Dopamine induces cell death, lipid peroxidation and DNA base damage in a catecholaminergic cell line derived from the central nervous system.

Dopamine can be autoxidized to superoxides and quinones. Superoxides can form hydroxyl radicals that are highly reactive with lipids, proteins and DNA leading to neuronal damage and cell death. We used a clonal catecholaminergic cell line (CATH.a) derived from the central nervous system to evaluate the effects of dopamine on cell death, lipid peroxidation and DNA base damage. Dopamine produces cell death in CATH.a cells and this is associated with an increase in annexin binding, which is an early indicator of apoptosis. Incubation of CATH.a cells with deferoximine, an iron chealator, partially antagonizes dopamine-induced cell death. In CATH.a cells, dopamine produces an increase in both lipid peroxidation, as measured by cis-parinaric acid fluorescence, and DNA oxidative base damage, as measured by 8-hydroxy-2'-deoxyguanosine formation. Cell death was inhibited 84-92% by the hydrophilic antioxidants, dithiothreitol, L-cysteine, and N-acetylcysteine. The lipophilic vitamins, retinol and vitamin E and the vitamin E analog, Trolox, inhibited dopamine-induced cell death by 18-33%. The lipophilic antioxidants probucol, propyl glycol and butylated hydroxyanisone had no inhibitory effect on dopamine-induced cell death. These data suggest that damage to DNA and lipids may be partially responsible for dopamine-induced cell death in CATH.a cells.

Brain-derived and glial cell line-derived neurotrophic factors protect a catecholaminergic cell line from dopamine-induced cell death.

Brain-derived neurotrophic factor (BDNF) promotes the survival of dopaminergic neurons in primary cultures and protects these neurons from the neurotoxic effects of 6-hydroxydopamine. The protective mechanism of BDNF on neurotoxicity was evaluated using CATH.a cells, a clonal catecholaminergic cell line derived from the central nervous system. Dopamine produced a dose-dependent cell death in CATH.a cells. Treatment of CATH.a cells with BDNF or glia cell line-derived neurotrophic factor (GDNF) reduced dopamine-induced cell death by approximately 60-70%. Nerve growth factor, basic fibroblast growth factor, neurotrophin-4/5 and insulin had no protective effect on dopamine-induced cell death. Dopamine decreased the activity of superoxide dismutase and the levels of glutathione in the CATH.a cells and these decreases were reversed by BDNF. In addition, BDNF treatment alone increased superoxide dismutase activity by 108%. These results suggest that BDNF may safeguard CATH.a cells from dopamine-induced cell death by maintaining or enhancing components of the cell, which protect from oxidative stress.

SR 141716A, a CB1 cannabinoid receptor antagonist, potentiates the locomotor stimulant effects of amphetamine and apomorphine.

The intraperitoneal (i.p.) injection of apomorphine or d-amphetamine significantly increased locomotor activity in Sprague-Dawley rats. Prior administration of the cannabinoid receptor antagonist, SR 141716A, significantly enhanced the stimulant effect of both d-amphetamine and apomorphine in a dose-dependent manner. Administration of SR 141716A alone had no effect on locomotor activity. These data indicate that endogenous cannabinoids exert an inhibitory action on the increase in locomotor activity produced by amphetamine and apomorphine.

Nitric oxide induces cell death in a catecholaminergic cell line derived from the central nervous system.

The nitric oxide (NO) donors, sodium nitroprusside (SNP), 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate] (DETA NONOate), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) produce a dose-dependent increase in cell death in a catecholaminergic cell line (CATH.a) derived from the central nervous system. Cell death is associated with a decrease in mitochondrial membrane potential. Dopamine also induced cell death of CATH.a cells and this was potentiated by concentrations of SNP which alone did not produce cell death. Hemoglobin, a scavenger of NO radicals, blocked SNP- and SNAP-induced cell death. Catalase and superoxide dismutase, enzymes that metabolize H2O2 and superoxide, respectively, did not inhibit SNP- or SNAP-induced cell death. These data indicate that NO donors produce cell death in CATH.a cells through a mechanism related to the production of NO and the loss of the mitochondrial membrane potential but unrelated to the production of H2O2.

