Regulation of cellular calcium metabolism and calcium transport by calcitonin.

Calcitonin was studied in isolated kidney cells and in isolated mitochondria. A concentration of 10 ng/ml of synthetic calcitonin increases the cellular accumulation of 45Ca and the total cell calcium. The mitochondrial pool is increased several-fold. Kinetic analysis of the data shows that although the total cellular exchangeable calcium pool is enlarged, calcium influx and efflux are significantly depressed by calcitonin. The absence of phosphate or the presence of inhibitors of mitochondrial calcium transport completely abolish the effects of the hormone. In isolated mitochondria, the hormone stimulates the active calcium uptake and depresses the extramitochondrial calcium activity. Calcitonin counteracts the effects of cyclic AMP which stimulates the release of calcium from mitochondria and increases the extramitochondrial calcium activity. These data indicate that cellular calcium homeostasis is controlled by the mitochondrial calcium turnover. They suggest that calcitomin regulates the cell calcium metabolism and inhibits the transcellular calcium transport by stimulating the rate of calcium uptake by mitochondria which depresses cytoplasmic calcium activity.     

Kinetic analysis of calcium movements in cell culture

Summary The distribution of intracellular calcium was determined in isolated kidney cells by kinetic analyses of⁴⁵Ca fluxes. Isotopic desaturation curves reveal an intracellular calcium compartment with a very slow time constant. The size of this calcium compartment is markedly increased by raising the extracellular calcium, by increasing the extracellular phosphate and may contain up to 99% of the intracellular exchangeable calcium. Accumulation of calcium in this pool is completely abolished by two specific inhibitors of mitochondrial calcium uptake, Antimycin A and Warfarin^®. These results suggest that this compartment represents a pool of calcium in the cell mitochondria. The sudden removal of phosphate from the medium immediately stimulates calcium efflux from the cell. Conversely, an increase in medium phosphate immediately inhibits calcium efflux. Both effects are rapidly reversible. Finally, calcium efflux from the cells is stimulated after the cells are exposed to low temperature suggesting that calcium transport out of the cell may be regulated by the cytoplasmic calcium activity. These experiments are consistent with the view that mitochondria play an important role in the control and regulation of cytoplasmic calcium activity and of calcium transport.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

Comparative effects of the ionophore A23187 and vitamin D metabolites on 45Ca desaturation curves derived from bone cells: interaction of a calcium channel channel inhibitor diltiazem

The effects of diltiazem, a calcium channel inhibitor, on the cellular transport of calcium were studied in isolated heterogenous rat bone cells. Efflux was measured after equilibrating the cells with 45Ca and adding the vitamin D metabolite (1,25dihydroxycholecalciferol-1,25(OH)2D3 or 24,25dihydrocholecalciferol-24,25(OH)2D3), the ionophore A23187 and/or diltiazem. Results were analysed by fitting the desaturation curve to a model of two exponential terms. Kinetic analyses of curve indicated the presence of 2 exchangeable pools with different rate constants of exchange between the medium and cells (expressed by K.). After incubation of bone cells with diltiazem (20 nmol/10(6) cells) the following changes were recorded: a marked decrease in the rate constant of efflux from the fast turnover calcium pool (K12) and a reduction of the calcium pool sizes. Incubation of 10(6) cells with 0.5 ng 1,25(OH)2D3 plus diltiazem significantly reduced K12 compared to incubation with 1,25(OH)2D3 alone. In presence of 24,25(OH)2D3, diltiazem did not significantly alter K12 which was raised by incubation with the metabolite alone. Ionophore A23187 (0.5 micrograms/10(6) cells) increased the value of slow turnover constants of efflux whose values were affected by diltiazem. The possible involvement of Ca movements in bone resorption does not seem confirmed in the present experiment since in vitro effects of diltiazem in organ culture (observed in an initial previous experiment) were not reflected in the calcium 45 desaturation kinetics in heterogenous bone cells.     

Effect of pH on the calcium metabolism of isolated rat kidney cells

Summary The effects of metabolic and respiratory acidosis and alkalosis on cellular calcium metabolism were studied in rat kidney cells dispersed with collagenase. In both types of acidosis, the intracellular pH, total cell calcium, and the cell relative radioactivity after 60 min of labeling are significantly depressed. Kinetic analysis of⁴⁵Ca desaturation curves shows that acidosis decreases all three cellular calcium pools and depresses calcium fluxes between the superficial and cytosolic pools and between the cytosolic and mitochondrial pools. In alkalosis the intracelluar pH, the total cell calcium, and the cell relative radioactivity are significantly increased. Kinetic studies show that in alkalosis, only the mitochondrial pool is consistently increased. Calcium exchange between the mitochondrial and cytosolic pool is increased in metabolic alkalosis only. These results suggest that hydrogen ion is an important modulator of calcium metabolism, and that the intracellular pH rather than extracellular pH is the critical factor in determining the calcium status of cells during altered acid-base conditions.     

