The effect of cotinine on nicotine- and cocaine-induced dopamine release in the nucleus accumbens.

Cotinine is the major metabolite of nicotine. Nicotine is rapidly metabolized and has a short half-life, but cotinine is metabolized and eliminated at a much lower rate. Because of the resulting increase with time in the cotinine to nicotine ratio in the body, including in the brain, it is of interest to examine the effect of cotinine on nicotine-induced changes. In studies on conscious, freely-moving rats, intravenous administration of either nicotine or cocaine induced the release of dopamine in the nucleus accumbens, as assayed by microdialysis. Prior intravenous administration of a high dose of cotinine (500 microg/kg) inhibited this nicotine- or cocaine-induced dopamine release. The action of cotinine does not seem to occur through its effect on the metabolism of nicotine or on its binding at the receptor site, because cotinine, unlike nicotine, does not affect the binding of the nicotinic ligand cytisine. The findings suggest that cotinine affects a putative component of the reward mechanism, and as such could have therapeutic value.

Mutations that increase acidity enhance the transcriptional activity of the glutamine-rich activation domain in stage-specific activator protein.

Sea urchin stage-specific activator protein (SSAP) activates transcription of the late H1 gene at the mid-blastula stage of development. Its C-terminal 202 amino acids form a potent glycine/glutamine rich activation domain (GQ domain) that can transactivate reporter genes to levels 5-fold higher than VP16 in several mammalian cell lines. We observed that, unlike other glutamine-rich activation domains, the GQ domain activates transcription to moderate levels in yeast. We utilized this activity to screen in yeast for intragenic mutations that enhance or inhibit the transcriptional activity of the GQ domain. We identified 37 loss of function and 23 gain of function mutants. Most gain of function mutations increased the acidity of the domain. The most frequently isolated mutations conferred enhanced transcriptional activity when assayed in mammalian cells. These mutations also enhance the ability of SSAP to up-regulate the late H1 promoter in sea urchin embryos. We conclude that the GQ domain fundamentally differs from other glutamine-rich activators and may share some properties of acidic activators. The ability of acidity to enhance SSAP-mediated transcription may reflect a mechanism by which phosphorylation of SSAP activates late H1 gene transcription during embryogenesis.

Receptor systems participating in nicotine-specific effects.

It is generally accepted that self-administration of drugs is prompted primarily by a reward system driven by an increase in extracellular dopamine in the nucleus accumbens. Recent findings that dopamine increase in the accumbens can be caused by many other factors, among them stress, suggest a more complex mechanism, and possibly differences in the reward system for different compounds. In the present paper we compare the effects of receptor-specific antagonists on the increase of dopamine induced by nicotine with that induced by cocaine in the nucleus accumbens in conscious rats. The compounds alone or together were injected intravenously, and dopamine level changes were measured via microdialysis. When administered together the effect of nicotine and cocaine on the level of dopamine in the accumbens was additive. Apparently there is some interaction between the two compounds, since nicotine had no effect after combined nicotine and cocaine administration. Perhaps the available dopamine pool was exhausted by the prior administration. The nicotinic antagonist mecamylamine, the muscarinic antagonist atropine, and the NMDA glutamate receptor antagonist MK-801 each blocked nicotine-induced dopamine release in the accumbens, indicating the participation of more than a single receptor system in the nicotine-induced effect. These three antagonists did not inhibit cocaine-induced dopamine increase in the accumbens, indicating the lack of a role of these receptors in the cocaine effect under our experimental conditions. SCH-23390, a dopamine D1 receptor antagonist, blocked both nicotine- and cocaine-induced effects, indicating the possible role of this receptor in these reward effects. The results indicate that there are differences in some of the receptors mediating the central effects of the two compounds examined, nicotine and cocaine, although each influences dopamine levels, and that the two compounds interact.

Changes in brain protease activity in aging.

We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age-related changes in calpain specificity and substrate susceptibility were measured. Several age-related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).

Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats.