Effects of single and repeated footshock on dopamine release and metabolism in the brains of Fischer rats.

Changes in the tissue levels of 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and dopamine in the frontal cortex, hypothalamus, nucleus accumbens, and striatum were evaluated after 0.5-4 h of footshock (2 mA, for 3 s every 30 +/- 5 s) in Fischer rats. 3-MT, DOPAC, and HVA levels in the four brain areas peaked at 0.5 h and in most cases returned to baseline values within 4 h. No changes were found in dopamine levels. Repeated footshock stress was evaluated by administering 10 footshock sessions (0.5 h, two per day for 5 days). At the end of the 10th footshock session, 3-MT levels were higher than at the end of the first footshock session in three of the four brain regions, indicating sensitization of dopamine release. No differences were found between the first and 10th footshock sessions in DOPAC and HVA levels. Fourteen days after the 10th footshock session, the levels of 3-MT, DOPAC, and HVA were the same as in control rats in all four brain regions. A 0.5-h footshock challenge presented 14 days after the 10th footshock session attenuated DOPAC levels in the hypothalamus and nucleus accumbens. In contrast, DOPAC and HVA levels in the frontal cortex showed sensitization after footshock challenge, and a similar trend was apparent for 3-MT levels. These results indicate that repeated footshock stress induces generalized sensitization of dopamine release and turnover in some areas of the brain of Fischer rats. This sensitization may persist in the cortical but not subcortical dopamine neurons after discontinuation of the treatment.

Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics.

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E1- (PGE1), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE1, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism.

Dopamine induces apoptotic cell death of a catecholaminergic cell line derived from the central nervous system.

Dopamine produces a time- and dose-dependent increase in cell death in a clonal catecholaminergic cell line (CATH.a) derived from the central nervous system. Cell death also occurred after treatment with the catecholamines L-dihydroxyphenylalanine, norepinephrine, epinephrine, and isoproterenol, as well as the neurotoxic compound 6-hydroxydopamine. Cell death is not receptor mediated because selective noradrenergic and dopaminergic receptor agonists had no effect on CATH.a cell viability. Dopamine induces apoptotic cell death as indicated by DNA fragmentation measured by gel electrophoresis and by flow cytometric analysis. Apoptosis seems to be produced by dopamine autoxidation, because intracellular peroxides increase after dopamine treatment and cell death can be inhibited by catalase and N-acetylcysteine. N-acetylcysteine produced a dose-dependent decrease in dopamine-induced cell death; this correlated with a decrease in peroxide formation. In addition, antisense to the antioxidant protein bcl-2 increases the sensitivity of CATH.a cells to dopamine-induced cell death. These findings indicate that the oxidative products of dopamine cause neurotoxicity through apoptosis.

Chronic cocaine administration increases tyrosine hydroxylase activity in the ventral tegmental area through glutaminergic- and dopaminergic D2-receptor mechanisms.

Tyrosine hydroxylase activity was measured in the brain of rats treated chronically with saline or cocaine (10 mg/kg, 2 x day, for 7 days). Tyrosine hydroxylase activity was significantly increased in the ventral tegmental area 1, 6 and 12 weeks after the last treatment with cocaine. The increase in tyrosine hydroxylase activity at 6 weeks after the last cocaine injection was prevented by the prior administration of MK-801, haloperidol or clozapine but not by the D1 receptor antagonist, SCH-23390. SCH-23390 produced a significant increase in tyrosine hydroxylase activity when administered with saline. These data indicate that glutaminergic and dopaminergic D2-receptor mediated mechanisms are important in regulating the effect of cocaine on the ventral tegmental area.

Evaluation of cyclic AMP accumulation in EBV-transformed human B-lymphocytes: effects of dopamine agonists, isoproterenol, prostaglandin E1, cholera toxin, forskolin, and phorbol 12-myristate-13 acetate.

