Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones.
The influence of internal Ca2+ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca2+ current (ICa). Internal free Ca2+ ([Ca2+]i) was varied between 0.01 and 10 microM by addition of Ca(2+)-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca2+]i depressed the 5-HT effect. The dose/effect curve for the Ca2+ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants Kd1 = 0.063 microM and Kd2 = 1 microM. Addition of calmodulin (CM) antagonists (50 microM trifluoperazine or 50 microM chlorpromazine), phosphodiesterase (PDE) antagonists [100 microM isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 microM okadaic acid (OA)] in the perfusion solution caused "anticalcium" action and modified the Ca2+ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca2+ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with Kd1 = 0.04 microM and Kd2 = 0.69 microM were obtained describing the activation of the retained unblocked enzyme--PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 microM), a blocker of Ca(2+)-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca(2+)-induced suppression of the cAMP-dependent upregulation of Ca2+ channels is due to involvement of two Ca(2+)-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca2+ concentration of less than or equal to 10 microM.
Kostyuk
P G
PG
Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev, Ukraine.
Lukyanetz
E A
EA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology; D002746:Chlorpromazine / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004553:Electric Conductivity; D004789:Enzyme Activation; D004988:Ethers, Cyclic / Q000494:pharmacology; D006372:Helix, Snails; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology*; D013806:Theophylline / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D015220:Calcium Channels; D002147:Calmodulin; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D014268:Trifluoperazine; D012701:Serotonin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D013806:Theophylline; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases; D010727:Phosphoric Diester Hydrolases; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine; D002746:Chlorpromazine