Specific Activity Of Tyrosine Hydroxylase Research Articles

Neonatal overfeeding increases capacity for catecholamine biosynthesis from the adrenal gland acutely and long-term in the male rat.

A poor nutritional environment during early development has long been known to increase disease susceptibility later in life. We have previously shown that rats that are overfed as neonates (i.e. suckled in small litters (4 pups) relative to control conditions (12 pups)) show dysregulated hypothalamic-pituitary-adrenal axis responses to immune stress in adulthood, particularly due to an altered capacity of the adrenal to respond to an immune challenge. Here we hypothesised that neonatal overfeeding similarly affects the sympathomedullary system, testing this by investigating the biochemical function of tyrosine hydroxylase (TH), the first rate-limiting enzyme in the catecholamine synthesis. We also examined changes in adrenal expression of the leptin receptor and in mitogen-activated protein kinase (MAPK) signalling. During the neonatal period, we saw age-dependent changes in TH activity and phosphorylation, with neonatal overfeeding stimulating increased adrenal TH specific activity at postnatal days 7 and 14, along with a compensatory reduction in total TH protein levels. This increased TH activity was maintained into adulthood where neonatally overfed rats exhibited increased adrenal responsiveness 30min after an immune challenge with lipopolysaccharide, evident in a concomitant increase in TH protein levels and specific activity. Neonatal overfeeding significantly reduced the expression of the leptin receptor in neonatal adrenals at postnatal day 7 and in adult adrenals, but did not affect MAPK signalling. These data suggest neonatal overfeeding alters the capacity of the adrenal to synthesise catecholamines, both acutely and long term, and these effects may be independent of leptin signalling.

Angiotensin‐(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1-7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1-7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1-7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from (3)H-l-tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1-7) showed a significant decrease in TH specific activity. Ang-(1-7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT(2) receptor antagonist and not by an AT(1) or Mas receptor antagonist, suggesting the involvement of AT(2) receptors. The proteasome inhibitor MG132 blocked the Ang-(1-7)-mediated TH reduction. In addition, Ang-(1-7) increased the amount of TH-ubiquitin complexes, indicating that the Ang-(1-7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1-7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.

Microgravity decreases tyrosine hydroxylase expression in rat adrenals

During spaceflight, alterations in blood and urinary catecholamine (CA) levels have been observed, yet the cellular/molecular mechanisms leading to these changes are not known. We used molecular, immunological, and biochemical approaches to analyze in situ the expression of catecholamine enzymes in adrenal medullary chromaffin cells of rats flown for 6 days on board Space Shuttle mission STS-54. Exposure to microgravity (10(-6) g) resulted in a 35% inhibition of both the expression and the specific activity of tyrosine hydroxylase (TH), the rate-limiting step in the cascade of CA synthesis. By contrast, the expression, specific activity, and immunoreactivity of other catecholamine-synthesizing enzymes, e.g., phenylethanolamine-N-methyl-transferase (PNMT), were not altered. The total tissue CA contents were reduced, concomitant with a decrease in the epinephrine:norepinephrine ratio. These results are in line with reports of other gravity-sensitive cellular effects and suggest that the inhibition of TH expression might be due to a direct effect of microgravity on PKC-dependent signal transduction pathways in chromaffin cells.

Regulation of catecholamine-synthesizing enzymes in adrenals of Wistar rats under chronic stress

We have shown previously that chronic social stress has differential effects on adrenal weight and on tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) specific activity, depending on the experimental design. To determine the role of the sympathetic nervous system and of the hypothalamo-pituitary-adrenocortical axis (HPAA) in these modifications, we studied the mechanisms of regulation of these parameters in basal conditions as well as in response to reserpine treatment and chronic social stress in the Wistar strain of rats. We found that the adrenal weight is mostly dependent on the activity of the HPAA, which is increased in male rats living in mixed-sex colonies. PNMT specific activity is regulated by splanchnic innervation, confirming that its induction by social instability is a consequence of sympathetic nervous system hyperactivity. The increase of TH specific activity, as seen in unstable, mixed-sex colonies, is not under sympathetic control. However, we show that the pituitary may exert a tonic inhibitory influence, dependent on the sympathetic innervation. These data confirm that the HPAA and the sympathetic nervous system may be independently triggered in chronic social stress conditions.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in prenatal stage on the dopamine system in the postnatal mouse brain

A dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride (MPTP), administered to a pregnant female was found to affect postnatally the catecholamine metabolism of the pups. MPTP (5 mg/kg body weight/day) was administered to pregnant C57 Black BYA mice daily for 7 days between the 12th and 18th day of gestation. Dopamine levels and tyrosine hydroxylase (TH) activity were measured in the whole brain from the pups sacrificed after birth. In MPTP-treated pups at 7 days of age, TH total activity (TH activity/brain) did not change (92% of the control value), while TH specific activity (TH activity/mg protein) was increased to 163% of that in control mice. Thus, TH homospecific activity (TH activity/mg TH protein) doubled compared to the control mice. At 28 days of age, both the total activity and the specific activity of TH in the brains of postnatal mice were reduced to 50% and 78% of the control, respectively. Dopamine concentration in the striatum was also reduced significantly. Reduction in the TH activity and dopamine concentration were also observed at the age of 12 weeks. These data suggest that the prenatal exposure to MPTP induced a prolonged reduction of TH activity in the brains of mice with a transient increase of TH homospecific activity during the postnatal period.

Regulation of neurotransmitter expression by a membrane-derived factor

Cell-cell contact appears to play a critical role in the expression of transmitter traits in developing neurons. We have previously shown that cell membrane contact induced the de novo appearance of choline acetyltransferase (CAT) in virtually pure cultures of dissociated sympathetic neurons. A membrane-associated CAT-inducing factor has been extracted and purified 5000-fold. This factor exerts differential effects on transmitter traits in cultured sympathetic neurons. After 3 days in vitro, neurons exposed to the factor contained 40-fold higher levels of the neuropeptide substance P than controls. Somatostatin exhibited a similar dramatic elevation. In contrast, the factor had no effect on leucine-enkephalin. Further, the specific activity of tyrosine hydroxylase was reduced to 5% of control activity in treated cultures. These effects occurred in the absence of any increases in cell number. Thus, it appears that cell contact via membrane-associated factors may exert differential effects on phenotypic expression.

Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.

Cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by retinoic acid: increase of choline acetyltransferase activity and decrease of tyrosine hydroxylase activity

The effects of retinoic acid (RA), a naturally occurring metabolite of vitamin A, on the growth, morphology and neurochemical differentiation of the PC12 clone of rat pheochromocytoma cells were investigated. RA added to the medium inhibited the growth of PC12 cells in a dose-dependent manner up to 10 μM without affecting their morphology. In PC12 cells cultured in the presence of 10 μM RA for 8 days, the specific activity of choline acetyltransferase (ChAT) was increased2-fold, while the specific activity of tyrosine hydroxylase (TH) was decreased 0.5-fold compared with cells cultured in the absence of RA. Specific activities of acetylcholinesterase (AChE), glutamate decar☐ylase (GAD) and lactate dehydrogenase (LDH) were not affected by RA. Both the increase of ChAT and the decrease of TH induced by RA exhibited similar time and dose dependencies. RA inhibited the increase of TH activity induced by nerve growth factor (NGF), an adrenergic neuronotrophic factor on PC12 cells. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of PC12 cells independent of a morphological differentiation.

A new low-molecular-weight component promoting adrenergic development in cultured chick sympathetic neurons

A low-molecular-weight component present in medium conditioned by cultured chick liver cells (LCM) enhances the adrenergic properties of dissociated chick superior cervical ganglion (SCG) neurons in culture (Zurn and Mudry, 1986). This substance cannot replace NGF as a survival, growth, or differentiation factor. However, in the presence of NGF, it stimulates neuronal metabolism and catecholamine (CA), but not ACh production by the SCG neurons. The effect on transmitter production is greater than that on neuronal metabolism. Yet this is not due to an increase in the specific activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in CA synthesis. Interestingly, the effect of LCM on CA and ACh production, but not on neuronal metabolism, is potentiated in the presence of a large excess of NGF. The active component(s) present in LCM has a molecular weight lower than 500 Da and is not inactivated by heat or pronase treatment. So far, none of the small molecules tested (ascorbic acid, pyruvate, glucose, L-glutamic acid, glutathione, etc.) were able to mimic the effects of LCM on the SCG neurons. Thus this report describes a novel low-molecular-weight component different from NGF that promotes metabolism and adrenergic development in cultured chick sympathetic neurons.