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Summary Calcium efflux was measured in desheathed rabbit vagus nerves loaded with⁴⁵Ca²⁺. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of⁴⁵Ca²⁺ efflux profiles. The⁴⁵Ca²⁺ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min^–1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min^–1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm^–2 sec^–1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.Deceased 18 April 1988     

Sex difference in cellular calcium metabolism of rat hepatocytes and in α-adrenergic activation of glycogen phosphorylase

Three topics were the subject of these investigations: (i) the difference between males and females in the basal calcium metabolism of hepatocytes; (ii) the source of the calcium which triggers the phosphorylase a stimulation induced by epinephrine through alpha-adrenergic receptors; (iii) the time relation between the rise in phosphorylase activity and the increase in calcium efflux. We found that there was no difference between males and females in total or exchangeable cell calcium. However, there were significant differences in the mitochondrial calcium pool and fluxes measured by steady-state kinetic analyses: they were smaller and the rate constants of mitochondrial calcium influx and efflux were lower in males than in females. The ⁴⁵Ca content of isolated mitochondria and microsomes was also significantly lower in males than in females. In both males and females, epinephrine stimulated phosphorylase activity and calcium efflux even in the absence of extracellular calcium, indicating that the principal source of calcium which triggers the enzyme stimulation is intracellular. During the first 10 min following stimulation by 10^?6 M epinephrine, the total cell calcium, ⁴⁵Ca and the mitochondrial calcium were significantly depressed in male hepatocytes. After 10 min, these changes were reversed and the cell or mitochondrial calcium content was greater than in controls. In females, on the other hand, changes could only be detected if the cells were transferred to calcium-free media before the stimulation. In both males and females, there was a good temporal relationship between the stimulation of calcium efflux and the rise in phosphorylase a activity when hepatocytes were exposed to increasing concentrations of epinephrine: both rose at least 75% in less than 15 s. We conclude that there are important differences in cellular calcium metabolism between males and females. The rise in cytosolic calcium induced by alpha-adrenergic activation is principally due to a mobilization of calcium from an intracellular pool, probably the mitochondria.     

Kinetic Analyses of Calcium Movements in HeLa Cell Cultures : II. Calcium efflux

Calcium efflux was studied in monolayers of HeLa cells. The fast phase of exchange was studied in an open system by continuous washout. Its half-time was 1.58 min which is practically identical to the fast phase of calcium influx previously found to be 1.54 min. This suggests that the fast component of efflux represents calcium exchange from an extracellular compartment probably from calcium bound to the cell membrane surface. Dinitrophenol (DNP) and iodoacetate (IAA) do not inhibit calcium efflux from this compartment. The slow phase of calcium exchange was studied in a closed three compartment system. The half-time of calcium efflux measured under these conditions is almost identical to that obtained previously in studies of calcium influx: 33.0 and 37.0 min, respectively. This slow compartment is likely to be the intracellular exchangeable calcium pool. DNP and IAA inhibit calcium efflux from this compartment, lengthening the half-time from 33 min to 55.0 and 216 min, respectively. This suggests that calcium extrusion from the cell is an active process. Since calcium influx is not affected by metabolic inhibitors, the cellular calcium concentration increases as would be predicted under these conditions. Calcium efflux is also markedly depressed by lowering the temperature.     

Efflux of cholesterol from different cellular pools

Free cholesterol is very efficiently removed from cells by 2-hydroxypropyl-beta-cyclodextrins. The efflux of cholesterol occurs from two distinct kinetic pools: the half-times (t(1/2)) for the two pools in CHO-K1 cells are 15 +/- 5 s and 21 +/- 6 min and they represent 25% +/- 5% and 75% +/- 5% of the readily exchangeable cell cholesterol, respectively. In this study we have determined that the fast pool and the majority of the slow kinetic pool for cholesterol efflux are apparently present in the plasma membrane. Numerous agents that inhibit intracellular cholesterol trafficking are unable to affect either the size or the t(1/2) for efflux of either kinetic pool. In contrast, treatment of the cells with N-ethylmaleimide (NEM), exogenous lipases such as sphingomyelinase and phospholipase C, calcium ionophore A23187, or heat resulted in the dramatic increase in the size of the fast kinetic pool of cholesterol. These changes in the kinetics of cholesterol efflux are not specific to the nature of the extracellular acceptor indicating that they are a consequence of changes in the cell plasma membrane. The above treatments disrupt the normal organization of the lipids in the plasma membrane via either hydrolysis or randomization. The phosphatidylcholine and sphingomyelin present in the plasma membrane are critical for maintaining the two kinetic pools of cholesterol; any alteration in the amount or the location of these phospholipids results in an enhancement of efflux by redistributing cholesterol into the fast kinetic pool.     

Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.     

