Co-identity of brain angiotensin converting enzyme with a membrane bound dipeptidyl carboxypeptidase inactivating Met - enkephalin.

The distribution in rat brain of angiotensin converting enzyme (EC3.4.15.1) using hippuryl-His-Leu as substrate was identical to a dipeptidyl carboxypeptidase present in membranes assayed with Met-enkephalin as substrate. Highest activity occurred in pituitary, followed by cerebellum, corpus striatum, midbrain, pons-medulla, hypothalamus, cerebral cortex and spinal cord. The ratio of products His-Leu/Tyr-Gly-Gly was identical for all regions but differed from His-Leu/Tyr. Angiotensin converting enzyme purified by immunoaffinity chromatography gave a K_m for hippuryl-His-Leu of 0.5mM and for Met-enkephalin of 0.1 mM. In the presence of the specific inhibitor of angiotensin converting enzyme, SQ 14,225, the Ki value was 10^?7M. Present data point to the co-identity of brain angiotensin converting enzyme with the dipeptidyl carboxypeptidase inactivating enkephalin.     

Sodium-Sensitive Cocaine Binding to Rat Striatal Membrane: Possible Relationship to Dopamine Uptake Sites

Abstract: In rat striatal membranes, NaCl induced a twofold increase in the maximal number of cocaine binding sites but did not alter the affinity of these sites for cocaine. This effect was concentration-dependent, specific to sodium ions, and occurred in membranes prepared from corpus striatum but not from other brain regions. Lesions with 6-hydroxydopamine but not with kainic acid eliminated the sodium-induced increase in binding and produced a decrease in the B_max of binding measured in the presence of NaCl. The capacity of a series of drugs to interfere with Na⁺–dependent cocaine binding correlated well with their capacity to inhibit [³H]dopamine uptake into rat striatal synaptosomes. The present results suggest that Na⁺–dependent cocaine binding sites are localized presynaptically on dopaminergic nerve terminals in corpus striatum, and may be related to dopamine uptake sites.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.     

Isolation,partial purification,characterization, and tissue distribution of phenylmethylsulfonyl fluoride-inhibited feline carboxypeptidase

Phenylmethylsulfonyl fluoride (PMSF)-inhibited carboxypeptidase from cat liver was purified 148-fold by chromatography on CM- and DEAE-cellulose with 27.3% yield. Molecular weight of the enzyme is 100-110 kD as determined by gel filtration on Sephadex G-150. The enzyme has maximum activity at pH 5.50-5.75; its activity is completely inhibited by PMSF or p-chloromercuribenzoate and partially inhibited by iodoacetamide. EDTA, 2-mercaptoethanol, N-ethylmaleimide, Co²⁺ and Ca²⁺, basic carboxypeptidase inhibitor guanidinoethylmercaptosuccinic acid, and angiotensin-converting enzyme inhibitor captopril do not influence its activity. The enzyme cleaves arginine from enkephalin-Leu5-Arg6 and dansyl-Phe-Leu-Arg to form enkephalin-Leu5 and dansyl-Phe-Leu, respectively, and very slowly cleaves leucine from carbobenzoxy-Gly-Leu. Further cleavage of either enkephalin-Leu5 or dansyl-Phe-Leu was not detected. The highest activity of this enzyme was found in adrenal glands and testicles; this activity was 30% lower in hypophysis, and still lower in liver and kidney. The PMSF-inhibited carboxypeptidase activity in brain was about 6-16 times lower than that in adrenal gland. In brain regions, the highest activity was detected in gray matter of cerebral hemispheres and cerebellum, and slightly lower activity was found in thalamus/hypothalamus, striatum, and hippocampus. The lowest activity was found in quadrigeminal bodies, medulla oblongata, and white matter of cerebral hemispheres. The enzyme exists mainly in soluble form; the activity of membrane-associated enzyme is 7-25% of soluble enzyme activity depending on tissue type. We consider here a possible involvement of PMSF-inhibited carboxypeptidase in the metabolism of biologically active peptides.     

Dephosphorylation of purified brain tyrosine hydroxylase by rat striatal extracts

Tyrosine hydroxylase (TH) was purified from bovine brain and enzymatically phosphorylated in vitro. Radioactively phosphorylated TH was dephosphorylated by rat tissue extracts. Of tissues examined, rat corpus striatal extracts were highest in specific activity in the TH dephosphorylating assay. Phosphorylated histone did not inhibit dephosphorylation of TH by rat striatal extracts. The thermal decay of dephosphorylating activity of rat striatal extracts varied with substrate, with TH dephosphorylating activity most unstable of the activities assayed. The results suggest that TH can be enzymatically dephosphorylated and that, in corpus striatum, this process differs quantitatively from the dephosphorylation of phosphohistone and phosphoprotamine.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.     

