Cocaine: On-line analysis of an accumbens amine neural basis for psychomotor behavior

Dose-response studies on subcutaneous cocaine were done to ascertain its effects in nucleus accumbens in dopaminergic and serotonergic neuronal circuitry in the behaving rat with in vivo voltammetry. Simultaneously, and at each dose of cocaine, unconditioned psychomotor stimulant behavior induced by cocaine was studied in terms of multiple concurrent measures of spontaneous behavior and by activity patterns of locomotion. Time course studies showed that the neurochemical effects of cocaine (10, 20, and 40 mg/kg SC) significantly (p<0.001) increased accumbens synaptic concentrations of dopamine (DA) and concurrently and significantly (p<0.0001) decreased accumbens synaptic concentrations of serotonin (5-HT) in a dose response manner. Simulataneous behavioral time course studies showed that cocaine (10, 20, and 40 mg/kg SC) significantly (p<0.0001) increased ambulations (locomotor activity), fine movements (stereotypic movements of sniffing and grooming) and rearing behavior, while significantly decreasing agoraphobic behavior, as measured by a statistically significant increase in central ambulations (p<0.0001). The high dose of cocaine (40 mg/kg SC) significantly increased fine movements over those produced by the lower doses of cocaine (p<0.0002). One import of the findings is that the DA and 5-HT biogenic amine response occurs in a behavioral paradigm of psychomotor stimulation, which is a known measure of reinforcement. Another is that the biogenic amines DA and 5-HT are affected by cocaine in this reinforcement paradigm with exactly opposite directionality. Finally, acute cocaine administration is shown to produce a dose response inhibition of agoraphobia (fear),w hich is highly correlated (=.983, p<0.01) with the opposing effects of cocaine on the accumbens biogenic amines, DA and 5-HT.     

Modulation of the mesolimbic dopaminergic system by β-endorphin-(1-27) as assessed by microdialysis

In the present study we used in vivo microdialysis to examine the influence of β-endorphin-(1-27) (β-EP-(1-27) upon β-endorphin (β-EP)-induced dopamine (DA) release in the nucleus accumbens of anesthetized rats. Microdialysis probes were inserted into the nucleus accumbens and peifusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), using a reversed-phase HPLC system with electrochemical detection. Intracerebroventricular (i.c.v.) administration of β-EP-(1-27) (5-20 μg) resulted in a dose-dependent increase in DA release which was smaller than the β-EP-induced DA release, whereas metabolite levels were not altered. Pretreatment with β-EP-(1-27) (5-20 μg) significantly altered the β-EP (5 μg)-induced increase in DA release. These results indicate that β-EP-(1-27) antagonizes the β-EP-induced release of DA in the nucleus accumbens. In addition to its antagonistic properties at the β-endorphin binding site, β-EP-(1-27) appears to be a partial agonist, inducing increased DA release. These findings suggest a regulatory function for this naturally occurring β-EP fragment within the mesolimbic system.     

The effect of γ-acetylenic GABA, an enzyme-activated irreversible inhibitor of GABA-transaminase, on dopamine pathways of the extrapyramidal and limbic systems

γ-Acetylenic GABA (100 mg/kg i.p.) inhibited GABA-transaminase activity and caused a several-fold increase in the concentration of GABA in rat brain. This increased GABA concentration was associated with a decreased rate of dopamine depletion following -methyl-p-tyrosine treatment and a decrease in homovanillic acid in extrapyramidal and limbic structures suggesting a decrease in dopamine turnover in both pathways. In addition, γ-acetylenic GABA injected into the ventral mesencephalic tegmentum decreased dopamine turnover in the mesolimbic forebrain. These results are consistent with a modulatory function of GABAergic neurons on extrapyramidal and limbic dopamine pathways. Inhibitory effects on dopaminergic functions of the extrapyramidal and limbic systems were also indicated by the amphetamine and apormorphine-induced ipsilateral turning after unilateral substantia nigral injections of γ-acetylenic GABA and by the attenuation of dopamine-induced hypermotility after bilateral injections of γ-acetylenic GABA into the nucleus accumbens.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

