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EXPERIMENTAL STUDY

Potassium channel blocker activates extracellular signal-regulated kinases through Pyk2 and epidermal growth factor receptor in rat cardiomyocytes

Satoko Tahara, MD^*, Keiichi Fukuda, MD, PhD^*,, Hiroaki Kodama, MD^*, Takahiro Kato, MD^*, Shunichiro Miyoshi, MD, PhD and Satoshi Ogawa, MD, PhD^*

^* Cardiopulmonary Division, Department of Internal Medicine, Tokyo, Japan
Institute for Advanced Cardiac Therapeutics, Tokyo, Japan
Department of Physiology, Keio University School of Medicine, Tokyo, Japan

Manuscript received November 29, 2000; revised manuscript received June 27, 2001, accepted July 19, 2001.

^* Reprint requests and correspondence: Dr. Keiichi Fukuda, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
kfukuda{at}mc.med.keio.ac.jp

OBJECTIVES

We sought to determine whether potassium (K⁺) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes.

BACKGROUND

Because KBs attenuate K⁺ outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca²⁺) transient ([Ca²⁺]_i) in cardiomyocytes. Elevation of intracellular Ca²⁺ levels can trigger various signaling events. Influx of Ca²⁺ through L-type Ca²⁺ channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown.

METHODS

Primary cultured rat cardiomyocytes were stimulated with four different KBs—4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine—and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca²⁺)_i was monitored by the fluorescent calcium indicator Fluo-4.

RESULTS

E-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca²⁺)_i by 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not.

CONCLUSIONS

These findings indicate that KBs induce ERK activation, which starts with Ca²⁺ entry through the L-type Ca²⁺ channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.

Abbreviations and Acronyms   4-AP = 4-aminopyridine   APD = action potential duration   BAPTA-AM = 1,2-bis(2-aminophenoxy)-ethane- N,N,N',N'-tetraacetic acid tetrakis   Ca2+ = calcium   [Ca2+]i = Ca2+ transient   EGFR = epidermal growth factor receptor   ERK = extracellular signal-regulated kinase   HA = hemaggulutinin   K+ = potassium   KB = potassium channel blocker   LIF = leukemia inhibitory factor   MAPK = mitogen-activated protein kinase   MEK = MAPK/ERK kinase   Na+ = sodium   PKC = protein kinase C   Pyk2 = proline-rich tyrosine 2   TEA = tetra-ethylammonium

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