Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

Effects of denzimol on benzodiazepine receptors in the CNS: Relationship between the enhancement of diazepam activity and benzodiazepine binding sites induced by denzimol in the rat

Denzimol, a new anticonvulsant drug with a pharmacological profile similar to that of phenytoin, enhances the ataxic and antimetrazol activity of diazepam in rats without affecting its activity against picrotoxin-induced seizures. $\text{In vivo}$ and $\text{ex vivo}$ denzimol enhances the binding of ³H-flunitrazepam in cortex and in hippocampus but not in cerebellum.The possibility of this increase in the number of benzodiazepine binding sites contributing in some way to enhancement of the depressive and anticonvulsant activity of diazepam is discussed.     

Identification of diazepam binding in intack animals.

An $\text{in vivo}$ method for labeling specific benzodiazepine (BDZ) binding sites in brain was developed using intravenously injected [³H]diazepam. Labeling of these sites is blocked by pretreatment of animals with high doses of pharmacologically active BDZs (but not by an inactive BDZ). Using this $\text{in vivo}$ binding technique, specific BDZ binding is enhanced by pretreatment of rats with the GAB?A agonist muscimol or with amino-oxyacetic acid, which increases GABA levels in brain.     

Putative endogenous ligands for the benzodiazepine receptor.

The recent discovery of pharmacologically relevant, high affinity, stereospecific binding sites for the benzodiazepines in the central nervous system (CNS) has rekindled investigations concerning the mechanism of action of these drugs. It has become increasingly clear that elucidation of benzodiazepine action will provide new and important insights into the neurochemical substances of seizure activity, centrally mediated muscle relaxation and anxiety, three major actions of this class of drugs.The existence of a functional receptor for the benzodiazepines, compounds not present $\text{in vivo}$, suggests that endogenous substances exist that serve as natural substrates for this receptor. Furthermore, the characterization of endogenous benzodiazepine receptor ligands affords an opportunity to determine the neurochemical mechanisms underlying the pharmacologic and behavioral effects manifested by the benzodiazepines.Using receptor binding methodology to assay tissue extracts for [³H] diazepam binding inhibitory activity, putative endogenous ligands for the benzodiazepine receptor have been isolated and identified as the purine nucleosides. Compounds such as inosine and hypoxanthine exhibit competitive inhibition of [³H] diazepam binding. The low affinity purinergic inhibition of diazepam binding is consistent with their $\text{in vivo}$ concentrations. Distinct structure-activity relationships exist for the purines with subtle structural alterations having marked effects on diazepam binding inhibitory potency. The methylxanthine stimulants, caffeine, theophylline, and theobromine, also competitively inhibit diazepam binding, suggesting that some of their actions may be mediated by the benzodiazepine receptor.The purines also have “benzodiazepine-like” pharmacologic properties, since they have been shown to antagonize pentylenetetrazol induced seizures in mice in a dose dependent manner. Neurophysiologic studies have also shown that iontophoresis of inosine on cultured mouse primary neurons produce neurotransmitter like effects. Furthermore, these effects are similar to those observed with flurazepam, a finding that provides additional evidence for the “benzodiazepine-like” properties of the purines.The preliminary studies outlined below indicate that the purines are good candidates as putative endogenous ligands for the benzodiazepine receptor and provide a foundation for future studies that concern the homeostatic mediation of seizure activity and anxiety.     

Blockade of the picrotoxin-induced in vivo release of dopamine in the cat caudate nucleus by diazepam

The effects of picrotoxin and diazepam on the $\text{in}$$\text{vivo}$ release of DA in the caudate nucleus were examined in “encéphale isolé” cats. A push-pull cannula was introduced into the left caudate nucleus and the structure was continuously superfused with L-3, 5-³H-tyrosine. The ³H-DA endogenously synthesized and released in the superfusates was estimated in successive serial fractions. Picrotoxin (2.5 mg/kg) markedly enhanced the release of ³H-DA, as previously shown. Diazepam (10 mg/kg) had no effect on the spontaneous release of the labelled transmitter, but it did prevent the stimulating effect of picrotoxin.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

Effects of diazepam and muscimol on GABA-mediated neurotransmission: interactions with inosine and nicotinamide.

