Central GABAergic systems and depressive illness.

Clinical depression and other mood disorders are relatively common mental illnesses but therapy for a substantial number of patients is unsatisfactory. For many years clinicians and neuroscientists believed that the evidence pointed toward alterations in brain monoamine function as the underlying cause of depression. This point of view is still valid. Indeed, much of current drug therapy appears to be targeted at central monoamine function. Other results, though, indicate that GABAergic mechanisms also might play a role in depression. Such indications stem from both direct and indirect evidence. Direct evidence has been gathered in the clinic from brain scans or postmortem brain samples, and cerebrospinal fluid (CSF) and serum analysis in depressed patients. Indirect evidence comes from interaction of antidepressant drugs with GABAergic system as assessed by in vivo and in vitro studies in animals. Most of the data from direct and indirect studies are consistent with GABA involvement in depression.

Kinetics of inactivation of glutamate decarboxylase by cysteine-specific reagents.

Mercuric chloride, p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) irreversibly inhibited the activity of Escherichia coli glutamate decarboxylase. Their second order rate constants for inactivation are 0.463 microM(-1) min(-1), 0.034 microM(-1) min(-1), 0.018 microM(-1) min(-1), respectively. The characteristics of the inhibition by the three thiol-group reagents supports the idea that cysteinyl residues at the binding sites for the cofactor and/or the substrate are important for enzyme activity in E. coli.

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex.

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.

Actions of sodium valproate on the central nervous system.

The branched chain fatty acid, valproate, has a number of distinct pharmacological effects on the central nervous system. In experimental animals it showed clear anticonvulsant activity, an observation which led to its major clinical use as an antiepileptic agent, especially in petit mal seizures. More recently, valproate has shown its usefulness in treating mood disorders and migraine headaches. The basis for its clinical efficacy might be related to its ability to enhance central GABAergic neurotransmission or perhaps to its inhibition of Na+ channels. Whether each of the distinct therapeutic effects of valproate has the same molecular basis is not known.

The GABA(A) receptor complex as a target for fluoxetine action.

The clinically important antidepressant fluoxetine is established as a selective serotonin reuptake inhibitor. This study demonstrates that fluoxetine also interacts with the GABA(A) receptor complex. At concentrations above 10 microM fluoxetine inhibited the binding of both [3H]GABA (IC50 = 2 mM) and [3H]flunitrazepam (IC50 = 132 microM) to the GABA(A) receptor complex in brain cortical membranes. Low fluoxetine concentrations (1 nM) enhanced GABA-stimulated Cl- uptake by a rat cerebral cortical vesicular preparation. At higher concentrations (100 microM and 1 mM), however, fluoxetine inhibited GABA-stimulated Cl- uptake, an effect related to a reduction in Emax. These observations might assist in an explanation of the basis of the antidepressant action of fluoxetine.

Uptake of [(3)H]glycine by synaptosomes of channel.

It is known that channel catfish erythrocytes can take up glycine by several distinct transport systems. Further, glycine is an inhibitory neurotransmitter in mammalian brain and spinal cord. Consequently, the uptake of [(3)H]glycine by catfish brain was investigated and found to be a saturable process, dependent on the presence of Na(++) and Cl(--) and sensitive to temperature. A kinetic analysis of transport was performed at 22C. This showed that a high-affinity system existed which exhibited a K(m) of 5.1 (+/- 2. 1) microM. Several structural analogues of glycine were capable of inhibiting uptake in a competitive manner. The most effective inhibitor was sarcosine (IC(50) 5 36 microM). Uptake was also able to be inhibited by harmaline, a drug known to interfere with Na(+)-dependent transport processes. It is concluded that glycine transport by channel catfish brain has much in common with transport by mammalian nervous tissue which is carried out by the membrane carriers GLYT1 and GLYT2. On the other hand, synaptosomal transport differs somewhat from glycine transport by channel catfish erythrocytes.

Pharmacology and function of imidazole 4-acetic acid in brain.

1. Imidazole 4-acetic acid (IMA) is a naturally occurring metabolite in brain, although it is unclear what biochemical pathways are involved in its biosynthesis and breakdown. Some evidence, however, suggests that IMA is an oxidation product of histamine. 2. The compound has pronounced neuropharmacological properties, many of which are consistent with an activation of GABA(A) receptors. Indeed, IMA is able to displace [3H]GABA from GABA(A) sites in a potent manner. 3. IMA displays definite partial agonist characteristics as an enhancer of benzodiazepine binding to the GABA(A) receptor complex in membrane preparations. In addition, it has an affinity for GABA(C) receptors, where it seems to act as an antagonist, and perhaps as a weak partial agonist. A third recognition site for IMA in brain is the I1-imidazoline receptor. 4. Parenteral administration to experimental animals leads to a sleep-like state which can often be accompanied by seizures. In addition, central application of IMA has been associated with a dose-related reduction in arterial pressure and sympathetic nervous discharge. 5. No specific receptor site or uptake system for IMA has yet been discovered, adding uncertainty to its role in central nervous system function. Yet the possibility cannot be overlooked that IMA plays a role in regulating blood pressure.

