Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes

A. Mattiazzi, L. Hove-Madsen, Donald M Bers

Research output: Contribution to journalArticle

62 Scopus citations

Abstract

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca- adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin- permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 μM ruthenium red and 10 mM oxalate. Neither N6,2'-O- dibutyryl-cAMP (up to 500 μM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 μM) affected the maximum uptake rate (V(max)) or the dissociation constant (K(d)) for Ca uptake. However, the PKA inhibitor H-89 significantly increased K(d) (e.g., from 307 ± 67 to 826 ± 62 nM Ca at 40-65 μM H-89) without significantly affecting V(max). Both CaM-KII inhibitors, KN-62 (60 μM) and a CaM-KII inhibitory peptide (10 μM), significantly decreased V(max) from 11.95 ± 0.5 to 9.48 ± 0.6 nmol · mg-1 · min-1 and from 10.95 ± 1.72 to 7.37 ± 0.94 nmol · mg-1 · min-1, respectively, without consistently changing K(d). The effects of H-89 on K(d) and of KN-62 on V(max) were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca- ATPase). Clear effects of protein kinase inhibitors and lack of stimulatory effects lead us to conclude that the Ca pump may be close to maximal activation in our system (such that only inhibitory effects are apparent). The results suggest that phospholamban phosphorylation by PKA and CaM-KII, respectively, may produce functionally distinct effects on Ca transport by the SR. That is, phosphorylation of phospholamban by endogenous CaM-KII appears to increase the V(max) of the SR Ca-ATPase, whereas PKA increases the Ca affinity of the pump.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume267
Issue number2 36-2
StatePublished - 1994
Externally publishedYes

Keywords

  • adenosine 3',5'-cyclic monophosphate-dependent protein kinase
  • calcium uptake
  • calcium- adenosinetriphosphatase
  • calcium-calmodulin-dependent protein kinase
  • sarcoplasmic reticulum

ASJC Scopus subject areas

  • Physiology
  • Agricultural and Biological Sciences(all)

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