David C. Pang

Modulation of lipolytic activity in isolated canine cardiac sarcolemma by isoproterenol and propranolol

Abstract Cardiac sarcolemmal preparations isolated from dog were tested for membrane-associated phospholipase A and lipoprotein lipase activities. The sarcolemma hydrolyzed 1-acyl 2 14 C-linoleoyl 3-glycero-phosphorylethanolamine at pH 7.0 to form predominantly 14 C-lyso PE with 5 mM EDTA and 14 C-free fatty acid with 5 mM Ca 2+ suggesting the presence of both phospholipases A 1 and A 2 and/or lysophospholipase activities in these preparations. Sarcolemmal PLA activity was stimulated 300% by 10 −5 to 10 −6 M d1-isoproterenol; this stimulation was blocked by 10 −4 M d1-propranolol. Lipoprotein lipase activity associated with the sarcolemmal fraction was enhanced 10-fold by 10 −5 M d1-isoproterenol; stimulation was blocked by d1-propranolol. Thus, the activities of membrane-bound lipolytic enzymes appear to be modulated by β-adrenergic agents in canine cardiac sarcolemma and could affect lipid dependent enzymes and/or membrane permeability.

Phospholipase A activity of highly enriched preparations of cardiac sarcolemma from hamster and dog.

Abstract Cardiac sarcolemma was isolated from hamster and dog and was examined for phospholipase A activity using 1-acyl [2- 14 C]-linoleoyl3-glycerophosphorylethanolamine as substrate. Hamster sarcolemmal preparations contained phospholipases A 1 which had optimal activity at pH 6.0 and pH 9.0 in the presence of 5 m m Ca 2+ ; EDTA was a potent inhibitor of phospholipase activity at both pHs. The specific activities of the hamster sarcolemmal phospholipases A 1 were increased 5.9-fold (pH 6.0) and 8.4-fold (pH 9.0) over the homogenate, while the increase in specific activity of the sarcolemmal marker enzyme, ouabain-sensitive (Na + -K + )-Mg + -ATPase, was 7.5-fold; thus both the (Na + +K + )-Mg + -ATPase and phospholipase A 1 activities were associated with enriched cardiac sarcolemmal membranes. Canine myocardial sarcolemmal preparations also contained a phospholipase A that had optimal activity at pH 7.0 in the presence of 5 m m Ca 2+ and the enzyme exhibited apparent specificity for the 2-position. The phospholipase A and (Na + +K + )-Mg 2+ -ATPase activities were similarly enriched in the canine sarcolemmal preparation. These endogenous, calcium-stimulated phospholipase A activities may be important constituents of the myocardial sarcolemmal membrane; they may modulate the lipid environment of the sarcolemma and may regulate the activity of lipid-dependent enzymes as well as alter membrane permeability.

Differential actions of calcium antagonists on calcium binding to cardiac sarcolemma.

The action of four calcium antagonistic drugs, including verapamil, bepridil, nifedipine, and diltiazem, on calcium binding to cardiac sarcolemma from guniea pig was tested. It was found that verapamil (10(-6) to 10(-5) M) inhibited calcium binding to a great extent. Bepridil at the same concentrations was less potent than verapamil in the depression of calcium binding. Nifedipine and diltiazem did not affect sarcolemmal calcium binding. The differential action of the calcium antagonistic drugs was discussed.

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma.

Ca2+ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 . 10(-5) and 1.2 . 10(-3) M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca2+ binding were studied. All cations tested inhibited Ca2+ binding with the following order of potency: trivalent greater than divalent greater than monovalent cations. The order of potency for the monovalent ions was: Na greater than K+ greater than Li+ greater than or equal to Cs+ and for the divalent and trivalent ions: La3+ greater than or equal to Mn2+ greater than Sr2+ greater than or equal to Ba2+ greater than Mg2+. 1 . 10(-3) M caffeine and 1 . 10(-8) M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 . 10(-7) M verapamil, acidosis (pH 6.4), 1 -10(-5) M Mn2+ and 1 . 10(-4) M ouabain depressed the capacity of the low-affinity sites to a range of 154--291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.

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.

Veratridine stimulation of calcium uptake by chick embryonic heart cells in culture.

Veratridine (2 X 10(-5) to 1 X 10(-4)M) stimulated Ca uptake into monolayer cultures of chick embryonic heart cells in a dose-dependent manner. The stimulation of veratridine was independent of the age of the chick embryos from which the heart cells were taken, and was inhibited by tetrodotoxin (3 microM). The half-maximal effect of veratridine was 37 microM. Since young embryonic hearts have few or no functional fast Na channels (Sperelakis, N. (1981) Cardiac Toxicol 1, 39-108), the present results indicate that the action of veratridine is not dependent solely on fast Na channels, but could be related to other properties of the surface membrane, such as increasing the resting Na permeability and/or opening of the slow Na channels. The increased Ca influx could then be a consequence of the Ca0:Nai exchange reaction.

Production of lysophospholipids and free fatty acids by a sarcolemmal fraction from canine myocardium.

Abstract The potential for injury of myocardial sarcolemma by endogenous lipases was studied. The sarcolemmal fraction was incubated for 30 min under conditions found optimal for hydrolysis of exogenous phosphatidylethanolamine (5 mM calcium, pH 7.0, 37°C). Incubation of the sarcolemmal fraction increased significantly the level of total free fatty acids (14.1 to 31.1 nmoles/mg protein, P

Cardiac sarcolemma of the hamster. Enrichment of the (Na+ + K+)-ATPase.

Cardiac sarcolemma was prepared from normal hamsters using gentle homogenization, extraction with 0.6 M KCl and continuous density gradient centrifugation. The final fraction exhibited high (Na+ + K+)-ATPase activity (24 micromol/mg per h) and contained minimal contamination from mitochondria, myofibrils and lysosomes.

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.

Mechanism of propranolol inhibition of the cardiac sarcotubule-γ-AT32P reaction

Abstract Analysis of the mechanism by which propranolol inhibits calcium-stimulated, magnesium-dependent, ATPase activity of a sarcotubule-enriched fraction (SR) prepared from canine ventricle was undertaken with the aid of γ-labeled AT 32 P. When calcium was the dominant divalent cation during incubation, the level of an intermediate, a phosphoprotein (EP), in the hydrolytic reaction increased rapidly to 0.9 nmole/mg of SR protein and plateaued. However, when magnesium was the dominant cation, the level of EP was much lower and decreased as the incubation period increased. Propranolol (1 mM) had no influence on the levels of phosphoprotein formed in the presence of calcium but increased EP levels when excess magnesium was added. Since calcium is required for the formation of EP and magnesium for the hydrolysis of EP, these observations suggest that 1 mM propranolol is primarily limiting EP breakdown. This conclusion was confirmed in an experiment in which propranolol was shown directly to produce a 67 per cent inhibition of the breakdown of EP to inorganic phosphate. Since 5 mM propranolol was found to depress the level of EP formed during incubation of SR with calcium, it appears that higher doses of the drug may also interfere with EP formation.