Changes in in vivo binding of 3H-Ro 15-1788 in mouse brain by reserpine

The effects of reserpine on the in vivo binding of ³H-Ro 15-1788, (Ro 15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) a selective benzodiazepine antagonist, in the mouse brain were investigated. The biodistributions of tracer amounts of ³H-Ro 15-1788 in mice were significantly altered by pretreatment with reserpine (2.5 or 5.0 mg/kg, 24 h before the tracer administration). The time courses of radioactivity in the brain and the blood following i.v. injection of ³H-Ro 15-1788 with carrier Ro 15-1788 were not changed by pretreatment with reserpine, which suggested that the specific binding process might be altered by reserpine. The degree of alteration in the in vivo binding of ³H-Ro 15-1788 seemed to be dependent upon the dose of reserpine and the duration after the treatment of reserpine. The maximum changes in the biodistribution of ³H-Ro 15-1788 were observed at 1 day after injection of reserpine. The body temperature and the brain monoamine contents (dopamine, norepinephrine and 5-hydroxytryptamine) in mice were measured as indicators of pharmacological effects of reserpine, and good relationships to the degree of changes in the biodistribution of ³H-Ro 15-1788 and either the body temperature or brain monoamine contents, were observed. Furthermore, the changes in the biodistribution of ³H-Ro 15-1788 in the reserpinized mice were significantly suppressed by antidepressant imipramine treatment. These results suggest that it would be possible to detect the in vivo drug interaction with brain benzodiazepine receptors in the living human brain using ¹¹C-Ro 15-1788 and positron emission tomography (PET).     

“Specific” [3H]8-OH-DPAT binding to glass fiber filter paper: Implications for the analysis of serotonin binding site subtypes

The effect of buffer constituents on [³H]8-OH-DPAT binding to glass fiber filter paper was examined. Apparent “specific” [³H]8-OH-DPAT binding to glass fiber filter paper can be demonstrated when the radioligand binding assay is performed in the absence of 0.1% ascorbate. This artifactual “specific” binding is time dependent and appears to saturate. In addition, drug competition studies reveal complex interactions with [³H]8-OH-DPAT binding to glass fiber filter paper in the absence of ascorbate. Both 5-HT and chlorimipramine appear to “complete” for the sites labeled by [³H]8-OH-DPAT while both d-LSD and methysergide cause an increase in the [³H]8-OH-DPAT binding to glass fiber filter paper at micromolar concentrations. These data indicate that [³H]8-OH-DPAT binding to glass fiber filter paper may lead to misinterpretation of radioligand data obtained using brain homogenates in the absence of ascorbate.     

Suppression of lordosis in guinea pigs by ethamoxy-triphetol (MER-25) given at long intervals (34-46 hr) after estradiol benzoate treatment

Ovariectomized guinea pigs were given estradiol benzoate (EB) followed 40 hr later by progesterone (P). Behavioral testing commenced 1 hr after P injection and continued at hourly intervals for 8 hr. This treatment activated lordosis in almost 100% of animals. Administration of the antiestrogen MER-25 (75 mg/kg body wt per injection) between 2 hr before and 6 hr after EB treatment did not cause a significant decline in proportion of animals displaying lordosis, but did cause a decrease in length of time the lordosis position was held (maximum lordosis, sec). In contrast, $\text{13}\text{14}$ animals given MER-25 at 2 hr before and 2 hr after P and $\text{8}\text{10}$ animals given MER-25 simultaneously with and 2 hr after P, failed to show lordosis. Administration of supplementary EB at around the time of P injection, partially alleviated these behavior-blocking effects of MER-25. When MER-25 was given 2–6 hr after administration of P there was a significant decrease in duration of heat (hr). These results suggest that in addition to its early “triggering” effects, estrogen has important “maintenance” effects which determine the character of heat in guinea pigs. Continued presence of estrogen in the nervous system may be a requirement for the facilitatory actions of P on sexual behavior in guinea pigs, but such a requirement may not exist in other rodents such as rats.     

Morphine tolerance and naloxone receptor binding.

³H-naloxone specific binding studies have confirmed the induction of receptor expansion after an acute injection of morphine, as reported by Pert et al (3) as well as the lack of expansion in chronically morphinized rats shown by Klee and Streaty (4) using dihydromorphine. With a challenging test dose of morphine given to rats maintained drug free after acute and chronic regimens of morphine, the lack of expansion as measured by 3H-naloxone specific binding persisted up to at least 4 weeks. Between 4–8 weeks receptor expansion can be re-induced with a challenging test dose. This “physical binding tolerance” is dose related. That this persistant “tolerance” is not attributable to the presence of dissociable morphine remaining after the drug regimen or challenge dose can be shown by detergent extraction and exhaustive dialysis of the standard buffer homogenate preparation as well as with fresh excised tissue.     

