Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes

Purified striatal synaptosomes were superfused continuously with L-[3,5-³H]tyrosine to measure simultaneously the synthesis ([³H]water formed during the conversion of [³H]tyrosine into [³H]DOPA) and the release of [³H]dopamine ([³H]DA). Glutamate (10⁻³ M) and NMDA (10⁻³ M, in the absence of Mg²⁺) stimulated the release of [³H]DA, but they reduced the efflux of [³H]water. This reduction of [³H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L--amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [³H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [³H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [³H]DA synthesis and release were mimicked by ionomycin (10⁻⁶ M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.     

Dopaminergic regulation of GABA release from the intact goldfish retina

The rate of release of [³H]GABA from intact goldfish retinas was studied using a modified superfusion technique. Small, significant increases in the rate of GABA release were observed when the retinas were exposed to dopamine (DA) (100–1000 μM); however, when free Ca²⁺ was removed from the medium, the basal rate of GABA release was increased and DA became inhibitory. Forskolin, a non-specific stimulator of adenylate cyclase in intact cells, also inhibited GABA release in the absence of Ca²⁺. There was no significant effect of forskolin in the presence of Ca²⁺; however, (+)-butaclamol, a dopamine antagonist, increased basal GABA release under these conditios. l-glutamic acid (l-Glu) (1–10 mM) causes up to a 10-fold increase in GABA release. In the presence of Ca²⁺, DA did not significantly alter the effects of l-Glu; however, in the absence of Ca²⁺ a significant inhibition of the effects of l-Glu by DA was observed. Forskolin, on the other hand, inhibited the effects of l-Glu both in the presence and absence of Ca²⁺. Finally, EGTA (0.3–1 mM) produced a large release of GABA: this release was inhibited by DA, forskolin, theophylline and 8-bromo cyclic AMP. These results suggest a model wherein DA stimulates Ca²⁺-dependent GABA release from one site and inhibits Ca²⁺-independent GABA release from another site via cyclic AMP-mediated event.     

[H]Nipecotic acid binding to GABA uptake sites in human brain

The binding of [³H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with γ-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [³H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to high affinity site. The dissaciation constants (K_d) measured in kinetic and equilibrium experiments were in the same range (0.5–0.6 μM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [³H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.     

Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling

The ability of astrocytes to sequester MeHG may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10⁻⁵ M) significantly inhibited the initial rate (5 min) of uptake of⁸⁶RbCl, used as a tracer for K⁺.⁸⁶RbCl uptake was also sensitive to the omission of medium Na⁺. MeHg (10⁻⁵ M) also markedly inhibited the initial rate of uptake (1 min) of the Na⁺-dependent uptake of [³H]l-glutamate. A second neurotoxin, MnCl₂ (0–5 × 10⁻⁴ M), did not alter [³H]glutamate or⁸⁶RbCl uptake. MeHg, but not MnCl₂, also stimulated the release of intracellular⁸⁶Rb⁺ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K⁺ gradient. This would be expected to compromise the ability of astrocytes to control extracellular K⁺ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [¹⁴C]3-O-methyl-d-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0–5 × 10⁻⁴ M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.     

The neuroleptic-like peptide desenkephalin-γ-endorphin does not antagonize the dopamine receptor agonist-induced inhibition of the release of [H]dopamine from rat nucleus accumbens slices in vitro

In rats, the non-opioid β-endorphin (βE) fragment desenkephalin-γ-endorphin (DEγE, βE_6–17) antagonizes the hypomotility induced by a small dose of dopamine (DA) receptor agonists. It has been suggested that DEγE might act in this respect by a direct or indirect blockade of presynaptically located DA receptors in the nucleus accumbens, thereby causing an increase of DA release. Therefore in the present study the effect of DEγE was examined on DA receptor agonist-induced inhibition of the electrically evoked release of previously accumulated [³H]DA from rat nucleus accumbens slices in vitro. The DA receptor agonists apomorphine, LY 171555 andn,n-di-n-propyl-7-hydroxy-2-aminotetralin (DP-7-AT) inhibited in a concentration-dependent manner the electrically evoked release of [³H]DA. The selective D₂ receptor antagonist (−)-sulpiride blocked the effects of apomorphine, corroborating that the DA receptor involved is of a D₂ type. DEγE was tested at several concentrations (10⁻⁹–10⁻⁶) and under various experimental conditions. DEγE, by itself, did not affect either the electrically stimulated or the basal release of [³H]DA. The inhibiting effect of DA receptor agonists was slightly reduced by DEγE, but this effect was present in some experiments only. It is concluded that DEγE does not function as an antagonist for the DA receptor mediating DA release and that the interaction observed in behavioural experiments between DA agonists and DEγE does not occur at the level of this receptor.     

