Stimulation Of Calcium Uptake Research Articles

Angiotensin-mediated calcium efflux from adrenal glomerulosa cells.

The effects of angiotensin II on efflux of radiocalcium and production of aldosterone from dispersed bovine adrenal glomerulosa cells were studied using a flow-through system. Concentrations of angiotensin II between 1.25 X 10(-10) and 1.25 X 10(-8) M were found to stimulate both radiocalcium efflux and the rate of aldosterone production. The increase in radiocalcium efflux occurred within 1.5-2.5 min after angiotensin addition, reached a peak in 3.0-4.5 min, and then declined to a value slightly greater than control. The initial increase in aldosterone production occurred 3-5 min after the peak of calcium efflux. In cells preloaded with [45Ca] and then perfused for 1 h with a medium containing no calcium, the basal rate of aldosterone production fell to zero. Angiotensin II (1.25 X 10(-8) M) caused no increase in aldosterone secretion rate but still caused an efflux of radiocalcium. Exposure of cells to 5 X 10(-5) M verapamil blocked the effect of 1.25 X 10(-10) M angiotensin on both radiocalcium efflux and aldosterone production, but only partially blocked the effects of 1.25 X 10(-8) M angiotensin. In addition to stimulating calcium uptake into adrenal glomerulosa cells, angiotensin II stimulates the mobilization of calcium from an intracellular pool. The precise location of this pool is not known.

Electromagnetic modulation of biological processes: bicarbonate effect and mechanistic considerations in the Ca-uptake by embryonal chick tibia in vitro.

Electromagnetically induced currents pulsating at very low frequency stimulated calcium uptake by chick embryo tibia rudiments only when bicarbonate was present in the culture medium. A bicarbonate-dependent Ca2+-ATPase might be implicated in coupling of the electromagnetic signal with processes that promote Ca-transport and storage in bone tissue.

Stimulation of calcium uptake by 1-alkyl-2-acetyl- sn-glycero-3-phosphocholine (platelet-activating factor) in rabbit platelets: Possible involvement of the lipoxygenase pathway

1-Alkyl-2-acetyl- sn-glycero-3-phosphocholine (platelet-activating factor) induces an increase of Ca 2+ uptake in rabbit platelets. This process depends upon the extracellular concentration of Ca 2+ with the maximum stimulation occurring at 1–3 m m; uptake under these conditions is blocked by verapamil, a calcium-entry blocker. Increase of calcium uptake by the bioactive phospholipid was independent of ADP-induced platelet responses and of metabolites of arachidonic acid metabolism formed through the cyclooxygenase pathway. However, mepacrine, p-bromophenacyl bromide, eicosatetraynoic acid, and nordihydroguaiaretic acid significantly or totally inhibited the stimulation of Ca 2+ uptake by 1-alkyl-2-acetyl- sn-glycero-3-phosphocholine. When arachidonic acid was given sufficient time to be metabolized to other products by the platelets, stimulation of Ca 2+ uptake also occurred. Arachidonic acid and platelet-activating factor did not produce an additive or synergistic effect. Our data suggest that a metabolite(s) generated from arachidonic acid through the lipoxygenase pathway may be the mediator(s) responsible for the action of platelet-activating factor in the induction of increased Ca 2+ uptake in rabbit platelets.

Stimulatory effect of calcitonin on calcium uptake and glucose production in isolated rat hepatocytes.

The biological effect of calcitonin (CT) was investigated in isolated rat hepatocytes. Addition of synthetic [Asu1,7] eel CT (20 and 40 ng/ml of medium with 2.5 mg dry weight of cells) into the medium containing calcium ion produced a marked elevation of calcium uptake in the hepatocytes, when calcium concentrations in the medium were monitored spectrophotometrically using arsenazo III. Intracellular calcium contents in the mitochondria and microsomes were significantly increased by CT addition. Meanwhile, glycogenolysis and gluconeogenesis in the hepatocytes were significantly stimulated by addition of CT (10, 10(2), and 10(3) ng/ml of medium with 5 mg dry weight of cells) into the medium containing calcium, although CT effects were less than those effects of 10(-7) M glucagon and 10(-5) M phenylephrine. These data clearly indicate that CT stimulates calcium uptake and glucose production in the hepatocytes, suggesting that CT has an effect in the metabolic function of rat liver cells.

On the Mechanisms of ATP-Induced and Succinate-Induced Redistribution of Cations in Isolated Rat Liver Cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.

Induction of calcium-binding protein before 1,25-dihydroxyvitamin D3 stimulation of duodenal calcium uptake.

Protein synthesis occurring before the onset of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) stimulation of calcium uptake was examined in embryonic chick duodena by double label autoradiography. Duodena from 19-day-old embryos were cultured for 24 h. Duodena exposed to a saturating concentration of 1,25-(OH)2D3 during the last 2, 4, or 6 h were either cultured with [3H]leucine for the final 2 h or used for calcium uptake assays. Duodena not exposed to the hormone were either cultured with [14C]leucine for the final 2 h or used for calcium uptake assays. Calcium uptake by nonradiolabeled duodena was increased (p less than 0.05) only in tissues exposed to the hormone for 6 h. Tissue uptakes of [3H]leucine and [14C]leucine were identical and linear with time. Soluble proteins were extracted from 3H- and 14C-labeled tissues, mixed, and simultaneously resolved by two-dimensional polyacrylamide gel electrophoresis. Analysis of fluorographs and autoradiographs prepared from the double-labeled gels revealed a protein induced by 1,25-(OH)2D3 within 2 h of exposure to 1,25-(OH)2D3 and at lest 2 h before the calcium uptake response. The induced protein co-migrated with purified chick calcium-binding protein during two-dimensional electrophoresis. These results demonstrate that 1) this protein is the calcium-binding protein and 2) calcium-binding protein is clearly synthesized in the embryonic duodenum before the calcium uptake response to 1,25-(OH)2D3 occurs.

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca +-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca 2+ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.

Receptor-mediated release of plasma membrane-associated calcium and stimulation of calcium uptake by thyrotropin-releasing hormone in pituitary cells in culture.

Receptor-mediated release of plasma membrane-associated calcium and stimulation of calcium uptake by thyrotropin-releasing hormone in pituitary cells in culture.

The effect of scorpion venom tityustoxin on the uptake of calcium in synaptosomes

The effect of tityustoxin on calcium uptake in synaptosomes incubated in hypotonie medium low sodium and physiological medium high sodium was studied. In the hypotonie medium, calcium uptake was ATP and temperature dependent and TsTX inhibits this uptake. The inhibition of calcium uptake was not due to an osmotic effect since it was also observed in conditions of physiological cell osmolarity. In the physiological medium high sodium, tityustoxin stimulated calcium uptake in synaptosomes. The effect was inhibited by tetrodotoxin and cocaine. The stimulating effect of tityustoxin was specific for sodium concentration in the medium and neither choline nor sucrose could substitute sodium in its activation of the stimulation of calcium uptake in synaptosomes by tityustoxin.

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake.

Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca2+ uptake by in vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca2+ accumulation was achieved between 6-10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca2+ uptake was inhibited by the presence of Mg2+ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg2+ of mitochondrial Ca2+ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V max for Ca2+ uptake from 292 +/- 22 to 377 +/- 34 nmoles Ca2+/min per mg protein (n = 8) but did not affect the K 0.5, (6.5-8.6 microM). Since the major kinetic change in mitochondrial Ca2+ uptake evoked by glucagon is an increase in V max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca2+ or in the rate of Ca2+ carrier movement across the mitochondrial membranes.

