During anesthetic-induced activation of hypothalamic pituitary adrenal axis, blood-borne steroids fail to contribute to the anesthetic effect.

Anesthetic doses of ethanol (100 mmol/kg p.o.), chloral hydrate (2 mmol/kg i.p.), and urethane (9 mmol/kg i.p.) induce sharp and sustained (6- to 10-fold) dose-dependent increase in rat brain pregnenolone and progesterone content. In contrast, other general anesthetics such as ketamine (0.7 mmol/kg i.p.) and pentobarbital (0.2 mmol/kg i.p.), and the sedative/hypnotic clonazepam (17 mumol/kg i.p.) decrease brain pregnenolone and progesterone content. The increase in brain pregnenolone and progesterone content fails to occur if ethanol, chloral hydrate, and urethane are administered to hypophysectomized-adrenalectomized rats suggesting that the increase of brain steroids requires the hypophysis and probably originates in peripheral tissues and not in brain. The administration to hypophysectomized rats of 5 IU/kg of ACTH produces a brain pregnenolone and progesterone accumulation by an extent comparable to that elicited by anesthetic doses of ethanol, chloral hydrate, or urethane in intact animals. However, the increase in brain pregnenolone and progesterone content induced by ACTH is devoid of anesthetic or sedative effects and does not appear to change central GABAergic tone. In fact, ACTH, unlike allopregnanolone and allodeoxicorticosterone, failed to delay the onset of isoniazid-induced seizures, to reduce the fear of novelty in the elevated plus maze test as inferred by the increase in the number of entries or the time spent in the open arm. Thus, the data suggest that blood-borne steroids cannot function as precursors of brain neurosteroid modulators acting on GABAA receptor.

Vasoactive intestinal polypeptide facilitates tyrosine hydroxylase induction by cholinergic agonists in bovine adrenal chromaffin cells.

The possibility that vasoactive intestinal polypeptide (VIP) may facilitate the nicotine-mediated induction of adrenal medullary tyrosine hydroxylase (TH) was investigated with primary cultures (5-7 days in vitro) of bovine adrenal chromaffin (BAC) cells. Exposure of BAC cells to 100 microM nicotine led to only a marginal increase in the amount of TH mRNA, TH protein, and TH activity. VIP, alone or in the presence of a phosphodiesterase inhibitor, produced a marked increase in TH mRNA, TH protein, and TH activity. Moreover, VIP together with nicotine, at concentrations that alone were devoid of effect, increased the amount of TH mRNA and TH activity. A synergistic effect of VIP and nicotine on cAMP accumulation in BAC cells was also apparent. The marginal effects of large doses of nicotine on both cAMP accumulation and TH induction were blocked completely by hexamethonium but were also partially inhibited by the VIP antagonist [p-chloro-D-Phe6,Leu17]-VIP. Nicotine may, therefore, stimulate the release of VIP from cultured BAC cells and VIP, in turn, by increasing cAMP, may synergize with nicotine to enhance TH gene expression.

Subcellular location and neuronal release of diazepam binding inhibitor.

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.

Studies of a brain polypeptide functioning as a putative endogenous ligand to benzodiazepine recognition sites in rats selectively bred for alcohol related behavior.

The brain content of Diazepam Binding Inhibitor (DBI), its cell location and that of its specific mRNA were studied immunohistochemically and by in situ hybridization. Various strains of rats were genetically selected for their alcohol tolerance and the above mentioned brain parameters were studied before and after chronic ethanol consumption. The DBI like immunoreactivity (DBI-LI) was found to be located in selected neuronal population and in non-neuronal cells. The DBI-mRNA was located in brain areas where DBI is abundant. It was immunochemically determined that the DBI content was increased in cerebellum and in hypothalamus of alcohol preferring rats after chronic ethanol consumption. DBI content was compared in the cerebellum of rats genetically selected for different alcohol sensitivity and it was significantly higher on the ethanol sensitive (ANT) rat strain.

Diazepam-binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain.