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [³H]ACHC and [³H]GABA into cultured neurons and astrocytes was studied. [³H]ACHC uptake was less efficient than that of GABA in both cell types and K_m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V_max values were 0.321 and 0.405 nmol·min⁻¹·mg⁻¹ cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K_i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K_i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.     

Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.

Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Electrically induced release of [H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with6.25 × 10⁻⁷ M [³H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of³H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water,1 NHCl and 6 M urea in1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [³H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with10⁻⁵ M desmethylimipramine (DMI),10⁻⁴ M nialamide and 10⁻⁴ M tropolone, before and during incubation with [³H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [³H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Physiological roles of glycine and γ-aminobutyric acid in dissociated neurons of rat visual cortex

The effects of glycine (Gly) and γ-aminobutyric acid (GABA) on the neurons acutely dissociated from rat visual cortex (VC) were investigated in the whole-cell mode using a conventional patch-clamp technique. GABA and Gly evoked Cl⁻ currents (I_Cl) in a concentration-dependent manner at a holding potential (V_H) of −50 mV. The half maximum effective concentrations (EC₅₀) were 4.64 × 10⁻⁶ M for GABA and 6.67 × 10⁻⁵ M for Gly. Strychnine and bicuculline reversively inhibited both 10⁻⁵ M GABA -and 10⁻⁴ M Gly-induced I_Cl in a concentration-dependent manner. The half maximum inhibitory concentrations (IC₅₀) of strychnine on GABA- and Gly-induced currents were 4.00 × 10⁻⁶ M and 8.26 × 10⁻⁸ M, respectively. The IC₅₀ values of bicuculline on GABA and Gly responses were 1.18 × 10⁻⁶ M and 2.97 × 10⁻⁴, respectively. GABA at 10⁻⁵ M, which is near the EC₅₀ of the GABA response, induced I_Cl in all neurons tested n = 83). However, Gly of 10⁻⁴ M, which is near the the Gly response, induced I_Cl in 34 out of 83 neurons tested (41%). Moreover, the maximum amplitude of the Gly response was about 60% of that of the GABA response. On the other hand, the enhancement of N-methyl-d-aspartate (NMDA, 3 × 10⁻⁴M) response by Gly (10⁻⁶ M) was observed in all neurons (n=36) whether they had the Gly-induced I_Cl or not. In conclusion, it was proved that the VC neurons of immature rat could be classified into two classes, i.e. Gly sensitive and Gly insensitive in respect to activation of Cl⁻ channels. There were no significant correlations between the Gly response and the facilitatory action of Gly on the NMDA response, nor between the Gly and GABA responses. The GABA response was closely correlated to the NMDA response, having a correlation coefficient of 0.86.     

The role of cyclic AMP in chemoreception in the rabbit caroid body

The present study identified physiological factors which influence the generation (and degradation) of cyclic AMP (cAMP) in the arterial chemoreceptor tissue of the mammalian carotid body. Experiments established a 3-way correlation between cAMP generation, neurotransmitter release from chemoreceptor cells, and carotid sinus nerve (CNS) activity. Incubation of carotid bodies in vitro for 10 min in media equilibrated with different low O₂ (‘hypoxic’) gas mixtures (5% O₂ or 10% O₂, balance N₂) elevated basal cAMP levels (100% O₂ media) in proportion to the stimulus intesity. Similar experiments using nodose sensory ganglia showed that low O₂ stimulation did not alter cAMP levels in this non-chemosensory tissue. However, the adenylate cyclase (AC) activator, forskolin (10 μM), evoked large increases in the cyclic nucleotide content in both carotid bodies and nodose ganglia. After chronic (10 days) CSN denervation or synpathectomy, the basal levels of cAMP in the carotid body were elevated; the cAMP response to low O₂ media (stimulus minus control) was increased after CSN denervation but remained unaltered after sympathectomy. The effects of zero Ca²⁺ media on cAMP generation was examined in order to assess whether feedback from released neurotransmitters acting on known (presynaptic) type I cell receptors could have contributed to the observed changes in cAMP. Basal levels of cAMP were increased 2.8-fold, and the response to hypoxic stimulation was elevated 5-fold, in the absence of extracellular Ca²⁺. Forskolin (10 μM) did not alter basal release of [³H]-catecholamines ([³H]CA: synthesized from [³H]tyrosine, or resting CSN discharge; however, stimulus-evoked [³H]CA release and CSN discharge were potentiated in the presence of forskolin. This increased release was primarily due to enhanced efflux of dopamine (DA). At increasing stimulus strengths, however, the relative effect of forskolin on [³H]CA release was diminished. The data suggest that the chemoreceptor type I cells in the carotid body generate cAMP in their transductive response to hypoxia, but that the net levels of cAMP in the tissue are also regulated by both feedback actions of released neurotransmitters and by the sympathetic and sensory innervation to the organ. The effects of forskolin on [³H]CA release and CSN activity, combined with the finding that hypoxia increases the cAMP content of the carotid body, suggest the immediate invlovement of this classical second messenger in chemotransduction and chemotransmission of natural carotid body stimuli.     