A COMPARISON OF THE DIFFERENTIAL EFFECTS OF NITROGLYCERIN, NIFEDIPINE AND PAPAVERINE ON CONTRACTURES INDUCED IN VASCULAR AND INTESTINAL SMOOTH MUSCLE BY POTASSIUM AND LANTHANUM

AbstractTo elucidate the mechanisms of smooth muscle relaxant effects of nitroglycerin, the effects of this substance on the potassium contracture (K+−contracture) and the lanthanum-induced contracture (La3+−contracture) were studied using smooth muscle preparations of the canine coronary artery and colon and the findings compared with the effects of nifedipine, a representative calcium antagonistic vasodilator. The effects of papaverine, a prototype smooth muscle relaxant, were also studied. La3+−contracture was induced in a calcium-free environment with the addition of lanthanum, an effective blocker of calcium influx. In the coronary artery, nitroglycerin produced a relaxation both of the La3+−contracture and the K+−contracture. However, neither La3+−contracture nor K-contracture in the colon was affected. Nifedipine did not relax the La3+−contracture in a range of doses at which K+−contracture of both types of smooth muscles was relaxed. Papaverine produced a relaxation of La3+−contracture as well as K+−contracture, in both types of smooth muscles. Unlike the mechanism related to the relaxant effects of nifedipine, which is generally admitted to be an inhibition of calcium influx, the relaxant effect of nitroglycerin was attributed to the suppression of calcium release from the intracellular store sites and/or stimulation of calcium uptake into the intracellular store-sites. Papaverine was assumed to produce a relaxation through augmentation of the calcium binding to the intracellular store sites for calcium as well as through inhibition of the calcium influx.

Effect of 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol) on the stimuli-induced calcium uptake in rat brain synaptosomes.

Effects of 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol), a nonspecific smooth muscle relaxant, on the uptake of calcium in the synaptosomes of rat brain were studied, using two stimuli i.e. high KCl and ATP. We also studied the effect of Aspaminol on the analgesic response of rat hindpaw using the Randall-Sellito test. The addition of Aspaminol inhibited the stimuli-induced calcium uptake in a concentration dependent manner. Aspaminol inhibited the high KCl induced calcium uptake competitively against the external calcium concentration. These findings suggest that Aspaminol may act on the surface of the synaptosomal membranes. In case of ATP stimulated calcium uptake, Aspaminol reduced the rate of uptake and maximal level of the synaptosomal calcium, and did not appear to have any effect on the rate of calcium release. These findings suggest that the inhibitory effect of Aspaminol can be mainly attributed to inhibition of the calcium influx, but not to acceleration of calcium efflux. In the five weeks old rats, Aspaminol did not influence the responses in the paw pinch test, but, in the weanling rats (about three weeks old). Aspaminol had a slight effect on the responses in the paw pinch test. This discrepancy may be due to difficulties in passing through the blood-brain barrier. Our findings suggest that a certain relationship may exist between the analgesic effect and the calcium uptake in the synaptosomes.

EFFECT OF 1,1-DIPHENYL-3-PIPERIDINOBUTANOL HYDROCHLORIDE (ASPAMINOL) ON THE STIMULI-INDUCED CALCIUM UPTAKE IN RAT BRAIN SYNAPTOSOMES

AbstractEffects of 1,1 -diphenyl-3-piperidinobutanol hydrochloride (Aspaminol), a nonspecific smooth muscle relaxant, on the uptake of calcium in the synaptosomes of rat brain were studied, using two stimuli i.e. high KCl and ATP. We also studied the effect of Aspaminol on the analgesic response of rat hindpaw using the Randall-Sellito test. The addition of Aspaminol inhibited the stimuli-induced calcium uptake in a concentration dependent manner. Aspaminol inhibited the high KCl induced calcium uptake competitively against the external calcium concentration. These findings suggest that Aspaminol may act on the surface of the synaptosomal membranes. In case of ATP stimulated calcium uptake, Aspaminol reduced the rate of uptake and maximal level of the synaptosomal calcium, and did not appear to have any effect on the rate of calcium release. These findings suggest that the inhibitory effect of Aspaminol can be mainly attributed to inhibition of the calcium influx, but not to acceleration of calcium efflux. In the five weeks old rats, Aspaminol did not influence the responses in the paw pinch test, but, in the weanling rats (about three weeks old), Aspaminol had a slight effect on the responses in the paw pinch test. This discrepancy may be due to difficulties in passing through the blood-brain barrier. Our findings suggest that a certain relationship may exist between the analgesic effect and the calcium uptake in the synaptosomes.