The effects of phencyclidine on the uptake of 3H-catecholamines by rat striatal and hypothalamic synaptosomes

In tests of the effects of the psychotomimetic agent, phencyclidine, 1-(1-phenylcyclohexl) peperidine (PCP), on the uptake of ³H-catecholamines by synaptosome-rich homogenates of rat striatum and hypothalamus, PCP was shown to be a potent, competitive inhibitor of both ³H-dopamine uptake in the striatal region and ³H-norepinephrine uptake in the hypothalamus. The implication of these findings are discussed in terms of a drug-induced imbalance of the catecholaminergic-cholinergic systems in those regions of the brain where activity has been correlated with emotional expression.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Enhanced dopamine D<Subscript>2</Subscript> receptor function in hypothalamus and corpus striatum: their role in liver,plasma and in vitro hepatocyte ALDH regulation in ethahol treated rats

Dopamine D₂ receptors are involved in ethanol self- administration behavior and also suggested to mediate the onset and offset of ethanol drinking. In the present study, we investigated dopamine (DA) content and Dopamine D₂ (DA D₂) receptors in the hypothalamus and corpus striatum of ethanol treated rats and aldehyde dehydrogenase (ALDH) activity in the liver and plasma of ethanol treated rats and in vitro hepatocyte cultures. Hypothalamic and corpus striatal DA content decreased significantly (P < 0.05, P < 0.001 respectively) and homovanillic acid/dopamine (HVA/DA) ratio increased significantly (P < 0.001) in ethanol treated rats when compared to control. Scatchard analysis of [³H] YM-09151-2 binding to DA D₂ receptors in hypothalamus showed a significant increase (P < 0.001) in B_max without any change in K_d in ethanol treated rats compared to control. The K_d of DA D₂ receptors significantly decreased (P < 0.05) in the corpus striatum of ethanol treated rats when compared to control. DA D₂ receptor affinity in the hypothalamus and corpus striatum of control and ethanol treated rats fitted to a single site model with unity as Hill slope value. The in vitro studies on hepatocyte cultures showed that 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in 10% ethanol treated cells to near control level. Sulpiride, an antagonist of DA D₂, reversed the effect of dopamine on 10% ethanol induced ALDH activity in hepatocytes. Our results showed a decreased dopamine concentration with enhanced DA D₂ receptors in the hypothalamus and corpus striatum of ethanol treated rats. Also, increased ALDH was observed in the plasma and liver of ethanol treated rats and in vitro hepatocyte cultures with 10% ethanol as a compensatory mechanism for increased aldehyde production due to increased dopamine metabolism. A decrease in dopamine concentration in major brain regions is coupled with an increase in ALDH activity in liver and plasma, which contributes to the tendency for alcoholism. Since the administration of 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in ethanol treated cells to near control level, this has therapeutic application to correct ethanol addicts from addiction due to allergic reaction observed in aldehyde accumulation.     

Separate metabolic pathways for Leu-enkephalin and Met-enkephalin-Arg6-Phe7 degradation by rat striatal synaptosomal membranes

Metabolism of Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷ was studied using synaptosomal plasma membranes prepared from rat corpus striatum and whole brain. Cleavage of the pentapeptide was mediated largely by an aminopeptidase leading to the release of Tyr and Gly-Gly-Phe-Leu. Bestatin, an aminopeptidase inhibitor, prevented the release of Tyr and the tetrapeptide, but not secondary cleavage at the Gly Phe site leading to the release of Tyr-Gly-Gly and Phe-Leu. Cleavage at the latter site was inhibited by low concentrations of Thiorphan, an inhibitor of a non-aminopeptidase enkephalinase. MK-421, an inhibitor of the angiotensin converting enzyme, acted only at high substrate concentrations of Leu-enkephalin, indicating that the converting enzyme has a relatively low affinity for the pentapeptide. In contrast to the pentapeptide the major products found upon incubation of heptapeptide with synaptosomal plasma membrane were Arg-Phe and Met-enkephalin. Product release was inhibited by low concentrations of MK-421 but not by Thiorphan, indicating that the cleavage of the heptapeptide was mediated by the angiotensin converting enzyme. This pathway may represent a mechanism for the formation of Met-enkephalin from larger precursors present in striatum and other regions of the central nervous system.     

