Doris I. Repke

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.

Tissue factor accelerates the activation of coagulation factor VII: The role of a bifunctional coagulation cofactor

One way in which coagulation may be initiated is via the tissue factor pathway in which a non-proteolytic enzyme activator, tissue factor, complexes with a plasma protein, factor VII. Factor VII is the zymogen of a serine protease, factor VIIa. While factor VII-tissue factor has coagulant activity, upon conversion to factor VIIa the activity increases about 120-fold. We now show that tissue factor markedly accelerates the conversion of factor VII to factor VIIa. Thus, this essential coagulation cofactor is clearly bifunctional.

Calcium-membrane interactions in the myocardium: Effects of ouabain, epinephrine and 3′, 5′-cyclic adenosine monophosphate

Abstract Cardiac microsomes, which represent an enriched but not pure preparation of the hearts sarcoplasmic reticulum, can remove calcium from solution by 2 kinetically dissimilar mechanisms. In the presence of adenosine triphosphate (ATP), Ca ++ is taken up by cardiac microsomes by a process designated Ca-binding , which exhibits saturation kinetics. The rate and extent of Ca-binding, and the high affinity of the Ca-binding sites could allow this process to cause the intact cell to relax. When anions that permit Ca ++ to be precipitated within the mlcrosomal vesicles are included along with ATP, much larger amounts of Ca ++ are taken up by cardiac microsomes. This second process, designated Ca-uptake , does not follow saturation kinetics. Instead, the rate of Ca-uptake increases linearly with increasing Ca ++ concentration until Ca-uptake becomes inhibited at higher Ca ++ concentrations. The finding of 2 kinetically distinct Ca ++ transport processes in cardiac microsomes, both of which are highly active in the micromolar range of Ca ++ concentration, suggests that Ca ++ movements in the intact myocardial cell may be controlled by 2 mechanisms. It is suggested that one of these, possibly manifest in vitro as Ca-binding, represents an intracellular release site that initiates systole by delivering Ca ++ to the contractile proteins. The second process, possibly manifest in vitro as Ca-uptake, is suggested to represent the uptake of Ca ++ into an intracellular storage site whose Ca ++ content indirectly determines the amount of Ca ++ that is delivered to the contractile proteins. These 2 intracellular Ca ++ pools can be tentatively related to Ca ++ movements into and out of the myocardial cell, permitting the formulation of a model by which a number of inotropic interventions might modulate myocardial contractility. Cardiac glycosides had no detectible effect on either cardiac microsomal Ca-binding or Ca-uptake. Cyclic adenosine monophosphate (cAMP), which by itself was without effects on cardiac microsomes, more than doubled the rate of Ca-uptake in the presence of a cyclic AMP-dependent protein kinase. The resulting increase in rate of Ca-uptake could explain the actions of epinephrine to enhance contractility at the same time that systole is abbreviated.

Effects of ethanol on calcium binding and calcium uptake by cardiac microsomes

Abstract Calcium uptake and Ca binding by cardiac microsomes enriched in fragmented sarcoplasmic reticulum were inhibited by ethanol. After 5 min of incubation, half-maximal inhibition of the former was seen at approximately 1.3 M ethanol, that of the latter was seen at approximately 1.9 M ethanol. Neither Ca uptake nor Ca binding was stimulated at lower concentrations of ethanol; Prolonged exposure of the microsomes to ethanol increased the extent of inhibition of both Ca uptake and Ca binding. These inhibitory effects were almost completely reversed when microsomes were washed after exposure to ethanol. Inclusion of 0.12 M Na+ instead of 0.12 M K+ increased the inhibitory effect of ethanol on Ca uptake, but did not significantly change the sensitivity of Ca binding to ethanol. These inhibitory effects of ethanol were seen at concentrations that generally stabilize membrane structures and are associated with an anesthetic action. Even though the concentrations of ethanol needed to inhibit Ca uptake and Ca binding exceeded those found in the blood of chronic alcoholic patients, the time dependence of these effects raises the possibility that prolonged exposure to concentrations of ethanol might contribute to the myocardial weakness of chronic alcoholics by interfering with the retention of Ca2+ within the cardiac sarcoplasmic reticulum.

