Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides.
It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.
Gao
Junyuan
J
Department of Physiology and Biophysics and Institute of Molecular Cardiology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.
Wymore
Randy S
RS
Wang
Yongli
Y
Gaudette
Glenn R
GR
Krukenkamp
Irvin B
IB
Cohen
Ira S
IS
Mathias
Richard T
RT
D000818:Animals; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002316:Cardiotonic Agents / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008956:Models, Chemical; D018485:Muscle Fibers, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D012260:Ribonucleases; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity
D002301:Cardiac Glycosides; D002316:Cardiotonic Agents; D007527:Isoenzymes; D010042:Ouabain; D012260:Ribonucleases; D000254:Sodium-Potassium-Exchanging ATPase