Neurosteroids modulate calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive G-protein-coupled mechanism.
The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)
ffrench-Mullen
J M
JM
Department of Pharmacology, Zeneca Pharmaceuticals Group, Zeneca Inc., Wilmington, Delaware 19897.
Danks
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P
Spence
K T
KT
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002552:Cerebral Ventricles / Q000187:drug effects / Q000502:physiology*; D003345:Corticosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology*; D066298:In Vitro Techniques; D007263:Infusions, Parenteral; D007700:Kinetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin; D011284:Pregnenolone / Q000494:pharmacology*; D017966:Pyramidal Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000008:administration & dosage / Q000494:pharmacology*; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; C018370:pregnenolone sulfate; C016957:adenosine-3',5'-cyclic phosphorothioate; C003676:tetrahydrocorticosterone; D011284:Pregnenolone; D020866:omega-Conotoxin GVIA; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium; D003345:Corticosterone