Harvey L. Wiener

Chronic L-deprenyl-induced up-regulation of the dopamine uptake carrier

L-Deprenyl is an inhibitor of monoamine oxidase B and dopamine uptake. Chronic L-deprenyl (10 mg/kg i.p., twice weekly for 4 weeks) was shown to inhibit monoamine oxidase B activity by 89%, and also to induce an up-regulation of the [3H]mazindol binding site associated with the striatal dopamine uptake carrier. Scatchard analysis indicated a 56% increase in the maximal number of [3H]mazindol binding sites in chronic L-deprenyl animals, but no effect on the affinity of these binding sites. The ability of L-deprenyl to up-regulate the [3H]mazindol-associated dopamine uptake carrier appears to be a result of its role as a dopamine uptake inhibitor.

Chronic treatment of aged mice with L-deprenyl produces marked striatal MAO-B inhibition but no beneficial effects on survival, motor performance, or nigral lipofuscin accumulation

Male C57BL/6J mice were provided I-deprenyl (at 0, 0.5 mg/kg or 1.0 mg/kg per day) in their drinking water beginning at 18 months of age. A battery of motor tests, including open-field, tightrope, rotorod, inclined screen, runwheel, and rotodrum tests, was administered before treatment and then 6 months later at 24 months of age. A subsample of mice was retested again at 27 months of age. An untreated group of 9-month-old mice served as young controls. Deprenyl treatment reduced striatal MAO-B activity by up to 60% after 6 months on treatment but had no significant effects on striatal catecholamine levels. No significant effects of deprenyl treatment were observed on body weight, fluid intake, or survival of the mice. Chronic deprenyl treatment also did not affect motor performance in any test, except rotodrum performance at 27 months of age, which was significantly better in the 1.0 mg/kg group treated group compared to controls. No age or deprenyl effects were observed with respect to cell counts in the substantia nigra. However, nigral cells containing lipofuscin increased with age, but this neurohistochemical parameter was also unaffected by deprenyl treatment.

Sertraline and cocaine-induced locomotion in mice. I: Acute studies

The present study assessed the behavioral and pharmacokinetic interaction between the serotonin uptake blocker sertraline and cocaine in C57BL/6ByJ mice. Pretreatment with sertraline (1–32 mg/kg IP) did not affect the total amount of spontaneous locomotor activity during 50 min following administration of cocaine (15–40 mg/kg IP). At doses of sertraline (16 and 32 mg/kg) much higher than those found to inhibit ex vivo neuronal uptake of serotonin by 50% (1–2 mg/kg), the peak of cocaine-induced locomotor activity was shifted towards a later time. A similar effect was seen after pretreatment with serotonin uptake blockers other than sertraline, and also after desipramine. Sertraline (16 and 32 mg/kg), given 60 min prior to cocaine, did not affect levels of cocaine in brain and plasma, and cocaine administration did not alter the brain level of sertraline. Although female mice were more responsive to cocaine than male mice, they were not different in their response to sertraline.

Correlation between cocaine-induced locomotion and cocaine disposition in the brain among four inbred strains of mice

BALB/cByJ, C57BL/6ByJ, CXBH/By, and CXBK/By mice differed in their locomotor response to cocaine measured 1-10 min after administration of 25 mg/kg IP of the compound. These differences were paralleled by differences in the disposition of cocaine (measured at 12 min) in the brain. Among all individual animals taken together, there was a significant correlation between locomotor stimulation and the brain concentration of cocaine. These results suggest that the differences between strains in their locomotor responsiveness to cocaine are determined, in part, by the disposition of cocaine in the brain following IP administration of cocaine.

Differential effects of daily administration of cocaine on hepatic and cerebral glutathione in mice.

Twenty-four hours after acute administration of cocaine HCl (25 mg/kg, i.p.) to male C57BL/6ByJ mice, there was no hepatotoxicity as measured by plasma aspartate aminotransferase (AST) activity. In contrast, daily administration of cocaine (25 mg/kg, i.p.) for 14 days induced marked hepatotoxicity, as characterized by a greater than 400% increase in plasma AST activity when assayed 24 hr after the last injection. Concomitantly, the liver had increased levels of cysteine, gamma-glutamylcysteine, glutathione, cysteinylglycine, glutamate, methionine, taurine, and aspartate. The effect appeared to be selective for compounds of the glutathione metabolic pathways, because repeated cocaine exposure did not affect other amino acids such as leucine, isoleucine, phenylalanine, serine, and valine. There was a positive correlation between the magnitude of the elevation of cysteine and the extent of liver damage. Daily cocaine administration did not affect striatal or frontal cortex glutathione. A final cocaine challenge (50 mg/kg, i.p.) did not affect either hepatic or cerebral glutathione metabolism. The increase in hepatic cysteine and glutathione upon daily cocaine administration is a potentially important compensatory mechanism against cocaine-induced hepatotoxicity.

