Effects of the protein kinase A inhibitor H-89 on Ca 2+ regulation in isolated ferret ventricular myocytes

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.

Restoration of diabetes-induced abnormal local Ca2+release in cardiomyocytes by angiotensin II receptor blockade

“…We previously reported that, in a rat model of STZ-induced diabetes, a prominent abnormal Ca 2ϩ leak occurs through the RyR2, owing to a partial loss of RyR2-bound FKBP12.6 and PKA-mediated hyperphosphorylation of RyR2 (51). Both protein kinases, PKA and PKC, are expressed in cardiac muscle (22), and they strongly controlled cardiac inotropic activity (17,19,45). PKA and PKC play a central role in transducing signal and potentiating intracellular cross talk by phosphorylating diverse substrates, including receptors, enzymes, and transcription factors.…”

The Many Faces of H89: A Review

“…Similar inhibitory actions of high H89 concentrations (100 ìM) on net Ca 2+ uptake by the SR and Ca 2+ sensitivity of the contractile apparatus in rat skinned fibers were observed by Blazev et al (7). The inhibitory effect of H89 on SR Ca 2+ uptake was also demonstrated in isolated ferret ventricular myocytes (36). In view of the above, the use of H89 as a tool to study the role of PKA-mediated phosphorylation on Ca 2+ regulation in myocytes, is limited by the non-specific effects of the drug on the SR Ca 2+ -ATPase.…”

H‐89 inhibits transient outward and inward rectifier potassium currents in isolated rat ventricular myocytes

“…The nature of such PKA-independent inhibitory actions of H-89 is unknown and may involve other kinases or even other direct/indirect nonspecific effects unrelated to channel phosphorylation. This is particularly likely given that the three K þ currents investigated in this study have markedly different structures at the molecular level and also because previous studies have already documented some other nonspecific effects of H-89 (Hussain et al, 1999;Blasev et al, 2001;Choi et al, 2001), which can only therefore be used as an invaluable tool if these other effects are carefully avoided.…”