Evaluation of macrocytosis by general practitioners

The activities of monoamine oxidases, MAO-A and MAO-B, were separately determined in the cerebellum (CE) from adult rats neonatally exposed to 5 Gy X-irradiation. They were found to be markedly reduced: 58% and 66% of values from nonirradiated, littermate controls. Since the specific activities of both isoenzymes (per mg tissue weight) were not significantly different from controls, the reduction of activity per CE is basically explained by the irradiation-induced cerebellar atrophy. The unmodified MAO-A specific activity makes it highly improbable that the increase in the cerebellar noradrenaline content, characteristic of neonatally X-irradiated rats, could be due to a decreased neuronal metabolism of noradrenaline by this enzyme.     

Tyrosine hydroxylase gene expression in the locus coeruleus of depression-model rats and rats exposed to short-and long-term forced walking stress

Abnormal brain noradrenergic function is thought to cause depressive illnesses which are sometimes manifested or aggravated under stressful conditions. To investigate the effect of chronic stress on noradrenaline (NA) synthesis in the brain we used in situ hybridization to examine the expression of tyrosine hydroxylase (TH) mRNA in the locus coeruleus (LC) of "depression-model rats" that exhibit reduced activity following exposure to long-term (14 days) forced walking stress (FWS). We also examined TH mRNA expression in rats stressed for 30 minutes, 3 hours and 1, 2 (short-term), 6 or 12 (long-term) days. The expression of TH mRNA increased markedly following 1 to 12 days of FWS, but not in rats exposed to FWS for 30 minutes or 3 hours. The expression also increased significantly in the depression-model rats, but not in the "spontaneous recovery rats" whose activity was restored after long-term stress. Our results suggest that NA synthesis remains high in the FWS-induced depression-model rats because of the high levels of TH mRNA expression in the LC. Our results also suggest that FWS is initially a mild stress but gradually becomes a severe form of unadaptable stress as reflected by delayed but persistent increases in TH mRNA expression.     

Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptamine in rat brain

In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats. Whereas administration of l-triiodothyronine (10 mug/100 g/day) for 30 days to 120-day-old rats increased the levels of tyrosine by 23% and of tryptophan by 43%, no appreciable change was noted in tryptophan hydroxylase activity. In contrast to neonatal hyperthyroidism, excess of thyroid hormone in adult rats failed to produce any change in motor activity and tended to decrease striatal tyrosine hydroxylase activity only slightly. The concentration of dopamine remained unchanged in all regions of the brain except in midbrain where it rose by 19%. Whereas norepinephrine concentration was altered in hypothalamus, pons-medulla and midbrain, the levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, were significantly decreased in striatum and cerebellum. Since dopaminergic and noradrenergic neurons are the critical components of the motor system, the possibility exists that elevated behavioral activity in young L-triiodothyronine-treated animals might be associated with increased turnover of catecholamines in neuronal tissue.     

Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long-Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+ buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+ buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.     

L-2-chloropropionic acid inhibits glutamate and aspartate release from rat cerebellar slices but does not activate cerebellar NMDA receptors: implications for L-2-chloropropionic acid-induced neurotoxicity

L-2-Chloropropionic acid (L-CPA), when orally administered at single high dose to rats produces a selective lesion in the cerebellum involving destruction of a high proportion of granule cells by a mechanism which involves N-methyl-D-aspartate (NMDA) receptors. Receptor binding studies demonstrated that L-CPA a had low affinity at the glutamate and glycine binding sites at NMDA receptors (530-660 microM), respectively, whereas L-CPA did not displace [3H]AMPA, [3H]NBQX or [3H]kainate from AMPA or kainate receptors. Whole cell-patch clamp experiments using cultured granule cells failed to demonstrate changes in membrane potential of cultured granule cells when either L-CPA (0.25 or 1 microM) was added alone to the bathing solution, or in combination with glycine (10 microM). Furthermore L-CPA did not alter the magnitude of the inward current produced by application of NMDA (100 microM)) to cultured granule cells, in the presence of glycine, as measured by patch clamp techniques. Experiments were also performed to discover whether L-CPA may alter the release of the excitatory amino acids from the cerebellum, which may then indirectly alter activity at glutamate receptors, leading to neuronal cell death. L-CPA (2 mM) did not affect either basal or stimulated (electrical or high potassium) endogenous aspartate release from superfused cerebellar slices nor did it alter the basal or stimulated release of [3H]aspartate from preloaded slices when introduced into the superfusion medium over 30 min. However, when cerebellar slices were preincubated with 2 mM L-CPA for 2 h at concentrations that are known to be neurotoxic to the brain in vivo, but not in vitro, the stimulated endogenous glutamate and aspartate net release was significantly attenuated, as compared to controls. Basal release was not significantly affected by the introduction of L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum. In conclusion, although L-CPA does not appear to directly alter NMDA receptor activity the L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum.     

