Tyrosine hydroxylase activity in rat brain following "REM" sleep deprivation

I n R ecent years biogenic amines have been implicated in the control mechanism for induction and maintenance of sleep processes (J ouvet , 1969). Investigators have looked for changes in the rate of synthesis of cerebral norepinephrine from [³H]tyrosine after REM sleep deprivation and reported increased rates of synthesis during REM sleep deprivation (M ark , H einer , M andel and G odin , 1969) and REM sleep rebound following 91 h of deprivation (P ujol , M ouret and G lowinski , 1968). Because tyrosine is thought to be the rate-limiting enzyme (U denfriend , 1966) in the synthetic pathways for norepinephrine and since the above-mentioned studies are suggestive of changes in the activity of the enzyme, we decided to measure tyrosine hydroxylase activity following REM sleep deprivation.     

Tyrosine hydroxylase expression and Cdk5 kinase activity in ataxic cerebellum

Ataxia has been associated with abnormalities in neuronal differentiation and migration, which are regulated by Cyclin-dependent kinase 5 (Cdk5). The cerebellum of mice lacking Cdk5 or its activator, p35, resembles those of ataxic reeler and scrambler mice, suggesting that Cdk5 may contribute to ataxic pathology. As with other ataxic mice, the pogo/pogo mouse shows aberrant cerebellar tyrosine hydroxylase (TH) expression. Since Cdk5 phosphorylates and upregulates TH expression, we sought to analyze (i) Cdk5 activity in the pogo cerebellum, which exhibits abnormal TH expression, and (ii) TH expression in the cerebellum of p35-/- and p39-/- mice, which display reduced Cdk5 activity. Interestingly, we found that increased TH expression in the pogo cerebellum coincided with reduced Cdk5 activity. However, reduced Cdk5 activity in both p35-/- and p39-/- cerebellum did not correspond to defects in TH expression. Together, these suggest that abnormal TH expression in the cerebellum might be regulated by mechanisms other than Cdk5 activity.     

Tyrosine hydroxylase regulation in the central nervous system

Tyrosine hydroxylase is considered to be the rate-limiting enzyme in the synthesis of catecholamines in both the central and peripheral nervous system. Increased or decreased neuronal activity, stress, lesions, drug effects, endocrinological manipulations and experimental models of hypertension are associated with alterations in tyrosine hydroxylase activity in the central nervous system. In many of these instances, the changes in the activity of tyrosine hydroxylase in the central nervous system that occur are localized to discrete catecholaminergic pathways and nuclei in the brain. The purpose of this review is to summarize and assess this information and to provide insight into the function of catecholamine systems in the brain and their interactions with other putative neurotransmitter systems.     

Tyrosine hydroxylase in rat brain dopaminergic nerve terminals. Multiple-site phosphorylation in vivo and in synaptosomes.

Tyrosine hydroxylase, which catalyzes the initial step in catecholamine biosynthesis, is phosphorylated at serines 8, 19, 31, and 40 in intact pheochromocytoma (PC12) cells (Haycock, J.W. (1990) J. Biol. Chem. 265, 11682-11691). After 32Pi labeling of rat corpus striata in vivo or rat corpus striatal synaptosomes, 32P incorporation into tyrosine hydroxylase occurred predominantly at serines 19, 31, and 40. Electrical stimulation (30 Hz, 20 min) of the medial forebrain bundle (containing the afferent dopaminergic fibers) increased 32P incorporation into each of the three sites. Brief depolarization of the synaptosomes with elevated [K+]o (20-60 mM, 5-30 s) or veratridine (50 microM, 2 min) produced a selective increase in 32P incorporation into Ser19. Phorbol 12,13-dibutyrate (1 microM, 5 min) increased 32P incorporation into Ser31, and cAMP-acting agents such as forskolin (10 microM, 5 min) increased 32P incorporation into Ser40. In contrast, 32P incorporation into Ser8, which was usually detectable but very low, was not regulated either in vivo or in situ by any of the activators of signal transduction pathways. In synaptosomes, the only treatment found to increase Ser8 phosphorylation was okadaic acid (a protein phosphatase inhibitor), which increased 32P incorporation into all four phosphorylation sites. Thus, three different signal transduction systems appear to mediate the physiological regulation of tyrosine hydroxylase phosphorylation at three different sites.     

