Temperature thresholds and degree-day model for Marmara gulosa (Lepidoptera: Gracillariidae).

The developmental thresholds for Marmara gulosa Guillén & Davis (Lepidoptera: Gracillariidae) were investigated in the laboratory by using 17, 21, 25, 29, and 33 degrees C. The lowest mortality occurred in cohorts exposed to 25 and 29 degrees C. Other temperatures caused >10% mortality primarily in egg and first and second instar sap-feeding larvae. Linear regression analysis approximated the lower developmental threshold at 12.2 degrees C. High mortality and slow developmental rate at 33 degrees C indicate the upper developmental threshold is near this temperature. The degree-day (DD) model indicated that a generation requires an accumulation of 322 DD for development from egg to adult emergence. Average daily temperatures in the San Joaquin Valley could produce up to seven generations of M. gulosa per year. Field studies documented two, five, and three overlapping generations of M. gulosa in walnuts (Juglans regia L.; Juglandaceae), pummelos (Citrus maxima (Burm.) Merr.; Rutaceae), and oranges (Citrus sinensis (L.) Osbeck; Rutaceae), for a total of seven observed peelminer generations. Degree-day units between generations averaged 375 DD for larvae infesting walnut twigs; however, availability of green wood probably affected timing of infestations. Degree-day units between larval generations averaged 322 for pummelos and 309 for oranges, confirming the laboratory estimation. First infestation of citrus occurred in June in pummelo fruit and August in orange fruit when fruit neared 60 mm in diameter. Fruit size and degree-day units could be used as management tools to more precisely time insecticide treatments to target the egg stage and prevent rind damage to citrus. Degree-day units also could be used to more precisely time natural enemy releases to target larval instars that are preferred for oviposition.

Expression of TIMP3 mRNA is elevated in retinas affected by simplex retinitis pigmentosa.

To explore the molecular and cellular mechanisms associated with photoreceptor death in retinitis pigmentosa (RP), we have investigated altered transcriptional activity in RP retinas by a differential cDNA screening approach. We identified a clone (K222) showing over-expression in simplex RP retinas compared with controls. K222 encodes a partial cDNA of the human tissue inhibitor of metalloproteinases-3 (TIMP3) gene, a member of a family of genes implicated in extracellular matrix (ECM) remodelling. Increased expression of TIMP3 in degenerating RP retinas may reflect restructuring of the ECM architecture, and disruption of photoreceptor-matrix interactions could contribute to activation of apoptotic cell death processes.

Analysis of differentially expressed genes in retinitis pigmentosa retinas. Altered expression of clusterin mRNA.

The molecular and cellular processes underlying photoreceptor degeneration in retinitis pigmentosa (RP) are unknown. We have investigated gene expression in diseased retinas using differential hybridization screening of a retinal cDNA library with probes derived from normal and RP retinal RNA. Most differential clones detected corresponded to transcripts absent from the dystrophic state, including e.g. opsin. However, one clone was noticeably increased in RP in comparison with the control: partial sequencing showed it encoded clusterin. Increased expression of clusterin has been identified in several cases of tissues undergoing apoptosis (programmed cell death), and our finding suggests that the degenerative changes in advanced RP may represent another example of apoptosis, possibly with common causative mechanisms.

Adenovirus-mediated gene transfer to murine retinal cells in vitro and in vivo.

Adenovirus-mediated gene transfer to retinal cells was evaluated using the replication-defective recombinant adenovirus vector Ad2/CMVlacZ-1 (coding for beta-galactosidase) both in an in vitro murine culture model and in vivo in adult mice. In vitro, no difference in infectability of neuronal and glial cells was observed, and 50% of neurons expressed the exogenous gene at low viral concentration (10 pfu/cell). In vivo, intraocular injection of 3 x 10(6) pfu Ad2/CMVlacZ-1 resulted in expression of the transferred beta-galactosidase gene in retinal pigment epithelium and ganglion cells. These results demonstrate that Ad2/CMVlacZ-1 is an effective vector for gene transfer into retinal cells.