Rapid modulation of Ca2+ uptake in human jejunal enterocytes

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria

The influence of pH on the kinetics of the initial rate of calcium uptake by isolated kidney mitochondria was studied using the ruthenium red-ethylene glycol bis(β-aminoethyl ether) N,N-tetraacetic acid quench method (K. Reed and F. Bygrave, 1975, Eur. J. Biochem.55, 497–504). In the absence of phosphate, the K_m is increased 50% and the V is decreased 57% when the pH is decreased from pH 7.4 to 7.0. Conversely, when the pH is increased to 7.8, the K_m is decreased 25% while the V is not affected. The presence of 0.1 or 0.4 mm phosphate in the incubation medium abolishes the change in K_m at a low pH while the V remains depressed by 36 and 25%, respectively. The presence of phosphate does not affect the decrease in the K_m seen with an increased medium pH. Mitochondria incubated in steady-state conditions with a medium free calcium of 0.7 μm also show significant changes in calcium exchange and distribution with pH. Two kinetic calcium pools are found in isolated mitochondria. Decreasing pH from 7.4 to 7.1 decreases mitochondria total calcium 32%, decreases the rapidly exchanging pool 28%, and depresses both the mitochondrial membrane and an intramitochondrial calcium exchange by 54 and 22%, respectively. Raising the pH to 7.7 increases both exchangeable pools (63 and 46%), and increases the mitochondrial membrane calcium exchange 44%. These results are consistent with previous studies on the influence of intracellular pH on calcium metabolism of kidney cells in which the mitochondrial pool was markedly affected by pH (R. Studer, and A. Borle, 1979, J. Membrane Biol.48, 325–341). Alterations in cellular pH may modify mitochondrial calcium transport and cellular calcium metabolism and thus affect cell functions which are calcium dependent.     

Inhibition of dibutyryl cyclic AMP induced steroidogenesis in rat adrenocortical cells by the putative calcium antagonist TMB-8

A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced a smaller but significant reduction of EGTA-facilitated 45Ca2+ efflux from a mitochondrial-enriched fraction. We interpret these results to mean that TMB-8 blocks the steroidogenic effect of dibutyryl cyclic AMP by interfering with the mobilization of a cellular pool of calcium that is probably localized to the endoplasmic reticulum. The physiological implications of these findings in relation to the complex interactions between calcium and cyclic AMP in adrenal steroidogenesis are discussed.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Calcium pools in saponin-permeabilized guinea pig hepatocytes

The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.     

Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes

The permeability of neuronal membranes to Ca2+ is of great importance for neurotransmitter release. The temporal characteristics of Ca2+ fluxes in intact brain neurons have not been completely defined. In the present study 45Ca2+ was used to examine the kinetics of Ca2+ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca2+ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t1/2 = 7 s) which represented about 10% of the total cellular Ca2+ and was unaffected by the presence of Co2+, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t1/2 = 360 s) which represented 42% of the total cellular Ca2+ and was inhibited by Co2+, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t1/2 = 96 min) which represented 48% of the total cell Ca2+ was observed only in the prolonged efflux experiments. The rate of exchange of 45Ca2+ in the unstimulated cells was dependent on the extracellular Ca2+ concentration (half-saturation at 70 microM). Depolarization of the neurons with elevated K+ causes a rapid and sustained 45Ca2+ uptake. The cellular Ca2+ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net 45Ca2+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 72 s) into a single additional compartment. The other cellular Ca2+ pools were apparently unaffected by stimulation. The stimulated 45Ca2+ uptake was inhibited by Co2+ and by the Ca2+ channel blocker verapamil but not by the Na+ channel blocker tetrodotoxin. Ca2+ uptake into this compartment was dependent on the extracellular Ca2+ concentration (half-saturation at 0.80 mM Ca2+). Predepolarization of the cells with high K+ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of 45Ca2+ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca2+ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca2+ ions through a single class of voltage-sensitive Ca2+ channels. These Ca2+ channels are inhibited by Co2+ ions and by verapamil and are not inactivated during depolarization of the brain neurons.     

Triamcinolone acetonide induces transient changes in accumulation and fluxes of calcium in cultured bone cells

The effects of triamcinolone acetonide on the pattern of ⁴⁵calcium efflux were investigated in cultured bone cells. Efflux was measured after equilibrating the cells with ⁴⁵calcium for 24 hours and a short (3 hours) or a long (24 hours) preincubation with the hormone. The results were analyzed by fitting them to a model of three exponential terms, using a computer program based on the non-linear least square method. As reported previously the results indicated the presence of three exchangeable calcium pools which differ in their rates of calcium exchange. A short preincubation with the hormone (3 hours) caused the following changes: (1) A marked increase in the amount of exchangeable calcium in the “slow turnover” calcium pool (S₃) which is probably in the mitochondria. (2) A lesser but significant increase in the amount of calcium in the “fast turnover” calcium pool (S₂) which is probably the calcium in the cytosol. (3) An increase in the rates of calcium exchange between S₂ and the medium (?₂₀) and between S₃ and S₂ (?₃₂). All these changes were transient. After 24 hours of preincubation with triamcinolone acetonide the amounts of calcium in S₂ and S₃ decreased below the control levels and the fluxes were not significantly different from those of the controls.