A rapid method for the preparation of sarcolemmal vesicles from rat aorta, and the stimulation of calcium uptake into the vesicles by cyclic AMP-dependent protein kinase

In vascular smooth muscle, extracellular Ca2+ is required for the maximal development of tension in response to hormonal or electrical stimulation [l-3]. During subsequent relaxation, Ca*’ must be transported back across the sarcolemma by an energydependent process [4]. This relaxation can be stimulated by hormones which elevate intracellular cyclic nucleotide concentrations [5,6). The known action of cyclic nucleotides in eukaryotes is to activate protein kinases leading to phosphorylation of substrate proteins [7]. We have therefore investigated the phosphorylation of sarcolemmal vesicles from rat aorta, and the effect of this on Ca2+ transport. A number of methods of preparation of sarcolemma1 vesicles from a variety of tissues have been reported. These inchrde enzymatic disaggregation and/or osmotic shock ]8,9], and various methods of homogenisation [ IO,1 11, in each case followed by sucrose density gradient centrifugation. With preparations of sarcolemmal vesicles from smooth muscle using these methods, contamination by sarcoplasmic reticulum has been reported [ 111. The effect of phosphorylation of sarcolemmal vesicles from smooth muscle by cyclic AMP-dependent protein kinase has also been studied. Using crude membrane fractions from trachea [ 121, it was reported that on incubation with [T-~~P]ATP and cyclic AMP-dependent protein kinase phosphorylation of sarcolemmal proteins did not occur, and there was no change in oxalatedependent Ca2+ uptake. In contrast, using a more purified preparation from rat aorta [ 133, a protein of&f, 44 000 was phosphorylated under similar conditions, and an increase in Ca2+ uptake was reported. We now report a new, rapid method for the preparation of purified sarcolemmal vesicles free of mitochondrial and cytoplasmic contamination using Percoll. The preparation also appears to have a low level of contamination by sarcoplasmic reticuium. On incubation with [T-“~P] ATP and cyclic AMP-dependent protein kinase, proteins of app. Mr 11 000, 2 1 000,44 000 and 110 000 are phosphorylated. The phosphorylation also causes a 2-3-fold stimulation of oxalate-independent Ca”-uptake which is inhibited by ouabain.

Parathyroid hormone as a calcium ionophore in bone cells: tests of specificity.

Parathyroid Hormone (PTH) stimulates calcium entry into bone cells in vitro, and this effect has been proposed to mediate the actions of PTH on bone. Using cells harvested from infant rat calvaria by collagenase digestion, we evaluated the sensitivity and specificity of this phenomenon. Calcium uptake was temperature dependent, and was increased by PTH over the concentration range 0.01 to 0.50 units/ml. A series of nonhormonal peptides obtained during the purification of a single batch of PTH was found to stimulate calcium uptake. In addition, isoproterenol, ACTH, calcitonin, and glucagon stimulated calcium uptake. In no case, however, was the degree of stimulation as great as that produced by biologically active PTH. Prostaglandins are also potent resorbers of bone, and a series of prostaglandins also stimulated calcium uptake into bone cells. These results are compatible with a calcium ionophore model of PTH and prostaglandin action on bone.

Molecular properties of the red cell calcium pump: I. Effects of calmodulin, proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg 2+-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V max/, decreases K Ca, and does not affect K ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V max and not affecting K Ca and K ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents. Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.

Calcium movements promoted by vesicles in a highly enriched sarcolemma preparation from canine ventricle. Calcium-calcium countertransport.

