Adrenal Tyrosine Hydroxylase Activity Research Articles

The Effects of Acute Glucoprivation on Adrenomedullary Function in SHR and WKY Rats

We have shown previously, acute intraperitoneal administration of 2-deoxy-d-glucose (2DG) into Sprague-Dawley rats led to activation of the adrenal medulla chromaffin cells, indicated with increased protein kinase activity and increased tyrosine hydroxylase (TH) phosphorylation, as well as increased plasma adrenaline and glucose levels. Here we have used spontaneous hypertensive (SHR) and Wistar Kyoto (WKY) rats to investigate whether hypertension alters basal adrenal chromaffin cell function, or the response of these cells to acute 2DG treatment. At basal level, we found no differences in adrenal medulla TH protein, TH phosphorylation, TH activity or catecholamine levels between SHR and WKY despite a significant difference in the level of systolic blood pressure; nor were there differences in plasma catecholamine levels or blood glucose (BG). Furthermore, the vehicle animals evoked no significant changes in any parameter measured in SHR, but evoked significant increases in pSer19TH, plasma adrenaline and BG in WKY. Single episode of glucoprivation evoked increases in PKA and CDK/MAPK, pSer40TH, pSer31TH, TH activity, and plasma adrenaline and BG in SHR, and in addition evoked increases in PKC, CAMKII, and pSer19TH in WKY. These findings are significant which indicates hypertension does not impact catecholamine function in the adrenal gland. It also appears that hypertension does not alter the adrenal response to glucoprivation. The findings are also significant as WKY showed greater adrenal activation of protein kinases and TH phosphorylation in response to saline and 2DG when compared to SHR and possible reasons for these findings are further discussed.

Early social instability affects plasma testosterone during adolescence but does not alter reproductive capacity or measures of stress later in life

The social environment plays an important role in modulating processes of the hormonal and behavioural profile of an animal in a variety of group-living species. In wild cavies for instance, unstable social environmental conditions during pregnancy and lactation lead to an infantilised biobehavioural profile of the male offspring. In the present study, the influence of the social environment during pregnancy and lactation on the male wild cavy offsprings' plasma testosterone development, reproductive capacity and stress system activity was investigated. To this purpose, 12 sons whose mothers had lived in an unstable social environment during pregnancy and lactation were compared with 12 sons whose mothers had lived in a stable social environment during the same time. Plasma testosterone (T) and plasma cortisol (C) concentrations were determined from days 20 to 107 of age. Adrenal tyrosine hydroxylase (TH) activity and different parameters of reproductive capacity (weights of testes, epididymides and accessory sex glands, cellular composition of the testes, DNA fragmentation indices and sperm motility parameters) were analysed at day 107 of age. TH activity and plasma C were unaffected by different social environmental conditions early in life. The developmental time course of T concentrations, however, was significantly different: Sons whose mothers had lived in an unstable social environment during pregnancy and lactation showed a delayed increase in T concentrations around adolescence compared to controls. In contrast, no reproduction-related parameters measured within this study differed significantly between the two groups. Thus, early social instability affects plasma testosterone development during adolescence in a significant way but does not alter reproductive capacity or measures of stress later in life.

Individual housing of mice — Impact on behaviour and stress responses

The replicability of results derived from studies in rodents might be influenced by stress caused by inappropriate housing conditions. Here we compared the experimental behaviour and stress response (circulating corticosterone level and adrenal tyrosine hydroxylase activity) of individually-housed male and female inbred mice with that of animals housed in social groups. All mice were behaviourally tested in the modified hole board test (mHB). Male C57BL/6, BALB/c and A mice housed in groups of 3 were compared with individually-housed mice. In a subsequent experiment female C57BL/6 and BALB/c mice were housed under similar conditions. To exclude the possible effects of within-cage order of testing, only one individual per group was behaviourally tested. Neither male nor female mice housed individually showed stronger signs of stress than their socially-housed counterparts. However, we observed a within-cage order effect on the hormonal stress response (corticosterone) in socially-housed female C57BL/6 mice. No effects of individual housing on behaviour in the mHB were found.

Evidence for regulation of tyrosine hydroxylase mRNA translation by stress in rat adrenal medulla

Long-term stress leads to the induction of tyrosine hydroxylase (TH) protein and enzymatic activity in the adrenal medulla. This adaptive response is necessary to maintain the catecholamine biosynthetic capacity of adrenal chromaffin cells during periods of sustained catecholamine secretion. In this report we demonstrate that when rats are subjected to short-term stress, TH mRNA is induced for at least 24 h, but TH protein and TH activity (assayed under Vmax conditions) are not increased. In contrast, adrenal TH mRNA, TH protein and TH activity are induced in rats subjected to long-term stress. Using sucrose gradient fractionation, we show that the lack of induction of TH protein after one type of short-term stressor, a single 2-h immobilization stress is associated with a decrease in the percentage of TH mRNA molecules associated with polysomes. In contrast, after repeated immobilizations the polysome profile of TH mRNA is identical to that observed in control animals, even though TH mRNA is induced 2- to 3-fold. These results are consistent with the hypothesis that even though TH mRNA is induced by short-term stressors, mechanisms that control TH mRNA translation must also be appropriately regulated for TH protein to be induced.

The Effects of Carbon Dioxide Pneumoperitoneum on Tyrosine Hydroxylase Activity

The aim of this study is to investigate the effects of carbon dioxide (CO2) pneumoperitoneum on tyrosine hydroxylase (TH) activity and total protein (TP) levels. Forty male Sprague-Dawley rats were randomized into 10 groups, each consisting of 10 rats. Groups 1 and 2 consisted of anesthesia and sham-operated control rats, respectively. In the study groups, 10 mm Hg (group 3) and 15 mm Hg (group 4) pneumoperitoneum with CO2 were accomplished. At the end of the procedures, the brains and adrenals were removed quickly, and the hypothalamus and adrenal medulla separated, weighed, and homogenized. TH activity and TP levels were determined. The adrenal medulla TP and TH activity levels were decreased consistently and this decrease was significant in the sham and pneumoperitoneum groups compared with the control group (P<0.05). The adrenal medulla TP and TH activity levels were reduced significantly in group 4, as compared with the other groups (P<0.05). Elevation of hypothalamic TH activity in group 4 was significantly higher than in the other groups (P<0.05). These results indicate that CO2 pneumoperitoneum applied with 10 and 15 mm Hg pressure gradually decreases the adrenal medulla TH activity; TH is an indispensable enzyme for the biosynthesis of catecholamines. CO2 pneumoperitoneum with 15 mm Hg pressure significantly elevated hypothalamus TH activity.

Long-term effects of husbandry procedures on stress-related parameters in male mice of two strains.

In socially unstable groups of male laboratory mice, individuals may experience a chronic stress situation. Previous experiments have shown that the transfer of specific olfactory cues during cage cleaning, and the provision of nesting material decrease aggression and stress in group-housed male mice. In this study, the combined effect of these husbandry procedures were tested for their long-term effect on stress in groups of moderately aggressive (BALB/c) and severely aggressive (CD-1) male mice. The physiological and behavioural stress-related parameters used were body weight, food and water intake, spleen and thymus weight, adrenal tyrosine hydroxylase activity, urine corticosterone levels and behaviour in a cage emergence test. Long-term provision of nesting material and its transfer during cage cleaning was found to influence several stress-related physiological parameters. Mice housed in cages enriched with nesting material had lower urine corticosterone levels and heavier thymuses, and they consumed less food and water than standard-housed mice. Furthermore, marked differences were found between strains. CD-1 mice were less anxious in the cage emergence test, weighed more, ate and drank more, and had heavier thymuses but lighter spleens and lower corticosterone levels than BALB/c mice. We conclude that the long-term provision of nesting material, including the transfer of nesting material during cage cleaning, reduces stress and thereby enhances the welfare of laboratory mice.

Early social stress in male Guinea-pigs changes social behaviour, and autonomic and neuroendocrine functions.

The present study was undertaken to investigate the effects of pre- and early postnatal social stress on the functioning of hormonal, autonomic and behavioural systems, by studying the distribution of sex hormone receptors in limbic brain systems. Dams had either lived in groups with a constant composition (= stable social environment) or in groups with changing compositions, i.e. every third day, two females from different groups were exchanged (= unstable social environment). The subjects were male offspring of dams who had either lived in a stable social environment during pregnancy and lactation (= control males) or in an unstable social environment during this period of life (= early stressed males). From days 20-80, the spontaneous behaviour of control males and early stressed males was recorded in their home cages. Five control males and five early stressed males were killed at 75 days, and five control males and five early stressed males at 120 days. Blood samples were taken to determine serum concentrations of cortisol, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate and oestrogen. The adrenals were prepared to determine tyrosine hydroxylase activities and the brains were used to investigate the distribution of sex-hormone receptors in specific hypothalamic and hippocampal brain areas. Early stressed males showed a behavioural infantilization that was accompanied by significantly decreased adrenal tyrosine hydroxylase activities and dehydroepiandrosterone levels. Furthermore, early stressed males showed a down-regulation of androgen receptors in the medial preoptic area and the nucleus arcuatus of the hypothalamus, as well as of oestrogen receptor alpha in the hippocampus compared to control males. Thus, the present study provides clear evidence that early social stress induces changes in endocrine, autonomic and limbic brain function, which is mirrored by changes in male social behaviour.

Effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice

This study investigated effects of different forms of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice. For this purpose, animals of the inbred strain CS were kept in groups of four males under three different housing conditions: (A) nonstructured Makrolon type III laboratory cages (“standard-housing” = S); (B) equivalent laboratory cages that were enriched with a box and a scaffolding (“enriched-housing” = E); and (C) spacious terraria that were structured richly (“super-enriched-housing” = SE). Both forms of enrichment caused a sharp rise in aggressive behavior, though play behavior was increased in E and SE mice, too. Levels of sociopositive behaviors in S and SE mice were higher than those in E mice. Plasma corticosterone concentrations and adrenal tyrosine hydroxylase activities were significantly increased in male mice kept in both forms of enriched cages, indicating an activation of the pituitary–adrenocortical and the adrenomedullary systems. The behavioral and endocrinological differences were partly reflected by immunological parameters: SE mice had levels of IgG1 and ratios of IFN-γ/IL-10 and IL-2/IL-10 significantly lower than those of S mice. Ratios of IgG2a/IgG1 were significantly higher in SE mice. The absolute percentages of CD8 cells in E-mice were significantly lower than those in S mice. Despite the elevated levels of stress hormones under both forms of enriched housing, the behavioral parameters also indicate positive effects of the enrichment, especially on SE animals. Obviously, an environmental enrichment is beneficial for male mice as long as the spatial conditions are generous enough to allow coping with the increased aggression brought about by the enrichment.

Chronic nicotine treatment leads to sustained stimulation of tyrosine hydroxylase gene transcription rate in rat adrenal medulla.

Nicotine is a powerful stimulant of the sympathoadrenal system, causing the release of peripheral catecholamines and activation of catecholamine biosynthesis. In previous reports, we have studied the mechanisms by which short-term nicotine treatment regulates tyrosine hydroxylase (TH) in adrenal medulla. In this report, we study the effects of chronic nicotine treatment on adrenal TH gene expression. Rats were injected with either saline or nicotine twice per day for up to 14 days. Chronic nicotine treatment elicited long-lasting, dose-dependent increases in the levels of adrenal TH mRNA, TH protein, and TH activity. In contrast, a single injection of nicotine elicited only a small increase in adrenal TH mRNA levels, which was transient and did not result in the induction of TH enzyme. Chronic nicotine administration also elicited a sustained increase in adrenal TH gene transcription rate, which persisted for up to 7 days after the final nicotine injection. This sustained transcriptional response correlated with a modest sustained increase in adrenal TH AP1 binding, but not in the levels of Fra-2 or other fos or jun proteins. These results demonstrate that repeated nicotine injections administered chronically over 1 to 2 weeks lead to sustained stimulation of the TH gene and consequent induction of TH gene expression in rat adrenal medulla. These studies support the hypothesis that chronic nicotine administration produces long-lasting cellular changes in adrenal medulla that lead to sustained transcriptional responses.