Increase in dopamine turnover and tyrosine hydroxylase enzyme in hippocampus of rats fed on low selenium diet

We have studied the turnover of dopamine, noradrenaline, and serotonin and their metabolites in hippocampus of adult female rats that were fed control or selenium-deficient diets during 15 days. Under these circumstances, there was an increase of dopamine turnover (4-fold) in rats fed with selenium-deficient diet with respect to controls and also an increase in the tyrosine hydroxylase activity (75.8%), which was the result of the increase of the amount of the enzyme (2-fold), without significant change in the phosphorylation of the tyrosine hydroxylase. In addition the glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase activities have been studied. After selenium-deficient diet, the enzymatic activities of superoxide dismutase and catalase did not show change with respect to the controls; however glutathione reductase and glutathione peroxidase significantly decreased 15% and 29%, respectively. It is concluded that the increase in dopamine turnover seems to be associated with the induction of tyrosine hydroxylase enzyme. In these conditions the decrease in antioxidant capacity may produce a cascade of events, which accelerates the degenerative process, since the increase in dopamine turnover produces an increase in oxygen radical by monoamine oxidase activity.     

Pharmacology of descending noradrenergic systems in relation to motor function

The function of descending noradrenergic systems in the spinal ventral horn has not been fully elucidated. We have reviewed our own findings and those of others relating to motor function of these noradrenergic systems. We studied the effects of adrenergic drugs on spinal reflexes, decerebrate rigidity, and noradrenaline release from the spinal cord in rats, and motoneuron activity in spinal cord slices isolated from adult rats. It was shown that the descending noradrenergic systems were facilitatory to the motor system, and that alpha 1-antagonistic action at the spinal cord and alpha 2-agonistic action at the brainstem inhibited spinal motor activity by blocking spinal alpha 1-receptors and by reducing the release of noradrenaline in the spinal cord, respectively.     

Effects of exposure to an estrous female on forebrain monoaminergic neurotransmission in the non-copulating male rat

Regional changes in the rate of brain monoamine synthesis were monitored in male rats exposed to, but prevented from physical contact with, an estrous or an ovariectomized female. The in vivo rate of tyrosine and tryptophan hydroxylase activities were estimated by measuring the accumulation of DOPA and 5-HTP following inhibition of cerebral aromatic L-amino acid decarboxylase by means of 3-hydroxybenzylhydrazine (NSD-1015) treatment (100 mg/kg i.p.). 5 min upon NSD-1015 treatment, the males were exposed to an intact estrous female or an ovariectomized female for 20 min before decapitation and brain dissections. Exposure to an estrous female produced an increased rate of tyrosine and tryptophan hydroxylase activity in the medial prefrontal cortex, the dorso-lateral neostriatum and in the ventral neostriatum, in comparison with home-cage controls. By the same comparison, exposure to an ovariectomized female resulted in an increased rate of tyrosine hydroxylase activity in the medial prefrontal cortex, but not in the neostriatal areas, whereas tryptophan hydroxylase activity was unaffected. Finally, exposure to the empty test cage, with no stimulus females present, did not produce any statistically significant changes in the rate of tyrosine or tryptophan hydroxylase activity in any of the brain areas sampled. Taken together with recent findings from this laboratory, the present results demonstrate that the level of sexual motivation brought about by the olfactory, auditory and/or visual stimulation of a receptive female is associated with an increased demand on catecholamine and 5-hydroxytryptamine synthesis in the limbic forebrain of the male rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by alpha2-adrenoceptors

