Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

Pflugers Arch. 1999 Mar;437(4):529-37. doi: 10.1007/s004240050814.

Abstract

We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca2+ regulation in Fura-2-loaded ferret myocytes. H-89 (10 micromol/l) decreased the amplitude of the Fura-2 transient to 28. 2+/-4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca2+ to diastolic levels: t1/2 increased from 311+/-35 ms to 547+/-43 ms (P<0.001, n=7). Reduced Ca2+ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca2+ content, as assessed with caffeine. The apparent slowing of the SR Ca2+-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca2+ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca2+-ATPase. In electrophysiological experiments, 5.0 micromol/l H-89 decreased the L-type Ca2+ current (ICa) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca2+ ]i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of beta-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca2+-ATPase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caffeine / pharmacology
  • Calcium / metabolism*
  • Calcium-Binding Proteins / metabolism
  • Calcium-Transporting ATPases / antagonists & inhibitors
  • Calcium-Transporting ATPases / metabolism
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors*
  • Dogs
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Ferrets
  • Heart Ventricles / drug effects
  • Heart Ventricles / enzymology
  • Isoquinolines / pharmacology*
  • Male
  • Microsomes / enzymology
  • Muscle, Skeletal / ultrastructure
  • Myocardium / enzymology*
  • Phosphorylation
  • Rats
  • Sarcoplasmic Reticulum / drug effects
  • Sarcoplasmic Reticulum / enzymology
  • Sulfonamides*

Substances

  • Calcium-Binding Proteins
  • Enzyme Inhibitors
  • Isoquinolines
  • Sulfonamides
  • phospholamban
  • Caffeine
  • Cyclic AMP-Dependent Protein Kinases
  • Calcium-Transporting ATPases
  • N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
  • Calcium