Stimulation Of Tyrosine Hydroxylase Activity Research Articles

The effect of the DISC1 Ser704Cys polymorphism on striatal dopamine synthesis capacity: an [18F]-DOPA PET study.

Whilst the role of the Disrupted-in-Schizophrenia 1 (DISC1) gene in the aetiology of major mental illnesses is debated, the characterization of its function lends it credibility as a candidate. A key aspect of this functional characterization is the determination of the role of common non-synonymous polymorphisms on normal variation within these functions. The common allele (A) of the DISC1 single-nucleotide polymorphism (SNP) rs821616 encodes a serine (ser) at the Ser704Cys polymorphism, and has been shown to increase the phosphorylation of extracellular signal-regulated protein Kinases 1 and 2 (ERK1/2) that stimulate the phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. We therefore set out to test the hypothesis that human ser (A) homozygotes would show elevated dopamine synthesis capacity compared with cysteine (cys) homozygotes and heterozygotes (TT and AT) for rs821616. [18F]-DOPA positron emission tomography (PET) was used to index striatal dopamine synthesis capacity as the influx rate constant Kicer in healthy volunteers DISC1 rs821616 ser homozygotes (N = 46) and healthy volunteers DISC1 rs821616 cys homozygotes and heterozygotes (N = 56), matched for age, gender, ethnicity and using three scanners. We found DISC1 rs821616 ser homozygotes exhibited a significantly higher striatal Kicer compared with cys homozygotes and heterozygotes (P = 0.012) explaining 6.4% of the variance (partial η2 = 0.064). Our finding is consistent with its previous association with heightened activation of ERK1/2, which stimulates tyrosine hydroxylase activity for dopamine synthesis. This could be a potential mechanism mediating risk for psychosis, lending further credibility to the fact that DISC1 is of functional interest in the aetiology of major mental illness.

Calcium-dependent mechanisms involved in the modulation of tyrosine hydroxylase by endothelins in the olfactory bulb of normotensive rats

Endothelins (ETs) are widely expressed in the olfactory bulb (OB) and other brain areas where they function as neuropeptides. In a previous study we reported that in the OB ET-1 and ET-3 participate in the long-term regulation of tyrosine hydroxylase (TH), the key enzyme in catecholamine biosynthesis. ETs stimulate TH activity by increasing total and phosphorylated enzyme levels as well as its mRNA. ET-1 response is mediated by a super high affinity ETA receptor coupled to adenylyl cyclase/protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CaMK-II) activation whereas that of ET-3 through an atypical receptor coupled not only to these signaling pathways but also to phospholipase C (PLC)/protein kinase C pathway. Given the participation of PLC and CaMKII in the regulation of TH by ETs in the OB we sought to establish the contribution of calcium to ETs response. Present findings show that calcium released from ryanodine-sensitive channels and extracellular calcium were necessary to stimulate TH by ETs through CaMK-II. On the other hand, intracellular calcium released by the endoplasmic reticulum partially mediated ETs-evoked increase in TH mRNA but calcium influx and CaMK-II inhibition abolished the response. However calcium mechanisms were not involved in ETs-evoked increase in TH protein content. Present findings support that different sources of calcium contribute to the long-term modulation of TH activity and expression mediated by ETs in the rat OB.

Ciliary neurotrophic factor stimulates tyrosine hydroxylase activity

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.

W13.4 PARP overactivity mediates the vascular dysfunction in an animal model of PE through excess peroxynitrite production

Functional effects of prenatal cocaine exposure may be mediated in part by changes in catecholaminergic development. The present study examined whether cocaine administration influenced fetal brain activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Subcutaneous (s.c.) injection of pregnant rats with 40 mg/kg of cocaine HCl daily from gestational day (GD) 8 to GD20 resulted in an 8.7% stimulation of fetal whole-brain TH activity compared to controls. We then switched to a s.c. implantation procedure involving Silastic capsules filled with 80 mg of cocaine base dissolved in polyethylene glycol (PEG). Implantation of 2 such capsules on GD18 produced a 28% increase in fetal TH activity measured only 3 days later on GD21. Subsequent experiments demonstrated that GD14 implantation was equally effective in stimulating fetal TH activity on GD17, but that the enzyme was unaffected in the brains of the treated dams. When cocaine-containing capsules were implanted on GD18, removed on GD21, and the females were allowed to deliver normally, offspring TH activity was still elevated on postnatal day 10 but not later. Finally, the presence of cocaine implants from GD18 to GD21 had no influence on fetal brain neurotransmitter and metabolite concentrations, however, the treated dams exhibited significant reductions in dopamine (DA) and the serotonin metabolite, 5-hydroxyindoleacetic acid. We conclude that maternal cocaine implants rapidly but transiently stimulate TH activity in the fetal brain, and that such stimulation prevented the DA depletion observed in the dams.

