Aminopeptidase activity levels during axonal and dendritic growth in the rat brain.

Brain aminopeptidase activity has been suggested as a candidate for the regulation and biotransformation of several neuropeptides. In this paper, changes in Lys- and Leu-aminopeptidase activities in rat brain hemispheres, cerebellum and medulla were examined in 1-, 3-, 5- and 7-days postnatal subjects. Aminopeptidase activities were studied by measuring the rate of hydrolysis of the artificial chromogenic substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities show a decrease on the 3rd day of life followed by increases on the 5th and 7th day postbirth. It is suggested that these activities could play a part in the neurochemical changes that take place during axonal and dendritic growth in the rat brain.     

Serum and brain aminopeptidase activities in cyclic rats

Research into the functional role of brain peptides is performed, in part, by studying peptidase enzymes which might be involved in the processing or inactivation of the brain peptides. Aminopeptidase activity has been proposed as a candidate for the regulation of the degradation of these peptides. In this paper, acid (Asp-) and basic (Arg-) aminopeptidase activities were studied in several brain regions and in the serum during the estrous cycle of the rat. Asp-aminopeptidase activity did not significantly change at any point. However, a marked rise was found in Arg-aminopeptidase activity in all the brain areas studied and the serum during the proestrus. It is suggested that this activity plays a role in the hormonal changes that take place during the cycle, possibly in regulating the activity of several neuroactive peptides.     

Angiotensinase A in the renomedullary interstitial cells

Aminopeptidase A (angiotensinase A; APA) was demonstrated by histochemical means in the renomedullary interstitial cells of the golden hamster, rat, guinea pig and hare. The highest APA activities were shown in the interstitial cells of the hamster. Ultracytochemical studies of the kidney medulla of the hamster indicated that APA is localized mainly on the cell membranes of interstitial cells. Reaction product was also observed intracellularly in the nuclear membrane region. Besides the interstitial cells, APA was demonstrable ultracytochemically in the endothelial cell membranes of medullary blood vessels. Biochemical studies of APA in the renal medulla of experimental animals (sodium loaded and sodium depleted hamsters) have shown that significant changes of APA activities were available only after sodium loading, namely a decrease of APA activities in comparison to control animals. - APA through its degradation of angiotensin, is presumed, to have a bearing on the angiotensin induced prostaglandin biosynthesis of renomedullary interstitial cells.     

Histochemistry of proteases in ependyma, choroid plexus and leptomeninges

Aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Regional Distribution of Kininase in Rat Brain

Kininase activity, which inactivates kinins, was measured in seven regions of the rat brain (i.e., the cerebral cortex, cerebellum, striatum, midbrain, hippocampus, hypothalamus, medulla oblongata), and in the spinal cord with a bioassay method using bradykinin as the substrate. Specific kininase activities in the cerebellum and striatum were higher than those in the other five regions or the spinal cord. Angiotensin-converting enzyme activity, which was measured fluorometrically using Hip-His-Leu as substrate, showed high activity in the striatum and cerebellum. These findings suggest that the presence of high concentrations of peptidases plays a role in the degradation of kinins and/or other peptides in these areas.     

A calmodulin endoproteinase from mitochondrial membranes

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.     

Changes of monoamine and TRH contents in naloxone induced inhibited development of rat cerebrum and cerebellum

We have studied effects of an opioid antagonist, naloxone (NLX) on rat brain development. Newborn rats were given daily subcutaneous injection of 1 or 50 mg/kg NLX from birth until weaning (day 21). The 28 day-old rats were examined their brain development. Both doses of NLX reduced the cerebral and cerebellar weights of rats but the body weight loss was significant only in the higher dose (50 mg). However, there were neither morphological changes in the central nervous system nor movement disorders such as abnormal gait and involuntary movements in naloxone treated rats (NLX-rats). We found that serotonin content was decreased significantly in the cerebral cortex and medulla while it was significantly increased in the pons and striatum of NLX-rats. Noradrenaline was decreased significantly in the medulla while it was increased in the pons of the NLX-rats. In contrast, the concentrations of these monoamines did not show any changes in cerebellum and hippocampus of NLX-rats. On the other hand, thyrotropin-releasing hormone (TRH) was significantly decreased in cerebellum and hippocampus of NLX-rats, while it did not show any changes in cerebral cortex, medulla and pons of NLX-rats. These observations suggest that the neurotransmitters influencing the brain development, which are modulated by endogenous opioid systems, may play an important role in the development of rat brain; monoaminergic neurons play a significant role in the development of the cerebrum while TRH containing neurons may be involved in that of the cerebellum.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.     

