Voltage dependent activation of tonic contraction in cardiac myocytes.

Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.     

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.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

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+.     

γ-Aminobutyric Acid and Glycine Modulate Each Other''s Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

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)     

Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin

alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose that the type of membrane receptor does not determine the mechanism of GABA release evoked by the toxin.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

EFFECTS OF ELEVATED [K+]0 ON THE RELEASE OF NEUROTRANSMITTERS FROM CORTICAL SYNAPTOSOMES: EFFLUX OR SECRETION?

Abstract— Elevated K⁺₀ elicited a substantial Ca-independent efflux of accumulated GABA from cortical synaptosomal fractions. Efflux from tissue labelled with either NE or choline was affected considerably less by elevated K⁺ pulses in the absence of calcium. K-facilitated Ca-dependent efflux was large for all three of the accumulated substances. K-dependent (Ca-independent) efflux of accumulated GABA was associated with all subcellular fractions exhibiting GABA accumulation whereas K-facilitated Ca-dependent efflux was restricted to fractions containing synaptosomes. Eighty per cent of both GABA accumulation and K-dependent efflux was, however, recovered in a purified synaptosomal fraction. Alanine slightly decreased GABA accumulation, but % K-dependent efflux was not affected.
Elevated K⁺, in the absence of calcium, released GABA from accumulated pools in preference to endogenous pools, whereas the Ca-dependent efflux, facilitated by K⁺, was similar for both accumulated and endogenous GABA.
The Ca-independent efflux of accumulated GABA increased linearly with log [K⁺]₀ between 10 and 70 mM-K⁺ in sodium-containing media. Prior treatment with veratridine or Na-free medium substantially decreased the Ca-independent but not the Ca-dependent GABA efflux produced by elevated K⁺ pulses.
The Ca-dependent and Ca-independent efflux of accumulated GABA in response to elevated K⁺ pulses is suggested to arise not only via different flux mechanisms but also from different GABA pools. The Ca-dependent efflux is interpreted to reflect stimulus-secretion coupling processes whereas the Ca-independent efflux may reflect membrane transport processes.     

Multiple mechanisms of transmitter release evoked by "pathologically" elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro.

The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced Release of γ-Aminobutyric Acid by a Carrier-Mediated, High-Affinity Uptake of L-Glutamate in Cultured Chick Retina Cells

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.     

The Effects of Scorpion Venom Toxin on the Release of Amino Acid Neurotransmitters from Cerebral Cortex In Vivo and In Vitro

Tityustoxin, the active component of the venom of the Brazilian yellow scorpion Tityus serrulatus, caused specific release of the neurotransmitter amino acids glutamate, aspartate and GABA in vivo from the superfused sensori-motor cortex of conscious unanesthetised rats and in vitro from rat cortical synaptosomes. The effects on synaptosomes appear to be due to a depolarising action. Synaptosomal potassium levels were depleted by the toxin. The action was also blocked both in vivo and in vitro by tetrodotoxin and was Ca2+-dependent. The uptake of [U-14C]GABA was inhibited by tityustoxin but this action was prevented by tetrodotoxin (1 microM). Since the release of [U-14C]GABA from synaptosomes due to the tityustoxin was also prevented by tetrodotoxin under identical circumstances, it is concluded that the tityustoxin has a primary action on release of neurotransmitters rather than on uptake.     

Functional significance of nitric oxide in ionomycin-evoked [3H]GABA release from mouse cerebral cortical neurons

We investigated a role of nitric oxide (NO) on ionomycin-evoked [3H]GABA release using mouse cerebral cortical neurons. lonomycin dose-dependently released [3H]GABA up to 1 microM. The extent of the release by 0.1 microM ionomycin was in a range similar to that by 30 mM KCl. The ionomycin (0.1 microM)-evoked [3H]GABA release was dose-dependently inhibited by NO synthase inhibitors and hemoglobin, indicating that the ionomycin-evoked [3H]GABA release is mediated through NO formation. The inhibition of cGMP formation by 1H-[1,2,4] oxodizao [4,3-a] quinoxalin-1-one (ODQ), a selective inhibitor for NO-sensitive guanylate cyclase, showed no affects on the ionomycin-evoked [3H]GABA release. Tetrodotoxin and dibucaine significantly suppressed the ionomycin-evoked [3H]GABA release and ionomycin increased fluorescence intensity of bis-oxonol, suggesting the involvement of membrane depolarization in this release. The ionomycin-evoked [3H]GABA release was maximally reduced by about 50% by GABA uptake inhibitors. The concomitant presence of nifedipine and omega-agatoxin VIA (omega-ATX), inhibitors for L- and P/Q-type voltage-dependent calcium channels, respectively, caused the reduction in the ionomycin-evoked release by about 50%. The simultaneous addition of nifedipine, omega-ATX and nipecotic acid completely abolished the release. Although ionomycin released glutamate, (+)-5-methyl-1-,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) showed no effects on the ionomycin-induced [3H]GABA release. Based on these results, it is concluded that NO formed by ionomycin plays a critical role in ionomycin-evoked [3H]GABA release from the neurons.     

KCl stimulation increases norepinephrine transporter function in PC12 cells

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.     

4-Aminobutyrate Can Be Released Exocytotically from Guinea-Pig Cerebral Cortical Synaptosomes

Guinea-pig synaptosomes possess two functional pools of 4-aminobutyrate (GABA). One is rapidly labelled by added [14C]GABA, is steadily released in a Ca2+-independent manner when the Na+ electrochemical potential across the plasma membrane is collapsed, and is depleted by the GABA analogue 2,4-diaminobutyrate (DABA), all of which is consistent with a cytosolic location. A second, noncytosolic compartment only slowly equilibrates with exogenous [14C]GABA, is not depleted by DABA, but can release 350 pmol of endogenous GABA/mg of protein (8% of the total intrasynaptosomal GABA) within 15 s of depolarization in the presence of Ca2+. Ca2+-independent release occurs by thermodynamic reversal of the plasma membrane uptake pathway following artifactually prolonged depolarization, whereas Ca2+-dependent release is consistent with physiological exocytosis from vesicular stores.     

Further studies on the role of calcium in the regulation of glutamate decarboxylase activity in brain slices

[3H]GABA synthesis in brain slices was used as a model to study the role of Ca2+ in the regulation of GAD activity. Experimental conditions were chosen to increase and decrease the flux of Ca2+ and to promote the increase in free intracellular Ca2+. The blockade of electron transport and the inhibition of oxidative phosphorylation in the slices inhibited [3H]GABA synthesis. High K+ depolarization stimulated [3H]GABA synthesis and this effect was not blocked by lidocaine, trifluoperazine, or verapamil, but the stimulation was blocked by the intracellular Ca2+ antagonist TMB-8. The data do not differentiate between the relative contributions of extra- and intracellular Ca2+ but reflect that GAD activity is modulated by a dynamic balance between these two compartments as well as between stored and free Ca2+ within the cells.