Alejandro J. Daniels

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1‐methyl‐4‐phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 rag/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte‐localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS. In cultured bovine adrenal medullary cells, MPP+ (300 μM) slightly decreased catecholamine levels, but had no effect on tyrosine hydroxylase activity or cellular protein. However, the incubation of these cells with both MPP+ and DDC (1.5 or 3.0 mM) caused large decreases in all indicators of cell viability. The enhancement of MPP+ neurotoxicity by DDC in an in vitro system, where distributional factors are limited, raises die possibility that mechanisms in addition to altered kinetics may account for DDC‐induced potentiation of MPTP neurotoxicity in vivo.

Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors.

Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.

Potentiation by reserpine and tetrabenazine of brain catecholamine depletions by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the mouse; evidence for subcellular sequestration as basis for cellular resistance to the toxicant.

Administration to mice of the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) decreased striatal dopamine and, to a lesser extent, hippocampal noradrenaline levels when measured 2 weeks after the last dose of MPTP. Reserpine and tetrabenazine, inhibitors of catecholamine vesicular transporter, potentiated the catecholamine depletions produced by MPTP in the hippocampus and striatum, respectively. These results are compatible with our hypothesis that sequestration of the toxic MPTP metabolite MPP+ (1-methyl-4-phenylpyridinium) in the catecholamine storage vesicle retards the catecholaminergic toxicity of MPTP.

Mechanisms of Toxicity and Cellular Resistance to 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine and 1‐Methyl‐4‐Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures

Abstract: Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and 1‐methyl‐4‐phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19–6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4–1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration‐dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose‐free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days’exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic “buffer.”Furthermore, under conditions where MPP+ impairs mitochondrial respiration, cells survive by increasing their reliance on glycolytic metabolism.

Neuropeptide Y mobilizes intracellular Ca2+ and increases inositol phosphate production in human erythroleukemia cells

The intracellular concentration of free Ca2+ was monitored by measuring the fluorescence of fura-2 loaded Human Erythroleukemia Cells. Neuropeptide Y (NPY) increased intracellular Ca2+ in a dose-dependent manner and the 50% effective concentration was 2 nM. Chelation of extracellular Ca2+ by EGTA did not reduce the NPY-mediated increase in cytoplasmic Ca2+, indicating that the increase in fluorescence was due to the release of intracellular Ca2+. A second dose of NPY, after intracellular Ca2+ had returned to basal levels, failed to elicit a response, indicating that the NPY receptor had undergone desensitization. In similar experiments, NPY increased the formation of inositol phosphates, suggesting that the mobilization of Ca2+ from intracellular stores in HEL cells was secondary to the generation of inositol phosphates and stimulation of phospholipase C.

GW1229, a novel neuropeptide Y Y1 receptor antagonist, inhibits the vasoconstrictor effect of neuropeptide Y in the hamster microcirculation

We studied the effect of GW1229, a novel neuropeptide Y Y1 receptor antagonists, on the vasoconstriction induced by neuropeptide Y and structurally related analogs in the hamster cheek pouch microcirculation. Changes in arteriolar diameter and microvascular conductance were assessed by intravital microscopy and measurement of sodium22 clearance. GW1229 did not affect basal vascular conductance but inhibited, concentration dependently, the reduction in arteriolar diameter and vascular conductance induced by 100 nM neuropeptide Y. GW1229 also counteracted the vasoconstrictor effect of 100 nM [Leu31,Pro34]neuropeptide Y, and that of 300 nM neuropeptide Y-[(13-36). In contrast, GW1229 had no effect on the vasoconstriction induced by noradrenaline. We conclude that the vasoconstrictor effect on neuropeptide Y in the hamster cheek pouch is mediated by neuropeptide Y Y1 receptors. The maintenance of physiological tone in this vascular bed does not involve the participation of endogenous neuropeptide Y.

Effect of intranigral Mn2+ on striatal and nigral synthesis and levels of dopamine and cofactor.

A single injection of manganese chloride into the rat substantia nigra caused a significant and reversible drop in nigral and striatal dopamine and cofactor content ipsilateral to the lesion. Maximal decrease, in both tissues, was observed 60 days after the lesion, and showed complete recovery at 90 days. In vivo striatal tyrosine hydroxylation and GTP cyclohydrolase activities were also decreased maximally at 60 days and were recovered by 90 days after the lesion. No effects were observed on the side contralateral to the injection.

Carrier-independent entry of 1-methyl-4-phenylpyridinium (MPP+) into adrenal chromaffin cells as a consequence of charge delocalization.

The administration of 1-methyl-4-phenylpyridinium (MPP+) to cultures of adrenal medullary chromaffin cells resulted in time and concentration-dependent increases in the cellular content of MPP+. Co-incubation of cells with MPP+, in the presence of desmethylimipramine (DMI), reduced but did not prevent the accumulation of the pyridinium in these cells. Similarly, DMI and MPP+ co-administration reduced but did not prevent the neurotoxicant-induced release of a cytosolic marker, lactate dehydrogenase, into the media. Molecular orbital calculations reveal that the positive charge of MPP+ is highly delocalized throughout the pyridinium ring and consequently MPP+ may be able to diffuse down concentration or charge gradients. Thus, these data provide a basis for the entry of MPP+ into cells and subcellular organelles that lack a catecholamine transporter, e.g. mitochondria.

Discovery of small molecule agonists for the bombesin receptor subtype 3 (BRS-3) based on an omeprazole lead.

Starting from a weak omeprazole screening hit, replacement of the pyridine with a 1,3-benzodioxole moiety, modification of the thioether linkage, and substitution of the benzimidazole pharmacophore led to the discovery of nanomolar BRS-3 agonists.

Neuropeptide Y Y5 receptors inhibit kindling acquisition in rats

Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood-brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4-5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4-5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4-5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation.