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 (INa), although the mechanism of the inhibition is unknown. Kinases can modulate INa and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia-induced inhibition of INa. 2. Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. INa 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 INa to 38.1 +/- 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, N6,O2-dibutyryl, sodium salt (db-cAMP) resulted in reduction of INa to 62.8 +/- 5.5% without an effect on activation or steady-state inactivation. INa 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 microM 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-bromocinnamyl-amino) ethyl]-5-isoquinolinesulphonamide (H-89; 30 microM) and by 30 microM of PKA inhibitory peptide PKA5-24 (PKAi). PKC activation was blocked by the kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7; 100 microM), by the PKC inhibitor calphostin C (10 microM) and by 20 microM of the inhibitory peptide PKC19-31 (PKCi). 6. The hypoxia-induced inhibition of INa 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 INa.
O'Reilly
J P
JP
Department of Biology, Yale University and School of Medicine, New Haven, CT 06520, USA.
Cummins
T R
TR
Haddad
G G
GG
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology*; D007546:Isoquinolines / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D010100:Oxygen / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000502:physiology; D013449:Sulfonamides
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D007976:Leupeptins; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; D013449:Sulfonamides; C061465:calphostin complex; C489276:protein kinase C-binding protein Beta15, rat; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C032854:leupeptin; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D010100:Oxygen