The contrasting effects of neuroleptics on transmitter release from the nucleus accumbens and corpus striatum.

The effects of haloperidol, chlorpromazine and clozapine on transmitter release have been studied by measuring the simultaneous release of dopamine and acetylcholine from tissue slices of nucleus accumbens and striatum in vitro following in vivo drug application, either a single dose or daily for periods of up to 25 days. In the striatum, both single and chronic injections of haloperidol (1 and 2 mg. kg-1) caused a large and significant enhancement (up to 83%) of the K+-evoked release of [3H]acetylcholine synthesized from [3H]choline. In contrast, in the nucleus accumbens, the K+-evoked release of [3H]acetylcholine was not significantly affected by neuroleptics when compared with the controls which received injection of vehicle. Clozapine (50 mg.kg-1) also enhanced the K+-evoked release of [3H]acetylcholine relative to the resting release but the effect was smaller than that with haloperidol or chlorpromazine. The evoked release of preloaded [14C]dopamine from either nucleus accumbens or striatum was unaffected by treatment with haloperidol. However, chlorpromazine (25 mg.kg-1) and clozapine (50 mg.kg-1) significantly enhanced the evoked release of preloaded [14C]dopamine from tissue slices of striatum. A similar but reduced effect of enhancing release was also seen in the nucleus accumbens in response to chlorpromazine. In support of these results, dopamine itself applied in vitro induced an opposite effect to that of the neuroleptics. Thus in the striatum, dopamine (4 x 10(-4) M) markedly reduced the release of [3H]acetylcholine (45%). A smaller inhibition was also seen in the nucleus accumbens (25%).

GABA release from Xenopus retina does not correlate with horizontal cell membrane potential.

The relationship between horizontal cell membrane potential and the release of GABA was explored in the retina of Xenopus laevis. The intracellularly recorded membrane potential of horizontal cells was monitored while the retina was exposed to different concentrations of depolarizing agents. The dose-response curves obtained revealed a rise from 5 to 95% maximum depolarization in 0.5-1.5 log unit concentration change. The molar concentrations that elicited a 20 mV depolarization were 40 mM (potassium), 0.8 mM (glutamate), 0.8 mM (glycine), 5 microM (kainate) and 1.3 microM (quisqualate). Autoradiography revealed that radiolabel was accumulated almost exclusively by horizontal cells when isolated retinas were incubated in medium containing 1 microM [3H]GABA. Thus, retinal release of radioactivity was used as a measure of [3H]GABA release from horizontal cells. Endogenous GABA released from retinas was measured using high performance liquid chromatography and was taken to reflect both amacrine and horizontal cell GABA pools. The release of both [3H]GABA and endogenous GABA was stimulated by glutamate, kainate and potassium, but not by glycine or quisqualate. Similar dose-response curves for GABA release and for depolarization were obtained in the case of potassium and kainate but not for glutamate. Potassium-evoked release either of endogenous GABA or [3H]GABA was both calcium- and sodium-dependent, whereas kainate- or glutamate-evoked GABA release was sodium-dependent but calcium-independent. The results indicate that depolarization per se is not necessarily associated with transmitter release in Xenopus retinal horizontal cells. It is suggested that the action of a given neurotransmitter upon the efflux of GABA from horizontal cells may depend on the degree to which it modifies the sodium conductance of the horizontal cell.

Release of endogenous ascorbic acid preserves extracellular dopamine in the mammalian retina.