5‐Aminoimidazole‐4‐Carboxamide‐1‐β‐4‐Ribofuranoside Stimulates Tyrosine Hydroxylase Activity and Catecholamine Secretion by Activation of AMP‐Activated Protein Kinase in PC12 Cells

The activity of AMP-activated protein kinase (AMPK) is regulated by the metabolic and nutritional state of the cell. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is transformed into riboside monophosphate (ZMP) via phosphorylation by adenosine kinase inside the cell and exerts it effect by stimulating AMPK. AICAR significantly induces an increase in AMPK activity in a dose- and time-dependent manner in the rat pheochromocytoma cell line PC12. In addition, compound C, an AMPK inhibitor, as well as 5'-amino-5'-dAdo, an adenosine kinase inhibitor, inhibits the AICAR-induced AMPK activity. AICAR significantly stimulates tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamine) activity and the corresponding mRNA level, which closely matches with the TH protein level. In addition, AICAR provokes a rapid and long-lasting increase in the phosphorylation of TH at Ser19, Ser31 and Ser40. AICAR also markedly activates ERKs, JNK and p38. The MEK-1-inhibitor (PD-098059) causes a partial, but significant, inhibition of AICAR-induced TH enzyme activity by phosphorylation of Ser31 without affecting phosphorylation at the two other sites. By contrast, neither the JNK-inhibitor nor the p38-inhibitor affects TH enzyme activity and phosphorylation. Similarly, PD-098059 partially, but significantly, inhibits the AICAR-induced increase in the TH mRNA level. Furthermore, AICAR increases the level of cAMP in PC12 cells. The present study also shows that H89, a protein kinase A inhibitor, abolishes the AICAR-induced increase in the level of TH mRNA, as well as the corresponding enzyme activity and Ser40 phosphorylation. Finally, AICAR significantly increases dopamine secretion from PC12 cells. These findings indicate that AICAR activates catecholamine synthesis and secretion through AMPK activation in chromaffin cells.

Chronic depolarization stimulates norepinephrine transporter expression via catecholamines

Chronic depolarization increases norepinephrine (NE) uptake and expression of the norepinephrine transporter (NET) in sympathetic neurons, but the mechanisms are unknown. Depolarization of sympathetic neurons stimulates catecholamine synthesis, and several studies suggest that NET can be regulated by catecholamines. It is not clear if the depolarization-induced increase in NET is because of nerve activity per se, or is secondary to elevated catecholamines. To determine if induction of NET mRNA was a result of increased catecholamines, we used pharmacological manipulations to (i) inhibit tyrosine hydroxylase activity in neurons depolarized with 30 mm KCl, thereby preventing increased catecholamines, or (ii) stimulate tyrosine hydroxylase activity in the absence of depolarization. Inhibiting the depolarization-induced increase in catecholamines prevented the up-regulation of NET mRNA, but did not block the increase in tyrosine hydroxylase (TH) mRNA. Furthermore, stimulating catecholamine production in the absence of depolarization elevated NE uptake, NET protein, and NET mRNA in sympathetic neurons. Similarly, elevating endogenous catecholamines in SK-N-BE2M17 neuroblastoma cells increased NE uptake and NET expression. These data suggest that chronic depolarization of sympathetic neurons induces NET expression through increasing catecholamines, and that M17 neuroblastoma cells provide a model system in which to investigate catechol regulation of NET expression.

Cyclic AMP–Protein Kinase A and Protein Kinase C Mediate In Vitro T3 Activation of Brain Tyrosine Hydroxylase in the Female Catfish Heteropneustes fossilis