Evidence of the existence of two different intraneuronal pools from which pharmacological agents can release serotonin

The effect of various drugs on the release of [³H]-serotonin from synaptosomes of reserpine-treated rats was compared with that obtained with synaptosomes of untreated animals.The increase in [³H]-serotonin release induced by d-fenfluramine was virtually abolished by reserpine; the effect of d-norfenfluramine, the main metabolite of fenfluramine, was instead enhanced in synaptosomes of reserpine treated animals. [³H]-serotonin release induced by l-isomers of fenfluramine or norfenfluramine was increased or not affected, respectively, after reserpine treatment.The effects of other drugs, known to activate serotonin mechanisms such as metachlorophenylpiperazine and quipazine, like d-norfenfluramine, were increased by the reserpine treatment.The present data show that [³H]-serotonin can be released by drugs from two pools with different sensitivity to reserpine. The reserpinized synaptosomes could provide useful information on the mechanisms of action of drugs acting on brain serotonin.     

Emulsification and degradation of "Bunker C" fuel oil by microorganisms

An enrichment culture procedure has been used to isolate mixed culture systems which grow upon “Bunker C” fuel oil. When inoculated into a mineral salts aqueous medium containing Bunker C oil, the mixed cultures initiate oil emulsification. Emulsification usually is observed in 24–48 hr. The role of microbes in this emulsification will be discussed. It appears that certain metabolic products produced by the microbe possess properties of surfactants. Bacteria and fungi have been isolated which possess the ability to cause emulsification. Freeze-dried biomass is also capable of emulsifying oil. Chromatographic analyses of biodegraded Bunker C fuel oil show that microorganisms selectively metabolize the n-paraffin fraction.     

Endogenous Dopamine (Da) Modulates [3H]spiperone Binding in Vivo in Rat Brain

Abstract

[³H]spiperone (SPI) binding in vivo, biochemical parameters and behavior were measured after modulating DA levels by various drug treatments. DA releasers and uptake inhibitors increased SPI binding in rat striatum. In other brain areas, the effects were variable; but only the pituitary remained unaffected. Surprisingly, nomifensine decreased SPI binding in frontal cortex. The effects of these drugs were monitored by measuring DA, serotonin (5–HT) and their metabolites in the same rats. The increased SPI binding in striatum was parallel to the locomotor stimulation with the following rank order: amfonelic acid > nomifensine > D-amphetamine ± methylphenidate > amineptine > bupropion. Decreasing DA levels with reserpine or alpha-methyl-para-tyrosine reduced SPI binding by 45% in striatum only when both drugs were combined. In contrast, reserpine enhanced SPI binding in pituitary. Thus, the amount of releasable DA seems to modulate SPI binding characteristics. It is suggested that in vivo, DA receptors are submitted to dynamic regulation in response to changes in intrasynaptic concentrations of DA.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Increased acoplasmic transport of H3-dopamine in nigro-neostriatal neurons after reserpine

Recent evidence suggests that dopamine can undergo axoplasmic transport in nigro-neostriatal neurons by binding to amine storage granules. In the present experiments it was demonstrated that reserpine pretreatment (10 mg/kg) 24 hours before stereotaxic injections of ³H-DOPA or ³H-dopamine into the substantia nigra increases the amount of ³H-dopamine transported to the neostriatum by about 300 percent. The activity recovered from the substantia nigra was significantly reduced by reserpine pretreatment however. Stereotaxic injection of ¹⁴C-leucine into the substantia nigra indicated that neither fast nor slow axoplasmic transport of protein was influenced by reserpine pretreatment in these same neurons. The increased transport of dopamine appears therefore to be due to a relatively selective action of reserpine. The results suggest that reserpine either (i) increases the binding of dopamine to newly synthesized amine storage granules, (ii) increases the number of newly synthesized amine storage granules, or (iii) accelerates the rate of transport of amine storage granules. In addition, the results support the view that reserpine can increase the membrane permeability of adrenergic neurons to the outward movement of catecholamines.     

Antagonism of benzodiazepine binding in rat and human brain by Antilirium.

Physostigmine (Antilirium^R) has been reported to reverse benzodiazepine- induced sleep or coma in man and prevent death in animals. Accordingly, we investigated the effect of Antilirium upon benzodiazepine binding to both rat and human brain. We report that Antilirium inhibits ³H-diazepam and ³H-flunitrazepam binding in a dose-dependent manner. The degree of inhibition of binding by Antilirium correlates with the affinity of benzodiazepine for its “receptor” such that diazepam is more affected than flunitrazepam. The inhibition is rapid but the kinetics are complex with only doses of Antilirium showing competitive inhibition when studied at equilibrium. These results may explain, at least in part, the effectiveness of Antilirium at reversing benzodiazepine-induced hypnosis without necessarily implicating a cholinergic mechanism.     

An experimental model of tardive dyskinesias

Rats treated continually and chronically with trifluoperazine (ca 3 mg/kg/day) for six months initially developed mild catalepsy and an inhibition of spontaneous locomotor activity; both effects disappeared by three months. An initial increase in dopamine turnover (as measured by levels of homovanillic acid and dihydroxyphenylacetic acid) also disappeared by three months. Apomorphine-induced stereotypy was completely inhibited in drug-treated animals at two weeks, but progressively returned to normal after three months of drug intake. An exaggerated response to apomorphine developed in animals after six months of drug administration. Inhibition of striatal dopamine-stimulated adenylate cyclase found during the first month of drug intake was reversed at three months, a trend exaggerated after continuous drug administration for six months. Specific striatal ³H-spiperone binding affinity decreased acutely, but was increased after six months drug intake; no change in number of receptor sites occurred.These changes suggest that at least striatal dopamine receptors may become “supersensitive” during chronic neuroleptic treatment.     

Striatal and Urinary DOPAC/DA Ratio May Indicate a Long-Lasting DA Release Enhancement by MPP+ and MPTP

The DOPAC/DA ratio in mouse striatum, in striatal synaptosomes, and in rat urine after MPP⁺ and MPTP neurotoxin administrations to the animals was followed temporally. The neurotoxins were given intraperitoneally and, in some experiments, to enhance the sensitivity, the animals were subsequently reserpinized before either sacrifice or 24 hour urine collection. MPP⁺ treatment, followed by saline, weakly lowered mouse striatal DOPAC/DA ratio up to 6 hours; in reserpinized animals, however, the neurotoxin reduced striatal ratio potently and for longer periods. Similarly, MPP⁺ reduced rat (saline treated) urinary DOPAC level and DOPAC/DA ratio in the short term (1.0 hr) while the neurotoxin effects could still be detected following longer periods up to 27 days in reserpinized animals. A single MPTP treatment (90 min.), followed by preparation of striatal synaptosomal fraction and its incubation (37°C) with or without reserpine, also led to a reduced DOPAC/DA ratio. Although mainly the pooled peripheral effect is directly indicated by urinary DOPAC/DA ratio, MPP⁺ may reduce DA oxidation in the CNS and may similarly affect the amine oxidation in the peripheral tissues. The CNS and peripheral effects differ, however, in respect to dose-sensitivity and time course. The similarities between the CNS and peripheral effects suggest that a blunted rise of urinary DOPAC/DA ratio after reserpine challenge could be utilized as a peripheral marker of MPP⁺ action in the CNS, a marker that is not currently available.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

[³H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Developmental status of sympathetic innervation in relation to calcium accumulation by submandibular gland following reserpine, surgical sympathectomy or cyclocytidine

Sympathectomy (Sx) of the submandibular gland was induced at various postnatal ages by ip administration of a single dose of reserpine or by unilateral excision of a superior cervical ganglion. If animals were 12 days old or less at the time of drug administration, [Ca] of the submandibular gland was not measurably increased 24 hr later; if rats were 14 days of age or older, [Ca] of the gland 24 hr after reserpine injection was nearly double that of untreated controls. Two days after surgical Sx, [Ca] of the denervated submandibular gland was unchanged from that of the innervated member of a pair if animals were less than 14 days of age at the time of denervation; [Ca] was twice that of glands of control rats if animals were older than 14 days of age when the denervation was performed. The anti-tumor agent, cyclocytidine (CC), given daily for 3 days in an ip dose of 500 mg/kg, also caused a two- to threefold increase in [Ca] of the submandibular gland when rats were more than 12 days of age at the time of the initial injection of the drug, but in rats younger than this age, CC caused no change in the [Ca] of the submandibular gland. Present data show that there are age-related differences in the ability of the submandibular gland to accumulate calcium following sympathetic denervation or treatment with a norepinephrine-releasing drug. These differences may be attributed either to incomplete development of calcium transport mechanisms, or incomplete development of the sympathetic innervation before 14 days of age.     