PURPOSE: To investigate whether the inhibitory effect of nitric oxide (NO) on dopamine release from the retina is due to chemical oxidation of dopamine in the extracellular medium rather than to an inhibitory effect on dopamine release from retinal neurons. METHODS: Dopamine was incubated in Krebs bicarbonate medium and its rate of chemical degradation measured by high-performance liquid chromatography (HPLC). The effects of NO donors and antioxidants on dopamine were assessed by comparing dopamine degradation in the presence and absence of drug. The effects of NO donors on the K-evoked release of [3H]dopamine were measured from isolated superfused rabbit retinas. The release of ascorbic acid from the isolated rat retina and from an eyecup preparation in anesthetized rabbits was measured by HPLC. RESULTS: After 10 minutes' incubation in Krebs bicarbonate medium, the dopamine concentration decreased by 20%. This decline increased to 80% in the presence of S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP). The increased rate of dopamine degradation was abolished if retina was incubated in the medium and then removed before the incubation of dopamine. The protective effect of preincubation with tissue was lost in the presence of ascorbate oxidase suggesting the release of ascorbic acid. HPLC analysis confirmed a substantial release of ascorbic acid from both rabbit and rat retinas. The K-evoked release of [3H]dopamine from the rabbit retina was inhibited by SNP. CONCLUSIONS: NO can rapidly, oxidize dopamine in physiological medium, but in the presence of retina, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent this chemical reaction. Thus, the inhibitory action of NO on dopamine release results from an action on retinal neurons. Ascorbate release in the retina may have an important physiological role in prolonging the life of dopamine, which often has to diffuse long distances from axons in the inner plexiform layer to receptors in other retinal layers.

Metabolism of fluorescein after intravenous administration.

The measurement of plasma unbound (free) fluorescein is important in the study of blood-ocular barrier kinetics. The authors became concerned about the quantitative significance of the presumed glucuronide metabolite of fluorescein to the measurement of plasma fluorescence in diabetic and normal subjects. Fluorescein was given intravenously (14 mg/kg) to seven normal subjects and eight diabetic subjects. Plasma samples taken during 60 min were subjected to microfiltration, from which aliquots of ultrafiltrate were incubated with beta-glucuronidase. Samples were subjected to high-performance liquid chromatography, and fluorescence activity was measured in the eluent. All subjects showed an additional fluorescence peak to that of fluorescein in plasma and ultrafiltrate 5 min after fluorescein administration and increased thereafter. This additional peak was abolished by incubation of ultrafiltrate with beta-glucuronidase and resulted in a marked increase in fluorescence due to the liberation of fluorescein from its presumed glucuronide. There were no pharmacokinetic differences between normal and diabetic subjects in plasma-free fluorescein and fluorescein glucuronide pharmacokinetics or in their respective binding to plasma proteins. The glucuronide had only 4.5% of the fluorescence of fluorescein, but because more of the glucuronide was unbound (32%) compared with fluorescein (10%) and its concentration increased while that of fluorescein decreased, it constituted an increasing proportion of the fluorescence in the ultrafiltrate. At 60 min, 80% of the fluorescein was present as glucuronide and contributed 20% of the total fluorescence in the ultrafiltrate. Fluorescein-glucuronide is a potential source of variability in studies on blood-ocular barrier kinetics.

Altered expression of secreted frizzled-related protein-2 in retinitis pigmentosa retinas.

PURPOSE: Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique. METHODS: Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription-polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies. RESULTS: By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing protein beta-catenin. CONCLUSIONS: SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise.

Expression patterns of neurturin and its receptor components in developing and degenerative mouse retina.

PURPOSE: Neurturin (NTN) and its receptor components (GFRalpha2 and Ret) play an important role in the survival of different populations of neurons in the central and peripheral nervous systems. To gain insight into their possible functions throughout normal retinal development and during retinal neuronal apoptosis, the retinal distribution of expression of NTN and GFRalpha2 mRNAs and Ret protein were compared in control and retinal degeneration (rd) mice. METHODS: Eyes from control and rd animals were fixed in paraformaldehyde before sectioning. For in situ hybridization, retinal sections were hybridized with 35S-radiolabeled sense and antisense riboprobes for murine NTN and GFRalpha2 and were autoradiographed. Ret localization was detected by immunofluorescence. RESULTS: Neurturin mRNA expression was modulated through normal postnatal retinal development and was localized primarily to the inner retina and photoreceptor outer segments. GFRalpha2 mRNA displayed a diffuse developmental pattern of expression, but in the mature normal retina, NTN and GFRalpha2 mRNAs were more closely colocalized. Ret protein was localized particularly at the outer segments of photoreceptors, inner retina, and ganglion cell layers, but there were no prominent differences among genotypes. Increased NTN mRNA expression was detected in the retinal pigment epithelium and neural retina in concert with photoreceptor degeneration in rd mouse. In contrast, the level of GFRalpha2 mRNA was lower in rd compared with that in normal retina. CONCLUSIONS: These results suggest that NTN and its receptor are involved in retinal postnatal development and maintenance and that alterations in their transcription patterns are associated with inherited retinal degeneration.