The present in vitro study demonstrates an involvement of both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signal transduction mechanisms in the triiodothyronone (T(3))-activation of forebrain (telencephalon and hypothalamus) tyrosine hydroxylase (TH) activity in the female catfish Heteropneustes fossilis. Incubations of the enzyme preparations with different concentrations of T(3) (0.15-2.4 ng/ml) stimulated TH activity over the concentrations. Similarly, coincubations of the enzyme preparations with T(3) and cAMP (1.0 mM) or cAMP-elevating drugs such as 1-methyl-3-isobutylxanthine (1.5 mM) or theophylline (1.5 mM) increased TH activity significantly over that of T(3). The stimulatory effect of TH activity with T(3) or cAMP was coincident with a low apparent K(m) and high V(max) for the cofactor, suggesting a higher affinity of the enzyme. Incubation of the enzyme preparations with PKA (H-89) and PKC (calphostin-C) inhibitors decreased basal enzyme activity significantly, with the inhibition being greater in the former group. The incubations of the enzyme preparations with T(3) or T(3) + cAMP, followed by the different inhibitors, also decreased enzyme activity. Although T(3) could not reverse the inhibitory effect of H-89, it could over-ride the effect of calphostin-C to some extent. The suppressive effect of the inhibitors could be related to a high apparent K(m) and low V(max) for the cofactor. The evidence strongly suggests a nongenomic action of T(3) on TH activity via the cell signalling pathways, for which the cAMP-dependent PKA appears to be the major regulatory mechanism.

Estrogen regulation of in vitro brain tyrosine hydroxylase activity in the catfish Heteropneustes fossilis: interactions with cAMP-protein kinase A and protein kinase C systems in enzyme activation

In the present in vitro study, interactions of both cAMP-protein kinase A (PKA) and protein kinase C (PKC) systems were investigated in the estradiol-17β (E 2) regulation of forebrain (hypothalamus and telencephalon) tyrosine hydroxylase (TH) activity in the female catfish Heteropneustes fossilis in vitellogenic phase. E 2 produced biphasic effects on TH activity: low concentrations (10 −12–10 −5M) stimulated, and high concentrations (10 −3–10 −4 M) inhibited enzyme activity (Tukey’s test, P < 0.05). Co-incubations of the enzyme preparations with cAMP (1.0 mM), IBMX (1.5 mM) or theophylline (1.5 mM) and a low concentration of E 2 (10 −9 M) increased TH activity significantly. However, the co-incubations with a high concentration of E 2 (10 −3 M) decreased it significantly. Pre-incubations of the enzyme preparations with cAMP (0.1 mM), followed by different concentrations of E 2 (10 −12, 10 −9, 10 −4, and 10 −3 M) produced concentration-dependent biphasic effects. The pre-incubations with a low concentration of E 2 (10 −9 M), followed by different concentrations of cAMP (0.05–1.0 mM) produced a significant concentration-dependent stimulation of TH activity and that with a high concentration of E 2 (10 −3 M) produced a significant decrease in TH activity. Co-incubations of high and low E 2, with or without cAMP, and PKA inhibitor (H-89) decreased TH activity significantly. The incubations with H-89 abolished the stimulatory effect of low E 2 or low E 2 + cAMP and intensified the inhibitory effect of high E 2 or high E 2 + cAMP combination. Co-incubations with PKC inhibitor (calphostin C) did not influence the stimulatory effect of low E 2 but lowered the stimulatory effect of low E 2 + cAMP treatment. Kinetic studies showed that the stimulatory effect of a low E 2 concentration was due to a decrease in apparent K m and an increase in apparent V max for both cofactor and substrate, and the inhibitory effect of a high E 2 concentration was due to reverse changes in the kinetics. The stimulatory effect of cAMP alone or in combination with low E 2 was related to decreased K m and increased V max for the cofactor. The inhibitory effect of PKA and PKC blockers, alone or in combination with E 2 and/or cAMP was due to increased K m and decreased V max of the enzyme for the cofactor. The present data suggest that E 2 modulates the short-term activation of brain TH activity differentially and may involve mainly the cAMP-PKA system.

Effects of altered photoperiod and temperature, serotonin-affecting drugs, and melatonin on brain tyrosine hydroxylase activity in female catfish, Heteropneustes fossilis: a study correlating ovarian activity changes.