Acute changes in the state of dopamine receptors; in vitro monitoring with 3H-dopamine

Preincubation of homogenates of rat caudate nucleus at 37 degrees C induced a 3-fold increase in the saturable and stereospecific binding of 3H-dopamine in washed suspensions of the 20,000 × g pellet. EGTA prevented and CaCl2 or MgCl2 reinstated the increase in 3H-dopamine binding. Stereospecific binding was reduced by 53% in the caudate nuclei of animals given a single dose of reserpine which depleted brain dopamine. The addition of dopamine to depleted homogenates restored the effect of preincubation on 3H-dopamine binding. The binding of 3H-spiroperidol was unaffected by preincubation or by reserpine pretreatment. These results suggest that dopamine regulates the 3H-dopamine, but not the 3H-spiroperidol binding site in rat brain.     

Letter from the President: Expectations and Needs of Members of the International Society of Chronobiology

The focus of the reported work is investigation of disopyramide chronopharmacokinetics in the mouse. Different groups of male NMRI mice maintained under controlled environmental conditions (LD: 0600-1800) received a single intraperitoneal injection of disopyramide (30mg per kg of body weight) at one of four different fixed time points of a 24-h period, i.e. 1000, 1600, 2200 or 0400. Blood samples were taken 0.5,1,2,3,4 and 6 hr after drug administration and total and free plasma levels of disopyramide were measured by an immunoenzymatic method.

Our data showed statistically significant circadian rhythms in the following pharmacokinetic parameters: highest volume of distribution = 3.91 ± 0.211kg^?1 at 2200 (circadian amplitude, half the peak-to-trough difference relative to the 24-hr mean multiplied by 100, is 34%); highest area under concentration curves = 16.06 ± 1.03μgml^?1hr^?1 at 0400 (circadian amplitude = 43%) and highest clearance = 3.04 ± 0.191hr“kg”¹ at 2200 (circadian amplitude = 21%). Protein binding of the drug was shown to ^he circadian time dependent. Alpha and beta phase elimination half-lives were not found to be significantly circadian phase-dependent. Thus circadian changes in disopyramide clearance may represent circadian changes in the drug's volume of distribution.     

Subcellular distribution and chemical nature of the receptor for 5-hydroxytryptamine in the central nervous system

Abstract— In vitro binding experiments with 5-hydroxy[¹⁴C]tryptamine (3.3 × 10^?6 M) were carried out on subcellular fractions of the cat brain. The highest specific activity was observed in some fractions of nerve-ending membranes isolated from the hypothalamus, basal ganglia, and gray areas of the mesencephalon. The specificity of this high affinity binding was demonstrated by competition with reserpine, butanolamide of lysergic acid, and desmethylimipramine. With butanol-water extraction the [¹⁴C]5-HT was found in the butanol while the gangliosides were separated in the water phase. Several experiments with thin layer and column chromatography suggest that in the organic phase the [¹⁴C]5-HT is not bound to the lipids but to a special proteolipid. This proteolipid is different from that found in myelin and has similar chromatographic properties to that previously observed in the proteolipid which binds d-[¹⁴C]tubocurarine in nerve-ending membranes of the cerebral cortex.     

Multifactorial etiology of anemia in SIV-infected rhesus macaques: Decreased BFU-E formation,serologic evidence of autoimmune hemolysis,and an exuberant erythropoietin response

We attempted to define the etiology of anemia in SIV-infected rhesus macaques. Bone marrow culture showed significantly decreased (75% reduction) burst forming unit-erythroid (BFU-E) growth in end-stage SIV⁺ “sick” animals. Direct antiglobulin tests (DAT) were positive in nine of 35 SIV⁺ “well” and 14 of 14 SIV⁺ “sick” monkeys (0 of 25 control animals had positive DATs). In animals with a positive DAT, moderate to severe anemia was observed, as was increased LDH and spherocytosis. Erythropoietin was measured in four control, eight SIV⁺ “well” and five SIV⁺ “sick” animals with mean levels of 4.0, 15.4, and 1176 mU/mL (r = .94) in the three groups. These data suggest that the cause of anemia in the SIV-infected rhesus macaque is multifactorial, that there may be a defect in erythropoiesis, and that, serologically, an IgG mediated autoimmune hemolytic anemia is also present.