Excessive release of [3H] noradrenaline by veratridine and ischemia in spinal cord

In this study, the properties of ischemic condition-induced and veratridine-evoked [3H]noradrenaline ([3H]NA) release from rat spinal cord slices were compared. It was expected that ischemia mimicked by oxygen and glucose deprivation results in the impairment of Na+/K+ -ATPase with a consequent elevation of the intracellular Na+ -level which reverses the NA carrier and promotes excessive NA release, and veratridine, by the activation of Na+ channels, releases NA both carrier-mediated and Ca2+ -dependent, i.e. vesicular manner. In our experiments, veratridine (1-100 microM) dose-dependently increased the resting [3H]NA release, and its effect was only partially blocked by low temperature or the lack of external calcium, whereas the sodium channel inhibitor tetrodotoxin (TTX, 1 microM) completely prevented it, indicating that veratridine induces NA release via axonal depolarization and reversing the transporters by eliciting Na+ -influx. In contrast to TTX, the local anesthetic lidocaine (100 microM) only partially blocked the veratridine-induced [3H]NA release due to its inhibitory action on K+ channels. The ischemia-induced [3H]NA release was abolished at 12 degrees C, a temperature known to block only the transporter-mediated release of transmitters. However, lidocaine was also partially effective to reverse the action of ischemia on the NA release, indicating that lidocaine is not a useful compound in the treatment of spinal cord-injured patients against the excessive excytotoxic NA release.     

Differential effects of methylmercuric chloride and mercuric chloride on the L-glutamate and potassium evoked release of [3H]dopamine from mouse striatal slices

The effects of CH3HgCl and HgCl2 on the evoked release of 3H from mouse striatal slices prelabelled with [3H]dopamine have been examined. CH3HgCl (10 microM) was observed to increase the L-glutamate-evoked release of [3H]dopamine, while HgCl2 (10 microM) had no effect. In contrast, CH3HgCl at concentrations up to 100 microM had no effect on the 25 mM K+-stimulated release of [3H]dopamine, whereas HgCl2 (100 microM) significantly reduced the 25 mM K+-stimulated release of [3H]dopamine. Thus CH3HgCl and HgCl2 have differential effects on the L-glutamate- and K+-stimulated release of [3H]dopamine from mouse striatal slices, suggesting that these compounds may have different sites and (or) mechanisms of action in altering neurotransmitter release. It is suggested that CH3HgCl may act predominantly at intracellular sites or at the level of the L-glutamate receptor, whereas the major site of action of HgCl2 may be the voltage-operated calcium channel.     

Separation of carrier mediated and vesicular release of GABA from rat brain slices.

In this study the temperature dependence of [3H]GABA release from brain slices evoked by electrical field stimulation and the Na+/K+ ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S2/S1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 microM) inhibited stimulation-induced [3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 microM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

Nicotinic Acetylcholine Receptor Antagonistic Activity of Monoamine Uptake Blockers in Rat Hippocampal Slices

The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine-evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 microM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole-cell patch clamp experiments neither drug blocked Na+ currents at 1 microM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 microM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine-induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects.     

Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

[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.     

Halothane induces vesicular and carrier-mediated release of [3H]serotonin from rat brain cortical slices

The serotonergic system may play a role during general anesthesia but the effect of the volatile anesthetic halothane on the release of serotonin (5-HT) is not fully understood. Rat brain cortical slices were labeled with [3H]5-HT to investigate the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [3H]5-HT that was dependent on incubation time and anesthetic concentration (0.006, 0.012, 0.024, 0.036, 0.048 and 0.072 mM). This effect was independent of extracellular calcium and was not affected by tetrodotoxin (blocker of voltage dependent Na+ channels). In contrast, the halothane-evoked [3H]5-HT release was reduced by BAPTA-AM, a membrane-permeable BAPTA analog that chelates intracellular Ca2+. The anesthetic-induced [3H]5-HT release depends on the ryanodine-sensitive intracellular calcium store since it was blocked by dantrolene and azumolene (inhibitors of the calcium-release through ryanodine receptors) but was not affected by aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-triphosphate receptor. The [3H]5-HT release induced by halothane comes mainly from the vesicular pool since it was reduced in about 70% by reserpine, a blocker of vesicular monoamine transporter. The halothane-evoked release of [3H]5-HT release is reduced by fluoxetine, an inhibitor of 5-HT uptake, and the volatile agent also decreased the uptake of [3H]5-HT into rat brain cortical slices. Moreover, a decrease on halothane-induced release of [3H]5-HT was also observed when the brain cortical slices were incubated at low temperature, which is known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na+/K+ ATPase pump inhibitor, which induces 5-HT release through reverse transport, also decreased [3H]5-HT release induced by halothane, confirming the involvement of a carrier-mediated release of the neurotransmitter in the presence of halothane. In conclusion, these data suggest that halothane induces vesicular and carrier-mediated release of [3H]5-HT in rat brain cortical slices.     

New proline-rich proteins in isolated insect Z-discs.

1. Protoveratrine A increased the release of gamma-amino[3H]butyrate from small slices of rat cerebral cortex. This effect increased with increasing protoveratrine concentration, reaching a maximum at 100 microM. 2. Removal of Ca2+ from the superfusing medium did not change the increase in release due to 10 microM-protoveratrine; however, the Ca2+ antagonists, compound D-600, La3+, Mn2+, Mg2+ and also high Ca2+ concentration inhibited the effect of the alkaloid, as did procaine. 3. Protoveratrine A increased the uptake of 22Na+ into the slices with a similar dose-response curve to that found for gamma-aminobutyrate release. For the most part, the substances that inhibited protoveratrine-stimulated gamma-aminobutyrate release also inhibited 22Na+ uptake, although the correlation was not perfect. 4. Although extracellular Ca2+ is not required for protoveratrine-induced gamma-aminobutyrate release, an increase in Na+ influx that is susceptible to inhibition by some Ca2+ antagonists does appear to be associated with this phenomenon. However, the possibility remains that changes in the free intracellular Ca2+ concentration may be important for transmitter release induced by depolarizing veratrum alkaloids.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Differential Sensitivity to Blockade by 4-Aminopyridine of Presynaptic Receptors Regulating [3H]Acetylcholine Release from Rat Hippocampus

The inhibitory effect of an adenosine analogue, R-N6-phenylisopropyl adenosine (R-PIA), of the cholinergic agonist carbachol, and of morphine on 3H efflux from [3H]choline-labeled field-stimulated rat hippocampal slices was compared with that produced by two inhibitors of N- and L-type Ca2+ channels, omega-conotoxin (CgTx; conotoxin GVIA) and cadmium chloride. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release, with a maximal effect (an almost threefold increase) at 100 microM. 4-AP (100 microM) did not affect the actions of CgTx, cadmium chloride, and R-PIA but almost abolished the effect of carbachol and morphine. The present results indicate that presynaptic muscarinic and opiate receptors reduce acetylcholine release by a mechanism that is somewhat different from that used by adenosine A1 receptors. Furthermore, the results indicate that presynaptic A1 receptors on hippocampal cholinergic neurons do not primarily regulate 4-AP-dependent potassium channels, but that they might act directly on a Ca2+ conductance.     

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na+, Ca2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na+ (Na(i), as determined with SBFI), and on the rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by an increased permeability of presynaptic Na+ channels, with veratridine, or by an increased permeability of presynaptic Ca2+ channels with high K+, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC(50)'s for carbamazepine and vinpocetine to inhibit veratridine-induced [3H]Glu release are 200 and 2 microM, respectively. Consistently 150 microM carbamazepine and 1.5 microM vinpocetine reduce the veratridine-induced rise in Na(i) in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na+ channel mediated responses, namely the rise in Na(i) and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca2+ via presynaptic Ca2+ channels, such as the release of [3H]Glu and the rise in Ca(i) induced by high K+, are insensitive to 300 microM carbamazepine and slightly reduced by 5 microM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.     

4-Aminopyridine Stimulates B-50 (GAP43) Phosphorylation and [3H]Noradrenaline Release in Rat Hippocampal Slices

In situ phosphorylation of the presynaptic protein kinase C substrate B-50 was investigated in rat hippocampal slices incubated with the convulsant drug 4-aminopyridine (4-AP). Phosphorylation of B-50 was significantly enhanced 1 min after the addition of 4-AP (100 microM). This increase by 4-AP was concentration dependent (estimated EC50 30-50 microM). Concomitant with the changes in B-50 phosphorylation, 4-AP also dose-dependently stimulated [3H]noradrenaline [( 3H]NA) release from the slices. 4-AP stimulated [3H]NA release within 5 min to seven times the control level. The B-50 phosphorylation induced by 4-AP remained elevated after removal of the convulsant, this is contrast to B-50 phosphorylation induced by depolarization with K+. A similar persistent increase was observed for [3H]NA release after a 5-min incubation period with 4-AP. These results give more insight into the molecular mechanisms underlying 4-AP-induced epileptogenesis and provide further evidence for the correlation between B-50 phosphorylation and neurotransmitter release in the hippocampal slice.     

Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit

Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 microM cocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential kappa-agonists ethylketocyclazocine, dynorphin A1-13, dynorphin A, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] -benzeneacetamide (U-50,488), and (-)-5,9-dimethyl-2'-OH-2-tetrahydrofurfuryl-6,7-benzomorphan [(-)-MR 2034], whereas (+)-MR 2035 [the (+)-enantiomer of (-)-MR 2034] was ineffective. In contrast, the preferential delta-agonists Leu-enkephalin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalin (DADLE) as well as the mu-agonists morphine, normorphine, D-Ala2-Gly-ol5-enkephalin (DAGO), and beta-casomorphin 1-4 amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 microM) was much more potent than ethylketocyclazocine (1 microM) or DADLE (1 microM). (-)-N-(3-furylmethyl)-alpha-noretazocine [(-)-MR 2266], 1 microM, a preferential kappa-antagonist, antagonized the effect of ethylketocyclazocine more potently than (-)-naloxone or (+)-MR 2267 [the (+)-enantiomer of (-)-MR 2266]. Given alone, (-)-MR 2266 slightly and (+)-MR 2267 (1 microM each) greatly enhanced NA release, apparently due to alpha 2-adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The calcium-magnesium interaction in the process of release of noradrenaline by nicotine

The calcium-magnesium (Ca2+-Mg2+) interaction in the process of nicotine-induced release of [3H]noradrenaline ([3H]NA) from rat isolated vas deferens was studied. Increasing extracellular concentrations of Mg2+ caused a dose-dependent depression of release of [3H]NA by nicotine, and this inhibitory effect of Mg2+ was overcome by raising the concentration of CA2+. It is concluded that Mg2+ antagonizes the nicotine-induced increase in the Ca2+ influx into the adrenergic nerve terminals, and that nicotine acts on adrenergic neuronal membrane rather than intraneuronally to cause release of NA.