Exposure of female catfish, Heteropneustes fossilis to 30-day regimes of long photoperiod (16L), elevated temperature (28 +/- 2 degrees C), or a combination of both stimulated brain tyrosine hydroxylase (TH) activity significantly over that of control fish held in natural conditions in gonad resting (10.5L:13.5D, 10 +/- 2 degrees C) and preparatory (12.5L:11.5D,18 +/- 2 degrees C) phases. The response was high in the combination group in both phases. The increase in TH activity was higher in forebrain regions (telencephalon and hypothalamus) than medulla oblongata. Exposure of the fish to short photoperiod (8L:16D) and total darkness decreased the enzyme activity significantly in both resting and preparatory phases regardless of the temperature. The inhibition was high in fish held under total darkness. Gonadosomatic index (GSI) was significantly elevated in long photoperiod and high temperature groups, alone or in combination, and decreased significantly in short photoperiod (only in preparatory phase) and total darkness groups. Administration of the serotonin precursor 5-hydroxytryptophan (5-HTP; 5mg/100g body weight [BW], three daily intraperitoneal [i.p.] injections prior to sacrificing) stimulated TH activity in fish held under long and normal photoperiods in both phases. Three daily injections of the serotonin blocker parachlorophenylalanine (p-CPA; 10mg/100g BW) and melatonin (75 microg/100g BW) prior to sacrificing inhibited brain TH activity significantly in both phases. GSI was significantly stimulated by 5-HTP, and inhibited by both p-CPA and melatonin injections. Changes in TH activity and GSI can be correlated and explained on the basis of previous reports on changes in catecholamine activity that modulates gonadotropin secretion in the catfish. Further, the photoperiod and temperature-induced changes in TH activity may be modulated by alterations in serotonergic activity.

Citicoline: neuroprotective mechanisms in cerebral ischemia.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.

Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals.

Multiple types of voltage-activated calcium (Ca(2+)) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca(2+) influx through N- and L- type voltage-activated Ca(2+) channels in sympathetic neurons to the depolarization-induced activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine (NE) synthesis, and the depolarization-induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both omega-conotoxin GVIA (1 microM), a specific blocker of N-type channels, and nimodipine (1 microM), a specific blocker of L-type Ca(2+) channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K(+), indicating that Ca(2+) influx through both types of channels contributes to enzyme activation. In contrast, K(+) stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by omega-conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K(+) stimulation of NE release from both ganglia and irises was also blocked completely when omega-conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca(2+) influx through N- and L-type Ca(2+) channels.

Dexamethasone's influence on tyrosine hydroxylase activity in the chemoreflex pathway and on the hypoxic ventilatory response.

Catecholamines have been implicated in neuromodulation of peripheral chemosensitivity and central respiratory mechanisms. Because glucocorticoids can affect catecholamine metabolism in the carotid body and brainstem, this study explored the possibility that, in rats, dexamethasone or adrenalectomy affects catecholamine biosynthesis in carotid body chemoreceptors and the medullary areas (A2C2, A5, A6, A7) involved in the chemoreflex pathway and the hypoxic ventilatory response (HVR). One dexamethasone injection (1 mg/kg body wt.) stimulated tyrosine hydroxylase activity in the carotid body and had no effect in brainstem catecholamine areas, while HVR was reduced. Chronic dexamethasone (1 mg/kg body wt. daily for 10 days) had a stimulatory influence on tyrosine hydroxylase activity in the carotid body and an inhibitory effect on A2C2, A5 and A7 cell groups. Breathing pattern, but not HVR, was altered. Adrenalectomy elicited an increase in tyrosine hydroxylase activity in A2C2, which was accompanied by a decreased respiratory frequency in hypoxia. The data show that glucocorticoids have differential effects on catecholamine biosynthesis in peripheral and central structures involved in the chemoreflex pathway. Depending on the treatment, the neurochemical changes were accompanied by alterations of HVR or the breathing pattern, which are consistent with a neuromodulating influence of catecholamines on peripheral chemosensory inputs or the central respiratory network.

2-Phenylaminoadenosine stimulates dopamine synthesis in rat forebrain in vitro and in vivo via adenosine A 2 receptors

The adenosine agonist 2-phenylaminoadenosine (PAD) stimulated tyrosine hydroxylase activity in rat striatum in vitro. This effect was selectively blocked by the A 2 antagonist 8-chlorostyrylcaffeine (CSC), suggesting an A 2 receptor-mediated mechanism. PAD also produced a corresponding increase in striatal adenylyl cyclase activity. Using an in vivo model that measures presynaptic effects of drugs at dopamine nerve terminals, intracerebroventricular administration of PAD to rats stimulated tyrosine hydroxylase activity in striatum in a manner that was selectively blocked by CSC. These results suggest that PAD stimulates adenylyl cyclase and tyrosine hydroxylase activity, with a corresponding increase in dopamine synthesis, by activation of presynaptic A 2-type receptors in mammalian forebrain.