Recurrent laryngeal nerve activity exhibits a 5-HT-mediated long-term facilitation and enhanced response to hypoxia following acute intermittent hypoxia in rat

A progressive and sustained increase in inspiratory-related motor output ("long-term facilitation") and an augmented ventilatory response to hypoxia occur following acute intermittent hypoxia (AIH). To date, acute plasticity in respiratory motor outputs active in the postinspiratory and expiratory phases has not been studied. The recurrent laryngeal nerve (RLN) innervates laryngeal abductor muscles that widen the glottic aperture during inspiration. Other efferent fibers in the RLN innervate adductor muscles that partially narrow the glottic aperture during postinspiration. The aim of this study was to investigate whether or not AIH elicits a serotonin-mediated long-term facilitation of laryngeal abductor muscles, and if recruitment of adductor muscle activity occurs following AIH. Urethane anesthetized, paralyzed, unilaterally vagotomized, and artificially ventilated adult male Sprague-Dawley rats were subjected to 10 exposures of hypoxia (10% O(2) in N(2), 45 s, separated by 5 min, n = 7). At 60 min post-AIH, phrenic nerve activity and inspiratory RLN activity were elevated (39 ± 11 and 23 ± 6% above baseline, respectively). These responses were abolished by pretreatment with the serotonin-receptor antagonist, methysergide (n = 4). No increase occurred in time control animals (n = 7). Animals that did not exhibit postinspiratory RLN activity at baseline did not show recruitment of this activity post-AIH (n = 6). A repeat hypoxia 60 min after AIH produced a significantly greater peak response in both phrenic and RLN activity, accompanied by a prolonged recovery time that was also prevented by pretreatment with methysergide. We conclude that AIH induces neural plasticity in laryngeal motoneurons, via serotonin-mediated mechanisms similar to that observed in phrenic motoneurons: the so-called "Q-pathway". We also provide evidence that the augmented responsiveness to repeat hypoxia following AIH also involves a serotonergic mechanism.     

Activation of tyrosine hydroxylase by intermittent hypoxia: involvement of serine phosphorylation.

Regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC-12) cells by exposing them to alternating cycles of hypoxia (1% O2, 15 s) and normoxia (21% O2, 3 min) for up to 60 cycles; controls were exposed to normoxia for a similar duration. IH exposure increased dopamine content and TH activity by approximately 42 and approximately 56%, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH-bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells, suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho-TH (Ser-40) antibody, respectively. Compared with normoxic cells, total serine and Ser-40-specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and Ser-40-specific phosphorylation of TH were inhibited by Ca2+/calmodulin-dependent protein kinase (CaMK) and PKA-specific inhibitors but not by inhibitors of the extracellular signal-regulated protein kinase pathway, suggesting that IH activates TH in PC-12 cells via phosphorylation of serine residues including Ser-40, in part, by CaMK and PKA. Our results also suggest that IH-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH, leading to increased synthesis of dopamine.     

Pb-induced alterations in tyrosine hydroxylase activity in rat brain

It was concluded that cytochrome oxidase was a strange enzyme for three reasons. (1) The thermodynamic flux-force relationship of this enzyme was inverse in some conditions: flux decreased when force increased. (2) The flux-force relationship was not unique and depended on the way in which the thermodynamic span of cytochrome oxidase was changed. (3) The regulation of cytochrome oxidase was different in the same conditions when different external parameters (energy demand, oxygen concentration) were changed.It was also shown that the flux control coefficient of cytochrome oxidase, small at saturating oxygen concentration, increases when oxygen pressure diminishes, approaching unity at very low oxygen concentrations. (Mol Cell Biochem 174: 137–141, 1997)     

Gasping and autoresuscitation in the developing rat: effect of antecedent intermittent hypoxia.

Gasping is a critically important mechanism for autoresuscitation and survival during extreme tissue hypoxia. Evidence of antecedent hypoxia in sudden infant death syndrome suggests that intermittently occurring hypoxic episodes may modify gasping and autoresuscitation. To examine this issue, an intermittent hypoxia (IH) profile consisting of alternating room air and 10% O(2)-balance N(2) every 90 s was applied to pregnant Sprague-Dawley rats (IHRA; n = 50) and to pups after a normal pregnancy (RAIH; n = 50) as well as to control pups (RARA; n = 50). At postnatal day 5, pups were exposed to 95% N(2)-5% CO(2), and gasping and the ability to autoresuscitate were assessed. Compared with RARA, IHRA- and RAIH-exposed pups had a reduced number of gasps, decreased overall gasp duration, and were less likely to autoresuscitate on introduction of room air to the breathing mixture during the last phase of gasping (P < 0.001 vs. RARA). We conclude that both prenatal and early postnatal IH adversely affect gasping and related survival mechanisms.     