Neurotransmitter synthesis, storage, and turnover in neonatally deafferented sympathetic neurons

In the superior cervical sympathetic ganglion (SCG) of the rat, a significant amount of morphological and biochemical maturation occurs in the first few postnatal weeks. The specific activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine (NE), increases during this time and is subject to transsynaptic regulation by the preganglionic inputs. In the present study, we examined the normal postnatal development of NE stores in sympathetic neurons and the transsynaptic regulation of this development. NE content undergoes an 8-fold increase from the time of birth, and stabilizes at adult levels at one month. Following neonatal deafferentation, there is a temporary stunting of NE accumulation in sympathetic neurons and a permanent reduction in the activity of TH, whether or not regeneration of the afferents occurs. When regeneration is prevented, the turnover of NE is significantly reduced, while NE levels rise to near normal levels. When regeneration is permitted, however, both the stored amount and turnover of NE attain normal levels. These data suggest that there is a critical period during the first two postnatal weeks when transsynaptic influences from afferents are necessary for the induction of TH in sympathetic neurons. Levels and turnover of transmitter do not have this critical period, but appear to depend solely on the functional integrity of the system.

Long-term regulation of tyrosine hydroxylase activity in the superior cervical ganglion in organ culture: Effects of nerve stimulation and dexamethasone

The rat superior cervical ganglion was stimulated in vitro via its preganglionic trunk and then maintained in organ culture for 2–3 days. Following nerve stimulation, the specific activity of tyrosine hydroxylase (TH) increased with a lag period of at least 12 h, and this increased enzyme activity was maintained throughout the cultured period. The magnitude of the increase in TH activity depended on the frequency and duration of preganglionic nerve stimulation. This transsynaptic increase in enzyme activity could be completely blocked by the nicotinic antagonist, hexamethonium. The synthetic glucocorticoid dexathasone, at concentrations above 1 nM, increased TH activity in unstimulated ganglia with a lag period of at least 24 h. The increased in enzyme activity produced by dexamethasone was not affected by nicotine antagonists or by prior decentralization of the superior cervical ganglion, in contrast to the findings of previous workers. These data indicate that dexamethasone can stimulate ganglionic TH activity in the absence of cholinergic stimulation. In addition, experiments combining dexamethasone with nerve stimulation demonstrate that the steroid can potentiate the effects on TH activity of brief periods of nerve stimulation.

Short-term regulation of tyrosine hydroxylase in tonically-active and in tonically-inactive dopamine neurons: Effects of haloperidol and protein phosphorylation

Dopamine (DA)-containing neurons of retina were employed as an experimental model for studying the short-term regulation of tyrosine hydroxylase (TH) in tonically-active and tonically-inactive neurons. These DA-containing neurons are trans-synaptically activated by light. Two mechanisms have been observed in this system for regulation of TH activity. A short-term activation of TH that is characterized by a decreased apparent K m for pteridine cofactors occurs in response to rapid increases of neuronal activity. A second mechanism occurs in response to prolonged, tonic changes of neuronal activity and is characterized by changes of V max. Both the K m changes and V max changes represent changes of specific activity of TH rather than enzyme induction. To determine the effects of short-term increases of neuronal activity on TH in tonically-active and tonically-inactive neurons, the effects of acute administration of haloperidol were examined in rats that were continuously light-exposed or light-deprived for 4 days. Haloperidol increased TH activity in both light-exposed and light-deprived retinas. The drug elicited the same percent stimulation in both experimental conditions. However, because the basal activity of TH was higher in the light-exposed than the light-deprived retinas, the absolute increase of TH specific activity was greater in the light-exposed samples. The effect of protein phosphorylation on TH activity in extracts of chronically light-exposed or light-deprived retinas was also examined to determine if the differences in the response to haloperidol might be due to a difference in the amount of TH available for short-term activation. Phosphorylation by endogenous cyclic AMP-dependent protein kinase (APK) or by purified catalytic subunit of APK resulted in larger increases of TH specific activity in extracts of light-exposed retinas than in those of light-deprived retinas. As was observed for haloperidol-induced activation, the percent stimulation elicited by phosphorylation was similar in extracts of light-exposed and light-deprived retinas. These observations suggest that more enzyme is available for short-term activation in tonically-active neurons than in those that are tonically inactive. A hypothetical model is proposed in which TH exists in active and inactive forms, the ratio of which depends on the tonic level of neuronal activity.

Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal chromaffin cells.

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: Dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10 5 cells/cm 2, to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant ( K D ) of 1 × 10 9, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.