The role of protein kinase C in nicotinic responses of bovine chromaffin cells

The effects of the protein kinase C inhibitor CGP 41251 (31-benzoyl-staurosporine) on nicotinic responses of cultured bovine adrenal chromaffin cells have been investigated. CGP 41251 inhibited tyrosine hydroxylase activation by phorbol 12,13-dibutyrate, with an IC 50 of < 0.3 μM and complete inhibition at 1 μM. In contrast, it had little effect on nicotine-stimulated tyrosine hydroxylase activity up to 1 μM, and did not fully inhibit it even at 10 μM. From 1 to 10 μM, CGP 41251 caused a similar concentration-dependent inhibition of tyrosine hydroxylase activity stimulated by nicotine, K +, forskolin and 8-Br-cyclic AMP. CGP 42700 (19,31-dibenzoyl-staurosporine), a structural analogue of CGP 41251 that lacks activity as a protein kinase C inhibitor, had no effect on tyrosine hydroxylase activity stimulated by any of the agonists. CGP 41251 had no effect on catecholamine secretion induced by nicotine. The results suggest phorbol ester-sensitive protein kinase C isozymes do not play a major role in nicotinic stimulation of tyrosine hydroxylase activity or catecholamine secretion in chromaffin cells.

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

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

Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats.

In the present study we investigated the regulation of tyrosine hydroxylase (TH) by angiotensin II (Ang II) in an attempt to provide cellular and molecular evidence that this hormone has increased neuromodulatory actions in the spontaneously hypertensive (SH) rat brain. Neuronal cells in primary culture from the hypothalamus-brain stem of both normotensive [Wistar-Kyoto (WKY)] and SH rats have been used. These cultures mimic in vivo situations. Ang II caused a time-dependent increase in TH activity in WKY rat brain neurons. A maximal increase of 2.5-fold was observed with 100 nM Ang II in an actinomycin- and cycloheximide-dependent process. In addition, Ang II caused a parallel increase in TH messenger RNA (mRNA) levels, with a maximal stimulation of 5-fold in 4 h by 100 nM Ang II in WKY rat brain neurons. The stimulation of TH mRNA was mediated by the AT1 receptor subtype, resulted from an increase in its transcription, and involved activation of phospholipase C and protein kinase C. Antisense oligonucleotide for c-fos attenuated Ang II stimulation of TH mRNA in a time- and dose-dependent fashion, indicating an involvement of c-fos as a putative third messenger in Ang II stimulation of TH. Ang II also caused stimulation of TH activity and its mRNA levels in neuronal cultures of SH rat brain by a mechanism similar to that observed for neuronal cultures of WKY rat brain, involving AT1 receptors, protein kinase C, and c-fos. However, the stimulation of TH activity and that of TH mRNA were approximately 30% and 80% higher, respectively, in the SH rat brain neurons than those in the WKY rat brain neurons. In vivo experiments have been carried out to validate the elevated response of TH gene expression to Ang II in SH rat brain neuronal cultures. Ang II stimulated both TH activity and TH mRNA levels in the hypothalami and brain stems of adult WKY and SH rats. The level of stimulation in the brain of the SH rat was significantly higher than that in the WKY rat. These observations are consistent with an increase in AT1, receptor gene expression and suggest that increased TH gene expression could be the cellular/molecular basis for the greater neuromodulatory action of Ang II in the SH rat brain.

The Ca++/Calmodulin-Dependent Protein Kinase II Inhibitors KN62 and KN93, and Their Inactive Analogues KN04 and KN92, Inhibit Nicotinic Activation of Tyrosine Hydroxylase in Bovine Chromaffin Cells

The possible role of Ca++/calmodulin-dependent protein kinase II (CAM-K-II) in the nicotinic activation of tyrosine hydroxylase in intact cultured bovine adrenal chromaffin cells has been investigated. Over the concentration range 3–30 μM, KN62, a specific CAM-K-II inhibitor, inhibited basal tyrosine hydroxylase activity and the activity stimulated by nicotine or K+depolarisation. KN04, a structural analogue of KN62 which does not inhibit CAM-K-II, produced an identical concentration-dependent inhibition of basal and nicotine-stimulated tyrosine hydroxylase activity. Another CAM-K-II inhibitor, KN93, also inhibited nicotine and K+stimulation of tyrosine hydroxylase activity; however, an inactive analogue of KN93, KN92, mimicked these effect. The results suggest that the inhibition of nicotine- and K+-stimulated tyrosine hydroxylase activity by KN62 and KN93 is not due to their ability to inhibit CAM-K-II.

