Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C

1. Hippocampal neurones respond to acute oxygen deprivation (hypoxia) with an inhibition of whole-cell Na+ current (I-Na), although the mechanism of the inhibition is unknown. Kinases can modulate I-Na and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia-induced inhibition of I-Na. 2. Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. I-Na was recorded at baseline, during exposure to kinase activators (with and without kinase inhibitors), and during acute hypoxia (with and without kinase inhibitors). 3. Hypoxia (3 min) reduced I-Na to 3.8 +/- 4.5% of initial values, and shifted steady-state inactivation in the negative direction. Hypoxia produced no effect on activation or fast inactivation. 4. Protein kinase A (PKA) activation with 2.5 mM adenosine 3',5'-cyclic adenosine monophosphate, N-6,O-2-dibutyryl, sodium salt (db-cAMP) resulted in reduction of I-Na to 62.8 +/- 5.5% without an effect on activation or steady-state inactivation. I-Na was also reduced by activation of protein kinase C (PKC) with 5 nM phorbol 12-myristate 13-acetate (PMA; to 40.0 +/- 3.7 %) or 50 mu M 1-oleoyl-2-acetyl-sn-glycerol (OAG; to 46.1 +/- 2.8 %). In addition, steady-state inactivation was shifted in the negative direction by PKC: activation. Neither the activation curve nor the kinetics of fast inactivation was altered by PKC activation. 5. The response to PKA activation was blocked by the PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H-89; 30 mu M) and by 30 mu M of PKA inhibitory peptide PKA(5-24) (PKAi). PKC activation was blocked by the kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7; 100 mu M), by the PKC inhibitor calphostin C (10 mu M), and by 20 mu m of the inhibitory peptide PKC19-31 (PKCi). 6. The hypoxia-induced inhibition of I-Na and shift in steady-state inactivation were greatly attenuated with H-7, calphostin C, or PKCi, but not with H-89 or PKAi. 7. We conclude that hypoxia activates PKC in rat CA1 neurones, and that PKC activation leads to the hypoxia-induced inhibition of I-Na.