The effect of short-term food deprivation on glutathione (GSH) and amino acid levels in brain regions of young and aged rats was compared with changes observed in liver. Animals aged 3 months and 24 months were deprived of food for 48 h. GSH and amino acid levels from cerebral cortex, cerebellum, pons medulla, and liver were assayed and compared with levels in animals of the same age fed normal diets. In liver in both young and old rats, GSH levels fell 30%, from 13 mumol/g tissue to 8.7 mumol/g tissue. Significant changes were observed in other amino acids, including an increase of 30-50% in methionine, glycine, and glutamine, and a decrease of 30-50% in alanine in liver of both young and aged rats, and a 4-fold increase in taurine in young. In brain, little change was observed upon food deprivation. No decrease was observed in GSH, and only small changes were observed in other amino acids. In the aged animal aspartate, glutamate, and alanine levels were slightly lower; tyrosine in cerebellum was reduced by 30%, and both glycine and tyrosine in the pons medulla were reduced by 20-30%. In the brain areas examined, levels of GSH ranged from 1-2 mumol/g in young and 0.8-1.4 mumol/g in old; with levels in pons medulla being lower than those in cerebral cortex. In brain, in contrast to liver, levels were scarcely affected by short-term food deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diet on tissue protease activity.

Rats 1, 3, 12, and 24 months old were fed diets low in protein (8% casein), and proteolytic activity in tissue from brain, liver, and lung was determined. After a low-protein diet was fed for 4 weeks to 1-month-old rats, there was a significant increase in cathepsin D activity in liver, and calpain activity was increased in lung. Little change was seen in proteolytic activity in brain. In 12-month-old rats, there was an increase in cathepsin D activity in brain and liver. In 24-month-old rats, cathepsin D activity in the liver and calpain activity in lung were increased. There was no change in proteolytic activity in the brain. When animals were fed diets supplemented with fatty acids or antioxidants for 2 months, in 3-month-old rats calpain activity was increased in brain but decreased in lung. Cathepsin D activity was significantly increased in young and adult animals in brain and in liver. These observations suggest that diet changes result in significant alteration in tissue calpain and cathepsin D levels, and possibly activity, in vivo. Generally, changes are greater for cathepsin D than for calpain, and are smaller in brain than in other tissues.

Proteolytic activity is altered in brain tissue of rats upon chronic exposure to ozone.

Tissue from pons medulla of rats exposed in vivo to various levels of ozone was assayed for calpain and cathepsin D activity. Chronic exposure to ozone increased calpain activity, which was 35% to 46% higher in the homogenates of animals exposed to 1.0 ppm ozone than in those of animals exposed to 0.5 ppm ozone or of controls. An increase in activity of 26% was also observed in the soluble supernatant. The increase in activity did not seem to be caused by ozone effects on calpastatin. Addition of 32 mM carnitine to the incubation mixture increased total activity 3-4 fold, making the differences in activity proportionately smaller. Cathepsin D activity was little altered. Changes in calpain activity and in the generation of free oxygen radicals have been implicated in the aging process, long-term exposure to ozone may magnify changes. Ozone exposure may cause changes in brain protein metabolism.

Peroxidative stress effects on calpain activity in brain of young and adult rats.

Three hours after administration of the pro-oxidant 2-cyclohexen-1-one, calpain activity was significantly reduced in the brain of young rats, but not in the brain of adult rats, and cathepsin D activity remained unchanged. Addition of isovalerylcarnitine to the incubation medium increased calpain activity 5-7-fold, counteracting the effect of the pro-oxidant.

Dopamine releasing effect of phenylbiguanide in rat striatal slices.

The present study explored the mechanisms underlying the dopamine releasing effect of phenylbiguanide, a compound commonly used as a 5-HT3 receptor agonist. Phenylbiguanide, and also serotonin and 2-methyl-serotonin, enhanced the outflow of radioactivity from superfused rat striatal slices preloaded with [3H]dopamine. The presence of the dopamine uptake blocker nomifensin prevented the increase in outflow. The effect of phenylbiguanide was not antagonized by 5-HT3 receptor antagonists, did not require the presence of Ca2+ in the superfusion buffer, and also occurred in reserpinized preparations with depleted dopamine stores. Phenylbiguanide caused a greater shift in the distribution of superfusate radioactivity from DOPAC to dopamine than did nomifensin. All these results are in agreement with an exchange mechanism by which phenylbiguanide promotes the efflux of dopamine by operation of the uptake carrier in the reversed direction. In consonance, phenylbiguanide, and also serotonin and 2-methyl-serotonin, inhibited the binding of [3H]CFT to dopamine uptake sites, although the rank order for promoting outflow, serotonin greater than phenylbiguanide greater than 2-methyl-serotonin, differed from that for inhibiting [3H]CFT binding to dopamine uptake sites, 2-methylserotonin approximately serotonin greater than phenylbiguanide. The present results raised the possibility that phenylbiguanide has an additional activity in releasing vesicular dopamine into the cytoplasmic pool.