Leonard P. Miller

STUDIES ON THE REGULATION OF GABA SYNTHESIS: SUBSTRATE-PROMOTED DISSOCIATION OF PYRIDOXAL-5′-PHOSPHATE FROM GAD

The association between glutamate decarboxylase (GAD) and its cofactor, pyridoxal‐5′‐phos‐phate (pyridoxal‐P), was studied using 20,0000 supernatant of rat brain. In this preparation GAD required added pyridoxal‐P to maintain a linear reaction rate beyond 5 min of incubation. Following exhaustive dialysis the enzyme was more than 83% saturated with cofactor indicating that the cofactor was tightly bound to the enzyme. When incubations were performed in the presence of glutamate and without added pyridoxal‐P there was a progressive inactivation of the enzyme which was dependent on the glutamate concentration. This lost activity was almost completely recovered by addition of pyridoxal‐P to the dialyzed glutamate‐inactivated enzyme. The results suggest that glutamate inactivates GAD by promoting the dissociation of pyridoxal‐P from the enzyme thereby producing inactive apoen‐zyme which can be reactivated by combining with available pyridoxal‐P. This interpretation is supported by the finding that progress curves for the reaction were accurately described over a 30 min incubation period and 10‐fold glutamate concentration range by an integrated rate equation which takes the glutamate‐promoted dissociation of cofactor into account. The progressive inactivation could not be attributed to denaturation of the enzyme, impurities in the substrate, effects of pH, depletion of substrate, protein concentration, sulfhydryl reagents or product inhibition. The results presented here also show that certain precautions must be adopted to accurately measure GAD activity in the absence of added pyridoxal‐P as has been widely done in studies of drug action. Specifically, measurements must be made at short times of incubation and low concentrations of glutamate to minimize the glutamate‐promoted inactivation of the enzyme.

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing ‐γ‐aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K+‐stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium‐dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (Km) was increased and maximal velocity (Vmax) was reduced. Sodium‐independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.

Lead, GABA, and seizures: Effects of subencephalopathic lead exposure on seizure sensitivity and GABAergic function

Abstract Seizures have been described as the endpoint of lead (Pb) intoxication in both humans and animals. Alterations in blood—brain barrier integrity may underlie some cases of Pb-induced seizures, but seizures can also occur in the absence of changes in blood—brain barrier. Exposure of rats to two levels of Pb below those producing overt encephalopathy sensitizes them to the behavioral effects of the convulsant agents picrotoxin, isoniazid, mercaptopropionic acid, and strychnine, but not pentylenetetrazol (PTZ). Pb exposure affects several aspects of regional GABAergic function: GABA-transaminase (GABA-T) and glutamic acid decarboxylase (GAD) activities, GABA levels, and apparent rate of GABA synthesis. The results indicated that Pb increased GAD activity, decreased GABA-T activity, and increased the apparent rate of GABA synthesis. However, these changes were not observed consistently in all regions studied. Pb exposure also inhibited the uptake and release (unstimulated and K-stimulated) of [ 14 C]GABA; this effect was observed in both Pb treatment groups and in all brain regions except the cerebellum. The results support an hypothesis of a neurochemical basis for Pb-induced seizures, involving inhibition of GABAergic neurotransmission.

EFFECTS OF AMINOOXYACETIC ACID AND l-GLUTAMIC ACID-γ-HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ‐aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2‐oxoglutarate aminotransferase (GABA‐T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA‐T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l‐glutamic acid‐hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.

Rat Pup Ultrasonic Isolation Calls and the Benzodiazepine Receptor

Since their first description by Anderson (1954), rodent ultrasonic vocalizations have been noted in three social contexts. Adult rats in aggressive encounters emit short (3–55 msec), high frequency (40–70 kHz) pulses when attacking and longer (300–500 msec), lower frequency (22–30 kHz) calls during submission (Sales, 1972). In addition, following ejaculation, male rats embark on a long (1–3 sec) 22 kHz “song” which is associated with the refractory period between ejaculations (Barfield and Geyer, 1972). Finally, in the young of a wide variety of myomorph rodents, ultrasonic calls have been described in association with social isolation (reviewed by Sales and Pye, 1974). These calls, variously denoted as “isolation calls” or “distress vocalizations,” are the focus of this chapter. We will briefly review some of the factors influencing these calls before describing several pharmacologic studies designed to test the hypothesis that these calls are mediated by the brain benzodiazepine receptor.

