Barbara Petrack

CGS 8216: Receptor binding characteristics of a potent benzodiazepine antagonist

CGS 8216 is a novel nonbenzodiazepine that inhibited 3H-flunitrazepam (3H-FLU) binding to rat synaptosomal membranes in vitro at subnanomolar concentrations. It prevented the in vivo labeling of brain benzodiazepine receptors by 3H-FLU with the same potency as diazepam when given orally to mice. Pharmacologic tests showed that it was devoid of benzodiazepine-like activity but it antagonized the actions of diazepam in these tests. It did not interact with alpha- or beta- adrenergic, H1-histaminergic or GABA receptors but it inhibited adenosine-activation of cyclic AMP formation. Studies with 3H-CGS 8216 demonstrated that it bound specifically and with high affinity to rat forebrain membranes and was displaced by drugs with an order of potencies similar to that observed when 3H-diazepam and 3H-FLU were used as radioligands. The regional distribution of 3H-CGS 8216 binding sites in the brain was also similar to that of 3H-FLU. Dissociation of 3H-CGS 8216 binding was slow at 0 degrees C but increased with temperature and was almost complete within 1 min at 37 degrees C. Scatchard analyses were linear, yielding KD values of 0.044, 0.11 and 0.18 nM at 0, 25 and 37 degrees C, respectively; the Bmax value did not change appreciably with temperature and was approximately 1000 fmoles/mg protein. Using 3H-FLU, the value for Bmax as well as for the KD increased with temperature. The total number of binding sites determined for 3H-FLU was greater than that for 3H-CGS 8216 at each temperature. CGS 8216 exhibited mixed-type inhibition of 3H-FLU binding. GABA did not stimulate 3H-CGS 8216 binding whereas it enhanced 3H-FLU binding. CGS 8216 may be a useful ligand for probing the antagonist properties of the benzodiazepine receptor and is likely to exhibit interesting therapeutic effects.

Characterization of central cholecystokinin receptors using a radioiodinated octapeptide probe

We have developed a binding assay for 125I-Bolton-Hunter-labeled cholecystokinin octapeptide (125I-(BH)CCK8) using mouse cerebral cortex membrane preparations. This ligand interacts with cortical membrane preparations in a saturable, high-affinity manner, satisfying the requirements for specific cholecystokinin receptor labeling. Salt is required for maximal binding and BSA is specifically inhibitory with cerebral cortical but not with pancreatic sites. Cholecystokinin peptides as small as CCK30-33 displace binding at low nanomolar concentrations. Dissociation of 125I-(BH)CCK8 is biphasic in both mouse and guinea pig cortex. Pretreatment of membranes at 37 degrees C results in a marked loss of recognition sites, suggesting that the sites may be rapidly metabolized in vivo. After 37 degrees C pretreatment, the loss of CCK recognition sites corresponds to a selective loss of the slow component of dissociation curves. This selective elimination of one dissociation population, as well as the biphasic dissociation kinetics, suggests that at least two distinct CCK receptor subtypes exist in the brain.

Regulation of Central Cholecystokinin Recognition Sites by Guanyl Nucleotides

Guanyl nucleotides affected the binding of radio‐labeled cholecystokinin (CCK) octapeptide to rodent cortical binding sites. Micromolar quantities of a stable GTP analogue, guanylyl‐imidodiphosphate (GppNp), resulted in a plateau where binding was decreased by 30%. In the presence of 25 μM GppNp, binding analysis revealed a decrease in affinity (increase in KD), without an apparent effect on the maximal number of binding sites. Ki values for CCK‐related peptides shifted up to 1.6‐fold. The rate of peptide association decreased by threefold, and the rapid component of peptide dissociation increased. The collective data suggest that a class of central CCK binding sites is linked to nucleotide regulatory proteins. The evidence is discussed with regard to multiple receptor populations and to possible interconversions between receptor types.

Potency of CR 1409, a new proglumide analog, on cholecystokinin-mediated behaviors and receptor binding.

Behavioral and receptor binding techniques were employed to evaluate the potency of CR 1409, a new analog of proglumide, as a cholecystokinin antagonist. CR 1409, at doses of 1 mg/kg i.p. in mice, effectively blocked the inhibition of feeding and exploratory behaviors induced by cholecystokinin. In rats, CR 1409 alone, at doses of 1 and 10 mg/kg, did not affect feeding or exploratory behaviors, but at 25 mg/kg alone, CR 1409 reduced food intake and exploratory behaviors, suggesting a mixed agonist-antagonist profile. On these several behavorial parameters, CR 1409 antagonized peripherally administered cholecystokinin with 10-1000 times greater potency than that reported for proglumide (Crawley et al., J. Pharmac. Exp. Ther.236, 320-330, 1986). In binding to pancreatic cholecystokinin membranes, CR 1409 was more than 100,000-times more potent than that reported for proglumide (Rovati, Scand. J. Gastroenterol.11, 113-118, 1976). CR 1409 inhabited binding of 125-I-cholecystokinin octapeptide in mouse parcreatic and brain membranes with IC(50) values of 13.7 nM and 2.6 ?M, respectively, demonstrating its selectivity for peripheral-type CCK receptors.

