Effect of parathyroid hormone on the transport of 45Ca2+ and 3H-GABA in nerve endings isolated from the rat cerebral cortex

Parathyroid hormone (PTH) (0.1-10 ng/ml) evokes a dose-dependent increase in 45Ca2+ accumulation in synaptosomes isolated from the rat brain cortex. In the presence of PTH the fast (I sec) potential-dependent 45Ca2+ uptake was less than in the control. PTH had no effect on 3H-GABA uptake by synaptosomes (P2 fraction). Synaptosomes preincubated in the presence of PTH in Ca2+-free medium and transferred into Ca2+-containing normal medium released more 3H-GABA than control synaptosomes. In this case depolarization-evoked 3H-GABA release was diminished.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Delta and sigma sites of clonal NCB20 cells do not modulate calcium uptake

Opiate alkaloids and peptides are reported to inhibit 45Ca2+ binding to synaptic plasma membranes and uptake into brain synaptosomes. We have examined the effects of a number of opiates on 45Ca2+ uptake in a clonal cell line NCB20 which expresses multiple opioid binding sites. The cells express voltage-dependent calcium channels that are blocked by verapamil and nifedipine. In contrast to brain, 45Ca2+ uptake in these cells, in normal or high potassium medium, is unaffected by opiates. This difference may be due to the particular receptor types; the delta and sigma sites of these cells do not inhibit 45Ca2+ uptake.     

Multiple calcium channels in synaptosomes: voltage dependence of 1,4-dihydropyridine binding and effects on function

The voltage dependence of binding of the calcium channel antagonist, (+)-[3H]PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on 45Ca2+ uptake have been investigated. Under nondepolarizing conditions (+)-[3H]PN200-110 binds to a single class of sites with a Kd of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K+ concentration, there was a decrease in affinity (approximately 7-fold) with no change in the number of sites. The effects of calcium channel ligands on 45Ca2+ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of 45Ca2+ whose magnitude was voltage-dependent. Verapamil (100 microM) almost completely inhibited calcium uptake at all potassium concentrations studied. In contrast, the effects of dihydropyridines (2 microM) appear to be voltage-sensitive. At relatively low levels of depolarization (10-25 mM K+) nitrendipine and PN200-110 completely inhibited 45Ca2+ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K+ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.     

Increase in the damaging effect of free fatty acids on brain synaptosomes induced by vitamin E deficiency

Using fluorescent probes it has been shown that free fatty acids cause depolarization of synaptosomes isolated from the rat brain. At the same time free fatty acids stimulated 45Ca2+ transport into synaptosomes. It has been demonstrated that synaptosomes isolated from the brain of E-deficient rats were more sensitive to the action of free fatty acids. Depolarization of synaptosomes isolated from the brain of both control and E-deficient rats were reduced by the addition of exogenous alpha-tocopherol.     

Brain synaptosomal Ca2+ uptake: comparison of Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats

1. K(+)-stimulated 45Ca2+ uptake by synaptosomes was measured with respect to the strain differences between Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. 45Ca2+ uptake by synaptosomes isolated from cerebral cortex of SD, WKY and SHR was measured at 15, 30, 60, 120 and 240 sec time periods. 3. The sequence of both the magnitude and rate of resting and depolarization-dependent 45Ca2+ uptake was SHR greater than WKY greater than SD. 4. The fastest rates of resting and depolarization-dependent 45Ca2+ uptake occurred in each rat during the first 15 sec and uptake rates dropped off quickly in both resting and depolarization states. 5. At 15 sec, there were significant differences between SHR and WKY, while there were no significant differences between WKY and SD. 6. The results suggest that an important alteration in Ca2+ channel characteristics may occur in SHR brain synaptosomes.     

