Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig

The relation of changes in internal, free Ca2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-S-C2(5) or 86Rb+ distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca2+ on the membrane potential between about -50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca2+ was reached when the membrane potential was depolarized from the resting level, about -64 mV, to about -40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca2+ channels concomitantly leads to an increase in cytosolic, free Ca2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.     

Quantitative measurements of the cytosolic Ca2+ activity within isolated guinea pig nerve-endings using entrapped arsenazo III and quin2

The absorbance changes of intrasynaptosomally entrapped arsenazo III have been converted into values of free Ca²⁺ concentration by correcting for the nonlinear response of arsenazo III at different concentrations of the dye as well as for changes in internal pH. An average resting value for free Ca²⁺ concentration around 0.4 μM is obtained. Depolarization with veratridine or gramicidin increases this value to around 3 μM. Measurements of cytosolic free Ca²⁺ with the quin2 method gives much lower values in similar conditions. The release of prelabelled [¹⁴C]noradrenaline from the nerve-endings is maximally activated when the internal free Ca²⁺ concentration rises as measured with arsenazo III to about 4 μM when titrated with increasing concentrations of ionophore A23187.     

Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms

Small molecules present during brain tissue homogenization are known to be entrapped within subsequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx-LC), pertussis toxin (PTx), anti-syntaxin, or anti-SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx-LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (-)-baclofen inhibited amino acid overflow more potently in standard than in PTx-containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti-syntaxin or anti-SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane-impermeant agents of different size in studies of presynaptic mechanisms.     

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.     

The stoichiometry of A23187- and X537A-mediated calcium ion transport across lipid bilayers

Initial rates of ionophore-mediated Ca2+ transport across egg phosphatidylcholine bilayers of large unilamellar vesicles were measured using the absorbance change of arsenazo III at 650 nm as an indicator of Ca2+ translocation. A23187 induced the movement of Ca2+ in a 2:1 ionophore: Ca2+ complex, whereas its methyl ester (CH3A23187) and X537A mediated Ca2+ movement in a 1:1 ionophore: Ca2+ complex. The relative potencies of these ionophores in transporting Ca2+ across lipid membranes were A23187 much greater than X537A greater than CH3A23187.     

Effect of the Ca ionophore A-23187 on the plasmatic and mitochondrial potentials of the brain synaptosomes in rats: fluorescence measurements

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Ionophore A23187, Verapamil, Protonophores, and Veratridine Influence the Release of γ-Aminobutyric Acid from Synaptosomes by Modulation of the Plasma Membrane Potential Rather than the Cytosolic Calcium

The release of GABA induced by veratridine shows no correlation with the synaptosomal Ca content and is therefore not mediated by the release of mitochondrial Ca. Instead, with both Ca-repleted and -depleted synaptosomes, the extent of GABA efflux is correlated with the decrease in plasma membrane potential. The slow release of GABA induced by protonophores and the Ca-dependent release induced by ionophore A23187 are also consequences of the depolarization of the plasma membrane, rather than of elevated cytosolic Ca. Finally, the ability of verapamil to inhibit the release of GABA induced by low veratridine concentrations is due to the ability of the Ca channel inhibitor to antagonize the action of veratridine, rather than to inhibit Ca entry into the synaptosome. It is concluded that it is essential to monitor plasma membrane potentials in experiments in which amino acid efflux from synaptosomes is induced.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

Pyruvate utilization by synaptosomes is independent of calcium

The significance of Ca2+ is assessed for the activation of pyruvate by intact nerve terminals (synaptosomes). Titration of glucose-depleted synaptosomes with pyruvate in the presence of either veratridine or uncoupler stimulates respiration in a Ca2+-independent manner. Additionally, the ability of exogenous pyruvate to support the mitochondrial membrane potential in situ is independent of Ca2+. It is concluded that Ca2+ does not regulate pyruvate oxidation in intact synaptosomes.     

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.     

Calcium efflux and cycling across the synaptosomal plasma membrane.

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Internalization of metallochromic Ca2+ indicators in mammalian cells

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.     

Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes

Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+-dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87-kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87-kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, gamma-aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+-dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus-secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Oxidation of sulfhydryl groups and inhibition of the (Ca2+ + Mg2+)-ATPase by arsenazo III

In the presence of divalent cations, the metallochromic Ca2+ indicator arsenazo III is reduced by sulfhydryl groups to form an azo anion radical. Reduced arsenazo III is reoxidized back to its original state by oxygen. The formation of the arsenazo III azo anion radical in the presence of sarcoplasmic reticulum vesicles leads to the rapid inhibition of the (Ca2+ + Mg2+)-ATPase. These data indicate that several factors should be considered when arsenazo III is used as a Ca2+ indicator; (1) Functionally important sulfhydryl groups may be oxidized by arsenazo III; (2) the generation of free radicals by arsenazo III reduction may be toxic to the system being studied; (3) the absorbance spectrum of arsenazo III is altered when reduced by sulfhydryl groups.     

Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons

Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2+ influx by 200-400 ms, and pH continued to decrease for several seconds after pulse termination which shut off Ca2+ influx. Trains of action potentials also produced an internal pH decrease. Recovery of the pH change required periods greater than 10 min. The magnitude of the pH change was largely unaffected by external pH in the range 6.8-8.4. The voltage dependence of the internal p/ change was similar to the voltage dependence of calcium influx determined by arsenazo III, and removal of calcium from the bathing saline eliminated the pH signal. In neurons injected with EGTA (1-5 mM), the activity- induced internal Ca2+ changes were reduced or eliminated, but the internal pH drop was increased severalfold in magnitude. After the injection of EGTA, voltage clamp pulses produced a decrease in arsenazo III absorbance instead of the normal increase. Under these conditions the dye was responding primarily to changes in internal pH. Injection of H+ caused a rise in internal free calcium. The pH buffering capacity of the neurons was measured using three different techniques: H+ injection, depressing intrinsic pH changes with a pH buffer, and a method employing the EGTA-calcium reaction. The first two methods gave similar measurements: 4-9 meq/unit pH per liter for pleural ganglion cells and 13-26 meq/unit pH per liter for pedal ganglion cells. The EGTA method gave significantly higher values (20-60 meq/unit pH per liter) and showed no difference between pleural and pedal neurons.     

Regulated plasmalemmal expansion in nerve growth cones

To study the mechanisms underlying plasmalemmal expansion in the nerve growth cone, a cell-free assay was developed to quantify membrane addition, using ligand binding and sealed growth cone particles isolated by subcellular fractionation from fetal rat brain. Exposed versus total binding sites of 125I-wheat germ agglutinin were measured in the absence or presence of saponin, respectively, after incubation with various agents. Ca2(+)-ionophore A23187 in the presence of Ca2+ increases the number of binding sites (Bmax) but does not change their affinity (KD), indicating that new receptors appear on the plasma membrane. Similarly, membrane depolarization by high K+ or veratridine significantly induces, in a Ca2(+)-dependent manner, the externalization of lectin binding sites from an internal pool. Morphometric analysis of isolated growth cones indicates that A23187 and high K+ treatment cause a significant reduction in a specific cytoplasmic membrane compartment, thus confirming the lectin labeling results and identifying the plasmalemmal precursor. The isolated growth cones take up gamma-amino-butyric acid and serotonin, but show no evidence for Ca2(+)-dependent transmitter release so that transmitter exocytosis is dissociated from plasmalemmal expansion. The data demonstrate that plasmalemmal expansion in the growth cone is a regulated process and identify an internal pool of precursor membrane.