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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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Related in: MedlinePlus

Effects of cAMP-related drugs on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. SQ-22536 (100 μM) (a) and dideoxyadenosine (100 μM) (b), both adenylate cyclase inhibitors, decreased the frequency of pacemaker potentials. Whereas rolipram (100 μM) (c), a cAMP specific PDE 4 inhibitor, and cell permeable 8-bromo-cAMP (100 μM) (d) increased the frequency of pacemaker potentials. The effects of these drugs on pacemaker potentials are summarized in e and f. Bars represent mean ± SE values (p < 0.05) con control, ddA didoxyadenosine
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Fig2: Effects of cAMP-related drugs on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. SQ-22536 (100 μM) (a) and dideoxyadenosine (100 μM) (b), both adenylate cyclase inhibitors, decreased the frequency of pacemaker potentials. Whereas rolipram (100 μM) (c), a cAMP specific PDE 4 inhibitor, and cell permeable 8-bromo-cAMP (100 μM) (d) increased the frequency of pacemaker potentials. The effects of these drugs on pacemaker potentials are summarized in e and f. Bars represent mean ± SE values (p < 0.05) con control, ddA didoxyadenosine

Mentions: The patch clamp technique was tested with ICCs that had network-like structures in culture (2–3 days). In the current clamp mode, the ICCs generated pacemaker potentials (Fig. 1a). Under control conditions in the current clamp mode, the resting membrane potential, frequency and amplitude were −58.7 ± 2.5 mV, 7.9 ± 2.6 cycles/5 min, and 41.4 ± 7.9 mV, respectively (n = 23; Fig. 1a, d, e). To evaluate whether voltage-dependent Na+ and Ca2+ channels were involved in generating pacemaker potentials, we first treated with tetrodotoxin (TTX, 1 μM; n = 10) and nicardipine (1 μM; n = 9). We found that the resting membrane potential, frequency, and amplitude of pacemaker potentials were not changed by TTX and nicardipine (Fig. 1b, c), indicating that voltage-dependent Na+ and L-type Ca2+ channels were not involved in generating pacemaker potentials. Since HCN channels were activated directly by cAMP, we next examined the involvement of cAMP using SQ-22536 (100 μM; n = 6), dideoxyadenosine (100 μM; n = 6) (both adenylate cyclase inhibitors), rolipram (a cAMP-specific phosphodiesterase inhibitor, 100 μM; n = 7), and cell-permeable 8-bromo-cAMP (100 μM; n = 8). Both SQ-22536 and dideoxyadenosine decreased the frequency of pacemaker potentials and hyperpolarized the membrane. In contrast, rolipram and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials and depolarized the membrane (Fig. 2a–d). The values of the frequency induced by above drugs were significantly different from the control values (Fig. 2f). However, the values of the resting membrane potential induced by these drugs were not significantly different from control values even though they changed membrane potentials (Fig. 2e). These results suggest that basal intracellular cAMP plays an important role in regulating the pacemaker frequency in colonic ICCs.Fig. 1


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 cAMP-related drugs on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. SQ-22536 (100 μM) (a) and dideoxyadenosine (100 μM) (b), both adenylate cyclase inhibitors, decreased the frequency of pacemaker potentials. Whereas rolipram (100 μM) (c), a cAMP specific PDE 4 inhibitor, and cell permeable 8-bromo-cAMP (100 μM) (d) increased the frequency of pacemaker potentials. The effects of these drugs on pacemaker potentials are summarized in e and f. Bars represent mean ± SE values (p < 0.05) con control, ddA didoxyadenosine
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Effects of cAMP-related drugs on the spontaneous pacemaker potentials in cultured ICCs of the mouse colon. SQ-22536 (100 μM) (a) and dideoxyadenosine (100 μM) (b), both adenylate cyclase inhibitors, decreased the frequency of pacemaker potentials. Whereas rolipram (100 μM) (c), a cAMP specific PDE 4 inhibitor, and cell permeable 8-bromo-cAMP (100 μM) (d) increased the frequency of pacemaker potentials. The effects of these drugs on pacemaker potentials are summarized in e and f. Bars represent mean ± SE values (p < 0.05) con control, ddA didoxyadenosine
Mentions: The patch clamp technique was tested with ICCs that had network-like structures in culture (2–3 days). In the current clamp mode, the ICCs generated pacemaker potentials (Fig. 1a). Under control conditions in the current clamp mode, the resting membrane potential, frequency and amplitude were −58.7 ± 2.5 mV, 7.9 ± 2.6 cycles/5 min, and 41.4 ± 7.9 mV, respectively (n = 23; Fig. 1a, d, e). To evaluate whether voltage-dependent Na+ and Ca2+ channels were involved in generating pacemaker potentials, we first treated with tetrodotoxin (TTX, 1 μM; n = 10) and nicardipine (1 μM; n = 9). We found that the resting membrane potential, frequency, and amplitude of pacemaker potentials were not changed by TTX and nicardipine (Fig. 1b, c), indicating that voltage-dependent Na+ and L-type Ca2+ channels were not involved in generating pacemaker potentials. Since HCN channels were activated directly by cAMP, we next examined the involvement of cAMP using SQ-22536 (100 μM; n = 6), dideoxyadenosine (100 μM; n = 6) (both adenylate cyclase inhibitors), rolipram (a cAMP-specific phosphodiesterase inhibitor, 100 μM; n = 7), and cell-permeable 8-bromo-cAMP (100 μM; n = 8). Both SQ-22536 and dideoxyadenosine decreased the frequency of pacemaker potentials and hyperpolarized the membrane. In contrast, rolipram and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials and depolarized the membrane (Fig. 2a–d). The values of the frequency induced by above drugs were significantly different from the control values (Fig. 2f). However, the values of the resting membrane potential induced by these drugs were not significantly different from control values even though they changed membrane potentials (Fig. 2e). These results suggest that basal intracellular cAMP plays an important role in regulating the pacemaker frequency in colonic ICCs.Fig. 1

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