Madeleine A. Kirchberger

Cyclic adenosine 3′,5′-monophosphate-dependent protein kinase stimulation of calcium uptake by canine cardiac microsomes

The rate of oxalate-dependent Ca-uptake by canine cardiac microsomes was unaffected by cAMP in concentrations up to 10−4m. In the presence of a cAMP-dependent protein kinase but in the absence of cAMP, slight enhancement of Ca-uptake was seen only after prolonged incubation. When the microsomes were preincubated with cAMP and the protein kinase, however, the Ca-uptake rate was approximately doubled. The threshold for this cAMP-dependent protein kinase stimulation was below 10−7m-cAMP, with an apparent Km of approximately 2 × 10−7m. Ca-binding, measured under similar conditions except that oxalate was omitted, was not significantly affected by cAMP and/or the protein kinase. These findings indicate that cAMP, by stimulating a cAMP-dependent protein kinase, can increase the rate at which Ca2+ is transported into the hearts sarcoplasmic reticulum without altering the number or Ca-affinity of the high-affinity Ca-binding sites. The resulting changes in intracellular calcium ion distribution may account for the ability of agents that increase adenylate cyclase activity, e.g. epinephrine, to abbreviate systole and to enhance myocardial contractility.

Adenylate cyclase: Its probable localization in sarcoplasmic reticulum as well as sarcolemma of the canine heart ☆

Abstract Cardiac microsomes prepared by two different methods were compared in terms of Ca-binding and Ca-uptake, believed to represent markers for fragmented sarcoplasmic reticulum, and the (Na+ + K+)-activated ATPase activity considered to be a marker for the plasma membrane. Microsomes prepared in dilute buffer (H2O-microsomes) contain two to four times the activity of sarcoplasmic reticulum markers when compared to microsomes prepared in 10% sucrose (suc-microsomes). Conversely, the plasma membrane marker was present in greater amounts in the suc-microsomes. Basal, epinephrine-stimulated and NaF-stimulated adenylate cyclase activities were slightly higher in H2O-microsomes, as was the degree of stimulation by the β-adrenergic agonist. These findings provide evidence that both β-receptor and adenylate cyclase activities may be present in the hearts sarcoplasmic reticulum, as well as in the sarcolemma.

Phospholamban: Dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Phospholamban, originally identified as a 22,000 dalton protein of cardiac sarcoplasmic reticulum where it is involved in the regulation of calcium transport, can be dissociated into 11,000 and 5,500 dalton forms as determined by polyacrylamide gel electrophoresis. The 22,000 dalton form is obtained by solubilizing microsomes in sodium dodecyl sulphate at 37°. Immersing sodium dodecyl sulphate-denatured microsomes in boiling H2O for one min or five min results in a shift to the 11,000 and 5,500 dalton forms, the latter predominating and presumably representing the monomeric form of phospholamban. These findings can reconcile some of the discrepant findings on phosphoproteins in cardiac sarcoplasmic reticulum reported by different laboratories.

Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22 000 dalton protein

Increases in protein kinase-catalyzed phosphorylation of a 22000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of less than 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.

Effects of ethanol on calcium transport by microsomes phosphorylated by cyclic AMP-dependent protein kinase

Abstract The sensitivity of cardiac sarcoplasmic reticulum to the inhibitory effects of ethanol was significantly increased after phosphorylation of these membranes by cyclic AMP-dependent protein kinase. Ethanol concentrations needed to inhibit oxalate-supported calcium uptake were reduced approximately 4-fold after phosphorylation. Ethanol decreased the extent of stimulation of calcium uptake by cyclic AMP-dependent protein kinase, but protein kinase-catalyzed phosphorylation of these membranes was not affected by concentrations of ethanol used in these studies. These findings suggest that ethanol interferes with the ability of protein kinase-dependent phosphoprotein to stimulate the calcium pump of the sarcoplasmic reticulum.

