Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation.
Antiarrhythmic peptides enhance gap junction current in pairs of cardiomyocytes and coupling in cardiac tissue. To elucidate the underlying mechanisms, we investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH2) on pairs of adult guinea pig ventricular cardiomyocytes and pairs of HeLa cells transfected with rat cardiac connexin 43 (Cx43). By using a double-cell voltage-clamp technique in pairs of cardiomyocytes, we found that under control conditions the gap junction conductance (gj) steadily decreased with time (by -0.292 +/- 0.130 nS/min). Use of 50 nmol/L AAP10 reversed this rundown and increased gj (by +0.290 +/- 0.231 nS/min, Pa). In HeLa-Cx43 cells, AAP10 exerted the same electrophysiological effect. In these cells, AAP10 activated PKC (determined by using ELISA) in CGP54345-sensitive manner and significantly enhanced incorporation of 32P into Cx43 with dependence on PKC. If G-protein coupling was inhibited with 1 mM GDP-BS, we found the effects of AAP10 on 32P incorporation were also completely abolished. Next, we performed a radioligand binding study with 14C-AAP10 as radioligand and AAPnat as competitor. We found saturable binding of 14C-AAP10 to cardiac membrane preparations, which could be displaced with AAPnat. The Kd of AAP10 was 0.88 nmol/L. We conclude that 1) AAP10 increases gj both in adult cardiomyocytes and in transfected HeLa-Cx43 cells, 2) AAP10 exerts its effect via enhanced PKC-dependent phosphorylation of Cx43, 3) AAP10 activates PKCa, and 4) a membrane receptor exists for antiarrhythmic peptides in cardiomyocytes.
Weng
Stephan
S
Clinic for Cardiac Surgery, University of Leipzig, Heart Center, 04289 Leipzig, Germany.
Lauven
Melani
M
Schaefer
Thomas
T
Polontchouk
Lioudmila
L
Grover
Rajiv
R
Dhein
Stefan
S
D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D004553:Electric Conductivity; D004789:Enzyme Activation; D017629:Gap Junctions / Q000187:drug effects*; D006168:Guinea Pigs; D006367:HeLa Cells; D006321:Heart / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha
C090806:AAP 10; D000889:Anti-Arrhythmia Agents; D018031:Connexin 43; D007527:Isoenzymes; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha