A pyridinium-substituted analog of the TRH-like tripeptide pGlu-Glu-Pro-NH2 and its prodrugs as central nervous system agents.

A metabolically stable and centrally acting analog of pGlu-Glu-Pro-NH2 ([Glu2]TRH, a tripeptide structurally related to TRH (thyrotropin-releasing hormone)) was designed by replacing the amino-terminal pyroglutamyl residue with a pyridinium moiety. The analeptic action of the analog was used to optimize the efficacy of this novel CNS agent when administered intravenously in its CNS-permeable prodrug forms obtained via the reduction of the pyridinium moiety to the nonionic dihydropyridine and esterifying the central Glu with various alcohols. The maximum effect in antagonizing pentobarbital-induced narcosis in mice was achieved with the hexyl ester that was used subsequently for a comparative evaluation with a prodrug of the parent neuropeptide in the Porsolt swim test as a paradigm for antidepressant effect. The novel analog maintained its antidepressant potency but showed reduced analeptic action compared to [Glu2]TRH; thus, an increase in the selectivity of CNS-action was obtained by the incorporation of the pyridinium moiety.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.

Identification of synaptic metabolites of dynorphin A (1-8) by electrospray ionization and tandem mass spectrometry.

Synaptic metabolism of the endogenous opioid octapeptide dynorphin (Dyn) A (1-8) (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile) was studied in vitro upon its incubation with synaptosomes and with synaptosomal plasma membranes isolated from rat brain tissue. Electrospray ionization (ESI) and tandem mass spectromety were performed using an ion trap instrument, and afforded the identification of Dyn A (2-8), Dyn A (1-6) and leucine enkephalin [Dyn A (1-5)] as major metabolites. Preliminary quantitative data on the kinetics of Dyn A (1-8) degradation and metabolite formation was obtained by size-exclusion chromatography/ESI tandem mass spectrometry, and revealed an apparent involvement of several enzymes in the metabolism upon incubation with synaptosomes. Predominant formation of Dyn A (1-6) was observed with the synaptosomal plasma membranes.

Protein phosphorylation of synaptic membranes isolated from the brain of ground squirrels during hibernation.

In vitro protein phosphorylation of synaptic membranes isolated from neocortex and hippocampus of ground squirrels was studied. Three functional states of animals were investigated: torpid, awakened and active normothermic. Phosphorylation of a protein with a mol. wt of 53 kDa was independent on the functional state of the animals. Incorporation of 32P into this protein was greater in membranes of torpid and awakened squirrels than in membranes of active animals. These results suggest that protein phosphorylation is involved in the maintenance of membrane functions during hibernation.

Reversible activation of GABA and L-glutamate uptake into synaptosomes isolated from the rat brain in response to a single carbacholine injection into hippocampus.

In the present investigation the functional activity of transport systems mediating the GABA and L-glutamate uptake into nerve terminals of the rat brain cortex and hippocampus in response to a single carbacholine administration to hippocampus was studied. It has been established that synaptosomes isolated from the brain cortex and hippocampus of rats used in the experiments 24 h after a single carbacholine injection possess an increased capability of GABA and L-glutamate accumulation, and 48 h later the GABA and L-glutamate uptake begins to return to its control level and was equal to it on seventh day after injection.

[The effect of beta-carotene on the cytoplasmic membrane lipids of cells in rats under chronic gamma irradiation]. / Vliianie beta-karotina na lipidy tsitoplazmaticheskikh membran kletok krys pri khronicheskom gamma-obluchenii.

