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.

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.

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.

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.

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.

Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol.

Sterol 27-hydroxylase, the mitochondrial enzyme that catalyzes the first step in oxidation of the sterol side chain in hepatic bile acid synthesis, also catalyzes the synthesis of 27-hydroxycholesterol from cholesterol. We have developed a high performance liquid chromatography (HPLC) assay for this enzyme, using either endogenous or exogenous cholesterol as substrate and cholesterol oxidase to convert 27-hydroxycholesterol to 4-cholesten-27-hydroxy-3-one. The alpha,beta-unsaturated ketone product was separated by normal phase HPLC and quantitated via absorption at 240 nm. Addition of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the assay mixture raised the enzyme activity of rat liver mitochondria more than 10-fold. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making more endogenous cholesterol accessible to the enzyme. Availability of cholesterol to the enzyme limits synthesis of 27-hydroxycholesterol in rat liver. Using our assay to simultaneously determine the activities of cholesterol 7 alpha-hydroxylase and cholesterol 27-hydroxylase in rat liver homogenates, we demonstrated that the two enzymes are separately regulated.

Regulation of central cholecystokinin recognition sites by guanyl nucleotides.

Guanyl nucleotides affected the binding of radiolabeled 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 microM 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.

Inhibition of climbing and mossy fiber input to mouse cerebellar Purkinje cells by cholecystokinin.

Cholecystokinin (CCK)-8S was found both to decrease basal and to antagonize harmaline-dependent increases in cerebellar cyclic GMP (cGMP) in the mouse. These actions were not blocked by parenteral pretreatment with naloxone, proglumide or CR-1409, indicating a central-type CCK receptor activation with no involvement of opioid systems. These data were further confirmed by the greater potency (200x) of intraventricular relative to s.c. doses for CCK-8S. The intraventricular administration of CCK-4, t.BOC.CCK-4 and CCK-8US also resulted in decreased cerebellar cGMP levels, consistent with a central-type CCK receptor action. Direct administration of CCK-8S into the cerebellum failed to alter either basal or harmaline-stimulated cerebellar cGMP levels, indicating that the actions of this peptide on cerebellar cGMP are not directly at the level of the cerebellum. The convulsants, picrotoxin and pentylenetetrazol, elevated cerebellar cGMP with no antagonism by pretreatment with s.c. CCK-8S. In marked contrast, the increases in cerebellar cGMP induced by treatment with amphetamine, apomorphine, DN 1417, oxotremorine and harmaline were all antagonized by pretreatment with s.c. CCK-8S. These data are consistent with CCK receptor involvement in the regulation of climbing and mossy fiber input to the cerebellum. These actions apparently involve the central-type CCK receptor that resides outside of the cerebellum proper. The exact site(s) of action for CCK-8S remain(s) to be defined.

Diazepam: in vitro 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 diazepam's 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.

Benzodiazepine interactions with central thyroid-releasing hormone binding sites: characterization and physiological significance.

Thyrotropin-releasing hormone (TRH) and several TRH analogs were examined in the [3H]-3-Me-His2-TRH ([3H]MeTRH) receptor-binding assay in rat amygdala, striatal and cortical membranes. The benzodiazepine, chlordiazepoxide, as reported in the literature was found to displace [3H]MeTRH with an IC50 value of 3.6 X 10(-7) M in amygdala membranes. Midazolam was, however, identified as being 6-fold more active than chlordiazepoxide with an IC50 value of 6.3 X 10(-8) M. The effect of these benzodiazepines on [3H]MeTRH binding did not appear to be related to their anxiolytic activity because the novel pyrazoloquinoline nonsedating anxiolytic, CGS 9896 was without effect on [3H]MeTRH binding at concentrations up to 1 X 10(-5) M. Chlordiazepoxide had similar activity in cortical membranes whereas midazolam was some 5 times less active in this preparation than in amygdala. Both compounds were weak displacers of [3H]MeTRH binding in striatal membranes, being at least two orders of magnitude less potent than in amygdala. In contrast TRH and its analogs, RX 77368 and DN-1417, were approximately 2 to 8 times more active in striatum than amygdala membranes. TRH and DN-1417 were less active in cortical membranes whereas RX 77368 was some three times more active than in striatum and amygdala. In three test procedures indicative of TRH agonist activity; thyroid-stimulating hormone release, reversal of pentobarbital sleeping time in mice and elevation of cerebellar cyclic GMP levels, the benzodiazepines were found to be devoid of activity, whereas TRH and related compounds produced their expected responses.(ABSTRACT TRUNCATED AT 250 WORDS)