Relationship between biochemical and functional effects of protein phosphatase 1 inhibitors in rabbit cardiac skinned fibers.
Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.
Berrebi-Bertrand
I
I
SmithKline Beecham Laboratoires Pharmaceutiques, 4 rue du Chesnay Beauregard, BP 58, 35762 Saint-Grégoire Cedex, France.
Brument-Larignon
N
N
Camelin
J C
JC
Quiniou
M J
MJ
Bril
A
A
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D003285:Contractile Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008387:Marine Toxins; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D010080:Oxazoles / Q000494:pharmacology*; D010210:Papillary Muscles / Q000187:drug effects / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1; D011714:Pyrans; D011817:Rabbits; D013141:Spiro Compounds; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D000935:Antifungal Agents; D002110:Caffeine; D002118:Calcium; D002135:Calcium-Binding Proteins; D003285:Contractile Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D008387:Marine Toxins; D010080:Oxazoles; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011714:Pyrans; D013141:Spiro Compounds; D000099211:Phospholamban; C053079:tautomycin; C059041:calyculin A