Retinal expression of gamma-crystallins in the mouse.

PURPOSE: High levels of expression of a form of gamma-crystallin mRNA in mouse retina have been identified. Because the six murine gamma-crystallins have generally been regarded as specific to the lens, the expression of these crystallins at the mRNA and protein levels in the retina were evaluated in more detail. METHODS: Expression of gammaE/F-crystallin mRNA was examined by northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis applied to murine retinal and lens total RNAs. For gammaA-D-crystallin mRNAs, a multiplex RT-PCR was used on total cDNAs. The detection of total gamma-crystallin protein in the retina was performed using an antibody to bovine lens gamma-crystallins, applied to protein extracts in immunoblot analysis and to cryostat sections of ocular tissues in immunofluorescence studies. RESULTS: By RT-PCR, we confirmed expression of both gammaE-and gammaF-crystallin as well as all four (gammaA-gammaD) remaining crystallins at the mRNA level in the mouse retina. Gamma-crystallin proteins were also detectable in murine retina by immunoblot analysis, although at a lower level than in the lens. By immunocytochemistry, gamma-crystallins were localized particularly to the inner retina, outer plexiform layer, and the photoreceptors during postnatal development. CONCLUSIONS: Our findings of gamma-crystallin mRNA and protein expression in the retina indicate that none of the major crystallin classes is uniquely expressed in the lens. The expression of gamma-crystallins in the developing murine retina suggests a role analogous to the anti-stress properties established for the small heat-shock protein alphaB-crystallin, perhaps in response to varying exposure to light.

Effect of sulphur containing amino acids on [3H]-acetylcholine release from amacrine cells of the rabbit retina.

1. The effects of the sulphur containing amino acids, homocysteic acid, homocysteine sulphinic acid, cysteic acid and cysteine sulphinic acid on the release of [3H]-acetylcholine ([3H]-ACh) from the cholinergic amacrine cells of the rabbit retina were examined. 2. All the compounds stimulated the spontaneous resting release and abolished the light-evoked release of [3H]-ACh. Except for homocysteine sulphinic acid these actions occurred at concentrations that did not affect the erg b-wave amplitude, indicating a site of action at the inner retina. 3. N-methyl-D-aspartate (in Mg(2+)-containing medium) clearly blocked the effects of homocysteic acid and homocysteine sulphinic acid on the resting release of [3H]-ACh but had no effect on the actions of cysteic acid and cysteine sulphinic acid. 4. Since N-methyl-D-aspartate is an antagonist of the light-evoked endogenous bipolar cell transmitter released onto cholinergic cells, these results are consistent with the suggestion that homocysteic acid or homocysteine sulphinic acid may be a transmitter released from this subpopulation of bipolar cells. 5. The present experiments indicate the existence of excitatory amino acids that have closer pharmacological properties to a bipolar cell transmitter than glutamate but it remains to be seen whether homocysteic acid or homocysteine sulphinic acid occur in these particular bipolar cells.

The effect of central stimulant drugs on acetylcholine release from rat cerebral cortex.

