Influx of calcium, strontium, and barium in presynaptic nerve endings

Depolarization-induced (potassium-stimulated) influx of 45Ca, 85Sr, and 133Ba was measured in synaptosomes prepared from rat brain. There are two phases of divalent cation entry, "fast" and "slow;" each phase is mediated by channels with distinctive characteristics. The fast channels inactivate (within 1 s) and are blocked by low concentrations (less than 1 micro M) of La. The slow channels do not inactivate (within 10 s), and are blocked by high concentrations (greater than 50 micro M) of La. Divalent cation influx through both channels saturates with increasing concentrations of permeant divalent cation; in addition, each permeant divalent cation species competitively blocks the influx of other permeant species. These results are consistent with the presence of "binding sites" for divalent cations in the fast and slow channels. The Ca:Sr:Ba permeability ratio, determined by measuring the influx of all three species in triple-label experiments, was 6:3:2 for the fast channel and 6:3:1 for the slow channel. A simple model for ion selectivity, based on the presence of a binding site in the channel, could account well for slow and, to some extent, for fast, channel selectivity data.     

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.     

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709- 728). This influx was blocked by multivalent cations with half- inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.     

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.     

The actions of diazepam and diphenylhydantoin on fast and slow Ca2+ uptake processes in guinea pig cerebral cortex synaptosomes

The activities of diazepam and diphenylhydantoin as inhibitors of the fast and slow phases of 45Ca2+ uptake in response to K+ depolarization and of [3H]nitrendipine binding were examined in guinea pig cerebral cortex synaptosomes. The slow phase of 45Ca2+ uptake was abolished in Na+-free media (choline substitution) and was more sensitive to inhibition by 3,4-dichlorobenzamil and represents a Na+-dependent Ca2+ uptake process. The fast component of uptake represents activation of voltage-dependent Ca2+ channels. Diazepam (to 300 microM) was selectively active against the fast component of 45Ca2+ uptake. The benzodiazepines Ro 11-3624 and Ro 11-3128 were similarly selective with a modest stereoselectivity against the fast component of 45Ca2+ uptake. Diphenylhydantoin (100 and 200 microM) blocked nonselectively both fast and slow phases of Ca2+ uptake. Diazepam (60 microM) and diphenylhydantoin (200 microM) blocked [3H]nitrendipine binding in a competitive manner. Diazepam and diphenylhydantoin probably exert at least part of their anticonvulsant activity by inhibition of voltage-dependent Ca2+ channels.     

Heterogeneity of presynaptic calcium channels revealed by species differences in the sensitivity of synaptosomal 45Ca entry to omega-conotoxin

The inhibition of K+-depolarization dependent Ca influx by omega-conotoxin GVIA was compared in the frog, chick, and rat brain synaptosomes. The toxin at concentrations greater than or equal to 0.3 microM completely inhibited Ca entry in the frog and chick preparations, but was only partly effective in blocking Ca influx in the rat brain synaptosomes. In chick synaptosomes the toxin's effect was biphasic: a small component (approximately equal to 15%) of total Ca influx was inhibited by the toxin with high affinity (I50 less than 0.002 microM); a major component (approximately equal to 80%) of Ca influx was inhibited with a moderate affinity (I50 approximately equal to 0.05 microM). In rat brain synaptosomes 40% of Ca influx was inhibited by the toxin with low affinity (I50 approximately equal to 0.3 microM), and 60% of Ca influx was unaffected by the toxin concentration of up to 10 microM. These data suggest a heterogeneity of voltage-sensitive Ca channels in vertebrate brain synaptosomes.     

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.     

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.     

'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.     

Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry

The effect of fendiline, a documented inhibitor of L-type Ca2+ channels and calmodulin, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca2+ probe. Fendiline at 5-100 microM significantly increased [Ca2+]i concentration-dependently. The [Ca2+]i rise consisted of an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca2+ influx and internal Ca2+ release. In Ca(2+)-free medium, pretreatment with 50 microM fendiline nearly abolished the [Ca2+]i rise induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca2+. This indicates that the internal Ca2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 microM, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La3+ by 50%, implying that fendiline-induced Ca2+ influx has two components separable by La3+. Consistently, 0.1 mM La3+ pretreatment suppressed fendiline-induced [Ca2+]i rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 50-100 microM fendiline in Ca(2+)-free medium. However, 50-100 microM fendiline inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 inhibited 50 microM fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 microM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca2+]i in MDCK cells by releasing internal Ca2+ in a manner independent of inositol-1,4,5-trisphosphate (IP3), followed by external Ca2+ influx.     

Oxidation of Analogs of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+-channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin-2-loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 microM EGTA or 100 microM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 microM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration-dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+-evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.     

The Suppression of Stimulus-Evoked Release of Amino Acid Neurotransmitters from Synaptosomes by Verapamil

Verapamil at 200 microM, prevented the respiratory stimulation, K+ loss, transmitter release, and 45Ca2+ entry into incubated synaptosomes evoked by veratrine (25 to 75 microM) or by high K+ (56 mM). Verapamil (100 microM) also blocked gamma-aminobutyric acid homoexchange, whilst tetrodotoxin was ineffective. Much lower concentrations of verapamil (less than 1 microM) blocked the 45Ca2+ entry caused by veratrine, but not its action in releasing neurotransmitter or K+. It is concluded that verapamil, at 30 to 200 microM, blocks active Na+ channels, thereby preventing depolarization. At greater than 1 microM, verapamil blocks Ca+ channels selectively.     

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.     

Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.

Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.     

Effect of cell ageing on Ca2+ influx into human red cells

The effect of cell ageing on Ca2+ entry was studied in this work, using sub-populations of young and old human red cells, separated by stringent percoll density gradients. Additionally, the influence of an osmotic gradient was investigated as a model for shear stress. Ca2+ entry was assessed at 37 degrees C, under conditions where the Ca2+ pump was either inhibited by NaVO3 (0.5-10 mM) or inactivated by ATP depletion. The entry was linear with time up to 1 h. No differences in Ca2+ influx between the two sub-populations were detected in isotonic Na(+)-medium. In contrast, after incubation in anisosmotic media, Ca2+ entry into old cells was significantly higher than into younger cells. In hypotonic Na(+)-medium, the entry into old cells was not affected by La3+ (10 microM) whilst it was partially blocked by Gd3+ at a similar level (half-maximal effect attained with about 1 microM Gd3+). The entry into young cells was only slightly stimulated by these lanthanides at low concentrations (10 microM), regardless of the tonicity of incubation medium. Further increasing Gd3+ levels above 10 microM markedly enhanced Ca2+ entry into both cell types. The selective blockade of Ca2+ influx by low Gd3+ concentrations suggests presence of mechano-sensitive channels, that become preferentially activated in old cells. Activation of these channels during in-vivo microcirculation may help to explain the increased Ca2+ content of senescent cells.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

BAY K 8644, a 1,4-Dihydropyridine Ca2+ Channel Activator: Dissociation of Binding and Functional Effects in Brain Synaptosomes

K+-stimulated 45Ca2+ uptake into rat brain and guinea pig cerebral cortex synaptosomes was measured at 10 s and 90 s at K+ concentrations of 5-75 mM. Net increases in 45Ca2+ uptake were observed in rat and guinea pig brain synaptosomes. 45Ca2+ uptake under resting or depolarizing conditions was not increased by the 1,4-dihydropyridine BAY K 8644, which has been shown to activate Ca2+ channels in smooth and cardiac muscle. High-affinity [3H]nitrendipine binding in guinea pig synaptosomes (KD = 1.2 X 10(-10) M, Bmax = 0.56 pmol mg-1 protein) was competitively displaced with high affinity (IC50 2.3 X 10(-9) M) by BAY K 8644. Thus high-affinity Ca2+ channel antagonist and activator binding sites exist in synaptosome preparations, but their relationship to functional Ca2+ channels is not clear.     

Effects of Lead Ions on Events Associated with Exocytosis in Isolated Bovine Adrenal Medullary Cells

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.     

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.     

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)