Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-controlling enzyme in melatonin synthesis is stimulated approximately equal to 100-fold by an adrenergic cyclic AMP mechanism in both neonatal and adult rat pineal glands. This stimulation is blocked in the adult gland by the depolarizing agents ouabain (1 microM) and K+ (80 mM) (Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H. (1975) Mol. Pharmacol. 11, 241-255). In the present study pineal glands obtained from prenatal to adult rats were used; it was found that K+ (80 microM) inhibited the adrenergic stimulation of N-acetyltransferase activity at all ages but that ouabain (1 nM to 1 mM) treatment was not inhibitory early in development. In contrast, in the neonate, ouabain (1-100 nM) enhanced adrenergic induction of N-acetyltransferase activity, and ouabain treatment alone (1-1000 nM) stimulated N-acetyltransferase activity. A small stimulation was also seen at one concentration (1 nM) in the adult. Analysis of the development of high affinity ouabain binding sites and Na+,K+-ATPase activity in the intact pineal gland indicated that the developmental pattern of both resemble the development of ouabain inhibition of the adrenergic stimulation of N-acetyltransferase activity. All are low for the first few days of life, gradually increase during the next 3 weeks of life, and then approach adult levels. Similarly, ouabain (1 nM to 1 mM) had no effect on 86Rb uptake in the 2-day-old gland but blocked (IC50 congruent to 20 nM) 86Rb uptake in the adult gland. These findings indicate ouabain probably has little inhibitory effect on the norepinephrine stimulation of N-acetyltransferase activity in the neonatal because a high affinity ouabain binding form of Na+,K+-ATPase activity, similar to the alpha + form identified in rat brain, is at very low levels in the pinealocyte. Accordingly, it appears that an ouabain-insensitive mechanism in the neonatal gland maintains membrane potential and that this mechanism plays a less important role in the adult. The explanation of why ouabain alone stimulates N-acetyltransferase activity and why it enhances the effects of norepinephrine in the neonatal pineal gland might be that ouabain acts on surviving neural elements present in the gland to cause the net release of a transmitter, perhaps norepinephrine, which then stimulates N-acetyltransferase activity.
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PMID:Developmental study of ouabain inhibition of adrenergic induction of rat pineal serotonin N-acetyltransferase (EC 2.3.1.87). 282 93

Ouabain inhibits (IC50 congruent to 200 nM) the congruent to 100-fold adrenergic cyclic AMP stimulation of rat pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87, serotonin N-acetyltransferase, NAT) activity in intact pineal glands. In the present study, ouabain binding sites in pineal membranes were characterized in detail and compared to sites in isolated pinealocytes, which mediate the inhibition of Na+,K+-ATPase, as indicated by 86Rb uptake and norepinephrine (NE) stimulation of NAT activity. High affinity ouabain-binding sites were identified in crude preparations of pineal membranes (Kd congruent to 14 nM; Bmax congruent to 4 pmol/mg of protein) and similar sites were also found in ovine and bovine pineal tissue. The ouabain Kd value for the rat pineal binding sites was similar to the estimated ouabain IC50 values for 86Rb uptake and the NE stimulation of NAT activity in intact rat pinealocytes. In addition, the relative orders of potency of four cardiac glycosides in displacing [3H]ouabain from high affinity binding sites and inhibiting both 86Rb uptake and NE stimulation of NAT activity were the same (acetyldigitoxin greater than ouabain greater than digitoxin greater than strophanthidin). The similarities in the characteristics of the high affinity [3H]ouabain-binding sites and the sites involved in the inhibition of 86Rb uptake and stimulation of NAT activity indicate that an alpha +-like Na+,K+-ATPase mediates the inhibitory effects of ouabain on the adrenergic induction of pineal NAT activity.
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PMID:Characterization of the alpha +-like Na+,K+-ATPase which mediates ouabain inhibition of adrenergic induction of N-acetyltransferase (EC 2.3.1.87) activity: studies with isolated pinealocytes. 282 93