1. The effects of central stimulant drugs injected intraperitoneally were examined on the release of acetylcholine (ACh) from the cerebral cortex of the anaesthetized rat. The effects of the drugs in increasing ACh release were approximately parallel to the increases produced in the electrical activity of the brain.2. Leptazol in a dose of 150 mg/kg increased the release of ACh by 2.9 times the resting release and in a dose of 300 mg/kg by 7.5 times; on the e.e.g. the injection produced a large increase in the electrical activity.3. Picrotoxin in a dose of 12 mg/kg increased the release of ACh by 7.5 times and on the e.e.g. caused a large increase in activity.4. Strychnine in doses of 8 mg/kg and 12 mg/kg increased the release of ACh by 2.0 and 2.4 times and produced a small increase in the activity of the e.e.g.5. Dexamphetamine in a dose of 100 mg/kg increased the release of ACh by 1.9 times and had no appreciable effect on the e.e.g.6. Nikethamide in a dose of 2 g/kg increased the release of ACh by 2.3 times and produced no appreciable change in the e.e.g.7. Caffeine in a dose of 100 mg/kg produced no significant effect on the release of ACh.

Release of [3H]-acetylcholine from the isolated retina of the rat by potassium depolarization: dependence on high affinity choline uptake.

1. The effect of potassium depolarization on the release of [3H]-acetylcholine ([3H]-ACh) from the isolated retina of the rat was studied. 2. Exposure of retinae to medium containing KCl (50 mM) evoked a large increase in the efflux of [3H]-ACh with only a small concurrent increase in the efflux of [3H]-choline. The KCl-evoked release of [3H]-ACh was almost abolished in calcium-free medium. 3. Incubation of retinae with [3H]-choline in sodium-free medium, or medium containing hemicholinum-3 (HC-3), procedures that are believed to inhibit selectively the high affinity choline transport system, reduced the retinal uptake of [3H]-choline by approximately 50% and the synthesis of [3H]-ACh by about 97%. 4. The potassium-evoked release of [3H]-ACh was almost abolished in retinae that had been loaded with [3H]-choline in sodium-free medium or medium containing HC-3, and subsequently superfused in normal medium. 5. It is suggested that as in other areas of the nervous system, a sodium-dependent, high affinity uptake system for choline is important in retinal cholinergic nerve terminals.

Potassium activation of [3H]-choline accumulation by isolated sympathetic ganglia of the rat.

1 The effect of K-depolarization on the uptake of low and high concentrations of [3H]-choline by isolated superior sympathetic ganglia of the rat has been studied. 2 In unstimulated ganglia, the uptake of [3H]-choline (0.1 microM) ('high affinity uptake') was unaffected by denervation or by hemicholinium-3 (HC-3), suggesting uptake by structures other than cholinergic nerve terminals. 3 K-depolarization of the ganglia increased [3H]-choline accumulation by the high affinity uptake process but in contrast the 'low affinity' accumulation of [3H]-choline (100 microM) was decreased. 4 The K-activated, 'high affinity' component of choline uptake was highly sodium-dependent, inhibited by HC-3, and was abolished by denervation. 5 In incubation conditions designed to prevent transmitter release (Ca-free medium and high-Mg medium), the K-activated uptake of [3H]-choline was abolished. 6 It is concluded that in unstimulated ganglia, there is little choline uptake by nerve terminals. However, when the terminals are depolarized, choline uptake is increased by the activation of a sodium-dependent, HC-3-sensitive transport process. The activation of this uptake process is apparently associated with the release of acetylcholine from the terminals, or by changes in ionic fluxes, and not by the depolarization per se.

Effect of potassium depolarization and preganglionic nerve stimulation on the metabolism of [3H]-choline in rat isolated sympathetic ganglia.

