Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes.
The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca(2+) content, Ca(2+) content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca(2+) stores as an index of sarcoplasmic reticulum (SR) Ca(2+) content. Caffeine-releasable Ca(2+) content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca(2+) content. Activation of the L-type Ca(2+) channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca(2+) influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca(2+) levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca(2+) content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca(2+) influx at the L-type Ca(2+) channel. The NCX inhibitor Ni(2+) was used to investigate whether the decrease in intracellular Ca(2+) content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni(2+) suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca(2+) channel activity.
McDowell
Susan A
SA
Cardiovascular Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0520, USA.
McCall
Eileen
E
Matter
William F
WF
Estridge
Thomas B
TB
Vlahos
Chris J
CJ
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles / Q000187:drug effects; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010879:Piperazines / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D016276:Ventricular Function
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002118:Calcium; D020746:Calcium Channels, L-Type; D000252:Calcium-Transporting ATPases; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010879:Piperazines; D010982:Platelet-Derived Growth Factor; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019831:Sodium-Calcium Exchanger; C093555:LY 303511; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one