Chemical inactivation of bacterial GABA aminotransferase.

The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme. The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner. Inhibition kinetics were consistent with a simple bimolecular reaction. The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1. In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site. p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme. 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA). The inhibitor constant (Ki) was 0.83 +/- 0.44 mM. We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.

Inhibitors of synaptosomal gamma-hydroxybutyrate transport.

Synaptosomes prepared from mouse brain possess a Na+-dependent transport system for gamma-hydroxybutyrate displaying saturation kinetics, the transport constant (Kt) for which was calculated as 31 +/- 9 micromol/l. Several gamma-hydroxybutyrate and gamma-aminobutyric acid (GABA) structural analogues were tested as potential inhibitors of gamma-hydroxybutyrate transport. The most effective inhibitor was harmaline (Ki = 94 +/- 21 micromol/l), a known competitive inhibitor of Na+ binding to certain transport proteins. 2-Hydroxycinnamic acid, 3-(2-furyl)acrylic acid and citrazinic acid also inhibited transport and were competitive with respect to gamma-hydroxybutyrate. The least effective gamma-hydroxybutyrate analogues were 3-hydroxypropane sulfonic acid (Ki = 4.1 +/- 0.8 mmol/l) 3,5-dihydroxybenzoic acid (Ki = 6.1 +/- 2. 8 mmol/l) and 3-hydroxybenzoic acid (Ki = 6.9 +/- 3.3 mmol/l), although 2-hydroxypropane sulfonic acid and kynurenic acid had no measurable effects. Four inhibitors of GABA transport - nipecotic acid, guvacine, ketamine and beta-alanine and GABA itself, were without effect on gamma-hydroxybutyrate transport. These results show that certain drugs that structurally resemble gamma-hydroxybutyrate have the capacity to compete with gamma-hydroxybutyrate at its recognition site on the transporter. By examining the structure of such inhibitors, we can learn more about the properties of the substrate binding site on the carrier protein. Moreover, the absence of inhibition by GABA uptake inhibitors shows that gamma-hydroxybutyrate transport is a separate entity from GABA transport.

Sites of action of gamma-hydroxybutyrate (GHB)--a neuroactive drug with abuse potential.

OBJECTIVE: This review highlights the biochemistry, pharmacology, and toxicology of the naturally-occurring fatty acid derivative, gamma-hydroxybutyrate (GHB). GHB is derived from gamma-aminobutyric acid (GABA) and is proposed to function as an inhibitory chemical transmitter in the central nervous system. CONTENT: When administered in pharmacological doses, its powerful central nervous system depressant effects are readily observed. Although some of the neurophysiological actions of GHB could involve alterations in dopaminergic transmission in the basal ganglia, both its physiological and pharmacological actions are probably mediated through specific brain receptors for GHB. In addition, GHB might mediate some of its effects through interaction with the GABA(B) receptor. Experimentally, GHB has been used as a model for petit mal epilepsy; clinically, it has been used as a general anesthetic and as a drug to treat certain sleep disorders and related conditions. Owing to the purported ability of GHB to induce a state of euphoria, recreational use of this substance is popular. Although no deaths or long-term problems have been associated with GHB abuse, symptoms of GHB intoxication can be severe. The continued potential for GHB abuse makes it imperative for clinical toxicologists to be aware of the effects of this agent. Future research on the mechanism of action of GHB is needed to elucidate both its central nervous system depressant properties and its ability to effect a state of well-being.

Basis of the antiseizure action of phenytoin.

1. Phenytoin has been used with much clinical success against all types of epileptiform seizures, except petit mal epilepsy, for over 50 years. Its mechanism of action, however, is still open to interpretation. 2. Several potential targets for phenytoin action have been identified within the central nervous system. These include the Na-K-ATPase, the GABAA receptor complex, ionotropic glutamate receptors, calcium channels and sigma binding sites. 3. To date, though, the best evidence hinges on the inhibition of voltage-sensitive Na+ channels in the plasma membrane of neurons undergoing seizure activity. Quieter nerve cells are far less affected. Moreover, the fact that phenytoin also has important cardiac antiarrhythymic effects and can inhibit Na+ influx into cardiac cells supports the idea that the primary target of phenytoin is, indeed, the Na+ channel.

