Synaptosomal Tyrosine Hydroxylase Activity Research Articles

Ameliorative role of Syzygium aromaticum aqueous extract on synaptosomal tyrosine hydroxylase activity, oxidative stress parameters, and behavioral changes in lead-induced neurotoxicity in mice.

This study reports the protective role of the aqueous extract of Syzygium aromaticum (ESA) against lead (Pb)-induced neurotoxicity in mice. Thirty male mice weighing between 18g and 25g were randomly divided into five groups. (1) Group 1 (control group), (2) group 2 (Pb-test group): was administered with a solution containing 0.1% (w/v) of lead acetate (PbAc), (3) group 3 (ESA+Pb100 group): was administered with 0.1% (w/v) of PbAc followed by 100mg/kg of S. aromaticum extract by gavage, (4) group 4 (ESA+Pb200): was administered with 0.1% (w/v) of PbAc followed by 200mg/kg of S. aromaticum extract, and (5) group 5 (ESA-group): was administered with 100mg/kg of S. aromaticum. Level of lead was determined by atomic absorption spectroscopy. Cerebral cortex synaptosomes prepared from mice administered orally with lead-acetate shown a significantly increased (p<.05) in tyrosine hydroxylase and protein carbonyl level and significantly decreased (p<.05) superoxide dismutase, glutathione reductase, and glutathione transferase activities. Also, there was a significant increase in brain lead concentration level, however, it was observed that S. aromaticum significantly reduced (p<.05) the level of lead at all tested doses. S. aromaticum rescued cerebral cortex synaptosomes from lead-induced neurotoxicity by relieving oxidative stress and abating elevated tyrosine hydroxylase activity. Moreover, S. aromaticum at the different dose grade (100mg and 200mg) abrogated the loss of motor performance in mice groups induced with lead. Altogether, our findings showed that S. aromaticum possesses antioxidant and neuro-modulatory potential against lead-induced neuronal damage. PRACTICAL APPLICATIONS: Environmental pollution with heavy metals is a known public health concern and their incremental concentrations in soil and water have risen to an unprecedented degree. Lead is one of the top 10 contaminants on the WHO's list of substances of greatest public health concern that impact the brain. However, exogenous natural bioactive supplements molecules could be one of the remedies to reduce Pb-induced toxicity. Our findings indicate therefore that, S. aromaticum could be a good fit for lowering Pb neurotoxicity and could be suggested as a neuroprotective molecule against neurodegenerative diseases involving catecholaminergic dysfunction induced by metallic elements.

CRF-like effects of sauvagine and urotensin I on synaptosomal tyrosine hydroxylase activity of mouse striatum

In the present study we have investigated whether sauvagine (SVG) and urotensin I (UT), two peptides displaying sequence homology with corticotropin-releasing factor (CRF), could affect synaptosomal tyrosine hydroxylase (TH) activity of mouse striatum in a manner similar to CRF. The enzyme activity was assayed in supernatants obtained following sonication and centrifugation of homogenates preincubated with the peptides. SVG and UT produced a concentration-dependent increase of TH activity with a half-maximal effect obtained at 5 and 10 nM, respectively. SVG and UT were as effective as CRF with maximal stimulations corresponding to 52–58% increase of basal enzyme activity, whereas the rank order of potency wasSVG>UT=CRF. Kinetic analysis of TH activity versus low concentrations of the pretin co-factor (0.05−0.4 mM) indicated that the stimulations elicited by CRF, SVG and UT were associated with an increase in theV max of the enzyme form with high affinity for the co-factor. The CRF receptor antagonist α-helical CRF 9–41 inhibited the effects of all 3 peptides. Moreover, the combined addition of CRF with either SVG or UT did not produce additive effects on TH activity. The stimulatory effects of CRF, SVG and UT were dependent on the concentration of extracellular free Ca 2+, being minimal in a Ca 2+-free medium and maximal at about 0.5 mM extracellular free Ca 2+. These results indicate that SVG and UT can mimic the effect of CRF on synaptosomal TH by acting on a common receptor site associated with a Ca 2+-dependent mechanism.

CRF stimulation of synaptosomal tyrosine hydroxylase activity in mouse striatum: possible role of phospholipid hydrolysis

CRF stimulation of synaptosomal tyrosine hydroxylase activity in mouse striatum: possible role of phospholipid hydrolysis

Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (−)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40–50% inactivation of G i and G o proteins, which is evident from the reduction of ADP ribosylation with [ 32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.

Presynaptic dopamine and serotonin receptors modulating tyrosine hydroxylase activity in synaptosomes of the nucleus accumbens of rats

Tyrosine hydroxylase (TH) activity was determined by measuring the formation of [ 3H]DOPA from [3,5- 3H]tyrosine in the crude synaptosomal fraction of the nucleus accumbens under conditions preventing dopamine reuptake in 30 mM K +-containing medium. TH seems to be allosterically activated under depolarizing conditions: a 4.4 fold decrease of the K m value for tyrosine of the synaptosomal TH was observed. Synaptosomal TH activity was inhibited concentration dependently by dopamine and apomorphine resulting in IC 50 values of 0.4 and 0.25 μM, respectively. The maximal inhibitory effects of dopamine as well as apomorphine were about 50% of the controls. The dopamine-induced inhibition was completely antagonized by neuroleptics. The rank order of antagonistic potencies was haloperidol > clozapine > sulpiride (with increasing EC 50); methiothepine was ineffective. Moreover, synaptosomal TH activity was inhibited by serotonin in a concentration-dependent manner (IC 50 = 0.8 μM). This inhibition was completely antagonized by methiothepine while, on the other hand, haloperidol was ineffective. The experimental system demonstrated here appears to be suitable for estimating the presynaptic dopamine and serotonin antagonistic potencies of drugs.

Striatal synaptosomal dopamine synthesis: Evidence against direct regulation by an autoreceptor mechanism

Regulation of the rate-limiting step in dopamine (DA) synthesis was estimated in striatal synaptosomes by measuring the rate of hydroxylation of L-4-[ 3H]phenylalanine, a substrate of tyrosine hydroxylase (TH). DA inhibited hydroxylation with an IC 50 of 0.2 μM. The concentration-response curve of DA-induced inhibition was not affected by the presence of 1 μM chlorpromazine, a phenothiazine DA antagonist. Sulpiride and haloperidol, DA antagonists of the benzamide and butyrophenone classes respectively, also failed to alter the inhibition of substrate hydroxylation by 1 μM DA, even at cocnentrations up to 10 μM. In contrast, a parallel 15 fold shift to the right in the concentration-response curve of DA-induced inhibition of hydroxylation was obtained when 10 μM nomifensine, a competitive DA uptake inhibitor, was added. Even in the presence of nomifensine, 1μM chlorpromazine had no effect on the DA concentration-response curve. The addition of DMPH 4, an artificial cofactor for TH, completely blocked DA-induced inhibition of enzymatic activity. These data suggest that direct autoreceptor control of synaptosomal TH activity does not exist in vitro, and that DA-induced inhibition of TH occurs subsequent to reuptake via classical feedback inhibition, presumably by competitive displacement of the necessary endogenous cofactor.

Effect of atypical neuroleptics carbidine and sulpiride on striatal synaptosomal tyrosine hydroxylase activity in the rat brain

Effect of atypical neuroleptics carbidine and sulpiride on striatal synaptosomal tyrosine hydroxylase activity in the rat brain

The effects of footshock and handling on tyrosine hydroxylase activity in synaptosomes and solubilised preparations from rat brain

The effects of mild noxious stimulation on central noradrenergic activity were investigated in rats, using low-intensity electrical footshock as the noxious stimulus. Synaptosomes were prepared from brain regions of control and experimental rats and the rate of tyrosine hydroxylation was assayed: this is a measure of transmitter synthesis in the isolated noradrenergic terminals. Single footshocks were found to elevate the synthesis rate in the hippocampus, the extent of the elevation depending on the baseline rate. This elevation was not significant in hypothalamus or cerebellum. Repeated handling over a period of 14 days was also found to elevate synthesis rate in hippocampus; but rats subjected to daily footshock for a week, in addition to handling, had synthesis rates similar to those of controls. Maximal tyrosine hydroxylase activity was measured by solubilizing the enzyme and assaying at saturating concentrations of cofactor and substrate: this is a measure of the amount of enzyme in the nerve terminals. No significant changes in maximal tyrosine hydroxylase activity were found in the brains of handled or shocked animals. These results suggest that the assay of tyrosine hydroxylase activity in a suspension of synaptosomes provides a sensitive indicator of changes in noradrenergic activity related to mild noxious stimulation. The biochemical responses are more pronounced in the hippocampus than in other noradrenergic terminal areas; this suggests that the synthesis rate of noradrenaline in terminals of central noradrenergic neurones could be regulated by local mechanisms at the terminal as well as by impulses transmitted from the cell body.

Pre- and postsynaptic neurochemical alterations following estrogen-induced striatal dopamine hypo- and hypersensitivity

The administration of pharmacologie doses of estrogen results in a biphasic response in striatal dopamine sensitivity, as measured by apomorphine-induced stereotypy. At 24 hr after the last dose of estradiol benzoate (EB) there is a suppression of apomorphine-induced stereotypy, which is followed by an increased sensitivity to apomorphine at 48 hr. The dopamine hyposensitivity is reflected postsynaptically by an increased K D (i.e., decreased affinity) for 3H-spiroperidol binding to striatal membranes, while the hypersensitive phase is reflected by an increased B max for 3H-spiroperidol binding to striatal membranes. Presynaptically during the hyposensitive phase the tyrosine hydroxylase displayed a decreased K M for the pterine cofactor. The decreased K M for the cofactor was retained in the hypersensitive animals, however the V max for tyrosine hydroxylase was decreased during the hypersensitive phase of the EB-induced changes in dopamine sensitivity. The presynaptic or autoreceptor sensitivity of the dopamine neurons projecting to the striatum was assessed by determining the apomorphine IC 50 value for the inhibition of synaptosomal tyrosine hydroxylase activity. Utilizing this assay the animals that were hyposensitive to dopamine showed a normal presynaptic sensitivity, while those animals that had developed a hypersensitivity to dopamine following EB were also hypersensitive to dopamine presynaptically.

