title: Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes.
abstract: 1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (ICa) in the absence of beta-adrenergic agonists. The increase occurred without effects on the peak current-voltage relation or voltage-dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (IK), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occurred without effects on voltage-dependent activation or reversal potential. 2. Analysis of the dose-response relations for OA and MC on ventricular cell ICa were best fitted with a single-site relationship with a K1/2 of 1.58 microM and 0.81 microM, respectively. These data suggest the predominant form of phosphatase active on ICa in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal ICa suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the ICa increase induced by OA or MC. This demonstrates that the effects of OA and MC on ICa are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished IK, suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was without effect on basal current levels of ICa or IK, suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of ICa to isoprenaline or forskolin, PKI does not affect the increase in ICa induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(Rp)-phosphothioate (Rp-cAMPS) also had no effect on the response to OA or MC. 5. Application of beta-adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK-A is not mediating the OA response and that phosphatase inhibition does not result in complete phosphorylation of PK-A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on ICa and IK, several types of protein kinase inhibitors were internally perfused.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.
authors.forename: A M
authors.initials: AM
authors.lastname: Frace
authors.forename: H C
authors.initials: HC
authors.lastname: Hartzell
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004988:Ethers, Cyclic / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D052998:Microcystins; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019319:Okadaic Acid; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D054645:Protein Phosphatase 1; D011892:Rana catesbeiana
chemical_list: D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D004988:Ethers, Cyclic; D052998:Microcystins; D010456:Peptides, Cyclic; D019319:Okadaic Acid; C078588:microcystin; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011188:Potassium; D002118:Calcium