Chronic gamma irradiation of rats at a dose rate of 12.9 rad/day for 155 days (total dose 20 Gy) did not change the cholesterol level in the brush border membrane of the small intestine. Maintenance of rats on a beta-carotene-enriched diet (daily throughout the irradiation period, at a diurnal dose of 3 mg/kg body weight produced an increase in cholesterol levels in the small intestinal brush border membranes. The phospholipid level after gamma irradiation alone did not change, but it was increased in gamma-irradiated rats fed with carotene, this increase being most pronounced for sphingomyelin. Beta-carotene decreased the cholesterol/phospholipid ratio in the brush border membrane of irradiated rats. Activation of lipid synthesis (as seen from inclusion of 2-14C-acetate in the epitheliocyte lipids of irradiated animals) and no influence of beta-carotene on the inclusion of the label in small intestine epitheliocytes of irradiated animals have been revealed. In the presynaptic membranes of brain nerve endings, chronic gamma irradiation of animals produced a deep fall of cholesterol and phospholipid levels. Also, a tendency was revealed for stimulation of transport of the neurotransmitter amino acids GABA and L-glutamate across the presynaptic membranes of nerve endings. Beta-carotene normalized the phospholipid and cholesterol levels in brain presynaptic membranes of irradiated rats, as well as decreased L-glutamate transport. It is assumed that the modifying and normalizing effects of beta-carotene on the lipid exchange of plasma membranes upon chronic irradiation of animals and permanent introduction of the drug are associated with the radioprotector activity of beta-carotene.

[Presynaptic autoregulation of dopamine release]. / Presinapticheskaia autoreguliatsiia vysvobozhdeniia dofamina.

Experiments in vivo with local perfusion of rat brain neostriatum revealed existence of autoregulation of dopamine release mediated by presynaptic dopamine autoreceptors. An increase in dopamine concentration in the perfusion medium (addition of exogenous dopamine 10(-7) M, or cocaine, the inhibitor of uptake of biogenous amines, 10(-6) M) leads to a decrease in K+--induced release into perfusate of 3H--dopamine preliminarily injected into neostriatum. Haloperidol (10(-6) M) abolishes the inhibitory effect of exogenous dopamine. Perfusion with dopamine--containing medium (10(-4) M) leads to a decrease in the amplitude of EPs recorded in the neostriatum upon electrical stimulation of zona compacta of substantia nigra, indicating that dopamine release from dopaminergic terminals of nigro--striatal neurons decreases in response to AP.

[Presynaptic regulation of dopamine release by beta-phenylethylamine]. / Presinapticheskaia reguliatsiia vysvobozhdeniia dofamina beta-fenilétilaminom.

Experiments with local perfusion of the rat neostriatum and subsequent chromatography of the perfusate have shown that addition of beta-phenylethylamine (beta-PEA) to the perfusion medium in a concentration of 10(-3) M enhanced spontaneous and inhibited the K+-induced release of 3H-dopamine preliminarily applied to the neostriatum. The stimulating effect of beta-PEA was Ca2+-dependent and was potentiated in sodium-free media. The inhibitory effect of beta-PEA on the K+-induced release of 3H-DA was abolished by haloperidol, a blocker of dopamine receptors. This fact allows one to suggest that this effect of beta-PEA is mediated by presynaptic dopamine autoreceptors. The data obtained indicate that beta-PEA can modulate the dopaminergic synaptic transmission depending on functional activity of dopaminergic neurons.

Role of presynaptic dopamine receptors in regulation of the glutamatergic neurotransmission in rat neostriatum.

In experiments with the use of a push-pull cannula and simultaneous recording of electrical activity at the site of perfusion, the release of L-[3H]glutamic acid from rat neostriatum induced by K+-depolarization (60 mM K+ in perfusate) has been shown to be inhibited by replacing Ca2+ in the perfusion medium by Co2+. In contrast, release of L-[3H]glutamate induced by electrical stimulation of frontal cortex is enhanced by replacement of these cations. Application of dopamine (10(-5)-10(-3) M). apomorphine (10(-4) M) or beta-phenylethylamine (10(-3) M) as well as stimulation of the substantia nigra enhanced the basal release of L-[3H]glutamate. Haloperidol (10(-4) M) completely abolished the effects of apomorphine and beta-phenylethylamine, and partially abolished the effect of dopamine. The enhancement induced by apomorphine is strongly dependent on the presence of Na+ in the perfusion medium. On the other hand, apomorphine (10(-4) M) and beta-phenylethylamine (10(-3) M) inhibited the release of glutamate induced by electrical stimulation of the frontal cortex and that by K+-depolarization (the latter was shown for apomorphine). This inhibition is also abolished by haloperidol. A possible functional role of endogenous dopamine in the regulation of glutamatergic neurotransmission in rat neostriatum is discussed.