Calcium uptake by vesicles in a highly enriched sarcolemma preparation from canine ventricle was found to be markedly stimulated by intravesicular calcium. Stimulation of calcium uptake appeared to be a saturable function of intravesicular calcium. Calcium efflux from the vesicles was stimulated by calcium in the reaction medium. Calcium uptake, supported by intravesicular calcium, and calcium efflux, stimulated by extravesicular calcium, were found to correspond on a one-to-one basis. Only small changes in net uptake or efflux were observed to occur in response to chemical gradients of calcium across the membrane. It was concluded, therefore, that under certain conditions, the major means for calcium movement across vesicles in the preparation is via a one-to-one exchange of calcium. Sodium was found to stimulate calcium uptake when present in the intravesicular space and to stimulate calcium efflux when present in the extravesicular space, but the effects of calcium plus sodium were not additive with respect to stimulating either calcium uptake or efflux. The effects of unlabeled calcium, strontium, barium, and magnesium on calcium uptake stimulated by intravesicular calcium and by intravesicular sodium were similar though not identical. The temperature dependence for calcium-stimulated and sodium-stimulated calcium movements was characterized by Q10 values of 1.27 and 2.06, respectively. Previous work has associated the sodium-calcium exchange reaction with the sarcolemma. It is argued that the present study, in turn, provides evidence that the calcium-calcium exchange reaction is also associated with the sarcolemma. In addition, the results of the study are consistent with the hypothesis that one membrane system can promote the exchange of either calcium for calcium or calcium for sodium.

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca 2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K + gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K + gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca 2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca 2+-activated ATPase protein.

Effects of diet calcium and 1,25-dihydroxyvitamin D3 on colon calcium active transport.

Unidirectional fluxes of calcium were studied in the absence of electrochemical gradients across rat descending colon segments in vitro. Dietary calcium restriction and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]enhanced absorption by increasing the mucosal-to-serosal fluxes, whereas secretory (serosal-to-mucosal) fluxes were unchanged. Low-calcium diet also stimulated calcium uptake by everted gut sac segments of ascending as well as descending colon, whereas transverse colon was unresponsive. These results show that the colon is a target organ for 1,25-(OH)2D3 and demonstrate the participation of colon in the intestinal adaptation to calcium deprivation.

Nucleotide-induced alteration of rat liver microsome calcium pump activity

Rat liver microsomes sequester calcium by an energy dependent process that may be a nonmuscle cell analog of the sarcoplasmic reticulum Ca 2+ pump of skeletal muscle (Moore, L., Chen, T.S., Knapp, H.R., Jr. and Landon, E.J. (1975) J. Biol. Chem. 250, 4562–4568). Homogenization of rat liver in the presence of ATP (5 mM) results in a 2-fold increase of the specific activity of the microsome Ca 2+ pump. The effect of ATP is concentration dependent and is detected at ATP levels as low as 0.1 mM. ATP will produce this effect if added before homogenization, after homogenization or after any of the centrifugation steps of microsome isolation. Homogenization of rat liver in the presence of ADP and AMP also increases specific activity of the microsome Ca 2+ pump, but to a lesser extent than ATP. Other nucleoside triphosphates have been tested and are in general less effective than ATP in increasing microsome Ca 2+ pump activity. The phosphate group of nucleotides appears to be important to this effect in that adenosine does not affect Ca 2+ pump activity, while sodium pyrophosphate will increase pump activity but to a smaller extent than ATP. The presence of nucleotides or pyrophosphate during microsome isolation results in the release of a small amount of protein material from microsomes. These proteins can be detected in 105 000 × g supernatant by SDS-polyacrylamide gel electrophoresis. Three bands of molecular weight 46 000, 42 000 and 36 000 comprise the majority of protein material released from microsomes. The ability of nucleotides to release one of the these proteins, the 42 000 molecular weight band, from microsomes correlates with the ability of the nucleotide to increase microsome Ca 2+ pump activity. Preliminary evidence indicates that the protein released from ATP-treated microsomes is able to suppress the stimulated calcium uptake measured in ATP-treated microsomes. It is possible that this protein functions to regulate Ca 2+ pump activity in the endoplasmic reticulum of liver.