Acute administration of cocaine regulates the phosphorylation of serine-19, -31 and -40 in tyrosine hydroxylase

Acute cocaine can inhibit catecholamine biosynthesis by regulating the enzymatic activity of tyrosine hydroxylase via alterations in the phosphorylation state of the enzyme. The mechanisms underlying acute cocaine-dependent regulation of tyrosine hydroxylase phosphorylation have not been determined. In this study, 0, 15 or 30 mg/kg cocaine was administered intraperitoneally to rats and the phosphorylation state of tyrosine hydroxylase in the brain was examined using antibodies specific for the phosphorylated forms of serine-19, -31 and -40 in tyrosine hydroxylase. In the caudate and nucleus accumbens, cocaine dose-dependently decreased the levels of phosphorylated serine-19, -31 and -40. In the ventral tegmental area, the levels of phosphorylated serine-19, but not serine-31 and -40, were decreased by 15 and 30 mg/kg cocaine. In the amygdala, the levels of phosphorylated serine-19, but not serine-31 or -40, were decreased. The functional effects of these alterations in phosphorylation state were assessed by measuring tyrosine hydroxylase activity in vivo (accumulation of DOPA after administration of the decarboxylase inhibitor NSD-1015). Acute administration of 30 mg/kg cocaine significantly decreased l-DOPA production in caudate and accumbens but not in amygdala. These data suggest that the phosphorylation of serine-31 or -40, but not serine-19, is involved in the regulation of tyrosine hydroxylase activity by acute cocaine.     

Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 +/- 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 +/- 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge.     

Characterization of [(125) I]epibatidine binding and nicotinic agonist-mediated (86) Rb(+) efflux in interpeduncular nucleus and inferior colliculus of beta2 null mutant mice

The beta2 nicotinic acetylcholine receptor subunit null mutation eliminated most high affinity [(3) H]epibatidine binding in mouse brain, but significant binding remained in accessory olfactory nucleus, medial habenula, inferior colliculus and interpeduncular nucleus. Residual [(125) I]epibatidine binding sites in the inferior colliculus and interpeduncular nucleus were subsequently characterized. Inhibition of [(125) I]epibatidine binding by 12 agonists and six antagonists was very similar in these regions. Most acetylcholine-stimulated (86) Rb(+) efflux is eliminated in thalamus and superior colliculus of beta2 null mutants, but significant activity remained in inferior colliculus and interpeduncular nucleus. This residual activity was subsequently characterized. The 12 nicotinic agonists tested elicited concentration-dependent (86) Rb(+) efflux. Epibatidine was the most potent agonist. Cytisine was also potent and efficacious. EC(50) values for quaternary agonists were relatively high. Cytisine-stimulated (86) Rb(+) efflux was inhibited by six classical nicotinic antagonists. Mecamylamine and D-tubocurarine were most potent, while decamethonium was the least potent. Agonists and antagonists exhibited similar potency in both brain regions. Alpha-bungarotoxin (100 nm) did not significantly inhibit cytisine-stimulated (86) Rb(+) efflux, while the alpha3beta4 selective antagonist, alphaConotoxinAuIB, inhibited a significant fraction of the response in both brain regions. Thus, beta2 null mutant mice express residual nicotinic activity with properties resembling those of alpha3beta4*-nAChR.     

Correlation of Increased Grooming Behavior and Motor Activity with Alterations in Nigrostriatal and Mesolimbic Catecholamines After α-Melanotropin and Neuropeptide Glutamine–Isoleucine Injection in the Rat Ventral Tegmental Area

1. We wished to further study the behavioral effects of -melanotropin (-MSH), melanin-concentrating hormone (MCH), and neuropeptide glutamine–isoleucine (NEI).2. To this effect we administered -MSH, MCH, and NEI in the ventral tegmental area of the rat, a structure where these neuropeptides are highly concentrated. To further elucidate the biochemical mechanisms of the behavioral effect of these neuropeptides, we determined the degree of grooming behavior and the levels of catecholamines, after neuropeptide administration.3. We preselected those animals responding to the central injection of -MSH with excessive grooming behavior. We administered the neuropeptides at the dose of 1 g/0.5 L, in each side of the ventral tegmental area, bilaterally. We studied grooming behavior, locomotor activity, and total behavior scores, 30 and 65 min after administration of the peptides.4. Three groups of animals were decapitated immediately after the injection of the neuropeptides, and 30 or 65 min after injection. We measured dopamine (DA), noradrenaline (NA), and the dopac/dopamine ratio (DOPAC/DA) to determine steady state levels of catecholamines and an indirect measure of DA release and metabolism, respectively.5. Injections of -MSH produced significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with significant decreases in DA content, increases in DOPAC content, and increases in the DOPAC/DA ratio. In the caudate putamen, changes in catecholamines occurred both at 30 and 65 min after injection. In the nucleus accumbens, changes were present at 65 min after injection. Conversely, there were no alterations in NA content, either in the caudate putamen or in the nucleus accumbens, at any time after the injection.6. Injections of NEI resulted in significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with increased DOPAC/DA ratio in the nucleus caudatus but not in the nucleus accumbens. Conversely, NEI produced increased NA concentrations in the nucleus accumbens, but not in the nucleus caudatus.7. Injections of MCH did not produce significant changes in behavior or significant changes in nucleus caudatus or nucleus accumbens catecholamines.8. Our results indicate (a) There is a correlation with alterations in behavior as induced for the neuropeptides injected here, and changes in extrapyramidal catecholamines. (b) There is a correlation between alterations in behavior and increases in DOPAC/DA ratio in the nucleus caudatus. (c) There is a correlation between alterations in behavior and alterations in catecholamines in the nucleus accumbens. In the nucleus accumbens, DOPAC/DA ratio is changed after -MSH, and NA ratio is changed after NEI injection. (d) Absence of alterations in extrapyramidal catecholamines, and in particular in catecholamines in the nucleus accumbens, correlates with absence of behavioral alterations after neuropeptide administration to the ventral tegmental area.9. In conclusion, the behavioral effect of exogenous administration of neuropeptides in the ventral tegmental area is peptide-specific, and is probably associated with alterations in catecholamine metabolism and release in the nucleus caudatus and the nucleus accumbens. Both -MSH and NEI seem to stimulate the nigrostriatal DA system. While -MSH appears to stimulate the mesolimbic DA system as well, NEI may exert its actions not through the DA, but through the NA mesolimbic system. The precise contribution of DA and NA, and the relative role of the nucleus caudatus and nucleus accumbens in these behaviors remain to be elucidated.     

