac agonist (10 μM) (b), and l-cis-diltiazem, a CNG channel blocker (10 μM) (c) had no effect on the spontaneous pacemaker potentials. The effects of KT-5720, 8-pCPT-2′-O-Me-cAMP and l-cis-diltiazem on pacemaker potentials are summarized in d and e. Bars represent mean ± SE values (p < 0.05)
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Fig3: Effects of protein kinase A (PKA) inhibitor, cAMP-regulated guanine nucleotide exchange factor (Epac) agonist, and cyclic nucleotide gated (CNG) channel blocker on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. KT-5720, a PKA inhibitor (10 μM) (a), 8-pCPT-2′-O-Me-cAMP, an Epac agonist (10 μM) (b), and l-cis-diltiazem, a CNG channel blocker (10 μM) (c) had no effect on the spontaneous pacemaker potentials. The effects of KT-5720, 8-pCPT-2′-O-Me-cAMP and l-cis-diltiazem on pacemaker potentials are summarized in d and e. Bars represent mean ± SE values (p < 0.05)

Mentions: The cellular action of cAMP is mediated mainly by protein kinase A (PKA) or cAMP-regulated guanine nucleotide exchange factor (Epac) [27]. Thus, to assess whether basal cAMP action was mediated by either PKA or Epac, we examined the effect of KT-5720 (a PKA inhibitor, 10 μM; n = 7) or 8-pCPT-2′-O-Me-cAMP (an Epac agonist, 100 μM; n = 7), and found that both KT-5720 and Epac agonist had no effects on pacemaker activity (Fig. 3a, b). Since cyclic nucleotide gated (CNG) channel is another downstream target of cAMP [28], we treated with l-cis-diltiazem, a CNG channel blocker (10 μM; n = 7). We found that l-cis-diltiazem also had no effect on pacemaker activity (Fig. 3c). The values of the resting membrane potential and frequency induced by the above drugs were not significantly different from control values (Fig. 3d, e). These results suggest that cAMP-dependent regulation of pacemaker frequency was not mediated by PKA, Epac or CNG channels.Fig. 3


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

Shahi PK, Choi S, Zuo DC, Kim MY, Park CG, Kim YD, Lee J, Park KJ, So I, Jun JY - J. Gastroenterol. (2013)

Effects of protein kinase A (PKA) inhibitor, cAMP-regulated guanine nucleotide exchange factor (Epac) agonist, and cyclic nucleotide gated (CNG) channel blocker on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. KT-5720, a PKA inhibitor (10 μM) (a), 8-pCPT-2′-O-Me-cAMP, an Epac agonist (10 μM) (b), and l-cis-diltiazem, a CNG channel blocker (10 μM) (c) had no effect on the spontaneous pacemaker potentials. The effects of KT-5720, 8-pCPT-2′-O-Me-cAMP and l-cis-diltiazem on pacemaker potentials are summarized in d and e. Bars represent mean ± SE values (p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048466&req=5

Fig3: Effects of protein kinase A (PKA) inhibitor, cAMP-regulated guanine nucleotide exchange factor (Epac) agonist, and cyclic nucleotide gated (CNG) channel blocker on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. KT-5720, a PKA inhibitor (10 μM) (a), 8-pCPT-2′-O-Me-cAMP, an Epac agonist (10 μM) (b), and l-cis-diltiazem, a CNG channel blocker (10 μM) (c) had no effect on the spontaneous pacemaker potentials. The effects of KT-5720, 8-pCPT-2′-O-Me-cAMP and l-cis-diltiazem on pacemaker potentials are summarized in d and e. Bars represent mean ± SE values (p < 0.05)
Mentions: The cellular action of cAMP is mediated mainly by protein kinase A (PKA) or cAMP-regulated guanine nucleotide exchange factor (Epac) [27]. Thus, to assess whether basal cAMP action was mediated by either PKA or Epac, we examined the effect of KT-5720 (a PKA inhibitor, 10 μM; n = 7) or 8-pCPT-2′-O-Me-cAMP (an Epac agonist, 100 μM; n = 7), and found that both KT-5720 and Epac agonist had no effects on pacemaker activity (Fig. 3a, b). Since cyclic nucleotide gated (CNG) channel is another downstream target of cAMP [28], we treated with l-cis-diltiazem, a CNG channel blocker (10 μM; n = 7). We found that l-cis-diltiazem also had no effect on pacemaker activity (Fig. 3c). The values of the resting membrane potential and frequency induced by the above drugs were not significantly different from control values (Fig. 3d, e). These results suggest that cAMP-dependent regulation of pacemaker frequency was not mediated by PKA, Epac or CNG channels.Fig. 3

Bottom Line: CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity.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.HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, College of Medicine, Chosun University, Sesuk-dong, Dong-gu, Gwangju, 501-759, Korea.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus