F.Norman Briggs

Calcium Requirements for Cardiac Myofibrillar Activation

The amounts of calcium required to achieve various levels of myofibrillar activation in the dog heart were determined by measuring the dependence of myofibrillar calcium binding, myofibrillar adenosinetriphosphatase (ATPase), and isometric tension on free calcium concentration. Myofibrillar ATPase was half-maximal at 2.4 × 10−6M free calcium, and tension development was half-maximal at 2.0 × 10−6M free calcium. No simple relation between calcium binding and activation was found. For example, between 10−8M and 10−6M free calcium, an appreciable amount of calcium was bound to the myofibrils, but there was little activation of isometric tension. On the other hand, myofibrillar calcium binding was not saturated at levels of free calcium at which both tension and ATPase were maximal; therefore, it appears that only a portion of the total myofibrillar calcium binding sites control ATPase and tension. Using the information derived from the binding and activation studies together with our determination of the myofibrillar content of the dog heart, 47.5 mg myofibrillar protein/g wet heart, we calculated the calcium required to achieve various levels of myofibrillar activation in the intact ventricle. By this calculation method, development of half-maximal tension required 22.4 μmoles calcium/kg wet heart, and development of maximal isometric tension required 92.8 μmoles/kg wet heart.

Ca-ATPase isozyme expression in sarcoplasmic reticulum is altered by chronic stimulation of skeletal muscle

Chronic stimulation of a predominantly fast skeletal muscle enhanced the expression of type I (slow muscle) Ca‐ATPase and suppressed the expression of the type II (fast muscle) Ca‐ATPase. Monoclonal antibodies IID8 and IIH11 against type I (slow) and type II (fast) isozymes respectively, were used to type the Ca‐ATPases of the isolated SR (sarcoplasmic reticulum) by Western blots, and the Ca‐ATPases of the muscle fibers by immunohistochemistry. Of the fibers from control muscles 80% stained for the type II isozyme and 20% for the type I isozyme. Following chronic stimulation all fibers stained for type I isozyme and none stained for type II isozyme. Ca‐ATPase isozyme distribution in isolated SR confirmed this effect of chronic stimulation. The calcium uptake activities of homogenates of stimulated muscles were 22% of the control muscles. The Ca‐ATPase and calcium‐uptake activities of the isolated SR from stimulated muscles were, respectively, 32 and 45% of the control muscles.

Determinants of calcium loading at steady state in sarcoplasmic reticulum

The determinants of steady-state calcium loading by sarcoplasmic reticulum vesicles were evaluated by measuring the contribution of different pathways of calcium flux to the total calcium flux at steady state. The diffusional passive pathway was least significant at all calcium loads studied. Diffusional passive calcium flux was evaluated by a number of methods which gave comparable results and support its designation as passive and diffusional. These methods included (a) flux measurements with the simple pump-leak system which pertains when acetyl phosphate is used to load the vesicles; (b) flux measurements made after quenching the pump with EGTA; (c) flux measurements made after quenching the pump with glucose plus hexokinase; and (d) evaluation of the effect of pump activity on the efflux of mannitol. The calcium efflux not accounted for by the diffusional pathway was assigned to non-diffusional pathways. Efflux through the non-diffusional pathways required ATP, ADP and extravesicular Ca2+. The ADP-dependent, phosphoenzyme-independent pathway described by Beirao and DeMeis (Biochim. Biophys. Acta (1976) 433, 520-530) was not significantly involved in efflux. We propose that the level of calcium loading achieved at steady state is determined by the levels of the intermediates of the calcium pump which are established at this pseudo-equilibrium condition, these levels being determined by the concentrations of intravesicular and extravesicular calcium ([Ca2+]i and [Ca2+]), ATP and ADP. The different levels of calcium loading achieved by skeletal and cardiac sarcoplasmic reticulum are attributed to different nucleotide and calcium kinetics in these two types of sarcoplasmic reticulum and possibly to different intravesicular volumes. Differences in diffusional permeability are not responsible for differences in calcium loading.

The effect of calcium oxalate crystallization kinetics on the kinetics of calcium uptake and calcium ATPase activity of sarcoplasmic reticulum vesicles

Abstract The ionophore A23187 is a potent inhibitor of oxalate supported calcium uptake if added before uptake is initiated by ATP and is a much weaker inhibitor of uptake once uptake has been initiated. This observation is shown to be due to a failure of oxalate to capture the transported calcium at the beginning of uptake because the rate of calcium oxalate crystallization is initially slow, thereby allowing the ionophore to release the accumulated calcium. This hypothesis is supported by the observation that calcium oxalate crystallization shows a lag phase which is absent when calcium oxalate seeds are in the reaction system. Once calcium uptake has progressed, calcium oxalate seeds are present in the sarcoplasmic reticulum and calcium oxalate crystallization proceeds sufficiently rapidly that the ionophore cannot compete successfully for calcium. That A23187 and oxalate compete for intravesicular ionic calcium is shown by the stimulation which each produces in ATPase activity and by the dependence of ionophore activity on oxalate concentration. The failure of calcium oxalate crystallization to reach equilibrium during the early phase of calcium uptake caused us to examine whether at any time during calcium uptake, crystallization reaches equilibrium. Skeletal sarcoplasmic reticulum accumulated calcium at such a high rate that oxalate, in concentrations up to 20mM, was unable to clamp intravesicular calcium at equilibrium values. The lower rate of calcium accumulation by cardiac sarcoplasmic reticulum and/or perhaps its greater permeability to oxalate apparently allows intravesicular calcium to be clamped by oxalate.

Analysis of the ATP-induced conformational changes in sarcoplasmic reticulum

Abstract A series of group-specific spin-labeled compounds was used to investigate the mechanism of the ATP-induced conformational changes in rabbit skeletal sarcoplasmic reticulum. The spin labels used can be divided into three classes according to their specificities: (I) N (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide for SH groups; (II) N (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)isothiocyanate for amine or hydroxyl groups; and (III) N -oxyl-4′,4′-dimethyl-oxazolidine derivatives of stearic acid for fatty acids. Of the three classes of compounds tested, only the mobility of probe (I) changed upon addition of ATP to the spin-labeled sarcoplasmic reticulum. This ATP-induced conformational change could be depressed by 5 m m propranolol, a concentration which by itself had no effect on the mobility of the spin label. Since similar concentrations of propranolol inhibited the breakdown but did not influence the formation of a phosphorylated intermediate during the hydrolysis of ATP, these observations suggest that the conformational change takes place at a step in ATP hydrolysis beyond the formation of the phosphorylated intermediate. The same basic series of experiments was also performed with the purified sarcoplasmic reticulum enzyme. Even though similar results were obtained, the sensitivity of the enzyme toward propranolol and also the mobility of probe (I) in the enzyme were different from that of the sarcoplasmic reticulum. Large doses (10–20 m m ) of propranolol, however, were found to directly alter the mobilities of all the classes of probes used. The effect of 20 m m propranolol on probe (III) in the sarcoplasmic reticulum was equivalent to a 10 °C rise in temperature of the membrane.

Mechanism of quinidine and chlorpromazine inhibition of sarcotubular ATPase activity

Abstract Quinidine and chlorpromazine in concentrations ranging from 0–25 to 1–2 mM inhibited sarcotubular ATPase activity by distinctly different mechanisms. The noncompetitive inhibition produced by quinidine was entirely due to its effects on hydrolysis of the phosphorylated intermediate. Formation of the phosphorylated intermediate from γ-AT 32 P was unaffected by quinidine. In contrast to these results, chlorpromazine was found to have no effect on the hydrolysis of phosphorylated intermediate but to depress its levels. Using the β,γ-methylene analogue of ATP. it was possible to show that chlorpromazine lowers the phosphorylated intermediate levels by depressing the affinity of the enzyme for its substrate. Chlorpromazine inhibition of substrate binding was non-competitive.

Primary structure of the nucleotide binding domain of the Ca,Mg-ATPase from cardiac sarcoplasmic reticulum.

The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.

The effect of Gram negative endotoxin on the calcium uptake activity of sarcoplasmic reticulum isolated from canine myocardium.

Abstract Gram negative endotoxin (Salmonella abortus equi) significantly inhibited the ATP dependent calcium uptake of canine myocardial sarcoplasmic reticulum (SR). The degree of inhibition was dependent upon the concentration of the endotoxin and its time of exposure to the SR. Significant inhibition was seen at endotoxin concentrations ranging between 0.024 mg/ml and 0.24 mg/ml. Inhibition, at a dose of 0.24 mg/ml, varied between 10.1 ± 0.1 percent with no preincubation to 61.0 ± 5.0 percent after fifteen minutes of preincubation. The relation between depression of the sarcotubular calcium pump and myocardial depression is discussed.

Transcription rates of SERCA and phospholamban genes change in response to chronic stimulation of skeletal muscle.

Chronic low frequency stimulation of predominantly fast-twitch skeletal muscles decrease the levels of SERCA1 (fast-twitch sarco(endo)plasmic reticulum Ca2+-ATPase) mRNA, and increase the levels of SERCA2 (slow-twitch sarco(endo)plasmic reticulum Ca2+-ATPase) and phospholamban mRNAs. To assess the role of transcription in these changes in mRNA levels, nuclei were isolated from chronically stimulated canine latissimus dorsi muscles and transcription rates were estimated by nuclear run-on assays. Decreases in the rates of SERCA1 gene transcription matched the fall in its mRNA level and increases in the rates of SERCA2 and phospholamban gene transcription matched the increases in their mRNAs.

Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

Abstract 8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca 2+ . The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca 2+ and Mg 2+ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.