Regional Distribution of Calmodulin Activity in Rat Brain

Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin-sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Immunochemical characterization of a proline endopeptidase from rat brain. Its relationship to proline endopeptidase from other tissues and from other species

Monospecific antiserum raised against rat brain proline endopeptidase is used to demonstrate the ubiquity of the enzyme and its unique role in the degradation of proline-containing peptides. All endoproteolytic activity directed toward proline residues in several rat tissues is shown to share one or more common antigenic determinants with rat brain proline endopeptidase. Similar activity from tissue of other species crossreacts with rat proline endopeptidase. The data presented suggest that proline endopeptidase is the sole cytoplasmic enzyme capable of degrading proline-containing peptides in every tissue examined and that previously reported proline-specific endoproteolytic activities observed in a variety of systems may be ascribed to proline endopeptidase. The putative role of proline endopeptidase in protein degradation is discussed.     

Venlafaxine Modulates Depression-Induced Oxidative Stress in Brain and Medulla of Rat

Venlafaxine is an approved antidepressant that is an inhibitor of both serotonin and norepinephrine transporters. Medical treatment with oral venlafaxine can be beneficial to depression due to reducing free radical production in the brain and medulla of depression- induced rats because oxidative stress may a play role in some depression. We investigated the effect of venlafaxine administration and experimental depression on lipid peroxidation and antioxidant levels in cortex brain, medulla and erythrocytes of rats. Thirty male wistar rats were used and were randomly divided into three groups. Venlafaxine (20 mg/kg) was orally supplemented to depression-induced rats constituting the first group for four week. Second group was depression-induced group although third group was used as control. Depressions in the first and second groups were induced on day zero of the study by chronic mild stress. Brain, medulla and erythrocytes samples were taken from all animals on day 28. Depression resulted in significant decrease in the glutathione peroxidase (GSH-Px) activity and vitamin C concentrations of cortex brain, glutathione (GSH) value of medulla although their levels were increased by venlafaxine administration to the animals of depression group. The lipid peroxidation levels in the three tissues and nitric oxide value in cortex brain elevated although their levels were decreased by venlafaxine administration. There were no significant changes in cortex brain vitamin A, erythrocytes vitamin C, GSH-Px and GSH, medulla vitamin A, GSH and GSH-Px values. In conclusion, cortex brain within the three tissues was most affected by oxidative stress although there was the beneficial effect of venlafaxine in the brain of depression-induced rats on investigated antioxidant defenses in the rat model. The treatment of depression by venlafaxine may also play a role in preventing oxidative stress. Abstract of the paper was submitted in 1st Ion Channels and Oxidative Stress Congress, 14–16 September 2006, Isparta, Turkey.     

Acetylcholinesterase activity in brain slices of rats subjected to cooling

The acetylcholinesterase (AChE) activity is studied in rat slices of the cerebral cortex, corpus striatum, hypothalamus and medulla oblongata of rats during hypothermia (20 degrees C) and also 1 and 7 days after the posthypothermal period. Cooling of animals down to 20 degrees C is accompanied by an increase in the AChE activity in the brain both under incubation temperature of 20 degrees and 37 degrees C. Under prolonged hypothermia the AChE activity in the investigated brain regions, except for corpus striatum, returns to the control level. By the 7th day of posthypothermal period the AChE activity in corpus striatum, hypothalamus and medulla oblongata does not restore completely. The most substantial changes in the AChE activity both under hypothermia and posthypothermal period occur in corpus striatum, which obviously reflects its complicated functional role.     