1. Phorbol 12-myristate-13-acetate (PMA), a protein kinase C activator, elevated cyclic AMP accumulation in EBV-transformed human B-lymphocytes, and potentiated isoproterenol-, prostaglandin- (PGE1), cholera toxin-, and forskolin-stimulated cyclic AMP accumulation. 2. The dopamine D1 receptor agonist, SKF38393 (10(-7) to 10(-5) MH, had no effect on cyclic AMP accumulation in transformed human B-lymphocytes. 3. The dopamine D2 receptor agonist, quinpirole (10(-7) to 10(-5) MH did not inhibit cyclic AMP accumulation even when cyclic AMP accumulation was maximized by the addition of PMA and forskolin. 4. These data suggest that dopamine D1- and D2-receptor coupling to a cyclic AMP generating system is not present at detectable levels in transformed human B-lymphocytes.

Age-related changes in tetrahydrobiopterin and GTP-cyclohydrolase activity in the brain and adrenal gland of rats.

In the present study, we observed that tetrahydrobiopterin (BH4) levels were decreased significantly in the striatum, substantia nigra, frontal cortex, hypothalamus, and cerebellum of 25-month-old and in the cerebellum only in case of 18-month-old rats as compared to 4-month-old rats. In contrast, BH4 levels in adrenal glands of 25-month-old rats were increased significantly. Kinetic analysis of GTP-cyclohydrolase revealed a decrease in the apparent Km along with an increase in Vmax value in the adrenal of 25-month-old rats compared to 4-month-old rats. Whereas, in cerebellum we observed that the apparent Km of the high affinity form of the enzyme was increased and a decrease in the Vmax value of the low affinity form only in case of 25-month-old rats compared to the young animals. These alterations in the BH4 levels and its synthesizing enzyme kinetics in specific brain areas and adrenal glands of aged rats are consistent with the reported changes in the catecholamine function in central and peripheral nervous system with aging.

Dopamine release in the rat cerebellum and hippocampus: a tissue 3-methoxytyramine study.

Multiple lines of evidence indicate dopamine is a neurotransmitter or neuromodulator in the cerebellum and hippocampus. In this study, we explored the utility of 3-methoxytyramine as an index of dopamine release in these regions. We found that: (1) cerebellar and hippocampal 3-methoxytyramine levels can be measured by combined gas chromatography-mass fragmentography with negative chemical ionization; (2) basal 3-methoxytyramine accumulation rates following monoamine oxidase inhibition, but not the steady-state tissue levels, are several times lower in these regions than in the frontal cortex; (3) accumulation of 3-methoxytyramine in the hippocampus and cerebellum can be enhanced following electroconvulsive shock, but not acute haloperidol (0.4 mg/kg) treatment. We conclude that 3-methoxytyramine accumulation may be a useful index of dopamine release in the cerebellum and hippocampus, but dopamine release is regulated differently in these regions than in the frontal cortex and striatum.

Effects of chronic cocaine administration on [3H]dopamine uptake in the nucleus accumbens, striatum and frontal cortex of rats.

The uptake of [3H]dopamine into synaptosomes obtained from the nucleus accumbens, striatum and frontal cortex was evaluated after chronic treatment with cocaine. Cocaine was administered in a concentration of 10 mg/kg, twice a day for 7 days. Fourteen days after the last injection, locomotor activity and [3H] dopamine uptake were evaluated. Base-line locomotor activity was significantly lower (29%) in rats treated chronically with cocaine compared with saline-treated rats. A challenge dose of cocaine (2.5 mg/kg or 5.0 mg/kg) or d-amphetamine (1 mg/kg) produced similar increases in locomotor activity above the corresponding base-line values in both saline- and cocaine-treated rats, indicating that behavioral sensitization had not occurred. Chronic cocaine administration produced a significant decrease in the uptake of [3H]dopamine into the frontal cortex (49%) with no significant differences in the nucleus accumbens or striatum. The decrease in [3H]dopamine uptake in the frontal cortex was due to a decrease in the Vmax with no change in the affinity of [3H]dopamine for the dopamine transporter. No differences were produced in the IC50 values of GBR 12909 or cocaine for [3H] dopamine uptake after chronic cocaine treatment. However, in all three brain regions, the IC50 values for cocaine were significantly greater than the values for GBR 12909. In addition, the IC50 values for GBR 12909 and cocaine in the frontal cortex were significantly greater than values for either compound in the nucleus accumbens or striatum. The administration of methamphetamine, using a similar treatment schedule, produced no changes in [3H]dopamine uptake in any of the three brain areas. These data indicate an inhibition effect of repeated cocaine administration on [3H]dopamine uptake in the frontal cortex of rats.

