Differential control of the hyperpolarization-activated current (i(f)) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes.
1. The actions of the phosphatase inhibitor calyculin A on the hyperpolarization-activated cardiac 'pacemaker' current (i(f)) were determined in single cells isolated from the sino-atrial (SA) node of the rabbit. 2. Cells were incubated for 8 min in Tyrode solution containing calyculin A (0.5 microM) and then superfused with normal Tyrode solution. The mean normalized i(f) measured in eight cells at mid-activation voltages during and after exposure to calyculin A increased maximally by 47% with a time constant of 466 s, a time much longer than that required for cAMP-mediated i(f) stimulation (about 8 s). 3. In two-pulse protocols, calyculin A treatment increased i(f) at full as well as at mid-activation voltages, indicating a higher i(f) conductance. 4. Measurement of the conductance-voltage (gf(V)) relation by voltage ramp protocols confirmed a conductance increase by calyculin A, with no significant change in the position of the activation curve on the voltage axis. Data pooled together from ramp and two-pulse protocols yielded a calyculin A-induced increase in fully activated i(f) conductance of 39.6 +/- 6.4% (n = 16 cells). 5. The positive and negative shift of i(f) voltage dependence in response to beta-adrenergic (1 microM isoprenaline) and muscarinic stimulation (1 microM acetylcholine), respectively, was preserved after the calyculin A-induced increase in conductance. The shift of the i(f) activation curve induced by 1 microM isoprenaline was significantly larger in calyculin A-treated cells (8.8 vs. 5.8 mV). 6. These data indicate that phosphatase inhibition increases i(f) in a manner distinct from the direct cAMP pathway and potentiates the beta-adrenergic-mediated i(f) modulation.
Accili
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Università di Milano, Dipartimento di Fisiologia e Biochimica Generali, Italy.
Redaelli
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DiFrancesco
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D000109:Acetylcholine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001683:Biological Clocks / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D008387:Marine Toxins; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D010080:Oxazoles / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011817:Rabbits; D012849:Sinoatrial Node / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D007545:Isoproterenol; D000109:Acetylcholine