Inosine and nicotinamide have been proposed as endogenous ligands for the brain benzodiazepine receptor. An $\text{in vivo}$ method for detecting drugs with GABA-mimetic properties was used to examine the effects of inosine, nicotinamide and diazepam in the rat globus pallidus. Inosine and nicotinamide completely prevented the GABA-mimetic action of diazepam but neither compound alone had any GABA-like activity. These findings suggest that inosine and nicotinamide are able to antagonize but are not able to mimic the GABA-like actions of diazepam at the benzodiazepine receptor.     

In vivo studies of dopamine receptor ontogeny

Studies of the ontogeny of dopamine and neuroleptic receptors in the central nervous system of the rat were carried out $\text{in vivo}$ using ³H-spiperone as ligand. It was demonstrated that intraperitoneal injections can be successfully used to label these receptors in rat pups up to at least 30 days of age. The time course and characteristics of ³H-spiperone binding in the brain of 5, 15 and 30 day old rat pups were determined and found to include appropriate regional distribution, saturability and appropriate pharmacology. The developmental pattern of ³H-spiperone binding paralleled what has been seen using $\text{in vitro}$ techniques. In addition preliminary autoradiographic studies describe the neuroanatomical pattern of dopamine receptor ontogeny in the striatum.     

Characterization of specific in vivo binding of neuroleptic drugs in rat brain

$\text{In vivo}$ binding of ³H-spiperone is saturable in the striatum, the limbic system and the frontal cortex but not in the cerebellum. A specific binding is different in all the brain regions thus the amount of labelling in the cerebellum may not be considered as a blank value.³H-spiperone binding revealed a specific subcellular distribution only when a very low dose was injected into rats.$\text{Ex vivo}$ experiments allow the assessment of biochemical profiles of neuroleptic drugs according to their relative affinity for dopamine or serotonin receptors.     

Stimulation of guinea-pig brain adenylate cyclase by adenosine analogues with potent pharmacological activity in vivo

1-Methylisoguanosine, a marine natural product with potent muscle-relaxant and cardiovascular actions $\text{in vivo}$, interacts directly with adenosine receptors in guinea-pig brain slices to stimulate adenylate cyclase. These effects are blocked by theophylline. Comparison of the $\text{in vivo}$ pharmacological activity of a number of synthetic analogues of 1-methylisoguanosine with $\text{in vitro}$ adenylate cyclase-stimulating ability indicates that compounds lacking the latter biochemical activity have little muscle-relaxant activity. Adenosine is a potent stimulator of adenylate cyclase but is inactive $\text{in vivo}$ because of rapid removal from the extracellular environment by uptake and deamination. Unlike adenosine, 1-methylisoguanosine is resistant to deamination and is only poorly accumulated by brain tissue slices or homogenates containing synaptosomes. Since it is an extremely weak competitive inhibitor of adenosine deaminase and only a weak inhibitor of adenosine uptake, it is unlikely to act by potentiating the effects of adenosine itself at extracellular receptors. Thus, the pharmacological effects of 1-methylisoguanosine are apparently due to its actions as a long-lasting adenosine analogue.     

Kyotorphin (tyrosine-arginine): Further evidence for indirect opiate receptor activation

Analgesia, opiate receptor binding, and neurochemical effects of kyotorphin (tyrosine-arginine) were studied in the rat. It was found that while kyotorphin, $\text{in vivo}$, causes naloxone reversible analgesia, and affects dopamine metabolism and acetylcholine turnover in the same manner as do morphine and other opiate agents, the dipeptide does not bind to mu, delta or kappa opiate receptors $\text{in vitro}$. Taken together, these data support the concept that there is an indirect action of kyotorphin on opiate receptors.     

Binding of opiates and endogenous opioid peptides to neuroleptic receptor sites in the corpus striatum.

Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with ³H-spiroperidol for common binding sites in rat striatum $\text{in vitro}$ (IC₅₀ in the range between 10^?6 and 10^?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since $\text{in vivo}$ studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites $\text{in vitro}$ and their potency to evoke “bizarre behavior” in rats $\text{in vivo}$. In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites $\text{in vitro}$ (IC₅₀ greater than 10^?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites.     

No changes in rat benzodiazepine receptors after withdrawal from continuous treatment with lorazepam and diazepam.

Five and 11 days after withdrawal from 8 weeks of treatment with 90 mg/kg/day of diazepam p.o. or 60 mg/kg/day of lorazepam p.o. there were no consistent changes in the number of benzodiazepine receptors or apparent affinity $\text{in vitro}$ for ³H-diazepam at 0°C in rat forebrain membranes. Daily exposure of rats from 10 days before birth until 7 days after birth was also without gross effects on the benzodiazepine receptor. Abstinence and tolerance to benzodiazepines were thus not attributable to changes in brain benzodiazepine receptors.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Pharmacological effects of a specific dopamine D-1 antagonist SCH 23390 in comparison with neuroleptics

Neuroleptics such as thioxanthenes (cis($\text{Z}$)-flupentixol and cis($\text{Z}$)-clopenthixol) and phenothiazines (fluphenazine and perphenazine), which block both dopamine (DA) D-1 and D-2 receptors and the butyrophenones (haloperidol and spiroperidol), which block D-2 receptors only, are equipotent both behaviorally and clinically. A new compound SCH 23390 which selectively blocks DA D-1 receptors, resembles many neuroleptics in its pharmacological profile: antistereotypic effects in mice, rats and dogs, cataleptogenic effect and inhibitory effect on amphetamine circling. In contrast SCH 23390 has no effect on apomorphine-induced vomiting in dogs and little effects on 6-OHDA-denervated supersensitive DA receptors, stimulated by the DA agonist 3-PPP. In a series of experiments where methylphenidate-induced stereotyped gnawing in mice was inhibited by neuroleptics, it was shown that concomitant treatment with scopolamine or diazepam attenuated the effect of butyrophenones (D-2 antagonists). The same treatment attenuated the effect of phenothiazines, to a lesser extent, and hardly attenuated the effect of thioxanthenes and SCH 23390 at all. It is concluded that DA D-1 receptors are as important as D-2 receptors for the expression of neuroleptic activity in most animal models believed to be predictive of antipsychotic and extrapyramidal side-effect potential. However, the D-1 antagonist is less sensitive than D-2 antagonists to antimuscarinic compounds and benzodiapines.     

The role of cAMP on neoplastic cell growth and regression. III. Altered cAMP-binding in DBcAMP-unresponsive Walker 256 mammary carcinoma.

An exogenous supply of N⁶,O^2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) $\text{in vivo}$ produces regression of one type of Walker 256 mammary carcinoma cell population (DBcAMP-responsive); a second type of cell population continues to grow despite DBcAMP treatment (DBcAMP-unresponsive). A correlation was found between altered cAMP-binding of the tumor cytosol and DBcAMP-unresponsiveness. It was found that there was: a) a higher apparent dissociation constant (Kd) for cAMP-binding in unresponsive tumor cytosol $\text{in vitro}$, and b) unsaturability of cAMP-binding by unresponsive tumor cytosol in response to elevated cAMP levels $\text{in vivo}$. Cycloheximide abolished the saturation of cAMP binding $\text{in vivo}$ as well as tumor regression produced by DBcAMP.     

Unmasking the mitochondrial K/H exchanger: tetraethylammonium-induced K+-loss.

Mitochondria respiring in media containing 80 mM tetraethylammonium ions lose all of their endogenous K⁺ within 7 minutes. K⁺-loss is associated with uptake of tetraethylammonium ions. K⁺ efflux under these conditions is energy-dependent and electroneutral. It is concluded that tetraethylammonium uptake unmasks the endogenous $\text{K}\text{H}$ exchanger. Considered in relation to the chemiosmotic theory, these results support the existence of a “carrier-brake” mechanism which modulates $\text{K}\text{H}$ exchange to maintain volume homeostasis $\text{in vivo}$.