ATP-dependent Calcium Accumulation Research Articles

Relationship between blood pressure of spontaneously hypertensive rats and alterations in membrane properties of mesenteric arteries.

Spontaneously hypertensive rats (SHR) and two strains of normotensive rats were compared with respect to enzymatic activities and calcium accumulation of plasma membrane and endoplasmic reticulum enriched fractions from their mesenteric arteries. Increased specific activities of alkaline phosphatase, 5'-nucleotidase and Mg2+-ATPase, and increased ATP-dependent calcium accumulation were found in 5- to 6-month-old SHR as compared to both strains fo age-matched normotensive rats. Alkaline phosphatase was increased in 33-day-old "early hypertensive" and 3- to 4-month-old SHR, but 5'-nucleotidase, Mg2+-ATPase, and calcium accumulation were not. Hydralazine treatment of young SHR partially prevented the increase of both alkaline phosphatase activity and blood pressure that develops with age. The relationship between alkaline phosphatase activity and the alterations in vascular reactivity associated with hypertension remains to be determined.

ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate

1. 1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200–250 nmol Ca 2+/mg protein. 3. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent K m for calcium of 6.7 · 10 −5 and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg 2+ concentration was 2 mM. 4. 4. Dinitrophenol and NaN 3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. 6. The Mg 2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 μM CaCl 2. Greater calcium concentrations caused a progressive inhibition. 7. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.

Effect of diuretics on ion transport of kidney cortex mitochondria I. Inhibition of ion accumulation by ethacrynic acid and furosemide

Rat kidney cortex mitochondria accumulated calcium remarkably in the absence of phosphate in the medium. ATP-dependent calcium and magnesium accumulation in rat kidney cortex mitochondria were inhibited by ethacrynic acid and furosemide, and the potassium accumulation stimulated by valinomycin was also inhibited by both diuretics. Oxygen consumption of kidney cortex mitochondria, on the other hand, was depressed by ethacrynic acid, but not at all by furosemide. Studies of this report place major emphasis upon the relationship between the effects of both diuretics on ion accumulation and on electron transport system.

The effect of calcium ionophores on fragmented sarcoplasmic reticulum.

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.

Biochemical basis of heart function. IV. Energy metabolism and calcium transport in hearts of vitamin E deficient rats.

Feeding a vitamin E deficient diet to rats for 10 weeks was found to decrease myocardial creatine phosphate, ATP, ATP/ADP ratio, NAD+, NADP+, and NADPH, whereas the level of ADP was increased without any changes in the levels of AMP, total adenine nucleotides, NADH, and ATP/AMP ratio. The levels of ATP and pyridine nucleotides were restored fully, whereas creatine phosphate was restored partially on feeding a normal diet for 4 weeks to animals previously on the vitamin E deficient diet for 10 weeks. Vitamin E deficiency was found to increase cardiac lactate, pyruvate, and lactate/pyruvate ratio and decrease the activities of lactate dehydrogenase and malate dehydrogenase. The activity of Na+–K+-stimulated, ouabain-sensitive ATPase was markedly elevated in the hearts of animals on the vitamin E deficient diet. The ATP-dependent calcium accumulation by the sarcoplasmic reticular fraction in the absence and presence of P1 or oxalate was greater in the vitamin E deficient heart. Vitamin E deficiency also increased the Ca2+-stimulated ATPase activity of the cardiac sarcoplasmic reticulum. Although myocardial contractility of the hearts from vitamin E deficient rats was depressed, no damage to the ultrastructures of mitochondria and sarcoplasmic reticulum was apparent. These results indicate marked alterations in myocardial metabolism due to vitamin E deficiency and it is suggested that such changes are due to abnormalities in the processes of both energy production and utilization.

The influence of some cardioactive drugs on the energy-dependent uptake of calcium, potassium and adenine nucleotides by mitochondria

A number of cardioactive substances were studied for their influence on isolated mitochondria obtained from rat liver and rabbit heart. Among several drugs tested, quinidine, dl-propranolol, l-alprenolol, dl-oxyfedrin and tetracaine appeared to be the most active in decreasing or blocking the rate of energy-dependent uptake of calcium, potassium and adenine nucleotides in an incubation medium containing substrate (succinate or glutamate/fumarate), MgCl 2, phosphate and approximately 5 mg of mitochondrial protein. The effective concentrations (5 × 10 −5 m to 10 −3 m) were comparable to those employed by other investigators to block the effects on the ATP-dependent calcium accumulation and the ATPase-activity of isolated sarcotubular fractions of heart and skeletal muscle by means of quinidine and propranolol. It is concluded that the rate-inhibiting effects of the active drugs on the velocity and the extent of calcium, potassium, and adenine nucleotide uptake could determine their effect on cardiac contractility.

Trypsin digestion of fragmented sarcoplasmic reticulum

Short incubation of fragmented sarcoplasmic reticulum with trypsin produces inhibition of ATP dependent calcium accumulation, while ATPase is activated. The addition of trypsin to loaded vesicles causes a rapid calcium leak, suggesting that certain easily digestible peptidic bonds may contribute to the maintenance of semipermeability in sarcoplasmic membranes. A small amount of calcium is still taken up by partially digested membranes, independently of the presence of ATP. This uptake is due to binding to the membrane itself and is competitively inhibited by other cations. ATPase is inhibited by longer digestions, while obvious structural changes appear throughout the thickness of the membrane. This indicates that either ATPase is extended into the deeper layer of the membrane, or it is very resistant to trypsin attack. A parallel pattern of inhibition is produced by trypsin on ATPase and ADP-ATP exchange, indirectly suggesting interdependence of these two activities. Depending on the time of digestion, more or less marked alterations of the membrane structure can be observed on electronmicroscopy (negative staining with phosphotungstic acid): disappearance of an outer granular layer, irregular thickness of the membrane, opening of the vesicles and aggregation. Suspensions of fragmented sarcoplasmic reticulum digested with trypsin, undergo a rapid decrease in turbidity, likely due to a change in the state of aggregation of the vesicles.