Differential Calcium Dependence of γ-Aminobutyric Acid and Acetylcholine Release in Mouse Brain Synaptosomes

The dependence of gamma-aminobutyric acid (GABA) and acetylcholine (ACh) release on Ca2+ was comparatively studied in synaptosomes from mouse brain, by correlating the influx of 45Ca2+ with the release of the transmitters. It was observed that exposure of synaptosomes to a Na+-free medium notably increases Ca2+ entry, and this condition was used, in addition to K+ depolarization and the Ca2+ ionophore A23187, to stimulate the influx of Ca2+ and the release of labeled GABA and ACh. The effect of ruthenium red (RuR) on these parameters was also investigated. Of the three experimental conditions used, the absence of Na+ in the medium proved to be the most efficient in increasing Ca2+ entry. RuR inhibited by 60-70% the influx of Ca2+ stimulated by K+ depolarization but did not affect its basal influx or its influx stimulated by the absence of Na+ or by A23187. The release of ACh was stimulated by K+ depolarization, absence of Na+ in the medium, and A23187 in a strictly Ca2+-dependent manner, whereas the release of GABA was only partially dependent on the presence of Ca2+ in the medium. The extent of stimulation of ACh release was related to the extent of Ca2+ entry, whereas no such correlation was observed for GABA. In the presence of Na+, RuR did not affect the release of the transmitters induced by A23187. In the absence of Na+, paradoxically RuR notably enhanced the release of both ACh and GABA induced by A23187, in a Ca2+-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine in the brain of rats exposed to acute irradiation

A two-fold increase in acetylcholine, that can randomly be released by brain synaptosomes, is registered 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg; depolarization of the synaptosome membranes by potassium chloride increases the release of acetylcholine the augmentation of the release in this case being lower than that in the control. The initial rate of spontaneous neuromediator release from synaptosomes grows by 80 per cent whereas after depolarization of synaptosome membranes by potassium chloride, by 15 per cent. There is a 2.5-fold increase in the maximum rate of a highly specific uptake of choline with Km value being constant. Acetylcholine content of gray substance of irradiated rat brain is invariable.     

Ebselen Protects Ca2+ Influx Blockage But Does Not Protect Glutamate Uptake Inhibition Caused By Hg2+

The goal of this study was to investigate the isolated and combined effect of ebselen and Hg2+ on calcium influx and on glutamatergic system. We examined the in vitro effects of 2 phenyl-1,2-benzisoselenazol-3(2H)-ona), (Ebselen) on 45Ca2+ influx in synaptosomes of rat at rest and during depolarization and glutamate uptake into synaptosomes. Entry of 45Ca was measured during exposure to mercury in non-depolarizing and depolarizing solutions. Ebselen abolished the inhibition of 45Ca2+ influx on non-depolarizing conditions; however, ebselen did no modify inhibition uptake of 45Ca2+ caused by Hg2+ in high K+ depolarizing medium. Ebselen did not modify glutamate uptake inhibition caused by Hg2+ in synaptosomes. These results indicate that ebselen has an in vitro protective effect against Hg2+ induced inhibition of Ca2+ influx into synaptosomes, depending on the depolarizing conditions of the assay. The effects of Hg2+ on glutamate uptake were not modified by ebselen, suggesting that its protection is dependent on the target protein considered.     

Effect of Age on K+-Induced Cytosolic Ca2+ Changes in Rat Cortical Synaptosomes

45Ca2+ uptake and cytosolic Ca2+ concentrations [( Ca2+]i) were measured in synaptosomes prepared from the cerebral cortex of 3-, 16-, and 24-month-old male Charles River Wistar rats. Electron-microscopic examination demonstrated no morphological differences between the synaptosomes prepared from 3- and 24-month-old rats. The fast phase of Ca2+ uptake was reduced in the 24-month-old animals as compared to the 3-month-old ones (-23%, p less than 0.001), whereas no difference was found between the 16- and the 3-month-old rats. Age did not modify [Ca2+]i, as measured by the quin 2 technique, both at rest and immediately after depolarization with 50 mM K+. The Ca2+ load following depolarization was cleared in about 13 min in the 3-month-old rats. The rate of clearance was significantly slower both in the 16- (p less than 0.01) and in the 24-month-old rats (p less than 0.0001). The addition of verapamil (60 microM) after depolarization restored [Ca2+]i to resting level in aged rats at the same rate as in young rats. A prolonged Ca2+ influx, therefore, may be responsible for the slower clearance of Ca2+ load in aged rats.     

Early developmental changes in intracellular Ca2+ stores in rat brain.