Glutamate-Glutamine Cycle and Aging in Striatum of the Awake Rat: Effects of a Glutamate Transporter Blocker

This study investigated the effects of aging on the actions of a specific glutamate reuptake blocker, L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC), in extracellular glutamate and glutamine in striatum of the awake rat. Microdialysis experiments were performed on young (2–3 months), middle-aged (12–14 months), aged (27–32 months) and very aged (37 months) male Wistar rats. Local infusion of PDC (1–4 mM) in striatum increased the dialysate concentration of glutamate and decreased dialysate concentration of glutamine in all the age-groups. In young rats, decreases of dialysate glutamine were correlated with increases of dialysate glutamate. The same profile glutamine/glutamate as in young rats was found in middle-aged, aged and very aged rats, which suggests that the action of glutamate on the glutamate-glutamine cycle in striatum of the awake rat is not modified as a consequence of aging. We also found a significant correlation between the increases of glutamate produced by PDC and the basal dialysate concentration of glutamine, a relationship that did show a significant change with age. Although the significance of this latter finding remains to be elucidated, it may be important to understand the changes in glutamate-glutamine cycle during aging.     

心肌肥厚大鼠心肌细胞核三磷酸肌醇受体的特征

为了研究细胞核三磷酸肌醇受体在心肌肥厚中的作用,制备了腹主动脉缩窄大鼠心肌肥厚模型、用差速离心和密度梯度离心提纯心肌细胞核,以［3H］IP3为配基,采用放射受体分析心肌细胞核膜IP3R与其配体的最大结合容量(Bmax)和解离常数(Kd)。大鼠心肌细胞核上存在IP3R、CaM和PKC激动剂PMA,能显著抑制该受体与IP3的结合(P<0.05);核外［Ca2+］也能剂量依赖的抑制细胞核IP3R与IP3的结合。腹主动脉缩窄术后4周,大鼠心肌显著肥大,伴有明显的血流动力学异常,其心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1.217和2.149倍(P<0.01)。心肌细胞核上存在IP3R,并受CaM和PMA及核外［Ca     

Peptide-based targeting of fluorophores to organelles in living cells

Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).     

Single rays as indicators of fin vestigialization in coho salmon: patterns and perspectives

Although finrays in salmon normally contain a pair of elements (biramous), finrays with a single element (uniramous) occasionally develop. Exposure to chronic stress during character development has been shown to increase fluctuating asymmetry, suggesting the occurrence of single finrays may be stress‐induced. On the other hand, single finrays may be evolutionary atavisms, reflecting fin vestigialization caused by reduced selection pressure. To assess the merits of these hypotheses, cleared and stained paired and median fins were examined for single finrays in juvenile coho salmon (Oncorhynchus kisutch Walbaum) from two compatible hatchery stocks and their reciprocal hybrids which had been exposed to different patterns of chronic temperature fluctuation throughout embryogenesis. In the median fins, uniramous secondary finrays predominated, and single primary finrays were infrequent. Single finrays in the median fins did not respond to thermal treatment or cross, suggesting the fins were evolutionarily stable and under strong developmental control. The paired fins were observed to contain only primary finrays. Frequencies of single pelvic finrays increased under thermal stress, as did fluctuating asymmetry, suggesting increased sensitivity to stress due to reduced developmental control in this fin. However, the presence or absence of single finrays in the paired fins did not alter the statistical significance of the conclusions regarding levels of fluctuating asymmetry, the number of asymmetric fish, or the contribution to meristic variation from asymmetry. Locations of single finrays in the paired fins were unaffected by thermal treatment or cross. Single finrays were most commonly observed in the trailing margins of both paired fins, a finding consistent with vestigialization theory. Frequency histograms of single pectoral finray locations revealed a second peak in the leading quarter of the fin. The esults support the hypothesis that single primary finrays are evolutionary atavisms, and that reduced selection pressure is differentially influencing the paired fins.