JO1784, a novel σ ligand, potentiates [H]acetylcholine release from rat hippocampal slices

JO1784, a potent and specific σ ligand, potentiated the KCl-evoked release of [³H]acetylcholine (ACh) from rat hippocampal slices superfused in vitro at 10 and 30 μM. This effect was stereospecific and was antagonized by the presence of haloperidol (0.3 μM). Under similar conditions, (+)-SKF 10,047 also had a potentiating effect whereas di-o-tolyl-guanidine had an inhibitory effect. Phencyclidine was devoid of activity up to a concentration of 30 μM. These results show that σ compounds display differential effects on evoked [³H]ACh release in rat hippocampal slice preparations.     

Blocking effect of serotonin on β-adrenoceptor activity in follicle-enclosed Xenopus oocytes

The effects of serotonin (5-HT) on a β-adrenergic response were studied, using the voltage clamp technique, in Xenopus laevis oocytes surrounded by their follicular cells. noradrenaline induced marked hyperpolarization, with a specific increase in the permeability of the membrane toward K⁺. Application of 10 μM 5-HT had little effect on the resting membrane, but significantly depressed the response to 0.1 μM noradrenaline. The dose-response curve for noradrenaline showed a parallel shift to the right in the presence of 5-HT, suggesting that 5-HT competes with noradrenaline for common binding sites at the β-adrenoceptor.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Theoretical studies on the selective mechanisms of GSK3β and CDK2 by molecular dynamics simulations and free energy calculations

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase which is widely involved in cell signaling and controls a broad number of cellular functions. GSK3 contains α and β isoforms, and GSK3β has received more attention and becomes an attractive drug target for the treatment of several diseases. The binding pocket of cyclin‐dependent kinase 2 (CDK2) shares high sequence identity to that of GSK3β, and therefore, the design of highly selective inhibitors toward GSK3β remains a big challenge. In this study, a computational strategy, which combines molecular docking, molecular dynamics simulations, free energy calculations, and umbrella sampling simulations, was employed to explore the binding mechanisms of two selective inhibitors to GSK3β and CDK2. The simulation results highlighted the key residues critical for GSK3β selectivity. It was observed that although GSK3β and CDK2 share the conserved ATP‐binding pockets, some different residues have significant contributions to protein selectivity. This study provides valuable information for understanding the GSK3β‐selective binding mechanisms and the rational design of selective GSK3β inhibitors.     

An insulin analogue with γ-amino butyric acid substitution for A13Leu-A14Tyr

Abstract: An insulin A chain analogue, [A13–14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, γ-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13–14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50%in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C–N peptide bond by an unnatural C–C bond may have general meaning in structure and function studies of other proteins.     

Role of positively charged residues on the polar and non‐polar faces of amphipathic α‐helical antimicrobial peptides on specificity and selectivity for Gram‐negative pathogens

We have designed de novo and synthesized eight 26‐residue all D‐conformation amphipathic α‐helical cationic antimicrobial peptides (AMPs), four with “specificity determinants” which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin‐sensitive, and eight of which were methicillin‐resistant. The four AMPs without specificity determinants are extremely hemolytic. In contrast, the four AMPs with specificity determinants had dramatic improvements in therapeutic indices showing the importance of specificity determinants in removing eukaryotic cell toxicity. The specificity determinants combined with the location of positively charged residues on the polar face provide Gram‐negative pathogen selectivity between A. baumannii and S. aureus. Specificity determinants maintain excellent antimicrobial activity in the presence of human sera, whereas the AMPs without specificity determinants were inactive. This study clearly shows the potential of amphipathic α‐helical AMPs with specificity determinants as therapeutics to replace existing antibiotics.