Failure of carbachol to influence the release of somatostatin from slices of rat cerebral cortex

A[K⁺]-related, Ca²⁺-dependent efflux of immunoreactive somatostatin (IRS) from superfused slices of rat cerebral cortex has been observed; this release paralleled the release of both [¹⁴C]noradrenaline and [¹⁴C]GABA. However IRS release in this preparation was not stimulated by the muscarinic agonist carbachol at low (10 μM) or high (500 μM) concentrations. Furthermore, 100 or 500 μM carbachol did not affect the IRS efflux from rat cortex slices incubated in the presence of 12, 25 or 53 mM K⁺.     

Release of ( 14 C)adenine derivatives from isolated subsystems of the guinea pig brain: actions of electrical stimulation and of papaverine

Two subsystems prepared from guinea pig brain were maintained in isolation: one comprising the lateral olfactory tract and piriform cortex and the other the optic tract and superior colliculus. Apparatus for this purpose is described. On superfusion with oxygenated glucose bicarbonate salines these preparations showed responses to excitation by electrical stimulation of the lateral olfactory tract and optic tract. On stimulation, output of lactic acid by the preparations increased: when preincubation media included [¹⁴C]adenine, output of [¹⁴C]adenine derivatives was observed and this also increased on excitation. The effluent products then included adenine nucleotides, inosine, hypoxanthine and adenosine. Output of¹⁴C and of lactate was increased also by papaverine, in presence of which electrical excitation gave greater output of [¹⁴C]adenine derivatives. Results are appraised in terms of a neurohumoral role for adenine derivatives in the brain.     

Internal K and Na concentrations and the volume of hypothalamic synaptosomes

Hypothalamic synaptosomes, prepared by discontinuous sucrose density gradient centrifugation, were suspended in media of varying osmolarity and ionic composition and the internal concentrations of K⁺ and Na⁺ were measured. The intrasynaptosomal volume was determined using [¹⁴C]inulin,³⁵SO₄ or [¹⁴C]sucrose as extracellular markers. When the synaptosomes were suspended in 0.32 M sucrose the distribution volume of [¹⁴C]inulin and³⁵SO₄ were similar. However, when the medium contained 140 mM NaCl and 5 mM KCl the³⁵SO₄ space was equal to the total water space while the distribution volume of [¹⁴C]inulin corresponded to 35–51% of the total water space. [¹⁴C]sucrose distributed in a larger volume than did [¹⁴C]inulin, presumably due to intracellular permeation of sucrose. Using inulin as an extracellular marker the synaptosome volume was found to be inversely proportionate to the tonicity of the medium. [¹⁴C]inulin was considered a suitable marker of the extrasynaptosomal space and was used when determining intrasynaptosomal K⁺ and Na⁺ concentrations. The internal concentration of K⁺ was considerably higher than the external indicating that the synaptosomes were able to retain K⁺ against a concentration gradient. The synaptosomes gained Na⁺ when transferred to media containing 140 mM NaCl. The internal concentrations of K⁺ and Na⁺ were unaffected by glucose and elevated temperature and was only moderately changed during 90 min of incubation. The equilibrium potentials for K⁺ and Na⁺ were—68 and 13 mV respectively when the medium consisted of 0.03 M sucrose containing 140 mM NaCl, 5 mM KCl. It is concluded that hypothalamic synaptosomes can maintain a high transmembrane concentration gradient for K⁺ whereas the membrane is rather easily penetrated by Na⁺.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Interstitial potassium concentration, slow depolarization and focal potential responses in the dorsal horn of the rat spinal slice

Extracellular potassium concentration ([K⁺]_o) was measured, and intra- and extracellular recordings made, in the dorsal horn of rat spinal cord slices maintained in vitro during repetitive dorsal root stimulation. In about half of the dorsal horn neurons, the stimulation evoked a possibly substance P-mediated slow depolarization. [K⁺]_o increased during stimulation, reaching its highest values approximately 150 μm from the dorsal surface. The time course of Δ[K⁺]_o was different from that of the slow depolarization. Substance P itself evoked a much smaller Δ[K⁺]_o (0.4 mM) in the dorsal horn. It is concluded that the slow depolarization is not mediated by elevated [K⁺]_o.     

Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U-C]glutamate

The effect of methylmercury on glutamate metabolism was studied by ¹³C magnetic resonance spectroscopy. Cerebral cortical astrocytes were pretreated with methylmercury, either 1 μM for 24 h, or 10 μM for 30 min, and subsequently with 0.5 mM [U-¹³C]glutamate for 2 h. Labeled glutamate, glutamine, aspartate and glutathione were present in cell extracts, and glutamine, aspartate and lactate in the medium of all groups. HPLC analysis of these amino acids showed no changes in concentrations between groups. Surprisingly, the amounts of [U-¹³C]glutamate and unlabeled glucose taken up by the astrocytes were unchanged. Furthermore, the amounts of most metabolites synthesized from [U-¹³C]glutamate were also unchanged in all groups. However, formation of [U-¹³C]lactate was decreased in the 10 μM methylmercury group. This was not observed for labeled aspartate. It is noteworthy that both [U-¹³C]lactate and [U-¹³C]aspartate can only be derived from [U-¹³C]glutamate via mitochondrial metabolism. [U-¹³C]glutamate enters the tricarboxylic acid cycle (located in mitochondria) after conversion to 2-[U-¹³C]oxoglutarate and [U-¹³C]aspartate is formed from [U-¹³C]oxaloacetate, as is [U-¹³C]lactate. [U-¹³C]lactate can also be formed from [U-¹³C]malate. This differential effect on labeled aspartate and lactate indicates cellular compartmentation and thus selective vulnerability of mitochondria within the astrocytes to the effects of methylmercury. The decreased lactate production from glutamate might be detrimental to surrounding cells since lactate has been shown to be an important substrate for neurons.     

Pharmacological characterization of adenosine A1 receptors and its functional role in brown trout (Salmo trutta) brain

The adenosine receptor agonist N⁶-cyclohexyl[³H]adenosine ([³H]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout (Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [³H]CHA bound saturably, reversibly and with high affinity (K_d=0.69±0.04 nM; B_max=0.624±0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [³H]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5′-triphosphate (IC₅₀=0.778±0.067 μM). GTP (5 μM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K⁺ stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K⁺ as well as 1 mM glutamate stimulated the release of adenosine in a Ca²⁺-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K⁺ depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.     

Stimulation of GABA release from retinal horizontal cells by potassium and acidic amino acid agonists

The release of [³H]GABA from horizontal cells of goldfish retina was studied by biochemical analysis of perfused isolated retina. Retinas were incubated for 15 min in 0.72 μM[³H]GABA, rinsed for 30 min and then perfused with 1 min pulses of increasing concentrations of K⁺ and acidic amino acid agonists under a variety of conditions. Radioactivity in the perfusate was determined by liquid scintillation spectroscopy. The main findings are: (1) virtually all of the [³H]GABA released byl-glutamate (l-Glu) andl-aspartate (l-Asp) and 50% of the K⁺-evoked release, is calcium independent; (2) K⁺-evoked [³H]GABA release is only 10% of that released byl-Glu; (3) threshold [³H]GABA release occurs with 320 μMl-Glu, 1175 μMl-Asp, 4μM quisqualic acid (QA), 4μM kainic acid (KA) and 53 μM N-methyl-dl-aspartate (NMDLA); (4) the quisqualate antagonist glutamic acid diethyl ester (GDEE), has no specific inhibitory action on any of the agonists, whereasd-α-aminoadipic acid (DαAA), an NMDA antagonist, potently inhibits the action of NMDLA andl-Asp; (5) the presence of Mg²⁺, even at 1 mM, totally inhibits NMDLA and also inhibits the action ofl-Glu andl-Asp below 1 mM; (6)d-Asp potentiates the action ofl-Glu by 0.6–0.8 log units and completely inhibits the action ofl-Asp; (7)l-Asp at a ratio of 3:1 potentiates the effect ofl-Glu. From these and other results one concludes that: (a) [³H]GABA release from H1 cells is calcium independent and depends on factors other than passive depolarization, probably sodium; (2) the likely transmitter of red cones isl-Glu acting on quisqualate or kainate receptors, and (3)l-Asp acts predominantly on NMDA receptors and may provide a modulatory role in the outer retina by potentiating the action ofl-Glu.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

On the role of calcium ions in the presynaptic alpha-receptor mediated inhibition of [H]noradrenaline release from rat brain cortex synaptosomes

Rat brain cortex synaptosomes, previously labeled by incubation with [³H]noradrenaline ([³H]NA) were continuously superfused with Krebs-Ringer media. Release of [³H]NA was induced by superfusion with medium containing either 15 mM K⁺, 20 μM veratrine or 1 μM of the calcium-ionophore A 23187 and was strongly dependent on the concentration of Ca²⁺ in the medium. Noradrenaline (1μM, in the presence of the uptake inhibitor desipramine) inhibited K⁺-induced [³H]NA release by activation of presynaptic alpha-receptors. When the Ca²⁺-concentration in the medium was reduced, or the Mg²⁺-concentration increased, [³H]NA release appeared to be more susceptible to alpha-receptor mediated inhibition.Noradrenaline (1 μM) inhibited [³H]NA release induced by 15 mM K⁺, in the presence of 0.075 Ca²⁺ and 10 mM Mg²⁺, by 86%. Veratrine-induced release was also inhibited by alpha-receptor activation. However, [³H]NA release induced by the calcium-ionophore was not affected by alpha-receptor agonists. These results strongly support the view that alpha-receptor activation results in a decrease of the availability of Ca²⁺ for stimulus-secretion coupling processes. Presumably this is effected by an inhibition of voltage-sensitive calcium channels in the neuronal membrane associated with neurotransmitter release.     

Storage and release of endogenous and labelled GABA formed from [H]Glutamine and [C]Glucose in hippocampal slices: Effect of depolarization

To study the effect of depolarization on the synthesis, storage and release of GABA, hippocampal slices were incubated in 0.25 mM [³H]glutamine and 2.5 mM [¹⁴C]glucose in the presence of 3 or 50 mM K⁺. Total and labelled glutamine, glutamate and GABA contents were measured by high-performance liquid chromatography. Depolarization in the presence of Ca²⁺ led to a two-fold increase of labelled glutamate and a 3-fold increase of labelled GABA content originating from both labelled precursors. In the absence of Ca²⁺ and in the presence of 10 mM Mg²⁺, depolarization failed to increase labelled glutamate content and labelled GABA formation was increased by only 30%. Following superfusion with unlabelled 0.25 mM glutamine and 2.5 mM glucose a second depolarization with 50 mM K⁺ released twice as much labelled GABA from slices that had been incubated in the presence of 50 mM K⁺, than from those incubated in 3 mM K⁺. This difference remained unchanged in slices that were superfused with 1 mM aminooxyacetic acid, an inhibitor of GABA synthesis. The contribution of labelled GABA, especially of GABA derived from [³H]glutamine, to released GABA was significantly higher than to GABA stored in the slices. Results suggest that depolarization in the presence of Ca²⁺ results in increased glutamate and GABA synthesis from both glutamine and glucose and that part of GABA released by high K⁺ originates from preformed GABA stores.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.     

Sodium dependency of gamma-aminobutyric acid binding to particulate fractions of mouse brain

The Na⁺-dependency of γ-aminobutyric acid (GABA) binding to particulate fractions of mouse brain was studied, by increasing the concentration of Na⁺ in the homogenizing fluid the retention of both [³H] GABA and [¹⁴C] sucrose was increased in the crude nuclear fractions. But, at Na⁺ concentrations greater than 10 meq/l, the retention of both substances was decreased in the synaptosomal-mitochondrial fractions. Although the total [³H] GABA bound to all fractions was increased by increasing the Na⁺ concentration of the homogenizing medium, the total [¹⁴C] sucrose entrapped in these fractions remained rather constant. The changes in GABA binding caused by Na⁺ thus could not be accounted for by the presence of entrapped homogenizing fluid in pellets. The decreased binding of GABA to synaptosome fractions found in the presence of 40 to 100 meq/l Na⁺ was due to “clumping” effects caused by the excess salt. With high salt concentrations many of the particles which bind GABA and which are normally associated with the synaptosome fraction were sedimented in the crude nuclear fraction. In sum, these results indicated that the binding of GABA occurs maximally at a Na⁺ concentration of about 40 meq/l.     

Uptake of horseradish peroxidase at isolated nerve terminals in relationship to transmitter release; Biochemical results

The uptake of horseradish peroxidase (HRP) into isolated nerve terminals (synaptosomes) has been studied by using a spectrophotometric method to determine the enzyme activity. HRP is rapidly taken up by synaptosomes, it is not removed by multiple washes in iso-osmotic medium but is lost if the particles are ruptured by hypo-osmotic conditions. The uptake is not affected by metabolic poisons, is reduced at lower temperature and is not with any significant release of cytoplasmic lactate dehydrogenase suggesting an endocytotic mechanism. Intra-synaptosomal HRP can be released by a process that is similar to uptake and also not accompanied by any loss of synaptosomal lactate dehydrogenase exocytosis. Depolarization of synaptosomes (by high potassium concentrations) was found to release [¹⁴C]ACh but to have no effect on HRP uptake either simultaneouly or after recovery in an non-depolarizing medium. Absence of Ca²⁺ prevented depolarization evoked release of [¹⁴C]ACh but had no effect on the uptake of HRP. The release of HRP was not increased by depolarization even though [¹⁴C]choline taken up during the same period wasreleased as [¹⁴C]ACh. It is concluded that the endo-exocytotic cycle that transport HRP across the synaptosomal membrane is unrelated to tranmitter release. A discrete vesicular localization of HRP reaction product was only occasionally in the EM nor could consistent differences resulting from depolarization be observed. However, nthe untrastructural localization was found to be unreliable because glutaradehyde fixation irreversiy inactivated 80–90% of the HRP even when it was sequestered within synaptosomes and the insoluble reaction product precipitated from a supersaturated solution onto membranes.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Release of synaptosomal dopamine formed from tyrosine andl-dopa