Social stress during pregnancy and lactation affects in guinea pigs the male offsprings' endocrine status and infantilizes their behaviour.

This study investigated the influence of the social environment during pregnancy and lactation on the male offsprings' endocrine parameters and behaviour in guinea pigs. The subjects studied were sons whose mothers had either lived in a stable social environment during pregnancy and lactation or in an unstable social environment during this period of life. The stable social environment was made by keeping the group composition (one male, five females) constant; in the unstable social environment situation every third day, two females from different groups were exchanged. After weaning, seven groups of sons, whose mothers had lived in an unstable social environment and seven groups of sons, whose mothers had lived in a stable social environment, consisting of two males each, were established. From their 20th through their 100th day of age the spontaneous behaviour of the males was recorded in their home cages. On the endocrine level, cortisol- and testosterone-concentrations in serum as well as adrenal tyrosine hydroxylase activities were determined. Sons, whose mothers had lived in an unstable social environment, showed significantly higher amounts of resting with bodily contact than sons, whose mothers had lived in a stable social environment. Additionally, they displayed this behaviour to an older age than the latter sons. Further on, sons, whose mothers had lived in an unstable social environment, displayed significantly higher frequencies of courtship behaviour than sons, whose mothers had lived in a stable social environment, which was integrated into play behaviour. These behavioural data point to an infantilization of sons, whose mothers had lived in an unstable social environment. The behavioural patterns corresponded with a delayed development of the hypothalamo-pituitary-adrenocortical-axis and a lower activity of the sympathetic-adrenomedullary-system compared to sons whose mothers had lived in a stable social environment. Testosterone did not differ between both categories of males. Thus, the instability of the social environment during pregnancy and lactation had distinct and not yet described effects on the behaviour and endocrine system of the male offspring.

The Behavioral Endocrinology of Domestication: A Comparison between the Domestic Guinea Pig (Cavia apereaf.porcellus) and Its Wild Ancestor, the Cavy (Cavia aperea)

In this study spontaneous behavior and endocrine parameters were compared between the domestic guinea pig (Cavia apereaf.porcellus) and its wild ancestor, the cavy (Cavia aperea), to elucidate the process of domestication in this species. In 120 h of observation time the behavior of five groups of wild and seven groups of domestic guinea pigs, each consisting of one adult male and two adult females, was analyzed quantitatively. To assess the activities of the pituitary–adrenocortical (PAC), the pituitary–gonadal (PG), and the sympathetic–adrenomedullary (SAM) systems, serum cortisol, testosterone, epinephrine, and norepinephrine concentrations, as well as adrenal tyrosine hydroxylase activities, were determined in males of both forms. The following significant differences between wild cavies and domestic guinea pigs were found: the domesticated animals displayed less aggressive but more sociopositive and more male courtship behavior than their wild ancestors. In addition, they were distinctly less attentive to their physical environment than the wild cavies. The basal activity of the SAM system, as well as the reactivity of the SAM and the PAC systems, was distinctly reduced in the domesticated animals. In contrast, the basal activity of the PAC system did not differ between both forms. The activity of the PG system was significantly higher in males of the domestic guinea pig than in male wild cavies. Thus, in guinea pigs the process of domestication has led to typical behavioral traits—reduced aggressiveness, increased social tolerance—which have also been found in comparisons between wild and domestic forms of other species. The decreased reactivity of the organism's stress axes can be regarded as a physiological mechanism which helps domesticated animals to adjust to man-made housing conditions.