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [3H]noradrenaline was diminished by more than 80% under Ca2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [3H]noradrenaline, however, it was less effective in reducing the release of [3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 microg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 microM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (-)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [3H]noradrenaline release. While CH 38083, a non subtype-selective alpha2-antagonist and BRL44408, the subtype-selective alpha2AD antagonist augmented the evoked release of [3H]noradrenaline, ARC239, a selective alpha2BC antagonist was without effect. In contrast, neither of the alpha2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and alpha1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [3H]noradrenaline is inhibited by endogenous noradrenaline via alpha2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.     

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry

The aim of the present study is to examine whether noradrenergic neurons of the locus coeruleus (LC) of the rat contain monoamine oxidase (MAO) activity. Sections were processed initially for MAO enzyme histochemistry using tyramine as a substrate, followed by fluorescence immunohistochemistry for tyrosine hydroxylase (TH). In the LC, virtually all TH-immunoreactive neurons (i.e., noradrenergic neurons) were also positive for MAO. No MAO activity was found in any TH-negative neurons. Neurons in the LC have previously been shown to form dopamine during noradrenaline biosynthesis and to produce serotonin from exogenously administered l-5-hydroxytryptophan. Moreover, dopamine- and serotonin-degrading MAO activity has also been found in LC neurons. Therefore, our results indicate that MAO activity is localized within noradrenergic neurons in the LC and is likely involved in the degradation of dopamine that is endogenously synthesized, and also in the elimination of serotonin that is produced from exogenous precursors.     

Intramedullary fixation of unstable both-bone forearm fractures in children

Mesaconitine, one of the main alkaloids contained in Aconiti tubers, is a centrally acting analgesic without affinity to opioid receptors. It has been reported that the antinociception is due to an interaction with the noradrenergic system. In the present study, the effect of mesaconitine on the uptake of noradrenaline and on neuronal activity was examined in rat hippocampus. Experiments were performed as a study of [3H]noradrenaline uptake into rat hippocampal synaptosomes. Mesoconitine inhibited [3H]noradrenaline uptake in a concentration-dependent manner with a Ki of 111.95+/-18 nM. In a further series of experiments, the effects of mesaconitine on the extracellularly recorded population spike were investigated in rat hippocampal slices. At a concentration of 10 nM, mesaconitine increased the amplitude of the postsynaptic population spike by 31.10%+/-6.7% of control and elicited one or two additional spikes. The presynaptic fiber spike and the field excitatory postsynaptic potential were not affected by this alkaloid. The enhancement of neuronal activity was abolished by 1 microM propranolol as well as by 1 microM timolol. It is concluded that mesoconitine increased the excitability in rat hippocampal pyramidal cells by an involvement of the noradrenergic system, with at least one mechanism being inhibition of noradrenaline uptake leading to an enhanced extraneuronal noradrenaline level.     

Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.     

Evidence from microdialysis and synaptosomal studies of rat cortex for noradrenaline uptake sites with different sensitivities to SSRIs

1. Microdialysis of the frontal cortex of freely-moving rats and uptake of [3H]noradrenaline into cortical synaptosomes were used to evaluate changes in efflux of noradrenaline in vivo and uptake of [3H]noradrenaline in vitro, respectively, induced by the selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram, and the tricyclic antidepressant, desipramine. 2. Noradrenaline efflux was increased during local infusion into the cortex of each of these drugs. All three agents also inhibited synaptosomal uptake of [3H]noradrenaline; this inhibition was unaffected by a substantial (50%) lesion of central 5-hydroxytrytaminergic neurones induced by intracerebroventricular infusion of 5,7-DHT (150 microg). 3. A noradrenergic lesion (70%), induced by pretreatment with the selective neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 40 mg kg(-1) i.p.), 5 days earlier, abolished the increase in noradrenaline efflux caused by local infusion of fluoxetine. In contrast, the desipramine-induced increase in efflux was greater than in non-lesioned rats whereas the effect of citalopram on noradrenaline efflux was unaffected by DSP-4 pretreatment. 4. The combined results of all these experiments suggest that there could be more than one, functionally distinct, noradrenaline uptake site in rat frontal cortex which can be distinguished by their different sensitivities to desipramine and the SSRIs, fluoxetine and citalopram.     

Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies

Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and on forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX-forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the serine 40-phosphorylated form of tyrosine hydroxylase. Treated slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre plexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggests that antibodies directed against phosphorylated forms of tyrosine hydroxylase can be used to evaluate the state of tyrosine hydroxylase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo.     

Quantitative evaluation of the blood-brain barrier capacity to form dopamine from circulating L-DOPA

The ability of the blood-brain barrier to form dopamine from increasing doses of systemically administered L-DOPA has been studied in rats by a combination of chemical determination of dopamine, and histochemical and cytofluorometric measurements of L-DOPA and dopamine. The break-through of L-DOPA from the circulation into the brain parenchyma via the enzymatic blood-brain barrier was estimated by comparing the amount of newly formed dopamine in the caudate nucleus-putamen and in the cerebellum. The capillaries were found to efficiently trap L-DOPA in their walls, and an upper limit was reached (at an administered i.p. dose of 100 mg/kg of L-DOPA). It could be estimated that approximately 3% of the total dose of L-DOPA given was decarboxylated by the blood-brain barrier. The possible influence by the regional differences in perfusion of the two regions seen after administration of L-DOPA was ruled out in measurements of local cerebral blood flow using the 14C-ethanol technique.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

The serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine facilitates noradrenaline release from rat spinal cord slices and inhibits monoamine oxidase activity

1. The influences of the purported serotonergic agonist 5-methoxy-N,N-dimethyltryptamine (MeODMT) on noradrenaline release and metabolism were investigated in a rat spinal cord release model and a monoamine oxidase (MAO) assay. 2. MeODMT inhibited the basal outflow of tritium from rat spinal cord slices preincubated with [3H]noradrenaline and enhanced the electrically-evoked overflow. 3. Effects on basal outflow were not observed, when monoamine oxidase (MAO) was inhibited by pargyline. Effects on the evoked overflow were not observed in the presence of metitepine or phentolamine. 4. Preferential inhibition by MeODMT of MAO A-type enzyme activity was found in a direct assay. 5. The results provide evidence for two different effects by which MeODMT reinforces noradrenergic neurotransmission in the rat spinal cord: facilitation of stimulation-evoked noradrenaline release and inhibition of noradrenaline metabolism by MAO inhibition.     

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction

The effect of systemic administration of the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on noradrenaline efflux in the frontal cortex was studied in freely-moving rats using microdialysis in vivo. Five days after treatment with DSP-4 (40 mg/kg i.p.), the noradrenaline content of the frontal cortex was reduced by 75%. Yet, noradrenaline efflux in the frontal cortex was nearly two-fold greater in DSP-4 treated rats than in saline-injected controls. Local infusion of the noradrenaline-selective uptake blocker, desipramine (5 microM), via the microdialysis probe, increased noradrenaline efflux in rats from both groups. Perfusion of Ringer's solution, containing 80 mM K+, also increased noradrenaline efflux in both groups, but the increase after DSP-4 pretreatment was greater than in the controls. In contrast, removal of Ca2+ from the infusion medium reduced noradrenaline efflux in both treatment groups. These results indicate that, at this dose, DSP-4 increases the extracellular concentration of noradrenaline in rat frontal cortex despite causing a partial lesion of noradrenergic neurones. This is due to an increase in the release of noradrenaline, although reduced clearance is also likely. These data challenge the assumption that depletion of noradrenaline content after treatment with DSP-4 invariably translates into diminished noradrenergic transmission.     

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.