Influence of thyrotropin-releasing hormone and catecholaminergic interactions on CNS ethanol sensitivity.

The role of catecholamine neuronal systems in mediating the analeptic and thermogenic effects of thyrotropin-releasing hormone (TRH) was examined in long-sleep (LS) and short-sleep (SS) mice. TRH [0.1 to 40 micrograms, intracerebroventricularly (icv)] was associated with a reduction in the sleep times of LS mice, but no dose of TRH had any effect on sleep times of SS mice. However, TRH (20 micrograms, icv) produced a 1.0 degree to 1.5 degrees C attenuation of the ethanol-induced hypothermia in both LS and SS mice. TRH did not change the rate of ethanol elimination in either line of mice, suggesting that the reduction in LS sleep times and attenuation of LS and SS hypothermia were due to decreased CNS ethanol sensitivity rather than an increase in the rate of ethanol metabolism. TRH (20 micrograms, icv) given alone produced an activation of central and peripheral catecholamine systems in LS, but not SS mice, as reflected by an increase in the in vivo tyrosine hydroxylase (TH) activity in the brain and adrenal gland. TRH, given with ethanol, prevented or attenuated ethanol-induced decreases in the brain and adrenal gland in vivo TH activity in LS mice but not SS mice. Thus, there was an association between the ability of TRH to produce an activation of catecholamine neuronal systems (increased rate of catecholamine biosynthesis) and the analeptic action of TRH to reduce the CNS depressant effects of ethanol (decreased sleep times).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of electroconvulsive shock and haloperidol on in vivo tyrosine hydroxylation and tetrahydrobiopterin in the brain of rats with 6-hydroxydopamine lesions.

We have evaluated the effects of electroconvulsive shock (ECS) and haloperidol treatment on the in vivo tyrosine hydroxylation rate and the tetrahydrobiopterin levels in the nigrostriatal system of 6-OHDA-lesioned rats. The rate of DOPA accumulation was significantly decreased by 96% in the ipsilateral striatum and by 50% in substantia nigra of the 6-OHDA-lesioned rats compared to the control activity of contralateral non-lesioned striatum and substantia nigra. The loss of total biopterin was found to be 75% and 50% in the ipsilateral striatum and substantia nigra, respectively. Following administration of haloperidol, the rate of DOPA accumulation increased significantly in the striatum and substantia nigra on the lesioned side compared to that in the vehicle treatment group. Application of ECS also significantly increased the rate of DOPA accumulation in the ipsilateral striatum and substantia nigra compared to that obtained in the non-shocked rats. The biopterin levels in the nigrostriatal system of 6-OHDA-lesioned were elevated significantly in the striatum after haloperidol treatment; in contrast the biopterin levels were unchanged in response to ECS. Our results show that both haloperidol and ECS significantly enhanced the rate of in vivo tyrosine hydroxylation in the striatum and substantia nigra of rats with greater than 90% lesions. These results suggest that the nigrostriatal system, although up-regulated following 6-OHDA lesions, still maintains the potential for further up-regulation of dopaminergic function in response to haloperidol and ECS treatment.

Effects of electroconvulsive shock on tetrahydrobiopterin and GTP-cyclohydrolase activity in the brain and adrenal gland of the rat.

The effects of a single and repeated electroconvulsive shock (ECS) (300 mA, 0.2 s) on tetrahydrobiopterin (BH4) levels and GTP-cyclohydrolase activity in the brain and adrenal glands of rats were examined. Twenty-four hours after the last ECS treatment (one/day for 7 days), biopterin levels were significantly elevated in the locus coeruleus, hippocampus, frontal cortex, hypothalamus, ventral tegmental area, and adrenal gland. There were no changes in biopterin levels after a single application of ECS. GTP-cyclohydrolase activity was significantly increased in the locus coeruleus, frontal cortex, hippocampus, hypothalamus, and adrenal gland 24 h after repeated ECS and remained elevated in certain tissues up to 8 days after the last treatment. Kinetic analysis of adrenal and locus coeruleus GTP-cyclohydrolase 1 day after 7 days of ECS showed significant changes in both Km and Vmax values. These data suggest that the long-term increases in BH4 levels and GTP-cyclohydrolase activity after repeated ECS may play a part in the mediation of the antidepressant effects of ECS.