Effects of repeated doses of azapirones on rat brain 5-HT1A receptors and plasma corticosterone levels.

Chronic buspirone or ipsapirone (3 mg/kg, twice daily) administration to rats for 10 days decreased the sensitivity of inhibition of single-unit activity of serotonergic dorsal raphe neurons to a challenge by each drug. The ED50 for buspirone was increased from 0.1 mg/kg to 1.8 mg/kg, and the ED50 for ipsapirone was increased from 0.7 mg/kg to 1.2 mg/kg. The binding properties (Kd and Bmax) of [3H]8-OH-DPAT to membranes of cerebral cortex and hippocampus were unaffected by chronic administration of either buspirone or ipsapirone. Chronic buspirone or ipsapirone administration increased the tolerance of the hypothalamic-pituitary-adrenal axis (HPAA) following a challenge by each drug. The ED50 for elevation of plasma corticosterone levels was increased from 4.0 mg/kg to 7.6 mg/kg for buspirone and 6.2 mg/kg to 8.0 mg/kg for ipsapirone. Chronic buspirone administration decreased the basal activity of the HPAA by 63%. Chronic buspirone administration did not alter the plasma corticosterone response of the HPAA to a 1-min episode of rotational stress. (Mg2+)-ATPase, (Na+ + K+)-ATPase, (Ca2+ + Mg2+)-ATPase and calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activities of erythrocyte plasma membrane were unaffected by either chronic or acute buspirone treatment, or by the addition of the drug to the in vitro assay systems.

Regulation of rabbit erythrocyte Ca(2+)-pump sensitivity to calmodulin in experimental hyperlipidemia.

Intracellular free calcium activity is in part determined by a calmodulin-regulated plasma membrane Ca(2+)-pump. Since changes in Ca2+ permeability have been implicated in atherosclerotic plaque formation, we initiated a lipid hyperalimentation protocol during which we measured various erythrocyte calcium flux parameters and early atheroma development. Adolescent New Zealand White rabbits were fed a diet with 0.5% cholesterol and 2.5% lard over a 3-month period. Plasma cholesterol and triacylglycerols increased on average 18.7- and 13.9-fold respectively, while erythrocyte membrane cholesterol content decreased 18% and total phospholipids by 54%. After 3 months of lipid hyperalimentation, 22% of the aortic arch was covered with large, early-stage, raised atheroma. Basal and calmodulin-activated (Ca2+ + Mg2+)-ATPase activities in erythrocyte membranes increased by 31% and 123%, respectively at 2 months, with a concomitant increase in calmodulin affinity (Km) from 15.6 to 4.2 nM. These differences were transient on account of changes in the control animals which exhibited a slowly developing sensitivity to calmodulin during maturation. Basal Ca2+ transport and passive Ca2+ permeability increased about 7-fold during the hyperlipidemic phase. This suggests that overt hyperlipidemia, leading to atherosclerotic plaque development, alters plasma membrane Ca2+ regulatory mechanisms including passive Ca2+ permeability. The changes in enzymatic function, membrane composition, and Ca2+ permeability seen in this red cell model system may be a reflection of early changes in cells that are directly involved in the development of atherosclerotic plaques.

Chemical modification of bacterial 4-aminobutyrate aminotransferase by phenylglyoxal.

4-Aminobutyrate aminotransferase (EC 2.6.1.19), obtained from Pseudomonas fluorescens, was irreversibly inhibited by phenylglyoxal, a reagent that specifically modifies arginyl residues. The inactivation appeared to be the result of a simple, bimolecular reaction since no evidence of a reversible complex between inhibitor and enzyme emerged. The second-order rate constant was 0.221 +/- 0.077 M-1 sec-1. The concentration of either substrate had no effect on the inhibition, but an increase in the concentration of pyridoxal 5'-phosphate reduced the rate of inactivation by phenylglyoxal. The data are consistent with the modification of amino acid residues at the cofactor binding site on the enzyme.

Stimulation of mouse brain gamma-hydroxybutyric acid transport by pyridoxal 5'-phosphate.

A synaptosomal preparation from mouse brain transported gamma-hydroxybutyric acid in a manner displaying saturation kinetics. A Kt of 48 +/- 11 microM was calculated. In the presence of pyridoxal 5'-phosphate, uptake was markedly enhanced. Stimulation of uptake was observed with increasing concentrations of pyridoxal 5'-phosphate, up to a maximum of about 3.5 mM. Increasing concentrations above this value led to a steady decrease in the stimulation of uptake, till at 7 mM pyridoxal 5'-phosphate and above there was a noticeable inhibition of uptake. The presence of increasing concentrations of gamma-hydroxybutyric acid gradually reduced the amount of stimulation, thus pyridoxal 5'-phosphate appeared to be producing its effect by reducing the Kt of the transport system rather than increasing its Vmax.