Developmental changes in intracellular Ca2+ stores in brain was studied by examining: (1) IP3- and cADPR-induced increase in [Ca2+]i in synaptosomes; (2) Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; (3) TG-induced inhibition of Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; and (4) gene expression of Ca(2+)-ATPase pump in neurons obtained from brains of the new-born and the 3-week-old rats. IP3 (EC50 310 +/- 8 nM, 200% maximum increase in [Ca2+]i) and cADPR (EC50 25 +/- 3 nM, greater than 170% maximum increase in [Ca2+]i) both were potent agonist of Ca2+ release from internal stores in synaptosomes obtained from the 3-week-old rats. However, IP3 (EC50 250 +/- 10 nM, 175 maximum increase in [Ca2+]i) was a potent, but cADPR (EC50 300 +/- 20 nM, 75% maximum increase) was a poor agonist of Ca2+ release from intracellular stores in synaptosomes obtained from the new-born rats. [3H]IP3, [32P]cADPR and [3H]Ry binding in the new-born samples were significantly less than that in the 3-week-old samples. [3H]Ry binding to its receptor was more sensitive to cADPR in microsomes from the 3-week-old rats than those from the new-born rats. Microsomes from the new-born rats exhibited TG-sensitive (IC50 30 +/- 4 nM) and TG-insensitive forms of Ca(2+)-ATPase, while microsomes from the 3-week-old rats exhibited only the TG-sensitive form of Ca(2+)-ATPase (5 +/- 1 nM IC50). Microsomes from the 3-week-old rats were more sensitive to TG but less sensitive to IP3, while microsomes from the new-born rats were more sensitive to IP3 but less sensitive to TG. The lower TG sensitivity of the new-born Ca2+ store may be because they poorly express a 45 amino acid C-terminal tail of Ca(2+)-ATPase that contains the TG regulatory sites. This site is adequately expressed in the older brain. This suggests that: (1) the new-born brain contains fully operational IP3 pathway but poorly developed cADPR pathway, while the older brain contains both IP3 and cADPR pathways; and (2) a developmental switch occurs in the new-born Ca(2+)-ATPase as a function of maturity.     

The Na(+)-Ca2+ exchanger activity in cerebrocortical nerve endings is reduced in old compared to young and mature rats when it operates as a Ca2+ influx or efflux pathway.

The activity of the Na(+)-Ca2+ exchanger, which regulates the entry and the extrusion of Ca2+ ions from nerve endings was investigated in Percoll-purified cerebrocortical synaptosomes of aged rats. 45Ca2+ uptake in a Na(+)-free medium and 45Ca2+ efflux in a 145 mM Na+ medium were significantly reduced in cerebrocortical synaptosomes from aged rats (24 months) as compared to those occurring in young (4 months) and mature (14 months) rats. 45Ca2+ influx induced by 55 mM K+, a concentration of K+ ions which selectively promotes Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), was significantly reduced in mature and aged rats as compared to that occurring in young rats. The impairment of these mechanisms in aged rats is not accompanied by any variation of fura-2 monitored Ca2+ levels under resting and depolarizing conditions.     

Transport of calcium to synaptosomes and subcellular membrane fractions of the brain: effects of opioid peptides

Highly purified synaptosomal and subcellular fractions identified as mitochondria and microsomes were obtained by fractionation of brain tissues. The greatest Ca-accumulating capacity and the highest rate of Ca2+ accumulation were revealed in the mitochondrial fraction. Upon further fractionation of the synaptosomal fraction the energy-dependent uptake (accumulation) of Ca2+ was revealed only in the mitochondria. It was demonstrated that opioid peptides accelerate Ca2+ uptake by the synaptosomes in a medium with physiological concentration of K+ and inhibit this process during K+-dependent membrane depolarization. It was shown that beta-endorphine, methionine-encephaline and leucine-encephaline (10(-8)-10(-5) M) inhibit the Ca-accumulating capacity of both mitochondria and microsomes from brain. The experimental data suggest that opioid peptides can modulate the release of neurotransmitters and/or neurohormones by inhibiting the potential-dependent Ca2+ influx into the nerve endings and by decreasing the intrasynaptosomal pool of Ca2+.     

