The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores

The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange.     

Liver mitochondria, confirmed as intact by complete suppression of succinate uptake and oxidation, possess a carnitine palmitoyltransferase I that is totally inhibited by malonyl CoA.

Succinate dehydrogenase activity in mitochondria, which were isolated by centrifuging partially purified mitochondria through 1. 315 M sucrose, was completely suppressed when [14C]succinate uptake was abolished by prior incubation of the mitochondria with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin. The conclusion that these mitochondria were intact was confirmed by the fact that, when these mitochondria were broken by a freeze-thaw cycle followed by sonication, such inhibition was totally abolished. The yield of mitochondria, microsomes, and peroxisomes from the initial homogenate was 17.8, <0.1, and 0%, respectively, indicating that the mitochondria were not only intact but also essentially free of contamination from microsomes and peroxisomes. The overt form of carnitine palmitoyltransferase (CPT I) in these intact and pure mitochondria was totally inhibited by malonyl CoA, indicating that previous reports of incomplete inhibition in mitochondrial preparations resulted from interference from CPT activity in the inner mitochondrial membrane (CPT II), microsomes, or peroxisomes.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats.

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.     

Inhibition of hepatic aldehyde dehydrogenase by carbon tetrachloride: an in vitro study

In vitro inhibition of rat liver mitochondrial and microsomal aldehyde dehydrogenase (ALDH) under conditions of active CCl4 metabolism was investigated. Incubation of microsomes or mitochondria in the presence of NADPH alone caused significant, time-dependent inhibition of mitochondrial and microsomal ALDH. EDTA partially protected ALDH from inhibition. Incubation of microsomes or microsomes plus mitochondria in the presence of NADPH and CCl4 resulted in marked inhibition of microsomal and mitochondrial ALDH activity. The inhibition was both dose- and time-dependent and was relatively less in the presence of EDTA. It is proposed that the inhibition of membrane-bound ALDH may be one of the early events responsible for the genesis of CCl4-hepatotoxicity.     

Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum.

Calcium uptake by the microsomal and mitochondrial fractions of pig coronary artery and guinea pig ileum was studied in the presence of ATP, ATP plus oxalate and without ATP and oxalate. Microsomes and mitochondria of both smooth muscles were found to be unable to accumulate appreciable amounts of calcium in the absence of ATP. Oxalate noticeably stimulated the calcium uptake of the mitochondrial fraction from pig coronary artery but had little effect on calcium uptake by the microsomal fraction of this smooth muscle. The calcium uptake of microsomes and mitochondria from guinea pig ileum was not or only slightly enhanced by oxalate. There are typical kinetics regarding the time course and the extent of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. In comparison, considerable qualitative and quantitative differences between both smooth muscles are observed. The high ATP-dependent calcium uptake capacity of the mitochondria from pig coronary artery and guinea pig ileum are a further argument for the hypothesis that these organelles may play an important role in the contraction-relaxation mechanism of smooth muscle.     

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

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. 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. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M 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. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 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. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 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.     

Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride.

ATP-dependent calcium uptake of rat liver microsomes is examined following ingestion of CC14 (2.5 ml/kg). Within 30 min there is an abrupt drop in calcium uptake activity of the liver microsomes. This activity remains down for 48 hours before slowly returning to normal levels. The effect is specific for CC14 as contrasted with CHC13 and CH2Cl2. The CCl4 does not affect similar calcium uptake activity of kidney microsomes. Calcium uptake activity of the liver mitochondria is unaffected. The first 12 hours after CCl4 ingestion there is a relatively slow rise in the calcium content of the liver tissue and mitochondria. After 12 hours a much larger influx of calcium into the tissue and the mitochondria takes place. Forty-eight hours after CCl4 ingestion the process begins to slowly reverse. The following postulated sequence may relate to the CCl4 hepatotocicity. CCl4 is activated to free radicals by the liver endoplasmic reticulum. The free radical inactivate calcium pump activity of the liver endoplasmic reticulum. Calcium levels of the cytoplasm increase and significantly modify ion permeability of the plasma membrane. High levels of external calcium enter the cytoplasm and are sequestered in the mitochondria. The high level of mitochondrial calcium uptake inhibits mitochondrial oxidative phosphorylation. The specific sensitivity of the calcium pump activity of liver microsomes to CCl4 further establishes the identity of a system seperate from the mitochondrial system. The above postulated sequence of events would suggest a critical role in liver metabolism for calcium pump activity of the endoplasmic reticulum.     

Induction of hepatic microsomal calcium uptake by glucocorticoids

Hepatic microsomes take up calcium in the presence of ATP and oxalate. In either fed or fasted adrenalectomized rats injections of dexamethasone 18 hours and then again 1 hour prior to sacrifice increased uptake of calcium by microsomes. Injections of estradiol had no similar effect indicating that the stimulation might be specific to glucocorticoids. Injection of Actinomycin D, an inhibitor of protein synthesis, 1 hour prior to dexamethasone administration resulted in a complete block of the stimulation. It is therefore likely that the increased calcium uptake is due to the induction of the microsomal calcium activated ATP-ase. The onset of this effect occurred later than the induction of tyrosine-amino transferase (TAT). The present data, in conjunction with the previous demonstration of glucagon stimulation and insulin inhibition of this system, indicates that microsomes might serve as a modifier of intracellular calcium distribution.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.     

