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.     

Aluminum Inhibits the Fast Phase of Voltage-Dependent Calcium Influx into Synaptosomes

Aluminum has been shown to have neurotoxic effects, but the mechanisms by which it acts are not well understood. Because it has been reported that aluminum can interact with Ca2+-binding sites, the possibility that aluminum might interfere with Ca2+ influx into synaptosomes was examined. At concentrations of 50 microM and greater, aluminum significantly inhibited the fast phase (0-1 s) of the voltage-dependent uptake of 45Ca2+ into synaptosomes. Higher concentrations of aluminum also reduced 45Ca2+ uptake measured at 1 s in nondepolarizing media and inhibited the slow phase of 45Ca2+ uptake into synaptosomes whether they were suspended in either low K or high K media. The possibility that aluminum competitively inhibits the fast phase of Ca2+ influx was investigated. Aluminum (250 microM) increased the apparent KT (concentration of Ca2+ at which Ca2+ transport is half maximal) for 45Ca2+ of fast phase voltage-dependent channels and slightly decreased the maximal influx (Jmax). These effects are characteristic of a mixed type inhibitor, and the apparent Ki for Al3+ is estimated to be 0.64 mM. The interaction of aluminum with the fast phase of voltage-dependent calcium influx may disrupt intraneuronal calcium homeostasis and may also represent a means by which aluminum could accumulate intraneuronally.     

Impact of external calcium and calcium sensors on ginsenoside Rb1 biosynthesis by Panax notoginseng cells

The effects of external calcium concentrations on biosynthesis of ginsenoside Rb1 and several calcium signal sensors were quantitatively investigated in suspension cultures of Panax notoginseng cells. It was observed that the synthesis of intracellular ginsenoside Rb1 in 3-day incubation was dependent on the medium Ca2+ concentration (0-13 mM). At an optimal Ca2+ concentration of 8 mM, a maximal ginsenoside Rb1 content of 1.88 +/- 0.03 mg g(-1) dry weight was reached, which was about 60% and 25% higher than that at Ca2+ concentrations of 0 and 3 mM, respectively. Ca2+ feeding experiments confirmed the Ca2+ concentration-dependent Rb1 biosynthesis. In order to understand the mechanism of the signal transduction from external Ca2+ to ginsenoside biosynthesis, the intracellular content of calcium and calmodulin (CaM), activities of calcium/calmodulin-dependent NAD kinase (CCDNK) and calcium-dependent protein kinase (CDPK), and activity of a new biosynthetic enzyme of ginsenoside Rb1, i.e., UDPG:ginsenoside Rd glucosyltransferase (UGRdGT), in the cultured cells were all analyzed. The intracellular calcium content and CCDNK activity were increased with an increase of external Ca2+ concentration within 0-13 mM. In contrast, the CaM content and activities of CDPK and UGRdGT reached their highest levels at 8 mM of initial Ca2+ concentration, which was also optimal to the ginsenoside Rb1 synthesis. A similar Ca2+ concentration-dependency of the intracellular contents of calcium and CaM and activities of CCDNK, CDPK, and UGRdGT was confirmed in Ca2+ feeding experiments. Finally, a possible model on the effect of external calcium on ginsenoside Rb1 biosynthesis via the signal transduction pathway of CaM, CDPK, and UGRdGT is proposed. Regulation of external Ca2+ concentration is considered a useful strategy for manipulating ginsenoside Rb1 biosynthesis by P. notoginseng cells.     

Somatostatin Release from Rat Cerebral Cortex Synaptosomes

Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin-like immunoreactivity (SRIF-LI) was measured by means of a radioimmunoassay procedure. High KCl (9-50 mM) concentration dependently evoked SRIF-LI release; the evoked overflow reached a plateau at 25 mM KCl and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KCl used. The 15 mM K(+)-evoked release of SRIF-LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K(+)-evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 microM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF-LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1-50 microM) induced release of SRIF-LI in a concentration-dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF-LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium-deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that greater than 90% of the SRIF-LI released either during the spontaneous outflow or by 15 mM KCl was represented by SRIF-14 (SRIF-28(14-28]. These values reflected the ratio SRIF-14/SRIF-28 found in synaptosomes at the end of the experiments.     

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.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

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)     

Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High-K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K(+)-stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na(+)-based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 microM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.     

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)     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Measurement of intrasynaptosomal free calcium by using the fluorescent indicator quin-2.

