Accumulation of noradrenaline and its oxidation products by cultured rodent astrocytes

The accumulation of [³H]noradrenaline ([³H]NA) and its oxidation products was studied in primary cultures of cerebral astrocytes. Astroglial accumulation of radiolabeled catecholamine ([³H] NA and oxidation products) was enhanced by manganese or iron, but it was inhibited by unlabeled NA, dopamine or ascorbate. Tissue:medium ratios of radioactivity increased as extracellular [³H]NA was oxidized. When extracellular oxidation was prevented by ascorbate, as confirmed by high performance liquid chromatography with electrochemical detection, either ouabain pretreatment or nominally Na⁺-free incubation medium inhibited approximately one-half of specific [³H]NA accumulation by rat (but not mouse) astrocytes. These observations suggest that neurological responses to trace metals and ascorbate may arise from the effects of these agents on the clearance of extracellular catecholamines. Astrocytes can accumulate oxidation products of NA more rapidly than they take up NA itself, but ascorbate at physiological concentrations prevents the oxidation process in extracellular fluid. Furthermore, in the presence of ascorbate, Na⁺-dependent transport mediates a significant component of NA accumulation in rat astrocytes.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Prostaglandins E₁ and E₂ are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E₂ binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine β-hydroxylase). The only other fractions enriched in prostaglandin E₂ binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E₂ binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.     

Effects of exercise on adiponectin and adiponectin receptor levels in rats

Adiponectin reportedly reduces insulin-resistance. Exercise has also been shown to lessen insulin-resistance, though it is not known whether exercise increases levels of adiponectin and/or its receptors or whether its effects are dependent on exercise intensity and/or frequency. Catecholamine levels have been shown to increase during exercise and to fluctuate based on exercise intensity and duration. In light of this information, we examined the effects of exercise on catecholamine, adiponectin, and adiponectin receptor levels in rats. Our data showed that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 min 2 days per week, but not 5 days, per week; no such increase was observed in rats that exercised at a rate of 25 m/min for 30 min. The effects of exercise on adiponectin receptor mRNA were variable, with adiponectin receptor 1 (AdipoR1) levels in muscle increasing up to 4 times while adiponectin receptor 2 (AdipoR2) levels in liver fell to below half in response to exercise at a rate of 25 m/min for 30 min 5 days per week. We also observed that urinary epinephrine levels and plasma lipids were elevated by exercise at a rate of 25 m/min for 30 min 2 days per week. Exercise frequency at a rate of 25 m/min for 30 min correlated with AdipoR1 and AdipoR2 mRNA expression in the muscle and liver, respectively (r=0.640, p<0.05 and r=-0.808, p<0.0005, respectively). Urinary epinephrine levels correlated with AdipoR2 mRNA expression in liver tissues (r=-0.664, p<0.05) in rats that exercised at a rate of 25 m/min for 30 min. Thus, exercise may regulate adiponectin receptor mRNA expression in tissues, which might cause increases in glucose uptake and fatty acid oxidation in the muscle. The effect of exercise on adiponectin levels depends on the specific conditions of the exercise.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Specific regulation of ACTH secretion under the influence of low and high ambient temperature—The role of catecholamines and vasopressin

The response of hypothalamo-pituitary-adrenocortical (HPA) axis to different stressors depends on numerous stimulatory and inhibitory signals gathering from various parts of the brain to the hypothalamic nuclei. The present study was aimed at determining whether catecholamines (CA) and vasopressin (VP) play the role in the specific regulation of adrenocorticotropic hormone (ACTH) secretion under the influence of thermal stressors, cold (+4 °C) and heat (+38 °C), applied acutely for 1 h or repeatedly during 7 and 14 day (1 h daily). The results showed that following acute exposure to those stressors, hypothalamic dopamine (DA), noradrenaline (NA) and adrenaline (ADR) concentrations were significantly decreased as compared to non stressed controls. The prolonged exposure to either of the two stressors left hypothalamic CA concentration unaffected. The amount of pituitary VP significantly increased only under the influence of acute heat stress. Prolonged exposure to both stressors induced significant decrease in the pituitary VP content. Unlike the heat, the cold-caused changes in circulating VP did not follow those in the pituitary. The applied stressors significantly increased the amount of the pituitary V1b receptor (V1bR) mainly present at the surface of corticotrophs, depending on both duration of exposure and nature of stressor. Additionally, both cold and heat specifically induced an increase in blood ACTH. In conclusion, this study's results suggest that the role of VP in the regulation of the ACTH secretion in response to cold and heat depends on the type of stressor, whereas the role of the CA depends on the manner of exposure.     

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: In adrenal chromaffin cells, depolarization-evoked Ca²⁺ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca²⁺ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca²⁺ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K⁺-depolarization-induced Ca²⁺ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1–1 μ M ) and FTX (3:3), a synthetic FTX analogue (1 m M ), blocked the [Ca²⁺]_i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca²⁺]_i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K⁺-evoked catecholamine release, as measured by HPLC. The ω-conotoxin GVIA (0.5 μ M ) was without effect on K⁺-stimulated ⁴⁵Ca²⁺ uptake. Our results indicate that FTX blocks dihydropyridine- and ω-conotoxin-insensitive Ca²⁺ channels that, together with L-type voltage-sensitive Ca²⁺ channels, are coupled to catecholamine release.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH⁻ substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Acute stimulation of ganglionic tyrosine hydroxylase activity by secretin,VIP and PHI

We have examined the ability of a number of neuropeptides to increase tyrosine hydroxylase (TH) activity in the superior cervical ganglion in vitro. Secretin and vasoactive intestinal peptide (VIP) both increased TH activity, whereas angiotensin II, bombesin, bradykinin, cholecystokinin octapeptide, insulin, luteinizing hormone-releasing hormone, [D-Ala², Met³]enkephalinamide, motilin, neurotensin, somatostatin, and substance P produced no effects. Secretin and VIP increased TH activity with an EC₅₀ of 5 nM and 0.5 μM, respectively. The effects of these peptides were not altered by prior decentralization of the ganglia, by addition of hexamethonium (3 mM) and atropine (6 μM), or by lowering the concentration of calcium in the medium to 0.1 mM. Addition of carbachol (3 μM) potentiated the effects of both secretin and VIP on TH activity. Several gastrointestinal peptides with structural similarities to secretin and VIP were examined for their ability to increase TH activity. Glucagon, gastric inhibitory peptide and human pancreatic tumor growth hormone-releasing factor produced no effect at a concentration of 10 μM, while PHI increased enzyme activity.     

The coexistence and release of bovine pancreatic polypeptide-like immunoreactivity from noradrenergic superior cervical ganglia neurons

The coexistence of bovine pancreatic polypeptide-like (BPP) immunoreactivity within the cell bodies and axons of the superior cervical ganglia (SCG) was studied. Adjacent sections stained by the indirect immunofluorescence technique for either BPP or dopamine-β-hydroxylase (DBH) revealed that virtually all (90–95%) SCG cells contained DBH and a subpopulation (30–50%) also contained BPP. Ligation of the pre- and postganglionic nerves of the SCG demonstrated BPP-positive fibers emanating from the SCG via both axonal trunks. BPP-containing fibers were also observed entering the SCG via the preganglionic nerve. The peptide was co-released with catecholamine by electrical stimulation of the preganglionic cervical sympathetic nerve trunk, but was not effected by reserpine. The extensive distribution and unique coexistence of a PP-like peptide with peripheral sympathetic nerves suggests a neuromodulatory role in autonomic functions.