Comparison of the Effects of Phospholamban and Jasmone on the Calcium Pump of Cardiac Sarcoplasmic Reticulum EVIDENCE FOR MODULATION BY PHOSPHOLAMBAN OF BOTH Ca2+ AFFINITY AND Vmax (Ca) OF CALCIUM TRANSPORT

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of β-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax(Ca) at 25°C and millimolar ATP; phosphorylation and trypsin decreased the Km(Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3.0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholambans effects on both Vmax(Ca) and Km(Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

A regulation of the level of phosphorylated phospholamban by inhibitor-1 in rat heart preparations in vitro

Inhibitor-1 following phosphorylation by protein kinase A inhibits phosphoprotein phosphatase-1. We have found that in the rat heart inhibitor-1 is present only in the cytosolic fraction and that its phosphorylation in ventricular slices was increased by isoproterenol but not by isoproterenol and propranolol together. Cardiac microsomal phosphoprotein phosphatase activity, with added phosphorylase a as the substrate, was inhibited 33% by phosphorylated inhibitor-1. Phosphorylated inhibitor-1 decreased the dephosphorylation by exogenous phosphoprotein phosphatase-1 of phospholamban present in the sarcoplasmic reticulum membranes. These results suggest an interaction of cytoplasmic inhibitor-1 with either cytoplasmic or membrane-bound phosphoprotein phosphatase-1 with a subsequent effect on the level of phosphorylated phospholamban and the probable involvement of this interaction in the cardiac response to beta-adrenergic hormones.

Evidence for catecholamine-stimulated adenylate cyclase activity in frog and rabbit corneal epithelium and cyclic AMP-dependent protein kinase and its protein substrates in frog corneal epithelium

Evidence was obtained for catecholamine-stimulated adenylate cyclase activity in particulate fractions of frog and rabbit corneal epithelium. Epinephrine (10(-5)M) stimulated adenylate cyclase by 22 and 53% in the frog and rabbit, respectively. The corresponding changes were statistically significant (P less than 0.01) when the data was analyzed using paired variates. Preincubation with 10(-4)M propranolol eliminated any stimulatory effect by 10(-5)M isoproterenol. Adenylate cyclase activity derived from either source was activated several fold by either 10 mM NaF or 10(-5)MGpp (NH)p. Soluble fractions of homogenized frog corneal epithelium contained cyclic AMP-dependent protein kinase activity which was half-maximally stimulated by about 6 nM cyclic AMP. Evidence was also obtained for the presence of protein substrates of cyclic AMP dependent protein kinase in frog corneal epithelium. With exogenous cyclic AMP and protein kinase, a rapid 32P labelling of proteins having approximate molecular weights of 56, 46, 23 and 21 K was obtained with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. A less marked and slower increase in phosphoprotein formation was observed when corneal membranes were incubated with cyclic AMP in the absence of added protein kinase.

Differences in calcium uptake in native canine cardiac microsomes are correlated with the ratio of unphosphorylated to phosphorylated phospholamban as determined by Western blot analysis.

Phospholamban (PLM) is detectable by Western blot analysis of canine cardiac microsomes using rabbit antiserum against a peptide containing the 2 to 30 amino acid sequence of PLM. Phosphorylated PLM is distinguishable from the unphosphorylated form by virtue of a reduced electrophoretic mobility. Utilizing digital image analysis to determine relative band densities, it was found that the ratio of unphosphorylated to phosphorylated PLM is correlated with the rate of calcium uptake in 5 preparations of native microsomes (r = 0.94, p less than 0.01). The present analysis may be useful for determining the phosphorylation state of PLM in microsomes obtained from animals in physiological states characterized by impaired sarcoplasmic reticulum calcium pump activity.

Polyphosphoinositide formation in isolated cardiac plasma membranes.

Phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase activities in plasma membranes isolated from canine left ventricle were partially characterized, and their sensitivity to a number of intracellular variables was established. PtdIns and PtdIns4P kinase activities were estimated by the formation of [32P]PtdIns4P and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PtdIns(4,5)P2), respectively, when membranes were incubated with [γ-32P]ATP and 0.1% Triton X-100. Unlike [32P]-PtdIns4P formation, [32P]PtdIns(4,5)P2 formation required exogenous (PtdIns4P) substrate. [32P]-PtdIns4P and [32P]PtdIns(4,5)P2 formation were insensitive to Ca2+ at concentrations ranging from 0.1–30 μM. The hydrolysis of [32P]PtdIns4P was less than 15% under standard assay conditions for measuring its formation, and was unaffected by any of the variables tested. The apparent Km of the PtdIns kinase for ATP was 53±13 (S.E.M.) μM (N=3). ADP inhibited [32P]PtdIns4P formation competitively with respect to ATP, the Ki being 0.4 mM. The data indicate that ADP is a poor competitive inhibitor of PtdIns kinase at the concentrations which are believed to be present intracellularly normally or which may be attained during mild hypoxia provided ATP levels are maintained in the millimolar range. Hence, any response of the myocardium to α-adrenergic hormones during mild hypoxia would be largely unimpaired by effects of Ca2+ on PtdIns and PtdIns(4,5)P2, or of ADP on PtdIns kinase activity.