Preincubation of homogenates from rat brain thalamus and striatum affects the rate of [3H]GABA uptake

The effect of preincubation of brain homogenates on the subsequent uptake of [³H]GABA was studied in rat thalamus and corpus striatum. The results show that in both brain regions preincubation causes a decrease in the initial rate of [³H]GABA influx, the phenomenon being most evident in the thalamus.     

Proteolytic conversion of [Met]enkephalin-Arg6-Gly7-Leu8 by brain synaptic membranes. Characterization of formed peptides and mechanism of proteolysis

The degradation of the enkephalin-containing octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL) was systematically investigated by incubating the peptide with synaptic membranes from rat striatum or with purified peptidases. The degradation products were derivatized with 4-dimethylamino-azobenzene-4'-isothiocyanate and then analyzed by high pressure liquid chromatography and by amino-terminal analysis. The incubation of YGGFMRGL with synaptic membranes yielded YGG, YGGF, YGGFM, and MR in a manner that was linear with respect to time. The corresponding carboxyl-terminal fragments FMRGL, MRGL, and RGL could not be detected, which suggests that the degradation of YGGFMRGL by synaptic membranes occurs by carboxypeptidase activity. The incubation of YGGFMRGL with different purified peptidases produced cleavage patterns unique from that seen with synaptic membranes. Enkephalinase recognized only the Gly-Phe bond to produce YGG and FMRGL. Thermolysin recognized the Gly-Phe bond and the Phe-Met bond to yield YGG, YGGF, FMRGL, and MRGL. Angiotensin-converting enzyme (ACE) produced primarily YGGF, MR, and lesser amounts of YGGFMR and YG. The formation of YGG, YGGF, and YGGFM by synaptic membranes could be stimulated 3-fold by the addition of 30 mM NaCl and inhibited by MK-422, an ACE inhibitor, with an IC50 of 3 nM. These data suggest that ACE, a dipeptidyl carboxypeptidase, is the primary enzyme involved in the degradation of YGGFMRGL in brain. ACE apparently works in concert with another carboxypeptidase in brain to yield YGGFM and YGG since the carboxyl-terminal peptides RGL and FMRGL could not be detected.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Effects of two-vessel forebrain ischemia and of administration of indomethacin and quinacrine on Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in various rat brain areas

The effect of a two-vessel forebrain ischemia (induced by occlusion of carotid arteries and hypotension), subsequent reperfusion, and administration of indomethacin and quinacrine on the Na⁺,K⁺-ATPase activity and diene conjugate content was studied in various rat forebrain fields. The most pronounced metabolic alterations were observed during ischemia and reperfusion. Under these effects, there was a statistically significant reduction of the Na⁺,K⁺-ATPase activity in the brain cortex and striatum and an increase of the diene conjugate content in the rat brain cortex in comparison with sham-operated animals. Injection of indomethacin, a cyclooxygenase inhibitor, to rats subjected to ischemia and reperfusion, resulted to a statistically significant increase of the Na⁺,K⁺-ATPase activity in the brain cortex, hippocampus, and striatum (p < 0.02) as compared with control animals. The diene conjugate content in the rat brain cortex during brain ischemia and reperfusion was statistically significantly lower in the rats injected with indomethacin. The effect of quinacrine (a blocker of phospholipase A₂) was similar to that of indomethacin in the rat cortex, whereas in the rat striatum and hippocampus, the quinacrine effect during ischemia and reperfusion was less marked than that of indomethacin. The obtained data indicate the ability of inhibitors of the arachidonic pathway of free radical formation to normalize the Na⁺, K⁺-ATPase activity during brain ischemia. There also revealed local peculiarities of metabolic disturbances in different regions of the rat forebrain during ischemia and reperfusion.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 33–38.Original Russian Text Copyright © 2005 by Molchanova, Moskvin, Zakharova, Yurlova, Nosova, Avrova.     

Changes in the activity of enzymes of renin-angiotensin and kinin systems in the rat brain after adrenalectomy and administration of hydrocortisone

It is shown that the activity of enzymes participating in renin-angiotensin and brain kinin systems' metabolism depends on functional state of hypothalamo-pituitary-adrenocortical system. Under experimental hypocorticism the activity of angiotensin-converting enzyme and kininase I in the hypothalamus, hippocamp, corpus striatum and rat pituitary decreases; the renin-like enzyme activity decreases in the corpus striatum but increases in the hypothalamus and hippocamp. After hydrocortisone administration to adrenalectomized rats the angiotensin-converting enzyme activity of the hippocamp and pituitary is shown to be normalized as well as renin-like enzyme and kininase I of the hippocamp and corpus striatum. The activity of the studied enzymes in the hypothalamus decreases in this case.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.