Down-regulation of protein kinase C activity preferentially attenuates high K +-stimulated tyrosine hydroxylase activity in adrenal chromaffin cells cultured with insulin-like growth factor-I

The purpose of this study was to determine whether the loss of protein kinase C (PKC) from adrenal chromaffin cells affected the enhancement of high K +- and forskolin-stimulated tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) activity observed in cells treated with insulin-like growth factor-I (IGF-I). Forskolin-stimulated tyrosine hydroxylase activation was not affected by down-regulation of PKC. High K +-stimulated tyrosine hydroxylase activity decreased substantially after treating the cells for ∼18 h with active, but not inactive, phorbol ester (300 nM). After down-regulation of PKC, high K +-stimulated tyrosine hydroxylase activity in cells cultured with IGF-I decreased by 61 ± 5% ( n = 14) compared to 36 ± 8% ( n = 14) in cells cultured without IGF-I. These data suggest that PKC is required for the enhancement of high K +-stimulated tyrosine hydroxylase activity observed with IGF-I treatment.

1-Phenyl-3-amino-1,2,3,4-tetrahydronaphthalenes and related derivatives as ligands for the neuromodulatory sigma 3 receptor: further structure-activity relationships.

A series of 1-phenyl-3-amino-1,2,3,4-tetrahydronaphthalenes (1-phenyl-3-aminotetralins, PATs) previously was found to stimulate tyrosine hydroxylase activity and dopamine synthesis in rat brain through interaction with a novel sigma 3 receptor. Specifically, the trans-1R,3S-(-) isomer of H2-PAT showed highest affinity for sigma 3 receptors and also produced maximal stimulation of tyrosine hydroxylase activity and dopamine synthesis, as compared to the trans-1S,3R-(+) isomer. Affinity for sigma 3 receptors and functional potency at stimulating dopamine synthesis were attenuated either by altering the position or dimethyl substitution pattern of the amino group or by hydroxylating the tetralin aromatic ring. A preliminary binding model can accommodate many PAT analogs and several non-PATs with a wide range of affinities for the sigma 3 receptor. Here, we report the synthesis and evaluation of additional analogs in order to expand previous structure-activity relationship studies. Further molecular modifications include synthesis of 1-phenyl-1-methyl-3-amino, 1-phenyl-2-amino, 1-phenyl-3-(trimethylammoniumyl), and 1-phenyl-3-(phenylalkyl) analogs, as well as ring-expanded tetrahydrobenzocycloheptenes. In general, the above modifications decreased sigma 3 receptor affinity and, in some cases, caused a reversal of the sigma 3 binding selectivity of trans- versus cis-PATs found previously. Most analogs were selective for sigma 3 receptors and showed little or no affinity for either sigma 1/sigma 2 or dopamine D1, D2, and D3 receptors. N-Phenylalkyl substituents, such as N-phenylethyl, however, endowed the 1-phenyl-3-aminotetralins with enhanced sigma 1/sigma 2 and dopamine receptor affinity while decreasing sigma 3 affinity, thus abolishing sigma 3 selectivity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases prolactin release and tuberoinfundibular dopaminergic neuronal activity

Pituitary adenylate cyclase activating polypeptide (PACAP) is a hypothalamic peptide that affects anterior pituitary cell function. This study examined the effects of PACAP on prolactin (PRL) release in vivo and on tyrosine hydroxylase (TH) activity in tuberoinfundibular dopaminergic neurons in vivo and in vitro. In ovariectomized rats, intravenous injection of PACAP increased circulating PRL levels 3-fold and TH activity in the stalk-median eminence (SME) by 30%. Incubation of the SME with 1 μM PACAP in vitro increased TH activity 2-fold. Intravenous infusion of ovine PRL (oPRL) by an osmotic mini-pump for 2 days in ovariectomized rats increased TH activity in the SME 1.7-fold and reduced circulating concentrations of endogenous rat PRL to 20% of control levels. PACAP induced a 4-fold rise in endogenous rat PRL levels in oPRL-treated rats and a 30% increase in TH activity that was additive to the elevation caused by hyperprolactinemia. In suckled lactating rats, PACAP did not alter circulating PRL levels or TH activity in the SME. When pups were removed from the dams for 4–5 h, systemic injection of PACAP stimulated PRL release without altering TH activity. However, PACAP, when administered in vitro, stimulated TH activity in the SME of lactating rats separated from their pups. These data indicate that PACAP may play a role in augmenting PRL release in female rats. The PACAP-induced rise in PRL release is modest and not due to a decrease in tuberoinfundibular dopaminergic neuronal activity. PACAP increases TH activity in the tuberoinfundibular dopaminergic neurons, possibly by a direct action on nerve terminals within the median eminence.