Chronicl-DOPA-pretreatment of rats: an electrophysiological and biochemical study in the basal ganglia

Treatment of rats with L-DOPA (251.25 mg as the methyl ester HCl/kg) plus benserazide (B, 50 mg/kg) (L-DOPA+B), twice daily (i.p.) for 5 days or 12 days resulted in the dopamine (DA) neurons of the substantia nigra pars compacta becoming subsensitive to the rate-depressing effects of D-amphetamine (i.v.) 16 to 24 h after the last chronic drug dose. In contrast, pretreated rats were significantly less sensitive than control rats to the rate depressant effects of apomorphine (i.v.) after 12, but not 5 days of L-DOPA+B-pretreatment. After 5, but not 12 days of L-DOPA+B-pretreatment, a significant increase in the number of spontaneously active DA neurons was noted in the substantia nigra pars compacta. Caudate tyrosine hydroxylase was examined and a significant increase in apparent Vmax was noted after 5 days of L-DOPA+B, with no apparent change being noted in Km for cofactor. At this time, no change was noted in caudate DA or HVA concentrations. Several distinct processes may be occurring in response to the L-DOPA+B-pretreatment: (1) the DA autoreceptors located on cell bodies in the substantia nigra have become subsensitive after 12 days of L-DOPA+B-pretreatment; (2) the subsensitivity to D-amphetamine seen after both chronic schedules is probably unrelated to the subsensitive DA autoreceptors and may depend upon homeostatic alterations in neurotransmitter systems other than those utilising DA; (3) the activation of tyrosine hydroxylase may be a reflection of the increase in the number of spontaneously active units.

The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0

The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 ?M to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K(m) values for the cofactor of 16 ?M (low K(m)) and 2.3 mM (high K(m)). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V(max) values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K(m) form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K(m) form with very little change in the overall V(max). A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.

The hydroxylase cofactor and catecholamine synthesis

Our concepts of the molecular mechanisms relating to the regulation of neurotransmitter synthesis in catecholamine neurons are in state of constant revision. Following the original description of tyrosine hydroxylase as the initial and rate-limiting enzyme in catecholamine biosynthesis, a rather simple regulatory system was proposed (Nagatsu et al. 1974). The finding that catecholamines were competitive inhibitors with regard to the reduced cofactor that served as an electron donor for the reaction suggested that the tyrosine hydroxylase reaction was controlled by simple end-product inhibition. In essence, as the concentration of neurotransmitters within a catecholaminergic neuron fell following neuronal firing, end-product inhibition would be relieved and synthesis of replacement transmitter would occur. Conversely, as neurotransmitter accumulated in the nerve terminal, synthesis of additional transmitter would be inhibited. This simple concept was attractive and certain pharmacological manipulations provided support for a physiological role for end-product inhibition.

A comparative analysis of the effect of actinomycin-d, α-amanitin and cordycepin on new RNA synthesis and N-acetyltransferase induction by isoproterenol in cultured rat pineals

Abstract 1. 1. Actinomycin- d (0.05 μg/ml) inhibited nuclear (n) and cytoplasmic (c) RNA synthesis by 70–80%. There was no effect, however, on isoproterenol mediated induction of N -acetyltransferase (NAT). 2. 2. Cordycepin inhibited (n) and (c) RNA synthesis by 1 and 63%, respectively, while NAT induction was inhibited by 23%. 3. 3. α-Amanitin inhibited (n) and (c) RNA synthesis by 47 and 45%, respectively, while NAT induction was inhibited by only 34%. 4. 4. These same three inhibitors were tested for their comparative effects on the synthesis of poly (A) rich messenger RNA and 3 H-leucine incorporation into TCA-precipitable protein. Results showed that inhibition of NAT induction correlated only with inhibition of total protein synthesis.

Ornithine dec arboxylase—apparent lack of involvement in the induction of n-acetyltransferase in rat pineal gland

Abstract The potential role ornithine decarboxylase in the induction of serotonin- N -acetyltransferase in the rat pineal gland has been studied. The injection of isoproterenol (5 mg/kg, s.c.) resulted in an apparent induction of serotonin- N -acetyltransferase within 1 hr with a maximal response at 4 hr. No change in ornithine decarboxylase was observed in the first hour following isoproterenol administration. Rat pineal glands in organ culture responded to addition of 2 μM isoproterenol with a similar induction of N -acetyltransferase. There was a small but significant decrease in ornithine decarboxylase in the cultured rat pineal glands in response to isoproterenol. Additional experiments indicated that isoproterenol also did not elevate ornithine decarboxylase in the superior cervical ganglia under conditions known to induce tyrosine hydroxylase in this organ. The results of this study are consistent with our previous finding that ribosomal RNA synthesis is unaffected during the induction process in the pineal gland.