Faculty Bibliography

The rates of protein synthesis can be measured by a variety of methods including pulse labeling, massive precursor administration, Scornik method, continuous feeding of labeled precursor, infusion, and pellet implantation. Each technique has some advantages and disadvantages. Massive precursor administration and infusion are the most widely used. The advantage of massive precursor administration is its simplicity, however, the amino acid concentration used is much higher than physiological levels. Infusion, however, is much more complicated as a technique and requires complicated calculations. The synthesis rates can also be calculated from degradation curves. Some of the above techniques can be used both in vivo and in vitro, and also for different organs (Shahbazian et al. (1987), Int. J. Dev. Neurosci., 5: 39-42). The brain has rapid rates of protein synthesis both in vivo and in vitro, the latter being much lower for adults

Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-alpha-benzoate binding in mouse brain synaptosomes

The behavioral and physiological effects of repeated nicotine administration are complex; sedation and hypothermia are present early but become attenuated while locomotor activity increases. Maximal blood levels and behavioral changes occur within 10 min of s.c. injection. We examined the effects of 10 nicotine injections (0.8 mg/kg) in 14 days on the levels of brain amines following challenge with either saline or nicotine on the 15th day. Dopamine, DOPAC, HVA, 3-methoxytyramine, norepinephrine, 5-hydroxytyramine, and 5-HIAA were measured in the frontal cortex, olfactory tubercle, nucleus accumbens, caudate-putamen, substantia nigra and ventral tegmental area. Ten minutes after nicotine was given to rats that had previously received only saline the levels of dopamine and its metabolite DOPAC indicated an increase in dopamine turnover in the nucleus accumbens. Of the areas examined the accumbens was the most sensitive to nicotine, with few significant amine changes in other regions. Twenty-four hours after the last nicotine injection the levels of dopamine and its metabolites indicated a sustained decrease in dopamine turnover in the accumbens induced by repeated administration. Following repeated nicotine a nicotine challenge still induced an acute increase in dopamine turnover in the accumbens, but the response was less than in animals not previously given nicotine. The results confirm earlier studies indicating that the accumbens is a major site of nicotine action

Specific binding of (3H) naloxone to opioid receptors in frog (Rana esculenta) brain membranes was irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Saturation analysis of (3H) naloxone binding revealed a marked reduction in the number of ligand binding sites after N-ethylmaleimide treatment. Pretreatment of the membranes with unlabelled opioid ligands, i.e. naloxone, morphine, or the kappa selective dynorphin (1-13), and sodium ions resulted in considerable protection of (3H) naloxone binding against the N-ethylmaleimide blockade

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10-18 areas, and increased in only 1-3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity

We examined the regional distribution in rat brain of calpain II, the calcium-activated neutral proteinase maximally active in the presence of 2mM Ca(2+), and of calpastatin, the endogenous inhibitor of the enzyme. A single-step chromatographic procedure was used to separate the constituents before determination. With [methyl-(14)C]?-casein as substrate, specific activity of calpain II was lowest in the cortex. The activity in the other areas tested was 10-50% higher, except pons-medulla > spinal cord > cerebellum > hypothalamus > striatum > hippocampus > cortex. When calpain II activity of the various areas was tested with endogenous brain protein substrates (the neurofilament proteins NF 200, 150 and 70 [NFT], glial fibrillary acidic protein [GFAP], desmin and actin), activity in each substrate was seen to be heterogenous, with a slightly different pattern of heterogeneity for each. The pons-medulla again was the highest in activity, but the cortex was usually not the lowest. Calpastatin was somewhat more evenly distributed in the various brain regions examined. Comparison of the enzyme activity of the crude supernatant with that in the purified fraction showed that at least 50% of the activity in the supernatant was inhibited by the calpastatin present. The regional differences in the substrate specificity of neutral proteolytic activity indicate that in vivo protein metabolism is influenced regionally by heterogeneity both in enzyme and in substrate distribution

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

Amiloride, an inhibitor of the Na+/Ca2+ exchanger, blocked the hydrolysis of inositol phospholipids in mouse cerebrocortical slices induced by the sodium channel activator veratridine, by KCl, or by the sodium ionophore monensin; there was no inhibition by A 23187, a Ca2+ ionophore, or by serotonin. It is concluded that agents that increase intracellular Na2+ stimulate inositide hydrolysis by an indirect effect via Na+/Ca2+ exchange

[3H]Mazindol was used to label the dopamine uptake complex in mouse striatum in vitro in the presence and absence of metaphit, an isothiocyanate derivative of phencyclidine. In some experiments, metaphit was present in the incubation fluid throughout the procedure; in other experiments it was eliminated by several washings and centrifugations. It was found that after removal of the metaphit by washing and centrifugation, the mazindol binding was not restored. Membranes that were pretreated with metaphit and washed had a lower density of mazindol binding sites than control membranes; the remaining mazindol sites had the same afinity for [3H]mazindol. These findings are in agreement with the previous studies on [3H]cocaine and [3H]methylphenidate binding. The following observations support that metaphit is irreversibly acting and not slowly dissociating from the mazindol recognition sites of the dopamine uptake carrier complex: 1) Metaphit did not change the off-rate of [3H]mazindol binding, arguing against an allosteric action at a distinct site. 2) The presence of cocaine protected the mazindol binding sites from the action of metaphit, supporting binding of metaphit and mazindol to the same site. 3) Nine hours after metaphit pretreatment and removal, the degree of inhibition of mazindol binding was the same as immediately after pretreatment, consonant with an irreversible effect of metaphit. 4) The potency of metaphit in inhibiting mazindol binding was greater under slightly alkaline conditions, consistent with acylation of the mazindol sites. Furthermore, it was found that intracerebroventricular application of metaphit did not result in a decrease in the binding of [3H]mazindol 5 hr after the administration

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