The present report compares the synaptosomal release of [³H]dopamine, continuously forming from [³H]DOPA, with that of [¹⁴C]dopamine, forming from [¹⁴C]tyrosine as a marker of dopaminergic nerve endings. For the purpose of the comparison, synaptosomal (P₂) preparations from rat caudate nuclei were incubated withl-[³H]DOPA and [¹⁴C]tyrosine for 10 min and the particulates were rapidly separated from the medium postincubation. The separated fractions were analyzed for the level of double labelled (¹⁴C/³H) dopamine and the synaptosomal content of the labelled substrates. Of the total labelled dopamine formed, the fraction that was present in the medium, following the synaptosomal release, was determined. Tested were the release enhancing effects of various additions which included several known dopaminergic agents, serotonin and 5-hydroxytryptophan. The data show that the addition of dopamine to the incubation mixture to either 0.5 or 1.0 μM concentration markedly enhanced the release of labelled dopamine. Serotonin when added to 5.0 μM concentration also raised the medium content of labelled dopamine but it was ineffective at 1.0 μM. 5-Hydroxytryptophan, 1.0 or 5.0 μM, had no effect. For the comparison of the release enhancing effects of an addition of [¹⁴C]dopamine and [³H]dopamine, the corresponding release indices (release index = medium/total ratio of labelled dopamine in the presence of an addition divided by the same ratio in control (no addition) sample) were determined. The data indicate that the index of [¹⁴C]dopamine did not differ significantly from that of [³H]dopamine following the addition of either dopamine, serotonin or 5-hydroxytryptophan at any of the concentrations tested. A similar lack of difference between the index of [¹⁴C]dopamine and that of [³H]dopamine was observed following the addition of reserpine (1.8 μM), although a considerable enhancement of labelled dopamine release occurred. The addition of either amphetamine (9.1 μM) or amfonelic acid (9.1 μM) also enhanced labelled dopamine release but in their presence the index of [³H]dopamine was significantly higher than that of [¹⁴C]dopamine. Amfonelic acid preferentially raised the [³H]dopamine index at the lowest concentration of 0.91 μM that we have tested and also after only 5 min of incubation; coaddition of reserpine increased the [¹⁴C]dopamine release, thus abolishing the preferential effect of amfonelic acid on [³H]dopamine. When the incubation was performed without an addition (control sample), no difference (NS) was observed between the particulate to medium distribution of [¹⁴C]dopamine and that of [³H]dopamine after either 5, 10 or 15 min. The results suggest that (a) dopamine synaptosomally formed froml-DOPA may exist in a pool distinct from that dopamine arising from tyrosine hydroxylation and (b) the observed dopamine compartmentation may be due to some mechanism distinct from any possible participation of serotoninergic partivles. Also, the present results support the previously suggested existence of synaptosomal DOPA in pools.     

The interaction between β- and α2-adrenoceptors in cerebral cortical membranes: Isoproterenol-induced increase in [H]clonidine binding in rats

The pretreatment of rat cerebral cortical membranes with 10 or 100 μM isoproterenol at 37°C for 40 min caused a significant elevation of the B_max value of [³H]clonidine binding but pretreatment at 4°C did not affect the value. The isoproterenol-induced increase in the B_max value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the B_max value of [³H]dihydroalprenolol binding but neitherK_d nor B_max of [³H]WB 4101 binding was affected by this treatment. Fitty μM propranolol or 100 μM GTP produced a significant reduction in isoproterenol-induced elevation of the B_max value of [³H]clonidine binding. In contrast, 100 μM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only B_max value in high affinity binding of [³H]clonidine was increased by isoproterenol.It is suggested that isoproterenol increases the density of α₂-adrenoceptors in a temperature-dependent manner. The direct interaction between β- and α₂-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.     

Anomeric specificity ofd-glucose metabolism in rat brain cells

Rat brain cells were incubated at 9 °C (and occasionally at 37°C) in the presence of the anomers ofd-[5-³H]glucose,d-[U-¹⁴C]glucose,d-[6-¹⁴C]glucose andd-[1-¹⁴C]glucose (1.0 mM). The utilization of β-d-[5-³H]glucose was slightly higher than that of α-d-[5-³H]glucose, a situation possibly attributable to the anomeric behaviour of hexokinase. However, the production of¹⁴CO₂ from the α-anomer always largely exceeded that from the β-anomer. The anomeric difference ind-[U-¹⁴C]glucose oxidation, relative tod-[5-³H]glucose utilization, was suppressed in the presence of NH₄Cl. Even at anomeric equilibrium, the relative contribution of α-d-glucose to¹⁴CO₂ output exceeded its relative abundance. The β/α ratio ford-[1-¹⁴C]glucose oxidation (ord-[U-¹⁴C]glucose oxidation) was higher than that ford-[6-¹⁴C]glucose oxidation. Comparable observations were made in brain cells from albino rats and either lean or obese Zucker rats. It is concluded thatd-glucose metabolism displays anomeric specificity in rat brain cells, even when the latter are exposed to equilibratedd-glucose. It is also speculated that anomeric differences in the phosphorylation ofd-glucose by bound hexokinase may directly influence mitochondrial oxidative events.