Striatal synaptosomal tyrosine hydroxylase activity: A model system for study of presynaptic dopamine receptors

Tyrosine hydroxylase activity determined in striatal synaptosomes by the formation of [ 3H]H 2O from [3,5- 3H]tyrosine was used as a system for determining receptor affinities of neuroleptic drugs for the dopamine autoreceptor. Four agonists were tested for their abilities to inhibit tyrosine hydroxylation, and the order of potencies was apomorphine > dopamine > norepinephrine and buspirone was inactive. The abilities of different neuroleptic drugs to shift the IC 50 of apomorphine (500 nM without additional drugs) were used to calculate K B values for each drug. The butyrophenones and +)butaclamol were the most potent of the drugs tested, while (−)sulpiride and thioridazine were very weak inhibitors of apomorphine. Clozapine, promethazine, (-)butaclamol and (+)sulpiride were all inactive. The order of potencies of antipsychotic drugs at the dopamine nerve-ending autoreceptor correlated closely with clinical dose, but it did not correlated with previous reports of displacement of radiolabeled dopamine or apomorphine striatal membrane binding. This observation combined with the fact that the IC 50 of apomorphine at this functional receptor was 100 times the concentration needed to saturates its binding site(s) in radioligand receptor assays suggests that the dopamine autoreceptor is not labeled by nanomolar concentrations of apomorphine or dopamine.

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus, hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of 3H 2O from L-4- 3H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of 3H 2O from L-4- 3H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank 3H 2O produced from L-4- 3H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank 3H 2O produced from L-3,5- 3H-tyrosine. The K m values of tyrosine hydroxylase for phenylalanine determined by the production of 3H 2O from L-4- 3H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of 3H 2O formed from L-4- 3H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.

The interactions of bromocriptine and lergotrile with dopamine and alpha-adrenergic receptors.

Bromocriptine and lergotrile, which are clinically used as antiparkinsonian (AP) agents, compete for the binding of H3-dopamine, H3-apomorphine, and H3-haloperidol to striatal membrane sites. Lergotrile has a higher affinity for the H3-dopamine binding to bovine striatal membranes than bromocriptine. Lergotrile and bromocriptine are almost equipotent in competing for the binding of H3-apomorphine to rat striatal membranes, but bromocriptine is more potent in competing for the binding of H3-haloperidol than lergotrile. These results indicate that lergotrile and bromocriptine are mixed putative agonist-antagonist with respect to the postsynaptic dopamine receptors. Lergotrile and bromocriptine at higher concentrations inhibit synaptosomal tyrosine hydroxylase activity, and reverse the apomorphine elicited enzyme inhibition. Thus, these ergot alkaloids behave as mixed agonist-antagonist also with respect to the presynaptic dopamine receptors. Bromocriptine and lergotrile, as well as other tested DH-ergot alkaloids and neuroleptics, compete for the binding of the alpha-antagonist H3-WB-4101 to rat cerebral cortical membranes. The displacing potencies of the tested DH-ergot alkaloids and of the neuroleptics indicate that they have a high affinity for the alpha-adrenoreceptors in the CNS.

Stimulation of tyrosine hydroxylase activity by cyclic amp in synaptosomes and in soluble striatal enzyme preparations

Dibutyryl cyclic AMP (dB-cAMP) elicits a concentration-dependent stimulation of tyrosine hydroxylase activity in the striatal and mesolimbic synaptosomes. The percent of stimulation is significantly higher in the mesolimbic synaptosomes than in the striatal synaptosomes. dB-cAMP and depolarizing agents (ouabain or veratridine) have an additive effect on synaptosomal tyrosine hydroxylase activity, indicating that they stimulate tyrosine hydroxylase activity by different mechanisms. cAMP does not stimulate soluble striatal tyrosine hydroxylase activity unless it is added in combination with ATP and Mg 2+, compounds required for the activity of cAMP-dependent protein kinase. The cAMP elicited per cent stimulation of soluble tyrosine hydroxylase activity is dependent upon the concentration of added protein kinase and upon the pH of the reaction. dB-cAMP has the same effect on the kinetic state of tyrosine hydroxylase in synaptosomes as cAMP on the soluble tyrosine hydroxylase. The nucleotieds does not alter tpparent Km for tyrosine, reduces the Km for the pteridine cofactor and increases the Ki for dopamine. Thus, cAMP increases the affinity of tyrosine hydroxylase for the pteridine cofactor and concomitantly decreases the affinity for the end-product inhibition.