Growth factor dependence of pheochromocytoma cells in chemically defined medium

We have previously described the isolation of a clonal pheochromocytoma cell line (PC-G2) which responds to nerve growth factor (NGF) [Goodman and Herschman, 1978] and epidermal growth factor (EGF) [Goodman et al, 1980] with increased specific activity of tyrosine hydroxylase (TH). This cell line thus can be used for studies of induction of a key enzyme in the biosynthesis of catecholamines using a homogeneous cell population under controlled environmental influences. However, the need for serum in the culture medium used to grow these cells still introduced a lack of total environmental control. Several laboratories have circumvented this problem by growing cells in chemically defined medium. In the present report we demonstrate that with a modification PC-G2 cells can survive and grow in the chemically defined N3 medium devised by Bottenstein and Sato [1979]. While another pheochromocytoma cell line, PC-12, was shown to grow in N3 medium without any supplement [Bottenstein et al, 1979], the PC-G2 cells exhibit an absolute requirement for NGF or EGF in order to grow in this chemically defined medium. As was the case in the induction of TH in serum-containing medium [Goodman and Herschman, 1978; Goodman et al. 1980], EGF was found to be 100 times more potent than NGF in the support of growth of the PC-G2 cells. In the present report we demonstrate not only that either of these growth factors will support the growth of the PC-G2 cells, but also that the relative potency of these two factors for support of the cell growth is similar to their relative potencies in the induction of tyrosine hydroxylase in serum-containing medium.

Ontogeny of neurochemical markers for noradrenergic, GABAergic, and cholinergic neurons in neocortex lesioned with methylazoxymethanol acetate.

Abstract: Methylazoxymethanol acetate (MAM), a potent, rapidly eliminated nucleic acid alkylating agent, produces microencephaly in rat pups when injected into their dams on day 15 of gestation. In the adult microencephalic rats, neuronal loss is largely confined to telencephalic structures, such as the superficial neocortical laminae, whose neuroepithelial progenitor cells were undergoing vigorous replication during the chemical exposure. Histological examination of the forebrain 2 days after injection revealed early selective damage to the ventricular geminal zone with relative sparing of cortical plate neurons generated on earlier days. The degree of specificity of MAM's action on neurochemically defined neuronal populations was examined by measuring presynaptic markers for GABAergic, noradrenergic and cholinergic neurons in atrophic lateral cortex from 20 days gestation to adulthood. Although treatment reduced GABAergic markers (GABA, its synthetic enzyme and synaptosomal uptake process) in proportion to loss of cortex mass (‐67%), the maturational pattern for remaining GABAergic neurons was virtually normal. Although the maturational sequence of noradrenergic markers was similar to control, the concentration of endogenous norepinephrine, [3H]norepinephrine uptake and tyrosine hydroxylase specific activity were two‐ to fourfold higher than control at each time. However, total noradrenergic markers per cortex section were nearly identical to control throughout development, indicating that development of the noradrenergic axonal arbor in neocortex was insensitive to loss of neurons in the terminal field. Maturation of cholinergic markers (endogenous acetylcholine, its synthetic enzyme and [3H]choline uptake) in the atrophic cortex was biphasic: concentrations were similar to control values for the first 12 postnatal days, but gradually rose to levels twofold higher than control. These results indicate that neurochemical alterations observed in cortex from prenatally MAM‐treated rats are primarily the result of early selective elimination of neuronal subpopulations. Fetal MAM exposure appeared to have minimal effects on biochemical differentiation of neurons remaining intact in the atrophic cortex. MAM appears to be a useful toxin for producing selective loss of neuronal groups based on their time of generation in the fetus.

Effects of synaptic and antidromic stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion.

1. The effects of orthodromic and antidromic stimulation of the rat superior cervical ganglion on the specific activity of the enzyme tyrosine hydroxylase have been studied. 2. Orthodromic stimulation of the ganglion via the sympathetic trunk produced an increase in the activity of tyrosine hydroxylase when measured 3 days later while causing no change in the protein content of the ganglion. This increase in the specific activity of tyrosine hydroxylase was blocked by administration of the nicotinic antagonist, hexamethonium. 3. Antidromic stimulation of the superior cervical ganglion by stimulating the internal carotid nerve, the external carotid nerve or both nerves simultaneously produced no change in the specific activity of tyrosine hydroxylase. 4. Parallel increases in tyrosine hydroxylase activity and protein content per ganglion were seen when the internal carotid nerve was stimulated but similar changes were seen in "sham-stimulated" animals. These "non-specific" changes were apparently produced by the trauma involved in the extensive dissection necessary to position electrodes on this nerve trunk. 5. We conclude that an increase frequency of firing in post-ganglionic neurones is not a sufficient stimulus to elevate the specific activity of tyrosine hydroxylase. Rather some other aspect of nicotinic receptor stimulation seems to be required.

Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: changes in specific activities of enzymes.

Abstract:A rat model was developed in which the comparative effects of malnutrition and ethanol on the neurochemical maturation of catecholamine neurons in brain were studied. The animals were offspring of rats optimally nourished (control pups), fed a diet with 35% of the calories supplied by ethanol (EtOH pups), or a diet calorically equivalent to the latter but lacking ethanol (isocaloric = IC pups). These latter two groups were calorically deprived since rats fed the diet containing ethanol ate 40–60% less than the optimally nourished group. The diets were administered to dams either during the last week of gestation (prenatal) or during lactation (postnatal). The protein content per gram wet weight was not significantly different between brains of control and experimental pups. Developmental changes in EtOH pups were noted in the activities of tyrosine hydroxylase (TOH) and dopamine β‐hydroxylase (DBH). The specific activity of TOH was increased in EtOH and IC pups preand postnatally. In the postnatal experimental group TOH activity in the brains of EtOH pups was higher not only when compared with control pups but also with IC pups. Prenatal caloric deprivation or ethanol exposure also caused increased specific activity of DBH in the neonatal rat brain. In contrast, exposure to ethanol postnatally caused a significant and persistent reduction in the specific activity of DBH, not only compared with control pups but also to matched IC pups exposed postnatally to the IC diet. The specific activity of 3,4‐dihydroxyphenylalaninedecarboxylase (DOPADC) was significantly elevated in the brains of prenatal and postnatal EtOH and IC pups, with little difference between the groups. Activities of monoamine oxidase (MAO) or catecholamine‐0‐methyltransferase (COMT) were generally lower in EtOH or IC pups than in control pups, again with little difference between EtOH and IC pups. These results suggest that an impaired metabolism of dopamine and norepinephrine is detectable in the brains of pups as a consequence of malnutrition, ethanol exposure, or both.

Nerve growth factor-mediated induction of tyrosine hydroxylase in a clonal pheochromocytoma cell line.

We have established a clonal cell line, PC-G2, from an experimentally induced rat pheochromocytoma. Administration of nerve growth factor to PC-G2 causes a 4- to 8-fold induction in the specific activity of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase(3-hydroxylating); EC 1.14.16.2]. The response is elicited in a dose-dependent fashion, at concentrations above 0.1 microgram/ml. Antiserum to nerve growth factor inhibited the induction of tyrosine hydroxylase. Dexamethasone enhances the nerve growth factor-mediated elevation of tyrosine hydroxylase. After 3--4 days of exposure to nerve growth factor the maximal induction of tyrosine hydroxylase is seen, although a significant increase can be observed after 24 hr. In contrast to the PC-12 cell line (derived from the same tumor), in which neurite outgrowth occurs in response to nerve growth factor, there is no morphological change or alteration in growth rate of PC-G2 cells after exposure to nerve growth factor.

Microinjection of kainic acid into the rat hippocampus

The effects of unilateral injection of kainic acid into the rate hippocampus have been examined in terms of morphologic, neurochemical and behavioral sequelae. Infusion of 10 nmoles if kainate causes a rapid and complete degeneration of neuronal perikarya in the entire hippocampal formation followed by gliosis and atrophy of the region. This unilateral neuronal loss is accompanied by a 50% decrease in the specific activity of the biochemical markers for GABAergic neurons including glutamic acid decarboxylase, endogenous GABA and synaptosomal uptake of [ 3H]GABA. The extrinsic hippocampal cholinergic and noradrenergic afferents also exhibit significant alteration. Although the specific activity of choline acetyltransferase is unaffected and the specific activity of tyrosine hydroxylase is significantly increased in the injected hippocampus, the synaptosomal high affinity uptake process for [ 3H]choline and [ 3H]norepinephrine are significantly reduced at 10 days after injection. Whereas the level of endogenous acetylcholine is elevated in the lesioned hippocampus at 2 days after injection, the level of endogenous norepinephrine is reduced. For several hours after intrahippocampal injections of 5 nmoles or more of kainate, rats exhibit epileptiform behavior. Intrahippocampal injection of kainate may be a useful rodent model for temporal lobe seizure disorders.