Methyl-beta-cyclodextrin influences glutamate transport in the rat brain nerve terminals by depletion of membrane cholesterol

Role of membrane cholesterol in direct and reversed function of Na+ -dependent glutamate transporters and exocytosis was investigated. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MebetaCD) resulted in a dose-dependent significant reduction of the L-[14C]glutamate uptake by synaptosomes. Treatment of synaptosomes with 15 mM MebetaCD caused a decrease in the velocity of L-[14C]glutamate uptake by 49 +/- 4% (P < or = 0.05). The depolarization stimulated Ca2+ -dependent glutamate release that occurred via reverse functioning of glutamate transporters decreased insignificantly for 1 min from 8.0 +/- 0.4% to 6.7 +/- 0.4% of total accumulated synaptosomal label after MebetaCD treatment. The depletion of membrane cholesterol resulted in a reduction of the depolarization evoked exocytotic release from 8.0 +/- 1.0% to 4.2 +/- 1.0% of total synaptosomal label. Thus, cholesterol depletion was found to decrease significantly the Na+ -dependent uptake and exocytotic release of glutamate.     

Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Reduction of K+-Stimulated 45Ca2+ Influx in Synaptosomes with Age Involves Inactivating and Noninactivating Calcium Channels and Is Correlated with Temporal Modifications in Protein Dephosphorylation

The voltage-dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage-sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of less than 500 microM, the initial (5-s) calcium uptake declined by approximately 20-50% in 12- and 24-month-old rats relative to 3-month-old adults. Depolarization of synaptosomes from 3-month-old rats in a calcium-free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage- or voltage-and-calcium-dependent inactivation) to approximately 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 microM nifedipine resulted in a 15-25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24-month-old rats, inactivation of 45Ca2+ uptake in calcium-free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3-month-old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 microM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24-month-old rats. These results indicate that the decrease in calcium channel-mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine-resistant and -sensitive calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple components of synaptosomal [3H]-gamma-aminobutyric acid release resolved by a rapid superfusion system

Release of [3H]-gamma-aminobutyric acid ([3H]GABA) from rat brain synaptosomes was studied with 60-ms time resolution, using a novel rapid superfusion method. Synaptosomes were prelabeled with [3H]GABA via an associated GABA uptake system. KCl depolarization stimulated at least three distinct components of GABA release: (1) a phasic Ca-dependent component, which develops rapidly and decays with a time constant of at most 60 ms; (2) a tonic Ca-dependent component that persists after KCl depolarization is ended; (3) a Ca-independent component. The three components of GABA release are pharmacologically distinct. The phasic component was selectively blocked by 50 microM Cd2+, while the tonic component was selectively blocked by 100 microM Ni2+. The Ca-independent component was selectively blocked by nipecotic acid (IC50 = 21 microM), a known inhibitor of Na+-dependent GABA uptake. The time course and amplitude of Ca-dependent GABA release evoked by the Ca2+ ionophore A23187 were nearly identical with Ca-dependent release evoked by depolarization. This result indicates that Ca-dependent GABA release depends primarily on Ca2+ entry into the nerve terminal, and not depolarization, per se. The properties of the phasic component suggest that it is normally initiated by a voltage-sensitive Ca2+ channel that is functionally and pharmacologically distinct from those previously described. The Ca-independent component of GABA release is probably mediated by reversal of the Na-dependent, electrogenic GABA uptake system. The ability to identify multiple components of GABA release on a physiologically relevant time scale may afford a more precise definition of the mechanism of action of drugs thought to affect neurotransmission in the brain.     

Ca2+-Dependent Regulation of Presynaptic Stimulus-Secretion Coupling

In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+]i) changes, (b) rapid 45Ca2+ uptake, and (c) Ca2+-dependent and -independent release of endogenous glutamate (Glu) and gamma-aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified nerve terminals (synaptosomes) from rat hippocampus. During stimulation, Percoll-purified synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+-dependent as well as a Ca2+-independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+-dependent (exocytotic) release. Ca2+-dependent transmitter release as well as [Ca2+]i elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+-dependent release of both Glu and GABA and elevation of [Ca2+]i. Ca2+-dependent release is maximally stimulated with an elevation of [Ca2+]i of 60% above steady-state levels, corresponding with an intracellular concentration of approximately 400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+-dependent release of both Glu and GABA. In contrast, Ca2+-independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)