Potentiation of arginine-induced glucagon secretion by adenosine

Abstract Adenosine and the synthetic adenosine agonists 2-chloroadenosine and N 6 -(L-2-phenylisopropyl)-adenosine were tested for effects on hormone secretion from the rat isolated perfused pancreas. These nucleosides, at concentrations of 5 μM, markedly potentiated both phases of arginine-induced glucagon release; the two synthetic agonists were more effective than adenosine. In the absence of arginine, each of the nucleosides induced a transient burst of glucagon. In contrast, adenosine and both synthetic agonists inhibited arginine-induced insulin secretion to varying degrees and caused only negligible insulin release when perfused without arginine. The adenosine antagonist 8-( p -sulfophenyl)-theophylline prevented the actions of adenosine on hormone release from the pancreas. Our data suggest that adenosine potentiation of arginine-induced glucagon release may be mediated via adenosine receptors on alpha cell membranes; such a mechanism could provide an important endogenous control over glucagon secretion.

Rapid isolation of total RNA from mammalian tissues

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.

Increases in rat striatal preproenkephalin mRNA levels following chronic treatment with the depot neuroleptic, haloperidol decanoate

Studies of the effects of chronic neuroleptic drug treatment have consistently demonstrated enhanced transcription and translation of the preproenkephalin gene in the rat striatum. However, all of these studies have used daily ip drug treatments and none have evaluated the effects of chronic depot neuroleptics. With these drug treatments, dopamine receptor blockade undergoes less variability as a result of sustained steady-state blood levels of the neuroleptic. Therefore, as a result of the increasing utilization of depot neuroleptics therapeutically, we examined the effects of haloperidol decanoate on striatal preproenkephalin mRNA levels. As with daily ip drug injections, the depot preparation was found to increase the levels of this mRNA to an apparent new steady-state level twice that of controls, by 3 days and sustaining this steady-state for the 14 day observation period. These data indicate that both continuous and fluctuating patterns of dopamine receptor blockade result in activation of the preproenkephalin gene.

Diazepam: Invitro effects on glucagon and insulin release

Diazepam suppressed arginine-induced glucagon release from the isolated perfused rat pancreas in a dose-dependent manner, with an IC50 of approximately 65 microM. In contrast, insulin release was enhanced by 10-50 microM diazepam, but inhibited by higher concentrations of drug. Thus, 50 microM diazepam simultaneously suppressed glucagon and increased insulin release in this model. The potentiation of insulin release may result from phosphodiesterase inhibition. The inhibitory effects on hormone release are discussed in terms of diazepams molecular conformation, which is similar to that of diphenylhydantoin, an inhibitor of both glucagon and insulin release in the isolated perfused rat pancreas. The possibility is also considered that the conformation of both compounds might be similar to the apparent active site of the hormone release inhibitor somatostatin.

Chapter 1. Anti-Anxiety Agents and Sedative-Hypnotics

Publisher Summary In this chapter, the pharmacological actions of different anti-anxiety agents and sedative-aypnotics have been discussed with current references. Current evidence indicates that most anxiolytic and sedative-hypnotic drugs exert their pharmacological actions by binding to discrete neuronal recognition sites, consisting of benzodiazepine (BZ) “receptors”, gamma-aminobutyric acid (GABA) receptors, and chloride ion channels. Binding of benzodiazepines (BZs) to the molecular complex increases the efficiency of GABA in opening chloride channels. Drugs acting at each component of the complex are known and have been used to label their respective recognition sites with either agonists or antagonists. The possibility of BZ receptor heterogeneity continues to be explored because it provides a mechanism for finding selective drugs. The ability of muscimol to enhance the binding affinities of ligands for the BZ receptor, rather than the affinities per se, correlates with their hypnotic activity. Ligands for BZ receptors can be characterized as agonists, antagonists, or inverse agonists on the basis of their behavior in three in vitro binding assays. Blockade of pentylenetetrazole (PTZ) interoceptive discriminative stimulus (IDS) as a model for anxiolytic activity has been discussed in the chapter and found to be reliable. The pharmacology of zopiclone, an anxiolytic-hypnotic BZ receptor ligand, has been discussed in this chapter. The pharmacological profile of suriclone is that of a typical agonist, but its binding characteristics differ suggesting that this compound might bind to a novel site linked allosterically to the BZ receptor. Sedatives like loprazolam and flurazepam have been equieffective in patients with disturbed sleep pattern. The relationship between the effects of muscimol on BZ receptor binding and the hypnotic activity of nine BZs has beenexplained in the chapter. BZs, whose receptor bindings are strongly modulated by muscimol, possess potent hypnotic activity, suggesting that the BZ-GABA receptor complex is involved in the hypnotic activity of the BZ drugs.

COMPARATIVE STUDIES ON BOVINE AND GUINEA PIG TYROSINE HYDROXYLASE

Publisher Summary This chapter compares some of the characteristics of bovine and guinea pig tyrosine hydroxylase as determined in experiments with 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine (DMPH4) and tetrahydrobiopterin (biopterin-H4) as cofactors. Native and trypsinised bovine adrenal medulla tyrosine hydroxylases were prepared. Tyrosine hydroxylase was partially purified from the supernatant of homogenized guinea pig adrenals by ammonium sulfate fractionation and Sephadex-G-200 chromatography, followed by a second ammonium sulfate step. The enzyme was partially purified from the supernatant of homogenized guinea pig brains by two fractionation steps with ammonium sulfate. L-Erythro-biopterin was reduced and was quantitated by spectrophotometry measurements. It was found that the activity of bovine adrenal medulla tyrosine hydroxylase, assayed at 38°C, was only slightly greater than at 25°C. This was observed with both native and trypsinised bovine adrenal enzyme, even when the latter was assayed after activation by preincubation with FeSO4 and mercaptoethanol. In contrast, guinea pig adrenal tyrosine hydroxylase is approximately fourfold more active at 38°C than it is at 25°C. This observation suggests an intrinsic difference between the enzymes from the two species.