Correlation between tyrosine hydroxylase activity and catecholamine concentration or turnover in brain regions.

Tyrosine hydroxylase activity correlated significantly with norepinephrine concentration and turnover, when results from regions containing predominantly noradrenergic terminals were compared, and with dopamine concentration and turnover when results from regions containing predominantly dopaminergic terminals were compared. Regions containing dopamine or norepinephrine cell bodies were characterized by higher tyrosine hydroxylase activities as compared to regions containing mostly nerve terminals. Higher levels of tyrosine hydroxylase activity and transmitter turnover were observed in regions containing dopaminergic terminals than in regions containing norepinephrine terminals. These findings are consistent with the view that tyrosine hydroxylase activity is linked to rates of catecholamine utilization by neurons in the CNS.     

Short-term regulation of tyrosine hydroxylase activity and expression by endothelin-1 and endothelin-3 in the rat posterior hypothalamus

Brain catecholamines are involved in several biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and -3 (ET-1 and ET-3) modulate norepinephrine release in the anterior and posterior hypothalamus. As tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, the aim of the present work was to investigate the effects of ET-1 and ET-3 on TH activity, total enzyme level and the phosphorylated forms of TH in the rat posterior hypothalamus. Results showed that ET-1 and ET-3 diminished TH activity but the response was abolished by both selective ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively). In addition ET(A) and ET(B) selective agonists (sarafotoxin S6b and IRL-1620, respectively) failed to affect TH activity. In order to investigate the intracellular signaling coupled to endothelins (ETs) response, nitric oxide (NO), phosphoinositide, cAMP/PKA and CaMK-II pathways were studied. Results showed that N(omega)-nitro-l-arginine methyl ester and 7-nitroindazole (NO synthase and neuronal NO synthase inhibitors, respectively), 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinozalin-1-one and KT-5823 (soluble guanylyl cyclase, and PKG inhibitors, respectively) inhibited ETs effect on TH activity. Further, sodium nitroprusside and 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and cGMP analog, respectively) mimicked ETs response. ETs-induced reduction of TH activity was not affected by a PKA inhibitor but it was abolished by PLC, PKC and CaMK-II inhibitors as well as by an IP(3) receptor antagonist. On the other hand, both ETs did not modify TH total level but reduced the phosphorylation of serine residues of the enzyme at positions 19, 31 and 40. Present results suggest that ET-1 and ET-3 diminished TH activity through an atypical ET or ET(C) receptor coupled to the NO/cGMP/PKG, phosphoinositide and CaMK-II pathways. Furthermore, TH diminished activity may result from the reduction of the phosphorylated sites of the enzyme without changes in its total level. Taken jointly present and previous results support that ET-1 and ET-3 may play a relevant role in the modulation of catecholaminergic neurotransmission in the posterior hypothalamus of the rat.     

Chronic intermittent hypoxia increases infarction in the isolated rat heart.

Coronary heart disease is frequently associated with obstructive sleep apnea syndrome and treating obstructive sleep apnea appears to significantly improve the outcome in coronary heart disease. Thus we have developed a rat model of chronic intermittent hypoxia (IH) to study the influence of this condition on myocardial ischemia-reperfusion tolerance and on functional vascular reactivity. Wistar male rats were divided in three experimental groups (n = 12 each) subjected to chronic IH (IH group), normoxia (N group), or control conditions (control group). IH consisted of repetitive cycles of 1 min (40 s with inspired O(2) fraction 5% followed by 20 s normoxia) and was applied for 8 h during daytime, for 35 days. Normoxic cycles were applied in the same conditions, inspired O(2) fraction remaining constant at 21%. On day 36, mean arterial blood pressure (MABP) was measured before isolated hearts were submitted to an ischemia-reperfusion protocol. The thoracic aorta and left carotid artery were also excised for functional reactivity studies. MABP was not significantly different between the three experimental groups. Infarct sizes (in percent of ventricles) were significantly higher in IH group (46.9 +/- 3.6%) compared with N (26.1 +/- 2.8%) and control (21.7 +/- 2.1%) groups. Vascular smooth muscle function was similar in aorta and carotid arteries from all groups. The endothelium-dependent relaxation in response to acetylcholine was also similar in aorta and carotid arteries from all groups. Chronic IH increased heart sensitivity to infarction, independently of a significant increase in MABP, and did not affect vascular reactivity of aorta and carotid arteries.     