1 The effects of potassium depolarization and preganglionic nerve stimulation on the metabolism of [(3)H]-choline in the isolated superior sympathetic ganglion of the rat have been studied.2 When unstimulated (resting) ganglia were incubated for 10 min with a low concentration (0.1 muM) of [(3)H]-choline (high affinity uptake), approximately 75% of the accumulated radioactivity was present as [(3)H]-phosphorylcholine, 11% was [(3)H]-acetylcholine ([(3)H]-ACh) and the remainder was unchanged [(3)H]-choline.3 Depolarization of the ganglia with K (46 mM) before their incubation with [(3)H]-choline, increased [(3)H]-choline uptake by 70% and increased [(3)H]-ACh synthesis by more than 700%, so that [(3)H]-ACh represented almost 50% of the total radioactivity recovered. In contrast, the proportion of [(3)H]-phosphorylcholine fell to 36% of the total radioactivity recovered.4 The striking effect of K-depolarization on [(3)H]-ACh synthesis in ganglia occurred at a concentration of 30 mM or above, and the maximum effect was seen at 45-50 mM.5 Chronic denervation of the ganglia abolished all the effects of high-K on [(3)H]-choline metabolism. In resting ganglia, [(3)H]-ACh formation was reduced by over 80% but [(3)H]-phosphorylcholine synthesis and the level of unchanged [(3)H]-Ch were not affected by denervation.6 Exposure of the ganglia to low-Na or hemicholinium-3 (HC-3) greatly reduced [(3)H]-ACh synthesis in control resting ganglia and almost abolished the effects of high-K on [(3)H]-ACh synthesis.7 Prevention of transmitter release with high-Mg or low-Ca medium also prevented K-depolarization from stimulating [(3)H]-ACh synthesis.8 Preganglionic nerve stimulation had an effect on [(3)H]-choline metabolism similar to that of K-depolarization. Thus, at all the frequencies studied (1-30 Hz), [(3)H]-ACh synthesis was greatly increased and [(3)H]-phosphorylcholine was reduced, the maximum effects occurring at 3 Hz.9 When ganglia were incubated with a high concentration (100 muM) of [(3)H]-choline (low affinity uptake), a different pattern of metabolism was observed. Most of the radioactivity in resting ganglia was present as unchanged [(3)H]-choline (70%) with [(3)H]-phosphorylcholine and [(3)H]-ACh representing 23% and 6% of the total radioactivity respectively. K-depolarization decreased [(3)H]-choline uptake but increased the proportions of [(3)H]-phosphorylcholine and [(3)H]-ACh to 32% and 24% of the total radioactivity respectively.10 It is concluded that in unstimulated (resting) rat sympathetic ganglia most of the [(3)H]-choline transport and metabolism occurs in postsynaptic structures. However, depolarization of the presynaptic nerve terminals appears to trigger a sodium-dependent, HC-3 sensitive, high-affinity uptake process, and causes a dramatic increase in presynaptic [(3)H]-ACh synthesis together with a fall in postsynaptic [(3)H]-phosphorylcholine synthesis. These changes in choline metabolism cannot be due to the depolarization of the nerve terminals per se, because they were abolished by high-Mg or low-Ca, i.e. when transmitter release was prevented. Thus, the increase in ACh synthesis may be triggered by a fall in the intraterminal concentration of ACh or by the changes in Ca flux induced by depolarization. Our experiments do not provide evidence on these possible mechanisms.

On the mechanism by which veratridine causes a calcium-independent release of gamma-aminobutyric acid from brain slices.

