We found that LY 294002, a specific inhibitor of PI3K, as well as specific inhibitors of each of the PI3K isoforms, i. e,B and catalytic PI3K subunits, and an inhibitor of Akt/PKB, significantly decreased i and abolished Ca2 transients or oscil lations. Moreover, inhibition of PI3K/Akt PKB signaling pathways abolished inward Ca2 current in the HL 1 cells, which likely results from L type Ca2 channels in HL 1 cells. Taken together we conclude that the PI3K/Akt PKB signaling pathway plays a role in sustaining the voltage activated Ca2 current contributing to the HL 1 cell action potential. Catalucci et al. have shown that Akt dependent phosphorylation of CavB2, the chaperone of the L type Ca2 channel pore forming subunit, Cav1, antagonizes Cav1 degradation and, as such, stabilizes the functional channel in the plasma membrane.

Inward Ca2 currents from action potential, via voltage activated membrane Ca2 channels, induce Ca2 release from the sarcoplasmic reticulum, which accounts for excitation contraction coupling in cardiomyocytes. We observed a two to five minute delay for various PIK3/Akt PKB inhibitors to reduce Ca2 transients, i and ICa. This is consistent with a time course for the mani festation of inhibition of an enzymatic signaling cascade. We conclude also that this delay is inconsistent with a dir ect inhibition of membrane Ca2 channels by the various inhibitors, which most likely would occur faster. The marked reduction of ICa by PI3K/Akt PKB inhibitors likely results from diminution of L type ICa. We cannot rule out involvement of T type ICa since both are expressed in HL 1 cells.

However, based upon our holding potential of ?50 mV compared with the more electronegative activating voltages for T type Ca2 channels and the relatively extended time course of our ICa, the effects measured here are likely those of L type ICa. Finally, we conclude that the large outward currents seen in the I/V plots at potentials 30 mV result Batimastat from K currents whose magnitude we have found to vary considerably among HL 1 cells in non confluent culture. These findings also have implications for our under standing of the role of PI3K/Akt PKB signaling in dis ease. As noted above, we have reported that sepsis results in decreased activation of the PI3K/Akt pathway in the myocardium. We have also discovered that constitutive up regulation of PI3K p110 in the myocar dium prevents sepsis induced cardiac dysfunction and improves survival outcome in septic mice. Although PI3K/Akt PKB inhibition in septic mice undoubtedly leads to increased cytokine production in these animals, the present findings also indicate that PI3K/Akt PKB inhibition directly decreases availability of Ca2 in the mouse cardiomyocytes.