Aromatase activity in the rat brain: Hormonal regulation and sex differences

The intracellular conversion of testosterone to estradiol by the aromatase enzyme complex is an important step in many of the central actions of testosterone. In rats, estrogen given alone, or in combination with dihydrotestosterone, mimics most of the behavioral effects of testosterone, whereas treatment with antiestrogens or aromatase inhibitors block facilitation of copulatory behavior by testosterone. We used a highly sensitive in vitro radiometric assay to analyze the distribution and regulation of brain aromatase activity. Studies using micropunch dissections revealed that the highest levels of aromatase activity are found in an interconnected group of sexually dimorphic nuclei which constitutes a neural circuit important in the control of male sexual behavior. Androgen regulated aromatase activity in many diencephalic nucleic, including the medial preoptic nucleus, but not in the medial and cortical nuclei of the amygdala. Additional genetic evidence for both androgen-dependent and -independent control of brain AA was obtained by studies of androgen-insensitive testicular-feminized rats. These observations suggest that critical differences in enzyme responsiveness are present in different brain areas. Within several nuclei, sex differences in aromatase induction correlated with differences in nuclear androgen receptor concentrations suggesting that neural responsiveness to testosterone is sexually differentiated. Estradiol and dihydrotestosterone acted synergistically to regulate aromatase activity in the preoptic area. In addition, time-course studies showed that estrogen treatment increased the duration of nuclear androgen receptor occupation in the preoptic area of male rats treated with dihydrotestosterone. These results suggest possible ways that estrogens and androgens may interact at the cellular level to regulate neural function and behavior.     

Tyrosine hydroxylase expression in differentiating neurons of the rat arcuate nucleus: inhibitory effect of serotoninergic afferents]

In vivo studies, serotonine synthesis in the rat fetal brain was inhibited by p-chlorphenylalanine from the 11th to the 20th embryonic day. Serotonine depletion significantly decreased thyrosine hydroxylase content in the neurones of males and females on the 21st embryonic day and in males--on the 35th postnatal day. In vitro, a co-culture of arquate nucleus' and raphe nucleus' embryonic neurones resulted in a sex-specific increase of the thyrosine hydroxylase level in the former neurones. The raphe nucleus' neurones manifested an increased level of serotonine. The findings suggest an activating long-lasting effect of serotonine afferents on the thyrosine hydroxylase expression in differentiating neurones of the arquate nucleus in rats during prenatal ontogenesis.     

Time-dependent modulation of carotid body afferent activity during and after intermittent hypoxia

The ventilatory response to several minutes of hypoxia consists of various time-dependent phenomena, some of which occur during hypoxia (e.g., short-term depression), whereas others appear on return to normoxia (e.g., posthypoxic frequency decline). Additional phenomena can be elicited by acute, intermittent hypoxia (e.g., progressive augmentation, long-term facilitation). Current data suggest that these phenomena originate centrally. We tested the hypothesis that carotid body afferent activity undergoes time-dependent modulation, consistent with a direct role in these ventilatory phenomena. Using an in vitro rat carotid body preparation, we found that 1) afferent activity declined during the first 5 min of severe (40 Torr Po(2)), moderate (60 Torr Po(2)), or mild (80 Torr Po(2)) hypoxia; 2) after return to normoxia (100 Torr Po(2)) and after several minutes of moderate or severe hypoxia, afferent activity was transiently reduced compared with prehypoxic levels; and 3) with successive 5-min bouts of mild, moderate, or severe hypoxia, afferent activity during bouts increased progressively. We call these phenomena sensory hypoxic decline, sensory posthypoxic decline, and sensory progressive augmentation, respectively. These phenomena were stimulus specific: similar phenomena were not seen with 5-min bouts of normoxic hypercapnia (100 Torr Po(2) and 50-60 Torr Pco(2)) or hypoxic hypocapnia (60 Torr Po(2) and 30 Torr Pco(2)). However, bouts of either normoxic hypercapnia or hypocapnic hypoxia resulted in sensory long-term facilitation. We suggest time-dependent carotid body activity acts in parallel with central mechanisms to shape the dynamics of ventilatory responses to respiratory chemostimuli.     

