Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate. The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using⁴⁵Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2mm calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.     

Effect of external sodium and calcium on calcium efflux in arteria carotis of cattle

The dependence of the 45Ca-efflux from the smooth muscle cells of the arteria carotis of cattle on external sodium and calcium was studied. In the absence of external calcium the replacement of NaCl by sucrose leads to a decreased 45Ca-efflux rate, replacement by LiCl to an increased efflux rate. With regard to the presence of sodium and calcium in the external medium, the 45Ca-efflux rate decreases in the following order: Na + Ca less than less than Na + Ca-free less than Na-free (Na substituted by sucrose) + Ca-free. LiCl considerably stimulates the 45Ca-efflux rate in the presence of external calcium. An inhibition of the Na, K-ATPase activity lasting for more than 20 min leads to a decrease of the 45Ca-efflux rate. The results obtained suggest the existence of a Na-Ca-exchange in the arteria carotis of cattle.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres.

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 micrometer. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 micrometer ionized Ca and and no ATP is 0.6 pmol-cm-2-s-1. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na+ and Ca2+ from the external medium causes a marked drop in the Ca2+ efflux in the presence of internal ATP.     

Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons.

The influx and efflux of calcium (as 45Ca) and influx of sodium (as 24Na) were studied in internally dialyzed squid giant axons. The axons were poisoned with cyanide and ATP was omitted from the dialysis fluid. The internal ionized Ca2+ concentration ([Ca2+]i) was controlled with Ca-EGTA buffers. With [Ca2+]i greater than 0.5 muM, 45Ca efflux was largely dependent upon external Na and Ca. The Nao-dependent Ca efflux into Ca-free media appeared to saturate as [Ca2+]i was increased to 160 muM; the half-saturation concentration was about 8 muM Ca2+. In two experiments 24Na influx was measured; when [Ca2+]i was decreased from 160 muM to less than 0.5 muM, Na influx declined by about 5 pmoles/cm2 sec. The Nao-dependent Ca efflux averaged 1.6 pmoles/cm2 sec in axons with a [Ca2+]i of 160 muM, and was negligible in axons with a [Ca2+]i of less than 0.5 muM. Taken together, the Na influx and Ca efflux data may indicate that the fluxes are coupled with a stoichiometry of about 3 Na+-to-1 Ca2+. Ca efflux into Na-free media required the presence of both Ca and an alkali metal ion (but not Cs) in the external medium. Ca influx from Li-containing media was greatly reduced when [Ca2+]i was decreased from 160 to 0.23 muM, or when external Li was replaced by choline. These data provide evidence for a Ca-Ca exchange mechanism which is activated by certain alkali metal ions. The observations are consistent with a mobile carrier mechanism which can exchange Ca2+ ions from the axoplasm for either 3 Na+ ions, or one Ca2+ and an alkali metal ion (but not Cs) from the external medium. This mechanism may utilize energy from the Na electrochemical gradient to help extrude Ca against an electrochemical gradient.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

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.     

Effects of calcium ionophores on the transport and distribution of calcium in isolated cells and in liver and kidney slices

Summary The effects of calcium ionophores on cellular calcium metabolism were studied in cultured kidney cells, in cells freshly isolated from rat kidney, and in liver and kidney slices. In isolated cells, these ionophores decreased the total cellular Ca content and the mitochondrial Ca.⁴⁵Ca efflux from prelabelled cells was also stimulated even in the absence of extracellular Ca. In slices, the ionophore A23187 increased the total slice Ca and the uptake of⁴⁵Ca. However, the mitochondria isolated from these slices treated with the ionophore had a lower total Ca and a depressed relative radioactivity. These results suggest that the increased cytosolic Ca produced by Ca ionophores may be due to mobilization of intracellular Ca stores rather than to a net shift of Ca from the extracellular fluids to the cell.     

Calcium export by sodium-calcium exchange in bovine chromaffin cells

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium Efflux from Internally Dialyzed Squid Giant Axons

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm²·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]_i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]_o and [Ca]_o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]_i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10^-7 mol Ca⁺⁺/mg of protein with an initial rate of 2.6 x 10^-8 mol Ca⁺⁺/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.     

Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells

When aequorin-loaded glomerulosa cells were incubated in isotonic Na2+-free medium containing N-methyl-D-glucamine instead of NaCl, there was an increase in cytoplasmic free calcium concentration, [Ca2+] c, which was not observed when extracellular calcium concentration was reduced to 1 microM. Upon removal of extracellular sodium, there was nearly five-fold increase in fractional efflux ratio of calcium. The reduction of extracellular sodium resulted in a stimulation of calcium influx rate, the magnitude of which was dependent on extracellular sodium concentration. Similar stimulation of calcium influx was observed when extracellular sodium was replaced with lithium. Nitrendipine did not affect the calcium influx induced by the reduction of extracellular sodium while a derivative of amiloride 3',4'-dichlorobenzamil, which inhibits Na-Ca exchange, attenuated calcium influx observed in sodium-free medium. These results indicate that removal of extracellular sodium leads to an increase in [Ca2+] c by stimulating calcium influx and that calcium enters the cell via Na-Ca exchanger.     

