David R. Burt

Dopamine receptor binding: Differentiation of agonist and antagonist states with 3H-dopamine and 3H-haloperidol

Abstract 3H-Dopamine and 3H-haloperidol bind with high affinity and selectivity to synaptic dopamine receptors in membrane preparations of the calf caudate. Binding of both ligands shows marked regional variations with greatest density in caudate, putamen, globus pallidus, nucleus accumbens and olfactory tubercle, areas rich in dopamine nerve terminals. The rank-order of phenothiazines and related agents as well as catecholamines in displacing both dopamine and haloperidol binding closely parallels their pharmacological potencies and affinities for the dopamine-sensitive adenylate cyclase. Dopamines affinity for specific 3H-dopamine binding sites is 100 times its apparent affinity for the dopamine sensitive adenylate cyclase. Agonists have about 50 times more affinity for dopamine than haloperidol sites, whereas antagonists display about 100 times greater affinity for haloperidol than dopamine sites.

Ethanol sensitivity of the GABAA receptor expressed in xenopus oocytes requires 8 amino acids contained in the γ2L subunit

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Thyrotropin releasing hormone (TRH): Apparent receptor binding in rat brain membranes

Thyrotropin releasing hormone (TRH) binds to membranes of rat brain tissue via high- and low-affinity binding components. The high-affinity binding of TRH to brain membranes resembles binding to pituitary membranes in terms of its affinity and specificity for most peptides. In equilibrium studies, the affinity and specificity for most peptides. In equilibrium studies, the dissociation constant for high-affinity binding to brain membranes is about 50 dissociation constant for high-affinity binding to brain membranes is about 50nM, which is about the same as for aat pituitary membranes, while low-affinity binding to brain membranes has a dissociation constant of about 5 muM. Liver membranes display low-affinity binding for TRH with a dissociation constant similar to the low-affinity binding component of brain membranes. No high-affinity binding can be detected with liver membranes. Negligible saturable binding to TRH can be detected with membranes of any tissues examined other than liver, pituitary and brain...

Generation of two forms of the gamma-aminobutyric acidA receptor gamma 2-subunit in mice by alternative splicing.

Abstract: γ‐Aminobutyric acidA (GABAA) receptors are multisubunit ligand‐gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (α, β, γ, and δ). The γ2‐subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine γ2‐subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids, LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the γ2‐subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight‐amino‐acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.

The dopamine receptor: differential binding of d-LSD and related agents to agonist and antagonist states.

Dopamine receptor binding is calf striatal membranes of 3H-dopamine and 3H-haloperidol appears to differentiate agonist and antagonist states of the receptor. Agonists and antagonists have selective affinities for dopamine and haloperidol sites respectively. In evaluating relative affinities for dopamine and haloperidol binding sites, we have observed that d-LSD interacts with considerable affinity at the dopamine receptor. Its similar competition petition for binding of the two tritiated ligands suggests that it is a mixed agonist-antagonist, which is consistent with its interactions with the dopamine-sensitive adenylate cyclase. The effects of LSD on dopamine receptor binding are stereospecific, with d-LSD being 1,000 times more potent than d-LSD. 2-Bromo-LSD has more of an antagonist profile than d-LSD for the dopamine receptor. In binding experiments methiothepin behaves like a potent and relatively pure antagonist at dopamine receptors.

Species differences in the brain regional distribution of receptor binding for thyrotropin-releasing hormone.

Abstract: A survey of the regional distribution of binding of 1 nM [3H](3‐MeHis2)thyrotropin‐releasing hormone ([3H]MeTRH) to TRH receptors in the brains of eight mammalian species revealed major species differences in both the absolute and relative values of TRH receptor binding in different brain regions. Several brain regions exhibited binding equal to or exceeding that in the anterior pituitary gland of the same species, including the amygdaia in the guinea pig and rat, the hypothalamus in the guinea pig, the nucleus accumbens in the rabbit, and all these and other regions in the cat and dog, for which pituitary binding was exceptionally low. Species could be divided into two groups according to which brain region appeared highest in binding: rabbits, sheep, and cattle had highest binding in the nucleus accumbens/septal area, whereas guinea pigs, rats, dogs, cats, and pigs had highest binding in the amygdala/temporal cortex area. The nucleus accumbens consistently exceeded the caudate‐putamen in receptor binding. For most brain regions, rabbits, rodents, and sheep tended to be higher than carnivores, cattle, or pigs. Further regions that exhibited appreciable binding in most species included the olfactory bulb and tubercle, hippocampus, and various cortical and brain stem areas. In fact, essentially all brain regions appeared to have detectable levels of TRH receptors in at least some species, but no rat peripheral tissues have yet shown detectable receptor binding. The species differences appeared to reflect largely if not entirely differences in receptor density, although this was not tested in every species.

Biochemical Actions of Neuroleptic Drugs: Focus on the Dopamine Receptor

Who cares how antipsychotic drugs act? What difference does it make whether the clinical effects of neuroleptics derive from one or another biochemical influence? Such questions are rarely asked by pharmacologists because they have taken as their vocation the study of how all drugs act. If it is a drug, the most sacred obligation of the pharmacologist is to find out how it does whatever it does. However, for drugs such as the neuroleptics, which have been the subject of massive amounts of biochemical investigation, one must seriously ask how one can justify such an expenditure of intellectual and financial resources. The obvious answer is that these are the drugs which alleviate the symptoms of schizophrenia, so that if we know how they act, we would know something about the aberrations which account for schizophrenic symptoms. Such an assertion involves certain assumptions. The most crucial assumption is that the drugs are in fact antischizophrenic. One can readily conceive of a drug that would make life more livable for schizophrenics and for the staff of mental hospitals without doing anything fundamental to the schizophrenic process. For many years sedatives, straitjackets, and wet sheets have “eased” the predicaments of schizophrenics and their keepers, but few people would argue that these treatments did anything fundamental to schizophrenic mechanisms.

Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.

The agonistic action of pentobarbital on GABAA β-subunit homomeric receptors

Murine γ-aminobutyric acid type A (GABAA) receptor β1, β2, and β3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three β-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in β2 and β3 homomers. The pentobarbital-induced currents in β3 homomers showed a dose dependence with an ED50 of 89 ± 8.9 μM and a Hill coefficient of 0.94 ± 0.08. Zinc (50 μM) blocked (61.1 ± 5.6% of control) and 200 μM lanthanum potentiated (139 ± 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an α-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a β2 or β3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous β-subunits suggests that the β-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.

Etomidate potentiation of GABAA receptor gated current depends on the subunit composition

The role of the gamma 2 subunit in etomidate potentiation of GABAA receptor-gated chloride current was studied by whole cell patch clamp experiments on H293 cells expressing GABAA receptors. The GABAA receptor subunits alpha 1 beta 1 with or without the gamma 2 subunit expressed well, with an overall peak current of 157 +/- 42 pA/pF. At a clinically relevant concentration, etomidate potentiates the peak current induced by GABA equally well in receptors with or without the gamma 2 subunit. In contrast, the time course of current decay was prolonged only in receptors with the gamma 2 subunit. This gamma 2 subunit-dependent prolongation of the current time course was not blocked by the benzodiazepine receptor antagonist flumazenil. These results show that etomidate, an imidazole general anesthetic, interacts with the GABAA receptor in a gamma 2 subunit-dependent manner.