Effect of intermittent high altitude hypoxia on gene expression in rat heart and lung

Hypoxia has been identified as an important stimulus for gene expression during embryogenesis and in various pathological situations. Its influence under physiological conditions, however, has only been studied occasionally. We therefore investigated the effect of intermittent high altitude hypoxia on the mRNA expression of different cytokines and protooncogenes, but also of other genes described to be regulated by hypoxia, in the left ventricle (LV), the right ventricle (RV), atria and the lung of adult rats after simulation of hypoxia in a barochamber (5000 m, 4 hours to 10 days). Heme oxygenase-1 as well as transforming growth factor-beta1 showed an increased expression in all regions of the heart and the lung at different periods of hypoxia. For lactate dehydrogenase-A, we found a significant up-regulation in the RV and the lung, for lactate dehydrogenase-B up-regulation in the RV, but down-regulation in the LV and the atria. Vascular endothelial growth factor was up-regulated in the RV, the LV and the lung, but down-regulated in the atria. Its receptor Flk-1 mRNA was significantly increased in the atria and RV only. Expression of c-fos was found in the LV and RV only after 4 hours of hypoxia. The level of c-jun was significantly increased in the LV but decreased in the atria. Our data clearly demonstrate that intermittent hypoxia is a modulator of gene expression under physiological conditions. It differently regulates the expression of distinct genes not only in individual organs but even within one organ, i.e. in the heart.     

Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat

The regulation of transmitter phenotype in primary sensory neurons remains poorly understood. However, recent studies of catecholaminergic (CA) sensory neurons suggest that expression of this particular phenotype may be related to innervation of specific peripheral tissues. In the glossopharyngeal petrosal ganglion (PG) of adult rats, for example, the vast majority of CA sensory neurons innervate a single target, the carotid body. The present study was undertaken, therefore, to begin investigating factors that underlie CA differentiation in sensory neurons, using the rat PG as a model system. Immunocytochemical, biochemical, and morphometric methods were used to investigate the normal time course of CA development in the PG in vivo, employing tyrosine hydroxylase (TH) as a phenotypic marker. These studies revealed two temporally distinct waves of TH expression during embryogenesis. TH immunoreactivity was initially detectable on Embryonic Day (E) 11.5; the number of stained cells increased markedly by E12.5 and then fell off sharply to near 0 by E15.5. Simultaneous immunostaining for TH and neurofilament proteins revealed a high proportion of double-labeled perikarya on E12.5, indicating that the transiently TH-positive cells are neurons. A second, sustained phase of TH expression began on E16.5, and by Postnatal Day 1 adult numbers of TH-containing ganglion cells were present. Western blot analysis demonstrated that TH levels per cell rose 3.5-fold in the perinatal period, indicating that maturation of this particular catecholaminergic trait in PG sensory neurons is highly regulated around birth. Morphometric techniques were used to define the relationship between neurons that transiently exhibit TH immunoreactivity early in gangliogenesis and those that maintain enzyme expression in the mature PG. These studies revealed separate and distinct growth curves for the early and late TH cells, respectively, demonstrating that the appearance, disappearance, and reappearance of immunoreactive cells reflects the differentiation of two separate populations of PG neurons. Moreover, these data indicate that TH expression in the population of CA cells that persists in the mature PG begins around E16.5. This is after peripheral target innervation has begun, raising the possibility that neuron-target interactions regulate biochemical differentiation of these CA sensory neurons.     

Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. I. Mechanical activity.

Long-lasting cardioprotection may be attained by chronic hypoxia. The basal parameters of contractile function and their response to hypoxia/reoxygenation were measured under isometric conditions, in papillary muscles isolated from left ventricle of rats that were submitted to 53.8 kPa in a hypobaric chamber from 7 wk of age and for their lifetime and of their siblings kept at 101.3 kPa. During acclimatization, hematocrit increased, body weight gain decreased, and heart weight increased with right ventricle hypertrophy. Papillary muscle cross-sectional area was similar in both control and hypoxic groups up to 45 wk of exposure. Developed tension (DT) was 34-64% higher in rats exposed to hypoxia for 10, 26, and 45 wk than in their age-matched controls, whereas resting tension was unchanged. Maximal rates of contraction and relaxation showed a similar pattern of changes as DT. Recovery of DT and maximal rates of contraction and relaxation after 60-min hypoxia and 30-min reoxygenation was also improved in adult hypoxic rats to values similar to those of young rats. Heart acclimatization was lost after 74 wk of exposure. Results are consistent with the development of cardioprotection during high-altitude acclimatization and provide an experimental model to study the mechanisms involved, which are addressed in the accompanying paper.