Nonspecific protease-catalyzed hydrolysis/synthesis of a mixture of peptides: product diversity and ligand amplification by a molecular trap.

We sought to develop a peptide library in solution and dynamically screen this library for peptides that would bind to macromolecules of interest. Peptide diversity was achieved in an initial stock solution of peptides by using proteases under conditions in which both hydrolysis and synthesis occurred. As an example, a simple reaction containing YGG, FL and thermolysin resulted in the synthesis of YGGFL as well as many other undefined products. When low molecular weight products of a reaction containing VA, AL, and thermolysin were subsequently exposed to dipeptidase, 7 out of 9 potential dipeptides were observed. Incubation of protease with an hydrolysate of albumin and a radiolabeled peptide resulted in the radiolabel participating in reactions other than simple hydrolysis and, after 24 h, the specific activity of radiolabel was shown by high performance liquid chromatography to disperse to a level that would be necessary in the event of maximum theoretical diversity. When a binding macromolecule was exposed to this system, ligand production was amplified relative to reactions run in the absence of binding macromolecule. This protease-based peptide scrambling and binding system was utilized for the discovery of novel peptides that bind to fibrinogen.

Benzodiazepines inhibit neutrophil chemotaxis and superoxide production in a stimulus dependent manner; PK-11195 antagonizes these effects.

Diazepam, which binds both central (neuronal) and peripheral (non-neuronal) benzodiazepine binding sites, and Ro5-4864, a ligand selective for benzodiazepine peripheral binding sites (PBS), both inhibited the FMLP induced chemotaxis in human neutrophils at concentrations as low as 10(-8) M. A selective peripheral benzodiazepine antagonist, PK-11195 (10(-5) M), partially reversed the benzodiazepine inhibition of chemotaxis. Diazepam also inhibited the superoxide production induced by FMLP, NaF, and A23187, but not that induced by PMA whose stimulant action was insensitive even to 10(-4) M diazepam. The FMLP-induced superoxide production was most sensitive to diazepam inhibition (ID50 = 2.25 x 10(-6) M diazepam); the effect of NaF was slightly less sensitive (ID50 = 1.34 x 10(-5) M diazepam); and the effect of A23187 was least sensitive as it was suppressed only at 10(-4) M diazepam concentrations. Like diazepam, Ro5-4864 inhibited the FMLP-induced superoxide production, and PK-11195 (10(-5) M) significantly antagonized both diazepam and Ro5-4864 inhibition. Binding studies showed the presence of a saturable benzodiazepine 'peripheral' type binding site (PBS) on human neutrophils with a Kd of 1.2 +/- 0.06 x 10(-8) M (+/- SEM), and a Bmax of 1028 +/- 86.2 fmol/10(6) cells (+/- SEM) for [3H]Ro5-4864; the binding was displaceable by PK-11195, Ro5-4864 and diazepam but not by clonazepam.

Chronic administration of flumazenil (Ro 15-1788) enhances non-appetitive exploratory behavior of rats.

The effects of chronic administration of the benzodiazepine receptor antagonist, flumazenil (Ro 15-1788; 4 mg/kg/day for 14 days in drinking water) on the performance of adult rats in the 12-arm radial maze were studied. Relative to controls, the animals treated with flumazenil showed an increase (P less than 0.002) in non-appetitively motived exploratory behavior, so called because it occurred in 88% of instances in non-baited alleys, facing the well-illuminated "enriched environment" of the center of the room, as opposed to the baited alleys, facing the "dull" corner of the room. This behavior emerged between day 5 and 7 of treatment with the drug, it continued to increase over the period of treatment with drug (P less than 0.002), and reached its peak at day 3, after withdrawal of the drug (P less than 0.008; a longer duration was not investigated). The occurrence of non-appetitively motivated exploratory behavior was inversely correlated with the scores for urination/defecation (P less than 0.003) and, therefore, most likely reflected the anxiolytic action of flumazenil. During treatment with drug or vehicle, the control and the drug groups made comparable numbers of "working memory" errors (P = 0.17). However, upon withdrawal of drug and introduction of alley gates (to confine the animal for 10 sec to the center platform, after an alley was explored), the working memory errors of the rats exposed to the drug, remained unchanged (P = 0.35), relative to the preceding three trials, while the performance of the control group was disrupted, as shown by an increase in the numbers of errors (P less than 0.004). At day seven of treatment with drug, the emergence of exploratory behavior was associated with an increased density and/or affinity of benzodiazepine receptors in cortex, hippocampus and brain stem, while three days after withdrawal of drug, when the exploratory behavior reached its peak, there was a reduction in GABA-enhanced binding of [3H]flunitrazepam in the cortex.

Chronic Ro 15-1788 treatment increases REM sleep in rats.

Administration of Ro 15-1788, a benzodiazepine antagonist (3.6 mg/kg/day in drinking water for 14 days), increased total sleep and rapid eye movement (REM) sleep in rats. Standard six-hour EEG recording periods were obtained on day 0, 1, 3, 7, 10, 14, as well as 24 and 72 hours following withdrawal. Enhanced REM sleep reached significance on day 7 of continuous drug treatment and remained significantly increased on day 10 and 14, as well as at 24 and 72 hours following drug withdrawal. The present data show that chronic administration of Ro 15-1788 increases total sleep time due to increases in REM sleep. The actions of Ro 15-1788 presumably occur through either adenosinergic or cholinergic mechanisms.