Characterization of Membrane-Bound Aminopeptidases from Rat Brain: Identification of the Enkephalin-Degrading Aminopeptidase

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.     

Regional distribution of neuropeptide-degrading enzyme activity in the rat brain: Effects of subacute exposure to carbon disulfide

Carbon disulfide, a volatile solvent, is widely used in industry. It has been demonstrated that it causes several neuropsychological symptoms. However, the neurochemical basis of its neurotoxic effect is relatively unknown. In this paper we have measured the effect of subacute i.p. administration on neutral and basic aminopepti-dase activities in discrete zones of the rat brain using lysine- and leucine-2-naphtylamides as substrates. Neutral aminopeptidase activity showed a significant decrease in the thalamus and cerebellum with marked (not significant) changes in the hypothalamus, hippocampus, medulla, and occipital cortex. There were no changes in basic ami-nopeptidase activity. It is suggested that amino-peptidase activity could play a role in carbon disulfide neurotoxic action in the aforementioned regions by generating changes in several neuropeptide levels.     

Changes in the catecholamine metabolism in the adrenal medulla of male hamsters with experimental hyperprolactinemia

1. Prolactin (PRL) can play a role as a physiological modulator of adrenal medulla function in several rodents. 2. We have examined the effects of hyperprolactinemia induced by ectopic pituitary grafts in Syrian hamsters on the adrenal medulla contents of catecholamines (CA) and their metabolites, as well as on the activities of several enzymes involved in the metabolism of these amines. 3. Increases in the peripheral levels of PRL in these animals were associated with decreases in adrenal medulla weight and increases in adrenal medulla contents of norepinephrine, epinephrine and vanilmandelic acid, the main degradative metabolite of CA, while adrenal medulla contents of the O-methylated derivatives of CA, normetanephrine and metanephrine, were unaltered. 4. These changes were correlated with increases in the adrenal medulla activity of monoamine oxidase, while the activities of tyrosine hydroxylase, phenylethanolamine-N-methyl transferase and catechol-O-methyl transferase were unaltered. 5. These results indicate that PRL is able to act on the adrenal medulla of hamsters by increasing the ability of these cells to metabolize CA via oxidative deamination.     

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.     

Nicotine-induced alteration in Tyr-Gly-Gly and Met-enkephalin in discrete brain nuclei reflects altered enkephalin neuron activity

Nicotine acts in CNS, but the pathways and mechanisms of its actions are poorly understood. Recent studies suggest an interaction between brain nicotinic receptors and endogenous opioid peptides. Acute administration of nicotine may alter enkephalin release without affecting brain enkephalin level. Tyr-Gly-Gly has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Concentrations of Tyr-Gly-Gly in brain were used to provide an index of enkephalin release in vivo. Thus we examined the thesis that nicotine alters brain neuronal enkephalin release, by measuring Tyr-Gly-Gly levels in specific brain nuclei from rats treated with nicotine 0.3 mg/kg SC 10 min before decapitation. Of 30 brain regions investigated, acute nicotine increased Tyr-Gly-Gly immunoreactivity in nucleus accumbens and in lower brain stem areas including dorsal raphe, pontine reticular formation, gigantocellular reticular formation, locus coeruleus, sensory trigeminal nucleus and the caudal part of ventrolateral medulla oblongata. Concomitantly, nicotine produced a significant decrease in native Met-enkephalin in central amygdala, flocculo-nodular lobe of cerebellum, caudal part of the ventrolateral medulla and intermediolateral cell column of the spinal cord. It is probable that the effects of nicotine to increase Tyr-Gly-Gly and alter Met-enkephalin concentration are mediated by nicotine-induced release of enkephalin at these brain sites. Furthermore, some of the physiologic and pharmacologic effects of nicotine may be mediated by such enkephalin release.