Differential responsiveness of the pituitary-thyroid axis to thyrotropin-releasing hormone in mouse lines selected to differ in central nervous system sensitivity to ethanol.

Long-sleep (LS) and short-sleep (SS) mice are genetic lines that differ in central nervous system sensitivity to ethanol. The possible role of TRH in mediating the difference in the thyroid status between these two lines was investigated. An increase in TRH gene expression in the paraventricular nucleus and TRH peptide levels in the hypothalamus between postnatal days 8-14 in both SS and LS mice coincided with increased circulating levels of thyroxine during this critical period of central nervous system development. No significant differences in TRH biosynthesis were observed between LS and SS mice during this time. Exogenous administration of TRH to LS and SS mice on day 8, when endogenous serum thyroxine levels were equivalent, resulted in a greater increase in serum thyroxine in SS mice (150%) than LS mice (51%). The differential response to the TRH stimulation test was also present on day 14 (SS, 43%; LS, 18%). The differential responsiveness of the pituitary-thyroid axis to exogenous TRH paralleled the differential increase in endogenous serum thyroxine observed between day 8 and 14 in these mice. Administration of TRH to day 20 and adult (60 days) LS and SS mice resulted in nearly equivalent (approximately 75%) increases in free thyroxine serum levels, yet the magnitude of thyroxine release was 50% greater in SS mice, due perhaps to between-line differences within the thyroid glands. It is unlikely that dissimilar endogenous levels of TRH account for the intrinsic difference in the thyroid status in LS and SS mice. Instead, the increased pituitary-thyroid responsiveness to TRH in SS mice during the second postnatal week may translate into increased functional capacity of the thyroid gland in adult SS relative to LS mice.

Effects of dopamine receptor agonists and antagonists on catecholamine release in bovine chromaffin cells.

Dopamine D2 receptors are known to regulate the release of catecholamines from neurons in the central and peripheral nervous systems. In the present study we have evaluated the effects of dopamine D2 agonists and antagonists on the release of endogenous norepinephrine and epinephrine stimulated by 50 microM nicotine in isolated bovine chromaffin cells in order to investigate whether isolated bovine chromaffin cells may contain functional dopamine D2 receptors. Dopamine (10(-4) and 10(-6) M), quinpirole (10(-5) M) and 2-amino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (10(-5) M) had no effect on the nicotine-stimulated release of norepinephrine or epinephrine from the bovine chromaffin cells. Pergolide (10(-6) M) and apomorphine (10(-4) M) produced a significant inhibition of nicotine-stimulated release of both norepinephrine and epinephrine from the chromaffin cells. The inhibitory effect of the selective dopamine D2 agonist pergolide on catecholamine release from the chromaffin cells was not reversed by 10(-6) M concentrations of the selective dopamine D2 receptor antagonists haloperidol, domperidone, metaclopramide, fluphenazine, flupentixol, (+)- or (-)-sulpiride, the dopamine D1 receptor antagonist SCH 23390 (10(-7) M) or the alpha receptor antagonist phentolamine (10(-6) M). These data suggest that the inhibition of nicotine-stimulated release of catecholamines from the bovine chromaffin cells is not a receptor-mediated effect. Further studies showed that the inhibitory effect of pergolide on catecholamine release in the bovine chromaffin cells was correlated with an inhibition of nicotine-stimulated 45Ca++ uptake and 22Na+ uptake into these cells. It is concluded that functional dopamine D2 receptors of the classical type do not exist on isolated bovine chromaffin cells.

The effects of electroconvulsive shock on catecholamine function in the locus ceruleus and hippocampus.

Repeated electroconvulsive shock (ECS) treatment (once per day for 7 days) produced a significant increase in tyrosine hydroxylase activity, GTP-cyclohydrolase activity and tetrahydrobiopterin (BH4) levels in the locus ceruleus and hippocampus from 1 to 4 days after the last treatment. These changes may be responsible for, or contribute to, the antidepressant effect of ECS treatment.

Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain.

The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with [3H]myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of [3H]inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.