Chronic exposure to flumazenil: anxiolytic effect and increased exploratory behavior.

The aim of the present study was to define the behavioral correlates of chronic exposure of adult rats to flumazenil (4 mg/kg/day X 21 days in drinking water). In the holeboard test, performed on day 13 of drug treatment, the animals showed a significantly greater interest for the holes under which objects were placed than for the holes without objects (p less than 0.03), while there was no such difference in the control group. In the plus-maze test, the flumazenil-treated animals spent significantly more time on open arms and left less fecal boluses than the controls when tested in the third week of treatment and 24 hours after flumazenil withdrawal. In the drinking-punishment test, conducted on days 3, 6 and 10 after drug withdrawal, the drug-exposed animals, following shock experience, did not significantly alter their unpunished drinking in subsequent trials, while the control rats significantly reduced (p less than 0.003) their unpunished drinking. Also, the punished drinking revealed a significant "anticonflict" effect of prior exposure to flumazenil (p less than 0.006) which was still observed 6 days after drug withdrawal. There were no group differences in the home-cage food and water consumption during flumazenil treatment; also, the drug treatment had no effect on nociceptive threshold. In summary, chronic treatment with a benzodiazepine receptor antagonist, flumazenil, increased exploratory activity and had a lasting anxiolytic effect.

Chronic flumazenil (Ro 15-1788) facilitates acquisition and retention of a swim-escape response in rats.

Since chronic flumazenil treatment was previously found to stimulate exploratory behavior in rodents, the aim of this study was to test the effect of chronic exposure to flumazenil on acquisition and retention of escape behavior. Adult rats were treated with flumazenil (Ro 15-1788; 4 mg/kg/day in drinking water) for 21 days (experiment 1) and for 17 days (experiment 2). In experiment 1 (a round water tank with one escape rope) conducted 24 h after drug/vehicle withdrawal, the time the animals needed to resolve a swim-escape task was significantly shorter in the drug group, compared to the controls. In the retention trial, 24 h later, the control group matched the performance of the drug group. In experiment 2, a water T-maze was used which was equipped with two ropes, one anchored to the bottom and the other unanchored and therefore was more difficult to climb. On day 14 of flumazenil/vehicle treatment, there were no differences between the groups in the time needed to escape from the maze. However, on day 15 and 16 of drug/vehicle treatment, the drug group made highly significant progress, while the control group showed no improvement of the escape behavior. The possible mechanisms of flumazenil-induced facilitation of escape behavior have been discussed.

Chronic exposure to Ro 15-1788: differential effect on flunitrazepam binding to cortex and hippocampus.

The time course of changes in specific [3H]flunitrazepam binding following 2 weeks of treatment with the benzodiazepine antagonist Ro 15-1788 (2.7 and 4 mg/kg per day in drinking water) was studied in the rat neocortical and hippocampal synaptosomal membranes. Such a treatment produced regional increases in the density of benzodiazepine sites, which remained for up to 24 and 48 h after drug withdrawal in the hippocampus and cortex, respectively; the dissociation constant (Kd) was unchanged. In addition, a significant reduction in GABA enhanced [3H]flunitrazepam binding to cortical, but not to hippocampal, membranes from Ro 15-1788-treated animals was found 72 h after drug withdrawal. These data show that there is a regional difference in responses of the GABA/benzodiazepine receptor complex following chronic in vivo exposure to Ro 15-1788 and that, beside the increases in the maximal binding (Bmax) of [3H]flunitrazepam the coupling between the GABA and the benzodiazepine recognition sites was also affected.

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain.

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.

Animal models of chronic anxiety and "fearlessness".

Three behavioral animal models have been described: a feline and a rodent model of chronic anxiety, and a rodent model of "fearless" behavior. The models have been obtained by pre- or perinatal exposure to diazepam (DZ) or RO 15-1788 which produced enduring postnatal deficits or enrichment, respectively, of brain benzodiazepine (BDZ) receptors. The receptor-deficient one-year-old cat progenies showed hyperarousal, unabated restless behavior, delayed acquisition of instrumentally conditioned behavior, bizarre escape responses and absence or reduced alpha-like EEG activity. The receptor-deficient rat progencies, studied at the age of 5-6 months, showed a reduction of time spent in deep slow wave sleep, and inability to habituate to novel environment, such as the radial arm maze. In the maze, the behavior of these progenies was characterized by delayed and incomplete exploratory activity often terminated by sudden escape, numerous fecal deposits and significantly more frequent than normal errors of "working memory." On the other hand, in all aspects, the receptor-enriched progenies were superior to the control animals as well as to the receptor-deficient group, particularly when the animals were challenged by novel and "intimidating" visual and/or auditory stimuli. In addition, 12 out of 51 receptor-deficient rats reared for 18 months developed mammary fibroadenomas, while no such tumors were found in the group of 44 vehicle-exposed control animals. Increased density and affinity of BDZ brain receptors was also observed after adult rats were treated with RO 15-1788 administered water for 7 or 14 days.