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.

Calcium-binding and calcium-uptake by cardiac microsomes: A kinetic analysis ☆ ☆☆

Cardiac microsomes, which contain membrane fragments derived from the sarcoplasmic reticulum, can remove Ca2+ from solution by two kinetically dissimilar mechanisms. Ca-binding, which occurs in the presence of ATP, accounts for the rapid sequestration of all smamounts of Ca2+. Ca-uptake, which is seen when a calcium-precipitating agent such as oxalate is included, permits much larger amounts of Ca2+ to be sequestered, though more slowly. The [Ca2+] dependence of these two processes was examined in the range of [Ca2+] between 1 × 10−7 m to 1 × 10−5 m. Ca-binding of microsomes prepared in sucrose (suc-microsomes) and in dilute buffers (H2O-microsomes) exhibited saturation kinetics. The number of sites (n) was approximately 13 nmol per mg protein for suc-microsomes and 45 nmol per mg protein for H2O-microsomes. The affinity constant for suc-microsomes was approximately 1.2 × 106 m−1 (half saturation at [Ca2+] = 8 ¢ 10−7 m-Ca2+) and approximately 2 × 106 m−1 (half saturation at [Ca2+] = 5 × 10−7 m) for H2O-microsomes. Ca-uptake did not show saturation kinetics. The rate of Ca-uptake (approximately 0.04 μmol/min/mg protein per μm Ca2+ for both types of microsomes) increased linearly with increasing [Ca2+] within this range of [Ca2+]. n nCa-binding is thus shown kinetically to involve a finite number of Ca-transport sites, whereas Ca-uptake, from a kinetic standpoint, resembles a non-facilitated diffusion process, possibly driven by an electrochemical gradient across the vesicular membrane that results from ATP hydrolysis. The rate limiting step in Ca-uptake does not involve the high affinity sites defined in the studies of Ca-binding.

Myosins from rat right and left ventricles. Comparison of ATPase activities and light fragments released by 8 M-urea.

Myosins prepared from rat right and left ventricles had similar levels of ATPase activity, measured in Mg2+ and in Ca2+. The low molecular weight fragments of these myosins, examined by SDS-polyacrylamide gel electrophoresis, also did not differ. It is concluded that the differences between the mechanical characteristics of right- and left-ventricular contraction are not reflected in differences between the myosins of the two ventricles. However, the ATPase activity of rat cardiac myosin was higher than that noted previously for comparable preparations from rabbit and canine ventricles, and the low molecular weight fragments of rat cardiac myosin differed from those reported for rabbit cardiac myosin. These findings suggest that shortening velocity, which has been reported to be greater in the hearts of smaller animals, may reflect a difference in the low molecular weight fragments and ATPase activity of cardiac myosin.

Oxalate dependence of calcium uptake kinetics of rabbit skeletal muscle microsomes (fragmented sarcoplasmic reticulum)

The initial rate of oxalate-facilitated Ca2+ uptake by skeletal microsomes depends on both Ca2+ and oxalate concentrations in the medium. The apparent Km for Ca2+ increases with increasing oxalate concentration, indicating that Ca2+ uptake can involve a carrier-mediated transport system.

Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca2+ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10−4 M CaCl2 (high ionized Ca2+) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca2+). In both high and low Ca2+, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca2+ (approx. 2.3 nmoles·mg−1·min−1) than in low Ca2+ (approx. 1.8 nmoles·mg−1·min−1). This difference, when analyzed by the Students t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na+ + K+)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca2+, measured with the use of CaEGTA buffers, was studied. The Ca2+-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10−7 M Ca2+. This corresponds to an apparent binding constant of 2.2·106 M−1, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca2+ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.