Comparison of the neuroendocrine responses to stress in outbred, inbred and F1 hybrid rats

In order to study the genetic factors involved in the neuroendocrine responses to stress, we have compared the intensity of the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system activation following a 60 minute-restraint stress or after a 10 minute-exposure to a novel environment in three rat strains: outbred Wistar, inbred Brown Norway and Fischer 344, and F1 hybrid Brown Norway x Fischer 344 rats. The basal activity of the HPA axis did not differ between the four groups of rats whereas Brown Norway rats had the lowest release of corticosterone following restraint stress. Although differences in plasma adrenocorticotropic hormone failed to reach significance after exposure to a novel environment, the lowest level of corticosterone was found in Brown Norway rats. This lower release of corticosterone in Brown Norway rats has probably an adrenal origin as suggested by the ratios of corticosterone to ACTH levels following exposure to a novel environment: 632 ± 222, 200 ± 45, 636 ± 89, 258 ± 65 in Wistar, Brown Norway, Fischer 344 and F1 hybrids, respectively. This trait was dominant over the “adrenal responsive” phenotype of the Fischer 344 rat strain. In response to novelty, the lowest levels of prolactin and renin activity were found in plasma of Brown Norway and Wistar rats and the highest in Fischer 344 and F1 hybrid Brown Norway x Fischer 344 rats, the “high response” phenotype of the Fischer 344 strain being dominant. No strain-related difference was found in plasma glucose and either adrenal tyrosine hydroxylase or phenylethanolamine N-methyl transferase activity. Taken together, these data suggest that 1) genetic factors might contribute to the interindividual differences in neuroendocrine responses to stress and 2) subsets of these responses are controlled by specific genetic factors.

The Social Environment During Pregnancy and Lactation Affects the Female Offsprings’ Endocrine Status and Behaviour in Guinea Pigs

KAISER, S. AND N. SACHSER. The social environment during pregnancy and lactation affects the female offsprings’ endocrine status and behaviour in guinea pigs. PHYSIOL BEHAV 63(3) 361–366, 1998.—This study investigated the influence of the social environment during pregnancy and lactation on the female offsprings’ endocrine parameters and behaviour. The subjects studied were daughters whose mothers had either lived in a stable social environment (SSE) during pregnancy and lactation (SE-mothers) or in an unstable social environment (USE) during this period of life (UE-mothers). The SSE was made by keeping the group composition (one male, five females) constant; in the USE situation every third day two females from different groups were exchanged. After weaning, 3 groups of daughters from UE-mothers (UE-daughters) and 3 groups of daughters from SE-mothers (SE-daughters), consisting of four females each, were established. From their 60th through their 80th day of age the spontaneous behaviour of the daughters was recorded in their home cages. On the endocrine level, cortisol and testosterone (T) concentration in serum as well as adrenal tyrosinehydroxylase activities in both adrenals and their weight were determined. UE-daughters displayed significantly higher amounts of male-typical courtship and play behaviour than SE-daughters. This behavioural masculinization corresponded with significantly higher T in UE- than SE-daughters. Cortisol levels did not differ between both categories of females. Significantly higher tyrosinehydroxylase activities and adrenal weight in UE- than SE-daughters pointed, however, to higher degrees of stress response in UE-daughters. Thus, the instability of the social environment during pregnancy and lactation has distinct effects on the behaviour and endocrine system of the female offspring.

The effect of chronic ethanol treatement and cold stress on catecholaminergic enzyme activity and mRNA in rat brain and adrenals