Stimulation of polymorphonuclear leukocytes by dicyclohexylcarbodiimide: calcium homeostasis

The involvement of calcium in N,N'-dicyclohexylcarbodiimide (DCCD)-mediated stimulation of guinea pig neutrophils was investigated. Exposure to DCCD resulted in a fast though moderate elevation of cytosolic calcium concentration. Exchange experiments indicated that DCCD enhanced 45Ca2+ efflux without affecting uptake of the radioisotope from the medium. Plasma membranes isolated from DCCD-stimulated cells failed to support ATP-dependent 45Ca2+ uptake indicating inhibition of their Ca-ATPase. The finding that the enhanced efflux of 45Ca2+ depended on the presence of Na+ ions in the medium implicated a Na+/Ca2+ exchanger in efflux of the ion observed in DCCD-stimulated neutrophils. This is the first indication for the participation of this carrier in calcium homeostasis in stimulated neutrophils. Experiments carried out with 14C-DCCD indicated covalent binding of the reagent to 20 and 150 Kd membrane proteins.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

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.     

Mechanism of sodium independent calcium efflux from rat liver mitochondria

On the basis of primarily two types of observations, it has been suggested that the Na+-independent Ca2+ efflux mechanism of rat liver mitochondria is a passive Ca2+-2H+ exchanger. First, when a pulse of acid is added to a suspension of mitochondria loaded with Ca2+, a pulse of intramitochondrial Ca2+ is often released, even in the presence of the inhibitor of mitochondrial Ca2+ influx, ruthenium red. Second, at a pH near 7, the stoichiometry of Ca2+ released to H+ taken up by Ca2+-loaded mitochondria, following treatment with ruthenium red, has been observed to be 1:2. This evidence for a Ca2+-2H+ exchanger is reexamined here by studying the release of Ca2+ upon acidification of the medium by addition of buffer, the dependence of liver mitochondrial Ca2+ efflux on external medium pH and intramitochondrial pH, and the Ca2+-Ca2+ exchange properties of the Ca2+ efflux mechanism. These studies show no pulse of mitochondrial Ca2+ efflux when pH is abruptly lowered by addition of buffer. The stoichiometry between Ca2+ and H+ fluxes is found to be highly pH dependent. The reported 1:2 stoichiometry between Ca2+ efflux and H+ influx is only observed at one pH. Furthermore, the rate of Ca2+ efflux from mitochondria is found to increase only very slightly at most as suspension pH is decreased. The rate of Ca2+ efflux is not found to increase with increasing intramitochondrial pH. Finally, no Ca2+-Ca2+ isotope exchange can be demonstrated over the Na+-independent efflux mechanism (i.e., in the presence of ruthenium red). It is concluded that these data do not support the hypothesis that the Na+-independent Ca2+ efflux mechanism is a passive Ca2+-2H+ exchanger.     

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 μM. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 μM ionized Ca and no ATP is 0.6 pmol · cm⁻² · s⁻¹. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na⁺ and Ca²⁺ from the external medium causes a marked drop in the Ca²⁺ efflux in the presence of internal ATP.     

Computer simulation and interpretation of45Ca efflux profile patterns

Summary Stimulations or inhibitions by various agents of⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries.     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

A study of calcium compartments in rat brain cortex thin slices: Effects of veratridine,lithium and of a mitochondrial uncoupler

The efflux kinetics of⁴⁵Ca from rat brain cortex thin slices previously equilibrated with it, was studied in a superfusion system. Two first order kinetic components of efflux from the tissue were found: k₂=0.0667 min^–1, that was unchanged by lowering the temperature from 37°C to 15°, and k₃=0.0167 min^–1 at 37°C, that was reduced to 0.0897 min^–1 at 15°C. This suggests that k₂ represents efflux from the extracellular space, and k₃ that from the cellular compartment. Addition of the mitochondrial uncoupler carbonyl cyanide,m-chlorophenylhydrazone (CCCP) (10^–5 M) increased the efflux fractional rate constant of⁴⁵Ca by 35%, while no change in efflux was induced by 10 mM caffeine. Veratridine (10^–5M) drastically reduced⁴⁵Ca efflux if superfusion was with physiological salt solution (150 mM sodium present), but not if 50 mM lithium replaced an equivalent amount of sodium in the superfusion fluid. This lithium-containing solution did not affect⁴⁵Ca efflux in the absence of veratridine. These results indicate that mitochondria accumulate only a minor fraction of intracellular⁴⁵Ca; that⁴⁵Ca possibly turns over very rapidly in the endoplasmic reticulum, and that most of⁴⁵Ca is present in a different, non-mitochondrial, non endoplasmic reticular compartment, the nature of which can be only conjectured.