[Effect of beta-phenylethylamine on synaptosomal and glial transport of labeled neuromediators]. / Vliianie beta-fenilétilamina na sinaptosomal'nyi i glial'nyi transport mechenykh neiromediatorov.

The effect of beta-phenylethylamine (beta-PEA) on the accumulation of 14C-serotonin, 14C-glutamate and 3H-gamma-aminobutyric acid (3H-GABA) by synaptosomes of the caudate nucleus and cerebral cortex of rats and rabbits was studied for the first time. beta-PEA inhibits 14C-serotonin influx and exerts no material effect on 14C-glutamate and 3H-GABA uptake. beta-PEA inhibits the synaptosomal and glinal accumulation approximately to the same extent. The data obtained are in good agreement with the concepts of the regulatory role of beta-PEA in the transport of biogenic monoamines in the nerve ending membranes. A similarity of the inhibitory effects of beta-PEA to those of amphetamine is suggested.

[Inhibition of serotonin uptake in synaptosomes and glia cells by some pharmacological substances]. / Ugnetenie nekotorymi farmakologicheskimi Veshchestvami zakhvata serotonina sinaptosomami i kletkami glii.

The uptake of serotonin -14 C by glial cells and synaptosomes of the rabbit brain cortex was studied. The Km value of the uptake of serotonin -14 C proved to be equal (0.83 + 0.02 microM) both for synaptosomes and glial cells. Synaptosomes of the rabbit brain cortex take up serotonin -14 C twice as fast as glial cells (uptake rates were compared from protein). Among psychotropic drugs studied the tricyclic antidepressant imipramine and psychostimulant cocaine turned out the most active inhibitors of both synaptosomal and glial uptake of serotonin -14 C. The drugs in 50 microM concentration inhibit the uptake of serotonin -14 C in synaptosomes and glial cells by 90 and 75-80%, respectively.

[Effect of beta-phenylethylamine on evoked potentials of the neostriatum of rats]. / Vliianie beta-fenilétilamina na vyzvannye potentsialy neostriatuma krysy.

The effect of intraperitoneal (70 mg/kg) and local (39 ug) administration of beta-phenylethylamine (beta-PEA) on evoked potentials (EP) in the caudate nucleus upon stimulation of substantia nigra zona compacta (SNC) and frontal cortex in rats has been studied. beta-PEA, glutamate and haloperidol were injected into the caudate nucleus by means of a system consisting of a pushpull cannule and an electrode for simultaneous registration of EP. Specificity in the effect of the drugs on EP in response to stimulation of the cortex and substantia nigra was revealed. The intraperitoneal injection of beta-PEA induced, comparatively to the application, more rapid and potent decrease in the amplitude of the component (N2-P2) as a result of the substantia nigra stimulation and slightly influenced the EP amplitude in stimulation of the frontal cortex. It was established using haloperidol that the component (N2-P2) of EP in response to the substantia nigra stimulation is of dopaminergic neuron function in the nigro-neostrital system of the rat brain.

[Effect of beta-phenylethylamine on the dopaminergic system of rat brain]. / Vliianie beta-fenilétilamina na dofaminérgicheskie sistemy golovnogo mozga krys.

Using a quantitative fluorescence-histochemical analysis the dynamics of changes of the dopamine level in dopaminergic neurons of the nigro-neostriatal and mesolimbic systems of the rat brain was studied during an hour after intraperitoneal injection of beta-phenylethylamine (100 mg/kg). The temporary increase in the dopamine level in the terminals coincided with a sharp decrease in the dopamine level in the neuron bodies, and conversely, the depletion of dopamine in the terminals after 30 min was accompanied by an increase in the dopamine level in the neuron bodies. The obtained results enable a suggestion that the increase in locomotor activity of animals after injection of beta-phenylethylamine described in the literature is due to the effect of phenylethylamine on catecholaminergic brain systems, the dopaminergic systems in particular.