The recently synthesized calcium indicator quin -2 was incorporated into synaptosomes from guinea-pig cerebral cortex following uptake and internal hydrolysis of quin -2 tetra-acetoxymethyl ester. Incubation in physiological media containing 1 mM- or 2 mM-CaCl2 led to equilibrium cytosolic ionized calcium concentrations of 85 +/- 10 nM and 205 +/- 5 nM respectively (mean +/- S.E.M. from eight and eighteen preparations respectively). Cytosolic Ca2+ was elevated following increases in external Ca2+ concentration, plasma membrane depolarization, mitochondrial inhibition, calcium ionophore addition or replacement of external sodium by lithium. Preliminary experiments were performed to assess changes in cytosolic Ca2+ accompanying the release of the neurotransmitter acetylcholine.     

Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.     

Regulation of Calcium Concentrations in Synaptosomes: α2-Adrenoceptors Reduce Free Ca2+ by Closure of N-Type Ca2+ Channels

The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K(+)-depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was approximately 10,000 times higher than [Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time- and dose-dependent manner. Maximum inhibition (40% at 100 microM) occurred at 2 min and the IC50 was 80 nM. The reduction caused by clonidine (1 microM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 microM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1-1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium regulation of magnesium dependent phosphorylation of human erythrocyte ghost spectrin

Phosphorylation of human erythrocyte ghost membrane proteins was found to be affected by micromolar calcium concentrations. Increasing Ca2+ concentration to 0.2 microM decreased spectrin (band 2) phosphorylation to 30 +/- 6% of control (to which no calcium was added). Decreasing calcium concentration by adding EGTA (0.2mM) to the standard membrane preparation increased spectrin phosphorylation to 575% control. This effect of Ca2+ was more pronounced at higher temperature. At 0 degree C, Ca2+ (0.05mM) had no effect on protein phosphorylation. Sodium fluoride like EGTA caused a four to five fold increase in phosphorylation. Pyrophosphate, a phosphoprotein phosphatase inhibator, had no effect. Once spectrin was phosphorylated in the presence of [gamma-32P]ATP the addition of Ca2+ or EGTA did not decrease or increase its phosphorylation. It is suggested that calcium regulates spectrin phosphorylation either by decreasing kinase activity or by decreasing substrate availability.     

'Slow' K+-stimulated Ca2+ influx is mediated by Na+-Ca2+ exchange: a pharmacological study

K+-stimulated 45Ca2+ influx was measured in rat brain presynaptic nerve terminals that were predepolarized in a K+-rich solution for 15 s prior to addition of 45Ca2+. This 'slow' Ca2+ influx was compared to influx stimulated by Na+ removal, presumably mediated by Na+-Ca2+ exchange. The K+-stimulated Ca2+ influx in predepolarized synaptosomes, and the Na+-removal-dependent Ca2+ influx were both saturating functions of the external Ca2+ concentration; and both were half-saturated at 0.3 mM Ca2+. Both were reduced about 50% by 20 microM Hg2+, 20 microM Cu2+ or 0.45 mM Mn2+. Neither the K+-stimulated nor the Na+-removal-dependent Ca2+ influx was inhibited by 1 microM Cd2+, La3+ or Pb2+, treatments that almost completely inhibited K+-stimulated Ca2+ influx in synaptosomes that were not predepolarized. The relative permeabilities of K+-stimulated Ca2+, Sr2+ or Ba2+ influx in predepolarized synaptosomes (10:3:1) and the corresponding selectivity ratio for Na+-removal-dependent divalent cation uptake (10:2:1) were similar. These results strongly suggest that the K+-stimulated 'slow' Ca2+ influx in predepolarized synaptosomes and the Na+-removal-dependent Ca2+ influx are mediated by a common mechanism, the Na+-Ca2+ exchanger.     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets

We have investigated the sub-second kinetics of changes in cytosolic free calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from internal stores. The delays in onset of both responses are sufficient for one or more biochemical steps to lie between ligand-receptor binding and Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were similar to those evoked by other agonists, but the response in the presence of external Ca2+ was markedly shorter, occurring without measurable delay at optimal ligand concentration. Analysis of this response showed some delay in ADP-evoked influx at lower concentrations, but this delay was markedly less than that observed with thrombin at doses giving the same elevation in [Ca2+]i. These results suggest that ADP evokes influx using a different transduction system, more closely coupled to the Ca2+ entry system than that used by other agonists. Differences between thrombin- and ADP-evoked influx were further demonstrated by the inhibitory actions of cAMP, which reduced and substantially increased the delay in onset of thrombin-evoked influx but did not measurably delay the influx evoked by an optimal concentration of ADP.