Godfrey Tunnicliff

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.

The Teaching of Complementary and Alternative Medicine in U.S. Medical Schools: A Survey of Course Directors.

Purpose The number of U.S. medical schools offering courses in complementary and alternative medicine (CAM) has risen sharply in recent years. This study gauged the current state of CAM instruction by gathering details about the specific topics being taught and the objectives behind the instruction. Method Data were collected from questionnaires mailed to 123 CAM course directors at 74 U.S. medical schools. Results Questionnaires were returned by 73 course directors at 53 schools. The topics most often being taught were acupuncture (76.7%), herbs and botanicals (69.9%), meditation and relaxation (65.8%), spirituality/faith/prayer (64.4%), chiropractic (60.3%), homeopathy (57.5%), and nutrition and diets (50.7%). The amounts of instructional time devoted to individual CAM topics varied widely, but most received about two contact hours. The “typical” CAM course was sponsored by a clinical department as an elective, was most likely to be taught in the first or fourth year of medical school, and had fewer than 20 contact hours of instruction. Most of the courses (78.1%) were taught by individuals identified as being CAM practitioners or prescribes of CAM therapies. Few of the courses (17.8%) emphasized a scientific approach to the evaluation of CAM effectiveness. Conclusion A wide variety of topics are being taught in U.S. medical schools under the umbrella of CAM. For the most part, the instruction appears to be founded on the assumption that unconventional therapies are effective, but little scientific evidence is offered. This approach is questionable, especially since mainstream medicine owes much of its success to a foundation of established scientific principles.

Significance of γ-hydroxybutyric acid in the brain

1. 1. Administration of the endogenous compound γ-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. 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. 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. 4. Brain contains a Na+-dependent GHB uptake system which exhibits two components, one with a Km of 46 μM and the other with a Km of 325 μM. 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 μM. Binding to both sites is inhibited in the presence of NCS-382, a GHB receptor antagonist. 5. 5. GHB might play a role as a neurotransmitter, particularly being involved in influencing dopamine release in the substantia nigra.

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.

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.

Submissive behavior in mice as a test for antidepressant drug activity

Previously, with the administration of antidepressant drugs, it has been demonstrated that the rat model of clinical depression, known as the reduction of submissive behavior model (RSBM), has considerable validity. The present study is an attempt to extend the model to mice. Several antidepressant drugs as well as a number of non-antidepressant agents were administered to mice that had been identified as submissive in a behavioral testing situation. Imipramine, desipramine, amoxapine and fluoxetine, representing three different classes of antidepressant drugs, were each able to increase competitive behavior in submissive mice and to decrease the dominance level between dominant and submissive mice in the behavioral tests. The stimulant amphetamine also reduced submissive behavior while yohimbine (also a stimulant), and the antianxiety agent diazepam had no such effect. The neuroleptic drug thiothixen had antidepressant-like effect on submissive C57BL/6J mice behavior. We conclude that like the rat model of depression from which it was developed, the mouse model responds to various antidepressants as predicted and thus may serve as a potential model of clinical depression.

Competitive Inhibition of γ‐Aminobutyric Acid Receptor Binding by N‐2‐Hydroxyethylpiperazine‐N′‐2‐ Ethanesulfonic Acid and Related Buffers

Abstract: Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high‐affinity and low‐affinity [3H]γ‐aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high‐ and low‐affinity binding sites, respectively. HEPES had Ki, values of 2.25 MM and 115 μM. The buffers had no appreciable effect on sodium‐dependent GABA binding or on γ‐aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high‐ or low‐affinity [3H]muscimol binding. Indeed, they were of the order of 105 times less effective in this case than against [3h]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.

Regulation of γ-aminobutyric acid synthesis in the vertebrate nervous system

The regulation of glutamic decarboxylase (GAD) activity is undoubtedly the key to the control of the steady-state concentrations of 4-aminobutyric acid (GABA) in the central nervous system. Those factors that might influence GAD activity are reviewed. They include repression and induction of GAD synthesis; the interconversion of the holo- and apo-form of GAD; the availability of substrate and cofactor; the competitive inhibition of GAD by endogenous substances, including GABA; and the involvement of calcium ions in whole-cell preparations. Where possible mechanisms of action are described, and the likelihood that each is of physiological importance is discussed. Experiments are suggested that would help clarify (1) the role of GABA in GAD repression; (2) the possible phosphorylation of GAD; and (3) the existence of multiple forms of the enzyme. In addition, a kinetic mechanism is proposed to explain the possible regulation of GAD by the interconversion of the holo- and apo-forms of the enzyme. It is concluded that the overriding factors responsible for GAD regulation are not yet understood. However, a possible mechanism relying on the direct feedback action of GABA on GAD activity has many attractive features.

The GABAA 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 GABAA receptor complex. At concentrations above 10 μM fluoxetine inhibited the binding of both [3H]GABA (IC50 = 2 mM) and [3H]flunitrazepam (IC50 = 132 μM ) to the GABAA 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 μM 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.

Kinetics of rat brain soluble catechol-O-methyltransferase and its inhibition by substrate analogues

1. The initial rates and inhibition of rat brain catechol-O-methyltransferase were studied. Double reciprocal plots of initial rates versus either S-adenosyl-L-methionine or 3,4-dihydroxybenzoic acid, in the absence of product, gave a series of lines intersecting to the left of the ordinate. 2. Inhibition in the presence of S-adenosyl-L-homocysteine was competitive but in the presence of vanillic acid was non-competitive if S-adenosyl-L-methionine was the varied substrate. 3. When 3,4-dihydroxybenzoic acid was the varied substrate, both S-adenosyl-L-homocysteine and vanillic acid gave rise to a non-competitive inhibition. 4. The initial rate and product inhibition patterns were consistent with an ordered BiBi mechanism with S-adenosyl-L-methionine being the first substrate and 3,4-dihydroxybenzoic acid the second substrate to combine with the enzyme. 5. In addition, these results suggest that vanillic acid is the first product and S-adenosyl-L-homocysteine the second product to dissociate from the enzyme. 6. The substrate analogues salsolinol and 3-carboxysalsolinol were competitive inhibitors with respect to 3,4-dihydroxybenzoic acid but were non-competitive with respect to S-adenosyl-L-methionine. For enzymes with an ordered mechanism an uncompetitive inhibition would be expected. 7. A possible explanation is that both substrate analogues can combine with either free enzyme with lower affinity or with an intermediary enzyme form with much greater affinity. 8. A scheme which is consistent with the data is presented.