Endothelin-1 and PKC induce positive inotropy without affecting pHi in ventricular myocytes.
It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.
Kang
Misuk
M
Department of Physiology, Molecular and Cellular Pharmacology Training Program, University of Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Walker
Jeffery W
JW
D000818:Animals; D002478:Cells, Cultured; D004558:Electric Stimulation; D019332:Endothelin-1 / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D019332:Endothelin-1; D011493:Protein Kinase C