Amino acid transport by human erythrocyte membranes.

The human erythrocyte plasma membrane is permeable to several free amino acids usually present in the bloodstream. Seven distinct routes of entry have been described which represent both secondary active transport and facilitated diffusion (passive transport). Additionally, certain amino acids can enter the cell by simple diffusion, at least to a limited extent. The function of most of these transport systems is unclear, although it has been suggested that the cell can take up certain amino acids and carry them to various parts of the body. In the case of glutamine, cysteine, and glycine, however, it is believed that the biosynthesis of the tripeptide glutathione is the primary reason for their uptake into the cell. Much of the amino acid transport probably has no function in mature red cells, but might be a remnant of the immature cell's needs. This review discusses the various amino acid transport systems known to be present in the red cell plasma membrane.

Inhibition of 4-aminobutyrate aminotransferase from Pseudomonas fluorescens by ATP.

The enzyme 4-aminobutyrate aminotransferase (EC 2.6.1.19) isolated from Pseudomonas fluorescens was inhibited by the nucleotide ATP in an apparent competitive manner (Ki = 10.4 mM). This reversible effect was antagonized by the substrate GABA, whose apparent Km was increased from 0.6 mM to 2 mM in the presence of 20 mM ATP, suggesting that ATP interferes with GABA binding to the active site of the enzyme. The apparent Km with respect to the second substrate alpha-ketoglutarate was also increased, although to a lesser extent, whereas the cofactor pyridoxal 5'-phosphate was unable to influence the inhibition by ATP. The ATP structural analogues ADP, CTP and XTP were also able to inhibit the enzyme to a similar extent. These data indicate that GABA concentrations within the bacterial cell can be regulated by the action of ATP on 4-aminobutyrate aminotransferase. In addition, because the inhibition by ATP is similar to the inhibition of the enzyme from mammalian brain, the bacterial enzyme could provide a convenient source of the enzyme for studies of drug effects on brain GABA metabolism in vitro.

Significance of gamma-hydroxybutyric acid in the brain.

1. Administration of the endogenous compound gamma-hydroxybutyric acid (GHB) can induce a sleep-like state in experimental animals and, indeed, it has been used as a general anaesthetic in clinical medicine. 2. Although GHB appears to be a CNS depressant, there is evidence it possesses epileptiform activity resembling petit mal epilepsy. In the brain GHB is evidently derived from GABA, the final step being catalyzed by succinic semialdehyde reductase, a cytosolic NADP(+)-dependent enzyme. 3. Two different oxidoreductases, GHB dehydrogenase and hydroxyacid-ketoacid dehydrogenase, acting independently, are responsible for the reverse reaction when GHB is being metabolically inactivated. 4. Brain contains a Na(+)-dependent GHB uptake system which exhibits two components, one with a Km of 46 microM and the other with a Km of 325 microM. GHB also binds to receptor sites in brain homogenates and exhibits two distinct affinities. One binding site displays a Kd of 95 nM whereas the second site has a Kd of 16 microM. Binding to both sites is inhibited in the presence of NCS-382, a GHB receptor antagonist. 5. GHB might play a role as a neurotransmitter, particularly being involved in influencing dopamine release in the substantia nigra.

Influence of repeated treatment with buspirone on central 5-hydroxytryptamine and dopamine synthesis.

The anxiolytic agent buspirone was administered subcutaneously twice a day for 10 days to Sprague-Dawley rats, at a dose of 3 mg kg-1. Controls were given saline. On the eleventh day, the rats were given an injection of NSD-1015, an aromatic L-amino acid decarboxylase inhibitor, 30 min before decapitation. To another group of rats, only one injection of buspirone was given, followed 30 min later by NSD-1015. After a further 30 min the animals were decapitated. The brains were rapidly removed and the raphe nuclei, striatum, hippocampus and cerebellum were dissected out on to dry ice. With the use of HPLC, the four regions of the brain were assayed for 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, reflecting the synthesis of 5-HT and dopamine, respectively. In those rats which had received an acute dose of buspirone, the synthesis of 5-HT was substantially reduced in all four regions of the brain. However, in those rats which had received buspirone for 10 days, no such alterations in the synthesis of 5-HT were observed. The synthesis of dopamine was unchanged in any of the regions of the brain, after the acute dose of buspirone. After 10 days of treatment with buspirone, however, the synthesis of dopamine in the striatum was significantly reduced. These findings suggest that repeated treatment with buspirone reduces the synthesis of dopamine in the striatum but that the synthesis of 5-HT is unaffected.