Sprague-Dawley male rats (150 g) were chronically treated with 5 v/v % ethanol admixed with nutritionally complete liquid diet and fed ad libitum for 3 weeks. One half of each group was exposed to cold stress at 4°C either for 24 h (for determination of mRNA by in situ hybridization) or for 48 h (for determination of enzyme activity). Chronic ethanol treatment (ethanol) did not affect tyrosine hydroxylase(TH) mRNA level in locus coeruleus(LC) of brain and adrenal medulla(AM) compared to controls. Cold stress showed strong increase of TH mRNA level in LC and AM compared to controls. Pretreated ethanol reduced the increased TH mRNA level by cold stress in LC and AM. Ethanol did not affect TH activity in LC and adrenal glands(adrenals). Cold stress increased TH activity in LC but not in adrenals. Pretreated ethanol did not reduce the increased TH activity by cold stress in LC but this result was not shown in adrenals. Phenylethanolamine-N-methyltransferase(PNMT) activity in C1C2 and adrenals increased only in ethanol treated group. These results suggest that ethanol does not affect TH mRNA level and activity in LC and adrenals, but increases PNMT activity in C1C2 and adrenals in normal rat. It is also suggested that pretreated ethanol reduces the magnitude of cold stress response, that is induction of TH mRNA in LC and AM, and does not reduce the protein activation of TH that is also cold stress response in LC.

Effects of streptozotocin-induced diabetes on the response of male rats to immobilization stress

The effects of streptozotocin-induced (STZ) diabetes on the response to immobilization stress were evaluated in adult male rats. Rats were injected with STZ or vehicle and handled daily to minimize stress. Four weeks later, half of the animals were lightly anesthetized with ether and immobilized for 20 min. At that time the stressed and nonstressed controls were sacrificed, and blood and tissue collected for hormone and amine determinations. Immobilization caused an increase in plasma glucose levels in the controls, but caused no further increase in the already high levels seen in the diabetic rats. Basal corticosterone levels did not differ between the STZ and control rats, and the increase after immobilization was of similar magnitude. The stress-induced increase in prolactin was attenuated in the diabetic rats. Immobilization caused a significant rise in plasma norepinephrine (NE) levels in control, but not in diabetic rats. Adrenal NE content and tyrosine hydroxylase activity were not significantly affected by stress or STZ treatment. Dopamine (DA) and NE content was increased in the hypothalamus of immobilized diabetic rats as compared to nondiabetic immobilized controls. These results demonstrate that diabetic rats respond to immobilization stress, but the endocrine and sympathetic nervous system response is impaired. Changes in the stress response may be related to changes in hypothalamic amine metabolism.

Dissociation between adrenal tyrosinehydroxylase and phenylethanolamine N-Methyltransferase activities following repeated experience of defeats in individually housed male DBA/2J mice

Adrenal activities of tyrosinehydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were compared between individually housed defeated intruders and socially housed residents (SR) (Experiment 1), and between individually housed defeated intruders and individually housed victor residents (Experiment 2). In addition, corticosterone titers were determined and dominance status of SR (dominant, subdominant active, and subdominant passive) considered. Defeats were induced twice a week for 6 consecutive weeks in short resident-intruder encounters. The main findings were:1) within the groups of SR, TH and PNMT activities were high in dominant and low in subdominant-passive mice; 2) in Experiment 1, TH activities of individually housed defeated intruders were low like those of subdominant-passive SR whereas their PNMT activities were high like those of dominant SR; 3) in Experiment 2, TH activities were low in both categories of individually kept mice whereas PNMT activities were significantly elevated in the defeated intruders; 4) in both experiments, PNMT activities of intruders correlated signficantly with the accumulated number of attacks they had received throughout all resident-intruder tests. Findings show separate effects of permanent social stimulation and of episodic experience of defeats on adrenal catecholamine synthesis. Lack of permanent social stimulation decreased adrenal TH and PNMT activities whereas repeated episodic experience of defeats specifically increased PNMT but not TH activities in individually housed mice.

Comparison of the effects of 2‐deoxyglucose and immobilization on secretion and synthesis rate of catecholamines in the adrenal gland: a microdialysis study in conscious rats