Functional damage of dopamine nerve terminals following intrastriatal kainic acid injection

The release of [³H] dopamine ([³H]DA) previously taken up into rat striatal slices was studied one week after a monolateral intrastriatal injectuion of kainic acid (KA). Different releasing stimuli (electrical pulses, veratrine, high-K⁺) were applied. The electrically evoked release in the KA-lesioned striata was drastically reduced with respect to the unlesioned contralateral striata. In contrast, KA had no effect on the release of [³H]DA evoked by veratrine or high-K⁺. In unlesioned striatal slices, depolarized with 15 mM KCl, apomorphine reduced and (−)sulpiride increased the release of [³H]DA. The effect of apomorphine was antagonized by (−)sulpiride indicating the presence of an autoreceptor system similar to that seen in unlesioned striata stimulated electrically. However, the effects of apomorphine and of (−)sulpiride were dramatically reduced in K⁺-depolarized slices prepared from KA-lesioned striata. The results suggest that the axon terminals in KA-treated areas remain intact in several of their properties but may be damaged in some critical processes.     

Cobalt ion enhancement of 2-chloro[H]adenosine binding to a novel class of adenosine receptors in brain: antagonism by calcium

We have recently reported the identification of a novel class of micromolar-affinity adenosine binding sites in rat brain membranes using the adenosine agonist 2-chloro[³H]adenosine (Cl[³H]Ado). These binding sites are distinguishable from the A1 and A2 adenosine receptors by a number of pharmacological criteria, and we have designated this new class of binding sites as the A3 adenosine binding sites. In the present study, the effects of a wide range of divalent and trivalent cations on micromolar Cl[³H]Ado binding to brain membranes were examined. Co²⁺, Ni²⁺ and La³⁺ markedly stimulated specific Cl[³H]Ado binding by 45–150% above control when tested at concentrations of 1–10 mM. Ca²⁺ had no significant effect on binding except at high concentrations where it depressed binding slightly. Ca²⁺, however, completely prevented the stimulation of Cl[³H]Ado binding by Co²⁺. These findings further distinguish the A3 class of adenosine binding sites from the previously characterized adenosine receptors and suggest that the A3 binding sites are associated with calcium systems in brain.     

Prostacyclin release from the coronary vascular wall by vasoactive substances

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI₂) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 μM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 μM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 μM acetylcholine, 25 μM noradrenaline, 25 μM isoproterenol, 10 μM adenosine triphosphate (ATP 5 μM adenosine, 1 μM angiotensin II, 25 μM histamine or 1 μM serotonin was infused for 30 sec, no inhibitory activity of platele aggregation was observed. Bradykinin (5 × 10⁻⁹ 5 × 10⁻⁶ M) and purified thrombin (1 × 10⁻⁹ 1 × 10⁻⁷ M) induced a dose-dependent release of PGI₂ which was assayed using a radioimmunoassay for 6-keto-prostaglandin F₁ (6-keto-PGF₁).     

Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain γ-aminobutyric acid_A (GABA_A) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABA_A-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABA_A-receptors would also exhibit a differential response to halothane as assayed by an in vitro³⁶Cl⁻ influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 μmol/1 GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC₅₀ 336±64 μmol/1 (S.E.M.) vs. 605±110 μmol/1, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7±0.4 nmol·mg⁻¹·3⁻¹, vs. DR 4.7±0.5nmol·mg⁻¹·3s⁻). Halothane (500 μmol/1) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC₅₀ for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC₅₀ 1.8±0.4 μmol/1vs.14.7±0.9 μmol/1 without halothane) than in the DR mice (GABA EC₅₀ 4.7±0.6 μmol/1vs.14.7±0.9 μmol/1 without halothane). This effect of halothane was highly significant, both when compared to control, and between the selected lines (P < 0.001). The findings that halothane enhances GABA-gated flux and enhances GABA's channel gating potency support the hypothesis that differential enhancement of agonist-stimulated chloride permeability at GABA_A receptors could be a mechanism underlying the differential obtunding potency of halothane in DS and DR mice. However, at high GABA concentrations halothane decreased maximal chloride flux, more in DS than in DR mice (P < 0.001), which is not consistent with such a mechanism.     

Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists

The release of immunoreactive cholecystokinin (CCK) and dopamine was monitored simultaneously from superfused rat striatal slices. Exposure of the tissue to medium containing elevated of dopamine (10⁻⁷ and 10⁻⁶)M), the dopamine agonist pergolide (10⁻⁷, M), the D₂-antagonist sulpride (1 μM) or the D₁-antagonist (SCH 23390) had no significant effect on basal overflow or on evoked release of CCK. On the other hand, preincubation of striatal slices withd-amphetamine (10⁻⁵ M) enhanced basal and veratrine-stimulated dopamine release but markedly suppressed evoked CCK release. Sulpiride blocked this action of amphetamine whereas SCH 23390 was ineffective. The data suggests that whereas it is difficult to observe any effects of exogenous dopamine agonists or antagonists on evoked CCK release, endogenously released dopamine appears to interact with D₂-receptors to suppress evoked CCK release from rat striatal slices.     

Phenytoin and transmitter release at the neuromuscular junction of the frog

The effects of phenytoin (diphenylhydantoin, DPH) on transmitter release were studied at the frog neuromuscular junction. It was found that in Ringer's solutions containing a normal concentration of Ca²⁺ ions, DPH (1−2 × 10⁻⁴ M) depresses neurally evoked transmitter release, whereas in Ca²⁺-deficient Ringer's solutions it produces an increase in evoked release. Spontaneous transmitter liberation is augmented by DPH under all the above conditions. An abrupt disappearance of the evoked response occasionally occured with stimulation at 0.5 Hz, but a normal response could be elicited by a second stimulus delivered shortly after the first. At 100–200 Hz, DPH regularly induced a partial block in synaptic transmission. In8mM MgCl₂, this phenomenon appeared at 50 Hz and developed into a total neuromuscular blockade.     

Stimulated release of [H]β-alanine from the rabbit retina

The efflux of [³H]β-alanine from rabbit retina after intravitreal injection has been studied.

The site of uptake of [³H]β-alanine into retina was checked by autoradiography and was found mainly in the inner plexiform layer and in cells with the position of amacrines and in some ganglion cells.

When the preloaded retina was stimulated by light flashes the release of radioactivity increased significantly. Chromatography of the superfusate demonstrated a single radioactive spot which cochromatographed with authentic β-alanine.

The efflux of [³H]β-alanine was affected by raising the K⁺ concentration. The rate of efflux was also immediately increased when unlabelled β-alanine or GABA was added to the superfusion medium. Glycine was much less effective.

The present study shows that light stimulation releases [³H]β-alanine from the retina and that β-alanine may use the same transport system as GABA. This further supports the suggestion that β-alanine may act as a ‘false transmitter’ replacing GABA in the retina.     

Neurotensin release by rat hypothalamic fragments in vitro

The release of neurotensin by hypothalami from male rats was investigated in vitro uding tissue fragments incubated in Krebs-Ringer bicarbonate-glucose buffer at 37°C. Neurotensin was measured by radioimmunoassay using an antibody directed toward the C-terminal portion of the peptide. Neurotensin-like immunoreactivity release into the incubation medium eluted from Sephadex G-25 in a position identical to that of the synthetic peptide and serial dilutions of incubation medium were parallel to those of synthetic neurotensin in the radioimmunoassay. Neurotensin-like immunoreactivity released into the incubation medium was degraded during the incubation period by released hypothalamic peptidases. The addition of bacitracin (0.5 mg/ml) to the medium partially prevented this degradation. Neurotensin release was stimulated by dibutyryl cyclic AMP (10⁻⁴ M) and by depolarizing concentrations of potassium. The latter effect was shown to be Ca²⁺-dependent. Dopamine (10⁻⁴−10⁻⁶ M) stimulated neurotensin release in a dose-dependent manner and this effect was blocked by the dopamine receptor antagonist, haloperidol. Neurotensin release was not stimulated by either norepinephrine (10⁻⁴ M) or serotonin (10⁻⁴ M). The results indicate that neurotensin is released by the hypothalamus in vitro; its release is stimulated by membrane depolarization in a Ca²⁺-dependent manner and may involve an adenylate cyclase mechanism; and dopamine appears to serve as a stimulatory neurotransmitter for neurotensin-containing neurons.