1 The mechanisms by which veratridine increases the release of gamma-aminobutyric acid (GABA) from brain slices have been studied.2 Exposure of superfused cerebro-cortical, nigral or cerebellar slices to veratridine (5 muM) or KCl (50 mM) caused large increases in the efflux of [(3)H]-GABA.3 Reduction of the external Ca concentration [Ca](o) to zero had strikingly different effects on the veratridine and K-evoked release of [(3)H]-GABA. The K-evoked release from all three areas was greatly reduced in Ca-free medium, but the veratridine-evoked release from cerebeller slices was not affected, and the release of [(3)H]-GABA from cortical and nigral slices was increased three fold. The potentiation of the veratridine evoked release of GABA which occurred in Ca-free medium was not due to the reduction in divalent ions, because it still occurred in medium in which the Ca was replaced by an equivalent amount of Mg.4 The veratridine-evoked release of [(14)C]-glycine from slices of spinal cord was also significantly increased in Ca-free medium. In contrast, the release of cortical [(3)H]-noradrenaline and [(14)C]-acetylcholine caused by the alkaloid was greatly diminished in Ca-free medium.5 The veratridine but not the K-evoked release of [(3)H]-GABA was abolished when the external Na concentration [Na](o) was reduced to zero and by tetrodotoxin (TTX) (0.2 muM). Cl-free medium did not affect the veratridine-evoked release of [(3)H]-GABA or its potentiation by Ca-free medium.6 Exposure of the tissue to depolarizing concentrations of external K ([K](o) = 120 mM) did not abolish the veratridine evoked release of [(3)H]-GABA or its potentiation by Ca-free medium.7 Pre-incubation of cortical slices with L-2,4, diaminobutyric acid (DABA), or substitution of Na in the superfusion medium with Li, did not affect the veratridine-evoked release of [(3)H]-GABA, indicating that the alkaloid does not stimulate GABA efflux by a carrier-mediated transport process.8 Exposure of the tissue to ruthenium red (10 muM) increased the veratridine evoked release of [(3)H]-GABA in both normal and in Ca-free medium but almost abolished the K-evoked release.9 It is suggested that veratridine causes GABA release by increasing the permeability of the nerve terminals to Na. In normal medium, the resulting influx of Ca(2+) ions through voltage-dependent Ca(2+) channels may be involved in triggering the release of GABA. However, a major part of the GABA efflux appears to be triggered by the release of Ca(2+) ions from intraterminal mitochondria, which results from the increase in[Na](i). Since Ca(2+) ions antagonize the action of veratridine, the potentiation of the drug-evoked release of GABA that occurs in Ca-free medium, might be due to the absence of the antagonistic Ca(2+) ions. The resulting greater increase in Na entry and [Ca](i) caused by Ca release from intracellular stores, must presumably more than balance the contribution normally made by any influx of extracellular Ca(2+).

Effect of electrical stimulation and high potassium concentrations on the effux of (14C) glycine from slices of spinal cord.

1. The effects of electrical stimulation and solutions containing a high concentration of potassium on the efflux of [(14)C] glycine from slices of rat spinal cord have been studied.2. Slices of cord were incubated with [(14)C] glycine which rapidly accumulated in the tissue. The slices were then superfused in a small chamber and the radioactivity released from the tissue was measured. After superfusion for 60 min, 98% of the radioactivity remaining in the tissue was present as unchanged glycine.3. The spontaneous efflux of [(14)C] glycine consisted of an initial rapid phase followed by a much slower release of [(14)C] glycine. After superfusion for 60 min, more than 65% of the radioactivity taken up during the incubation period was retained by the tissue.4. When the slices were depolarized by electrical stimulation or by solutions containing a high concentration of potassium (40 mM), a striking increase in the efflux of [(14)C] glycine was produced. This effect was not reduced by the absence of calcium ions in the superfusion medium.5. Electrical stimulation produced similar increases in the efflux of [(3)H] GABA and [(14)C] glutamate from slices of cord but had no significant effects on the efflux of [(3)H] alanine or [(14)C] urea.6. The results are consistent with the suggestion that glycine may be an inhibitory synaptic transmitter substance in the mammalian spinal cord.

The uptake of 3Hp -aminobutyric acid by the retina.