Oxidative Metabolism of Cocaine: Comparison of Brain and Liver

Abstract Norcocaine (NC) and N-hydroxynorcocaine (NHNC), products of the oxidative metabolism of cocaine, were examined in plasma, brain, and liver of mice injected intraperitoneally with cocaine. Plasma levels of NHNC were altered in vivo by inhibiting esterase activity with diazinon and chloral hydrate or activating esterase activity with phenobarbital, and activating the microsomal P-450 system with phenobarbital. Changes in plasma concentrations of NHNC resulted in similar changes in brain, which were often different from those in liver. After intracisternal administration of cocaine to mice, no appreciable amount of NC or NHNC could be detected in brain; the same results were obtained upon intracisternal and intraventricular administration to rats. Microsomal preparations from mouse brain were found to be considerably less active than those from liver in converting NC to NHNC. We conclude that the cerebral oxidative metabolism of cocaine is not appreciable and that most of the NC and NHNC found in the brain after systemic cocaine administration is derived from plasma rather than formed centrally by brain microsomal enzymes.

Determination of radioligand specific activity using competition binding assays

Radioligand binding assays are routinely utilized in laboratories throughout the world to study receptors and their related binding sites, carrier proteins, and enzymes. To accurately estimate equilibrium binding parameters, such as the equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax), the investigator must know the correct value of the specific activity of the radioligand. If the specific activity is overestimated the Kd and Bmax values will be underestimated, while underestimation of the specific activity results in an overestimation of the Kd and Bmax. The present communication describes a simple and rapid method for determining the specific activity of a radioligand using homologous competition binding assays. Performing the competition assays at two or more different concentrations of the radioligand allows the specific activity to be determined from the IC50 values without the need of analytical methods to quantify minute amounts of the radioligand. In addition to providing the specific activity, use of this method estimates the Kd for the radioligand. This method was utilized to determine the specific activity and Kd for two blockers of the dopamine uptake carrier, [3H]GBR-12935 and [3H]-CFT, which share a common binding site in the striatum.

Interactions between responses mediated by activation of adenosine A2 receptors and α1-adrenoceptors in the rabbit isolated aorta

1 This paper describes aspects of the functional antagonism between the responses mediated by activated α1‐adrenoceptors and adenosine A2 receptors in the adventitia‐ and endothelium‐denuded aorta of the rabbit. 2 Adenosine A2 receptor agonists relaxed aortic rings pre‐contracted with phenylephrine. The relaxation response was agonist concentration‐dependent and saturable. The respective contractile and relaxation responses were stable, reproducible, and reversible. 3 Increasing the phenylephrine concentration caused a progressive attenuation of the action of adenosine A2 receptor agonists, consisting of a decreased maximal response and a dextral shift of the adenosine agonist concentration‐response curve. This functional antagonism could be completely reversed upon removal of adenosine by either the addition of adenosine deaminase or by wash‐out of the adenosine agonist from the tissue. The relaxation response to the adenosine A2 receptor partial agonists, N6‐cyclohexyladenosine and R‐(−)‐N6‐(2‐phenylisopropyl)adenosine, was abolished at higher phenylephrine concentrations (e.g. 30 EC50). 4 A 1000 fold increase in the adenosine concentration was required to shift the value of the EC50 of phenylephrine six fold, while a similar increase in the value of the EC50 of adenosine could be elicited by only a 32 fold increase in the phenylephrine concentration. A 30 fold increase in the phenylephrine concentration shifted the value of the EC50 of 5′‐N‐ethylcarboxamidoadenosine four fold. 5 Analysis of the functional antagonism between the responses mediated by these receptors using the Black & Leff (1983) operational model of agonism allowed for the estimation of the agonist dissociation constant, KA, and the apparent efficacy, τ, for both phenylephrine and adenosine A2 receptor agonists. Increasing the concentration of phenylephrine reduced the value of τ for adenosine agonists in a concentration‐dependent and saturable manner. Similarly, increasing the concentration of adenosine reduced the value of τ for phenylephrine in a concentration‐dependent and saturable manner. The phenylephrine KA value obtained by the method of functional antagonism (1.9 μm) was similar to that obtained by the receptor inactivation method (2.1 μm). 6 Partial occlusion of the α1‐adrenoceptor by the alkylating agent, dibenamine, demonstrated that the magnitude of the adenosine A2 receptor‐mediated relaxation was inversely proportional to the number of functional α1‐adrenoceptors. 7 It is concluded that the magnitude of functional antagonism is proportional to the stimulus elicited through either receptor. We propose that this tissue preparation and pair of receptors is a good model to study quantitative aspects of functional antagonism between activated receptors.

Ascorbic Acid Inhibits [3H]sch-23390 Binding to Striatal Dopamine D1 Receptors

The present study describes the inhibition of [3H]SCH-23390 binding to striatal dopamine D1 receptors in the presence of ascorbic acid. Specific [3H]SCH-23390 binding was maximally inhibited by 0.1 mM ascorbic acid. As determined by Scatchard analysis the binding in the presence of 0.01, 0.1, or 10 mM ascorbic acid was consonant with non-competitive inhibition with a 26%, 38%, or 19% decrease, respectively, in the maximal number of binding sites; the affinity of these binding sites was not affected. Inhibition of [3H]SCH-23390 binding by ascorbic acid was reversible; striatal homogenates incubated with 0.1 mM ascorbic acid and subsequently washed free of ascorbic acid had the same Scatchard parameters as untreated preparations.