An endogenous polypeptide of rat brain has been identified that is capable of displacing 1,4-benzodiazepines and the esters of the 3-carboxylic acid derivatives of beta-carbolines from their specific synaptic binding sites. This polypeptide was termed diazepam-binding inhibitor (DBI). Previous studies have shown that DBI injected intraventricularly in rodents elicits "proconflict" responses and antagonizes the "anticonflict" action of benzodiazepines. An antiserum to this peptide, directed toward an immunodeterminant near its amino terminus, makes it possible to detect, measure, and study the neuronal location of this peptide in rat brain. In the rat cerebral cortex, DBI immunoreactivity is located in neurons that are not GABAergic (GABA, gamma-aminobutyric acid); in the cerebellum and hippocampus, however, it might be present also in GABAergic neurons.

On a brain polypeptide functioning as a putative effector for the recognition sites of benzodiazepine and beta-carboline derivatives.

Stimulation of benzodiazepine recognition sites by various ligands can elicit opposite types of responses such as proconvulsant and anticonvulsant or proconflict and anticonflict actions. The study of the pharmacological profile of various ligands makes it possible to distinguish three classes of compounds: (1) those that elicit anticonflict responses in the Vogel test, inhibit the convulsions due to an impairment of GABAergic transmission and increase the Bmax of the high affinity recognition site for GABA; (2) those that displace benzodiazepines from specific binding sites, facilitate convulsions due to impairment of GABAergic mechanisms, elicit proconflict responses in Vogel's test and inhibit the facilitation by benzodiazepines of GABA binding and (3) those that displace benzodiazepines and beta-carboline-derivatives from specific binding sites, antagonize the anticonflict, the proconflict, the anticonvulsant and the proconvulsant actions of the two preceding groups of substances and in very large doses elicit a small proconvulsant action. Examples of the latter are an imidazobenzodiazepine (RO 15-1788) and a pyrazolquinolinone derivative (CGS 8216). The nomenclature for these three classes of drugs should be kept flexible until the action of the endogenous ligand that functions as the physiological effector of the benzodiazepine/beta-carboline recognition site is known. A putative ligand for this site (DBI = diazepam binding inhibitor) has been isolated and purified to homogeneity. It includes 104 amino acid residues, the sequence of the last 45 amino acids has been determined. This compound elicits a proconflict action, displaces beta-carboline derivatives more than anxiolytic benzodiazepines, but its high molecular weight and relative low affinity for the binding sites in brain might suggest that it is a precursor, rather than the putative effector for benzodiazepine and/or beta-carboline recognition sites.

Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues.

A thermostable inhibition of ATP-protein phosphotransferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatography (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (Mr approximately or equal to 24,000) is specific for cAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985). The type II inhibitor (Mr approximately or equal to 15,000) competes for the enzyme with various substrate proteins (histone, alpha-casein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (cAMP- and cGMP-dependent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500-fold; this protein is thermostable, has acidic characteristics, and does not require Ca2+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.

Effect of thioridazine, clozapine and other antipsychotics on the kinetic state of tyrosine hydroxylase and on the turnover rate of dopamine in striatum and nucleus accumbens.

In rats, a single injection of antipsychotic drugs produced a transitory change in the kinetic state of tyrosine hydroxylase (TH) in striatum and nucleus accumbens. The affinity of TH for 2-amino-4-hydoxy-6,7-dimethyl-5,6,7,8-tetrahydropterine (DMPH4) and the Vmax with respect ot tyrosine were increased. The relative potencies of anti-psychotics to change the kinetics of TH in striatum and nucleus accumbens when injected into rats were measured in the presence of 0.4 mM DMPH4. The doses of methiothepin, pimozide and halopridol which increased the affinity for DMPH4 of striatal TH were lower than those required to produce a similar change in nucleus accumbens. In contrast, thioridazine and clozapine were more effective in nucleus accumbens than in striatum. Chlorpromazine was equally active in these two tissues. Haloperidol increased the turnover rate of dopamine in striatum with doses that are relatively smaller than those required in the nucleus accumbens. Clozapine was more active in increasing turnover rate of dopamine in nucleus accumbens; the activity of chlorpromazine in these two tissues was equal. These results suggest that antipsychotics with high incidence of extrapyramidal side effects affect the nigrostriatal dopaminergic pathway selectively.