1. The accumulation of (3)H-gamma-aminobutyric acid (GABA) by the isolated rat retina has been measured.2. When retinae were incubated at 37 degrees C in a medium containing (3)H-GABA, tissue:medium ratios of about 25:1 were attained after a 30 min incubation.3. After incubations of 40 min at 37 degrees C, almost all (98%) the radioactivity in the tissue was present as unchanged (3)H-GABA.4. The process responsible for (3)H-GABA uptake showed many of the properties of an active uptake system: it was temperature-sensitive, required the presence of sodium ions in the external medium, was inhibited by anoxia, dinitrophenol and ouabain, and showed saturation kinetics.5. The estimated Km value of GABA was 4.0 x 10(-5)M, and V(max) was 0.167 (mumoles/min)/g retina.6. The uptake of (3)H-GABA was not affected by the presence of large molar excesses of glycine, L-glutamate, L-aspartate, L-alanine, L-proline, or L-histidine, but was inhibited by DL-gamma-amino-beta-hydroxybutyrate, beta-guanidinopropionate, and L-2,4-diaminobutyrate.7. The retina was capable of achieving a large net uptake of GABA, indicating that the accumulation of (3)H-GABA by the tissue was not due only to an exchange process with the endogenous GABA pool.8. The uptake of (3)H-GABA occurred only in tissue from the central nervous system. Thus, retina and cerebral cortex rapidly accumulated radioactivity, but slices of cornea, posterior wall of the eye, and liver achieved tissue: medium ratios of approximately one.9. There was a rapid efflux of radioactivity from retinae placed in fresh medium and after 60 min, 90% of the radioactivity was lost from the tissue. The radioactivity released into the medium was present largely as (3)H-acidic and neutral metabolites. When the metabolism of GABA was inhibited by the presence of amino-oxyacetic acid in the medium, only about 10% of the radio-activity was lost from the tissue during a similar 60 min incubation, and the radioactivity released was present largely as unchanged (3)H-GABA.10. It is suggested that the GABA uptake process may represent a possible mechanism for the inactivation of GABA if this amino acid is released at inhibitory synapses in the retina.

Effect of inhibitors of -aminobutyrate aminotransferase on the accumulation of 3H- -aminobutyric acid by the retina.

1. Rat retinae pre-incubated and incubated at 37 degrees C in media containing amino-oxyacetic acid (AOAA) (0.1 muM to 1 mM) accumulated more (3)H-gamma-aminobutyric acid ((3)H-GABA) than control retinae incubated in the absence of AOAA. This increased accumulation of (3)H-GABA by tissue exposed to AOAA was not apparent at short incubation times (0-20 min), but became significant after incubations of 30 min, and maximal after incubation for 60 minutes.2. At a concentration of 10 muM, AOAA did not alter the apparent K(m) for (3)H-GABA uptake or V(max) for either the low or the high affinity GABA uptake systems present in retina.3. The potentiation of (3)H-GABA accumulation produced by AOAA appeared to parallel the inhibitory effect of this compound on 2-oxoglutarate-4-aminobutyrate aminotransferase (GABA-T). Similarly, hydrazinopropionic acid inhibited retinal GABA-T and potentiated the accumulation of (3)H-GABA, but hydroxylamine and thiosemicarbazide which did not affect GABA-T, were also without effect on the retinal accumulation of (3)H-GABA.4. In vitro incubation with AOAA did not increase the endogenous levels of GABA or other amino acids in the retina.5. AOAA did not significantly increase the retinal accumulation of radioactive L-glutamate, L-glutamine, taurine, glycine, alpha-aminoisobutyrate or dopamine: the accumulation of L-aspartate was increased by approximately 30%.6. The inhibition of retinal GABA-T by AOAA was time-dependent and was not reversed by pyridoxal-5'-phosphate or by repeated washing of the tissue with fresh medium.7. AOAA also inhibited glutamate decarboxylase (GAD) in retinae incubated in vitro. This inhibitory effect was partially reversed by pyridoxal-5'-phosphate.8. Efflux of radioactivity from the retina was strikingly reduced in the presence of AOAA at concentrations sufficient to inhibit GABA-T by 100%.9. These findings suggest that AOAA potentiates the accumulation of (3)H-GABA by isolated retina, not by increasing the exchange of (3)H-GABA with the endogenous GABA pools, but by reducing the metabolism of the amino acid and hence reducing the loss of radioactivity from the tissue in the form of tritiated metabolites.