Using microdialysis, extracellular 3,4-dihydroxyphenylalanine (DOPA), noradrenaline (NA) and adrenaline (AD) concentrations in the adrenal gland were monitored in conscious rats during and after 60 min of immobilization (IMM) as well as after injection of 500 mg kg(-1) 2-deoxyglucose (2-DG). IMM produced a rapid and transient increase in secretion of AD (20-fold), NA (13-fold) and DOPA (3.6-fold). This was accompanied by an increase in blood pressure (+18 mmHG) and heart rate (+146 b.p.m.). Repeated exposure to IMM (daily 60 min, for 5 days) had no influence on either catecholamine secretion of haemodynamic profiles, indicating the lack of habituation to stressful conditions. Unlike IMM, the stress of 2-DG-induced central neuroglucopenia stimulated the release of AD without affecting NA secretion. AD levels peaked (5.1-fold increase) 40-60 min after 2-DG injection and then slowly declined. 2-DG induced no changes in blood pressure but reduced the heart rate (-48 b.p.m.). In separate experiments, steady-state dialysate DOPA levels, reached during continuous infusion of decarboxylase inhibitor NSD 1015 into adrenal gland tissue through the dialysis probe, served as an index of adrenomedullary tyrosine hydroxylase (TH) activity. IMM evoked a marked response in TH activity (DOPA formation increased 2.7-fold), which remained elevated 60 min after the cessation of stress when AD and NA secretion had already fallen to baseline. After 2-DG, despite significant hormonal response, adrenal TH activity was unchanged. These results give clear evidence that IMM and 2-DG-induced neuroglucopenia may be considered as two different types of stressful stimuli.

Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase.

1. Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2. TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. 3. Nicotine (5 microM), forskolin (1 microM) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4. TOH activation by all three drugs was blocked by H89 (3-20 microM), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5. A structural analogue of H89, H85 (3-20 microM), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 microM) or basal TOH activity. Neither sodium nitroprusside (0.3-1O microM) nor 8-Br-cyclic GMP (1 mM) increased TOH activity.6. In digitonin-permeabilized chromaffin cells, forskolin (3 microM), cyclic AMP (10 microM) and Ca2+ (approx.2 micro M free Ca2+) each increased TOH activity. The response to all three drugs was blocked by H89(10 microM), which also reduced basal TOH activity in the permeabilized cells.7. Maximal activation of TOH by forskolin was achieved with 10 micro M forskolin. This concentration was less than the EC50 for forskolin-induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (1O microM) and nicotine (5 microM) were additive.8. The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels.It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.

Maturation of the behavioral and neuroendocrine differences between the Roman rat lines

The behavioral and/or neuroendocrine reactivity to psychological (open-field exposure) and physiological (CRF challenge) stimulations, as well as adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyl transferase (PNMT) activities were measured, at different ages, in the Roman high avoidance (RHA) and Roman low avoidance (RLA) rat lines that have been genetically selected on the basis of their divergent active avoidance behavior. The highest locomotor activity in the open field, associated to blunted prolactin and renin reactivity to an emotional stress and lower specific TH and PNMT activities, characterized the RHA rats of all ages. HPA axis reactivity to psychological and/or physiological stimulations was identical in young animals (14 weeks old) of the two lines. Nevertheless, it displayed with age maturation processes, since the amplitude of postopen-field secretion peak for ACTH was larger in RLA rats from 20 weeks on, the response to CRF being not increased until 42 weeks. These maturation processes could result from genetically influenced changes related to environmental stimulations. Therefore, the Roman lines may be an excellent model to study the interactions between the genetic and developmental factors controlling the coupling between both behavioral and neuroendocrine functions.

Acute effects of reserpine on tryptophan hydroxylase activity and mRNA in rat brain

The present study examined changes in tryptophan hydroxylase (TPH) activity in the rat brainstem 1–4 days after i.p. reserpine administration. TPH enzyme activity was significantly decreased day 1 posttreatment and followed by a small but significant increase on days 2–4. Maximal TPH activity on day 4 was elevated only 42% above control level. This small increase in TPH activity was not preceded by any change of in situ hybridization signal of TPH message on day 1. In contrast, adrenal tyrosine hydroxylase (TH) activity in these same reserpinized animals was 2.5-fold higher than control on 4 day posttreatment. In addition, TH message was correspondingly increased day 1 posttreatment. Taken together, these results indicate a trend of small increase in TPH activity and no alteration in message after reserpine treatment unlike catecholaminergic systems.