Abstract
Abstract The effects of an adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on Ca2+-activated adenosine triphosphatase (ATPase) activity and calcium uptake of canine cardiac microsomes were examined. The cardiac microsomes, which represent an enriched preparation of fragmented sarcoplasmic reticulum, were preincubated with varying concentrations of cyclic AMP-dependent protein kinase or cyclic AMP or both in the presence of Mg2+ and ATP. Ca2+-activated ATPase activity and calcium uptake were determined in the presence of oxalate and various concentrations of Ca2+. Ionized Ca2+ concentrations were maintained with Ca2+ buffers containing CaCl2 and ethylene glycol bis (β-aminoethyl ether)-N,N'-tetraacetic acid. The initial rates of both Ca2+-activated ATPase and calcium uptake were increased 2 to 3 times following 10-min preincubation with protein kinase and 1 µm cyclic AMP. Pretreatment with protein kinase in the absence of cyclic AMP caused a lesser degree of stimulation, whereas pretreatment with cyclic AMP alone had no detectable effect. Stimulation of the Ca2+-activated ATPase and calcium uptake by protein kinase was dependent on cyclic AMP concentration: maximal stimulation was seen at approximately 10-6 m with apparent half-maximal stimulation at approximately 10-7 m. Marked stimulation of both Ca2+-activated ATPase activity and calcium uptake by cyclic AMP and protein kinase was seen at a Ca2+ concentration of approximately 1 µm. The stoichiometric coupling of Ca2+-activated ATPase and calcium uptake was maintained at 2 moles of calcium taken up per mole of ATP hydrolyzed, following stimulation by protein kinase and cyclic AMP. The stimulatory effects on Ca2+-activated ATPase and calcium uptake of pretreatment with protein kinase and cyclic AMP could be shown after microsomes were washed with buffered 50 mm KCl. These findings indicate that protein kinase can increase the rate of calcium transport by the cardiac sarcoplasmic reticulum without altering the efficiency of the calcium pump. This effect may account for the abbreviation of systole that is caused by agents, like epinephrine, which increase cyclic AMP production, and resulting alterations in the distribution of Ca2+ within the myocardial cell may be responsible, at least in part, for the augmentation of myocardial contractility.
Highlights
AMP)-dependent protein kinase on Ca2+-activated adenosine triphosphatase (ATPase) activity and calcium uptake of canine cardiac microsomes were examined
ATPase and calcium uptake of pretreatment with protein kinase and cyclic AMP could be shown after microsomes were washed with buffered 50 mM KCI. These findings indicate that protein kinase can increase the rate of calcium transport by the cardiac sarcoplasmic reticulum without altering the efficiency of the calcium pump
We have previously reported that calcium transport by the cardiac microsomes, which consist largely of fragmented sarcoplasmic reticulum, can be influenced by a cardiac adenosine
Summary
ATPase AssayThe ATPase activity of canine cardiac microsomes was determined at 25” in standard reaction mixtures containing40 mhz histidine buffer (pH G.8), 5 mM MgC12, 5 mM [-,-3zP]ATP (1 to 3&i per umole). 125 mM KCI, 5 rnM NaNn. 2.5 mM Tris-oxalate, calc;umiEGTA’buffer containing125 PM CaCl,, and 20 to 50 $per ml of microsomal protein in a volume of 0.3 to 0.6 ml. The ATPase activity of canine cardiac microsomes was determined at 25” in standard reaction mixtures containing. Reaction mixtures were equilibrated at 25” for 5 min, after which reactions were started by the addition of microsomal protein, unless otherwise stated. To determine “basic” (Caz+-independent) ATPase activity, reactions were carried out in the presence of 0.5 mM EGTA instead of calcium-EGTA buffer. At various time intervals after the start of the reactions, 100.~1 aliquots were added to tubes (5 X 50 mm) containing 25 11 of 25yo (w/v) trichloroacetic acid and 125 PM carrier Pi. After centrifugation (1000 X g, 10 min at 4’), the amount of 32Pi in a 100-J aliquot of the supernatant was determined by the isobutanol-extraction procedure described by Siegel and Albers [24] with slight modifications.
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