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.     

Uptake and release of calcium by microsomes of nonvascular smooth muscle.

Microsomes prepared from guinea-pig ileum longitudinal smooth muscle and rat uterus continuously sequester calcium for a one hour period in the presence of Mg-ATP as an energy source and oxalate anion as a trapping agent. Dithiothreitol is essential for maximal calcium uptake activity of the rat uterus microsomes. On sucrose density gradients, calcium uptake of the smooth muscle microsomes appears to be associated with intracellular membrane (sarcoplasmic reticulum). Release of sequestered calcium from the longitudinal muscle microsomes is very slow (20% in 50 minutes). A small labile fraction (20%) is released by EGTA (1 mM) in 10 minutes. Rapid release of sequestered calcium (90% in 10 minutes) occurs in presence of the calcium ionophore A23187 (2 μM) or in the presence of chlorpromazine (1 mM).     

The effect of cytochrome P-450 and reduced glutathione on the ATP-dependent calcium pump of hepatic microsomes from male rats

In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.     

Hormone sensitive calcium uptake by liver microsomes

The effects of glucagon and insulin on hepatic microsomal calcium uptake were investigated. Microsomes isolated from perfused rat liver accumulated calcium in the presence of ATP and oxalate. Addition of glucagon to the perfusate significantly increased calcium uptake by microsomes subsequently isolated. In contrast, addition of insulin to the perfusate resulted in a decreased microsomal calcium uptake and inhibition of the glucagon effect. Because the effects of glucagon and insulin on hepatic microsomal calcium uptake are opposite, as are the metabolic effects of these hormones, it is likely that the observed differences are of physiological importance.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

Uptake of calcium in chromaffin granules of bovine adrenal medulla stimulated in vitro

The calcium content of bovine adrenal medulla perfused in vitro has been shown to increase about 30% in response to extensive acetylcholine stimulation. The calcium accumulated during secretion was mainly associated with the mitochondria and chromaffin granule fractions and to a lesser extent in the microsome fraction. While the calcium taken up by the mitochondria and microsomes was partly or totally removed by treatment with EDTA, the chelating agent had no effect on the granule content of calcium. The uptake of calcium in the mitochondria and microsomes during secretion is consistent with a function of these organelles in regulating the cellular calcium concentration. It is suggested that also the chromaffin granules may act as a “Ca-pump” in the chromaffin cell of the adrenal medulla.     

Alterations in platelet aggregation and microsomal benzo-α-pyrene hydroxylase activities after exposure of rats to a prudhoe bay crude oil

Administration of a Prudhoe Bay crude oil (PBCO) to rats has been shown to (a) inhibit platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid, or epinephrine and (b) induce benzo-α-pyrene hydroxylase (BPH) in the liver and small intestine. Maximum inhibition of aggregation (90%) was seen 12 to 16 hours subsequent to dosing. However, substantial inhibition was observed as early as four hours and as late as 48 hours after dosing. Of particular interest was the sensitivity of the platelet response compared with the putatively sensitive response of monooxygenase induction in liver. As little as 0.1 ml of PBCO per kilogram body weight (bw) caused an inhibition of aggregation with all three agonists. A similar inhibition of the release of ADP from platelets in the presence of arachidonic acid or epinephrine was also observed. In contrast, hepatic BPH activity showed only a modest increase (67%) over the control value even after administration of 2 ml of PBCO per kilogram body weight. Small intestine BPH activity was more sensitive, showing a gradual increase of up to 19-fold 24 hours after dosing with 2 ml of PBCO per kilogram body weight. The sensitivity of the platelet response is of general environmental interest and evaluating platelet aggregation in humans may be important as a noninvasive assay for exposure to either accidental or “acceptable” levels of petroleum hydrocarbons in the occupational environment.     

Functional coupling of the calcium pump and glucose 6-phosphatase activity in liver microsomes: preliminary results

The addition of G-6-Pi to the incubation system for MgATP-dependent calcium transport in liver microsomes results in a marked stimulation of Ca2+ uptake. At physiological pH values (7.2-7.4), the G-6-Pi stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. In the system for the G-6-Pi-stimulated calcium uptake, G-6-Pi is actively hydrolyzed by the glucose 6-phosphatase activity of liver microsomes. Such an activity is not influenced by the concomitant calcium uptake. After the incubation of the system for the MgATP-dependent microsomal calcium transport in the presence of G-6-Pi, Pi and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose 6-phosphatase multicomponent system.     

The calcium abnormality in cystic fibrosis mitochondria: Relative role of respiration and ATP hydrolysis

Calcium uptake by mitochondria isolated from skin fibroblasts of patients with cystic fibrosis and controls was studied in the presence and absence of inhibitors. Since mitochondrial calcium accumulation may be supported by ATP hydrolysis or respiration, inhibitors of each were used to characterize the basis of previously described alterations in calcium uptake by mitochondria from patients with cystic fibrosis. Calcium uptake measurements under the influence of oligomycin and antimycin A suggest that the increased calcium uptake by mitochondria from patients with cystic fibrosis is related to altered respiratory system activity. Binding constants of calcium to the carrier system in mitochondria were not different between genotypes.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.