Tryptophan hydroxylase (TPH) is the first enzyme in both serotonin and melatonin biosynthesis in neuroendocrine cells of the pineal gland. The lack of immortalized neuroendocrine pineal cell lines has been a major obstacle to the study of the tissue-specific and circadian regulation of TPH gene expression in the pineal gland. Previously, we demonstrated that a 6.1 kb 5' upstream region of the mouse TPH gene directs the restricted expression of a lacZ reporter gene to the pineal gland and the raphe nuclei of transgenic mice. Therefore, to develop TPH-expressing pineal cell lines we first established transgenic mice carrying a construct consisting of 6.1 kb of 5' flanking region fused to the SV40 T-antigen. These animals developed highly invasive pineal tumors and died at 12-15 weeks of age. The pineal tumors obtained from the transgenic mice were utilized to establish the immortalized pinealocyte-derived cell lines. These cells express two marker enzymes, TPH and serotonin N-acetyltransferase (NAT). In pineal gland TPH and NAT expressions have been known to be regulated during circadian cycle. The two established cell lines therefore promise to be a valuable in vitro model system for the study of the rhythmic nature of the pineal function at molecular level in mammal.
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PMID:Immortalization of neuroendocrine pinealocytes from transgenic mice by targeted tumorigenesis using the tryptophan hydroxylase promoter. 873 33

The circadian hormone melatonin is synthesized predominantly in the pineal gland by the actions of two pineal-specific enzymes: serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Pineal night-specific ATPase (PINA), another pineal- and night-specific protein we recently identified, is produced as a truncated form of the Wilson disease gene (Atp7b) product. To identify the regulatory elements required for pineal-specific gene expression, we isolated sequences upstream of the rat PINA gene and discovered a cis-acting element that is recognized by a novel pineal/retina-specific nuclear factor. This pineal regulatory element (PIRE) has a consensus of TAATC/T and is present in six copies in the 5' regulatory region of the PINA gene, at least three copies in the rat NAT promoter, and at least one copy in each of the putative HIOMT promoters A and B. A recently identified retina-specific protein, cone rod homeobox (CRX), binds to PIRE in vitro and transactivates PIRE-reporter constructs. These data suggest that Crx may play a crucial role in regulating pineal gene expression through interactions with PIRE.
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PMID:A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX. 946 10

Recent findings have clarified the mechanisms regulating the night- and pineal-specific transcription of serotonin N-acetyltransferase, the rate-limiting enzyme in melatonin formation. Norepinephrine, acting via beta-adrenoceptors and cAMP at night, stimulates the cAMP response element binding protein, which turns on the transcription of N-acetyltransferase and inducible cAMP early repressor, the major inhibitor of N-acetyltransferase transcription. The tissue-specific gene expression within the pineal gland and retina derives, in part, from a pineal/retina-specific transcription factor, cone-rod homeobox protein, which binds to a pineal regulatory element. This regulatory element is present in promoters of pineal-selective enzymes, such as N-acetyltransferase, hydroxyindole-O-methyl transferase, and pineal night-specific ATPase.
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PMID:Molecular rhythms in the pineal gland. 981 31

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.
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PMID:Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture. 1206 61

Long Evans cinnamon (LEC) rat is an animal model for human Wilson disease (WD) due to a deletion in Atp7b, the copper transporter defective in WD patients. Previously, we have demonstrated presence of an alternative product termed PIneal Night-specific ATPase (PINA) generated by an intronic promoter in Atp7b gene. Analysis of LEC rat in this study demonstrates that PINA is absent in the LEC pineal establishing its usefulness for investigating PINA function. Studies of the LEC pineal, however, revealed an additional defect in serotonin N-acetyltransferase (NAT), the key enzyme in melatonin production. Linkage studies confirm that the NAT phenotype is entirely independent of PINA mutation in the pineal gland of LEC rats, and sequence analysis demonstrates that NAT defect is due to a point mutation in NAT coding region. In addition, we demonstrate that the cinnamon coat color of the LEC rat is unlinked to PINA and NAT deficiencies in these animals. To facilitate further functional analysis of PINA in pineal physiology, we crossed LEC rats with PVG rats that are wildtype for PINA, NAT and coat color, and obtained rats that are defective only in PINA/Atp7b locus (termed LPP rats) and normal for NAT activity and coat color. Furthermore, we have identified the deletion breakpoint of Atp7b gene in LPP rats, which allows simplified genotyping of mutant animals. The separation of PINA mutation from both NAT and coat color mutations in the new LPP rats will permit better functional studies of PINA in pineal circadian physiology.
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PMID:A new strain of rat for functional analysis of PINA. 1595 Jul 62

Nocturnal melatonin production is reportedly controlled by the rhythms of serotonin N-acetyltransferase (NAT, or arylalkylamine N-acetyltransferase). While analyzing the melatonin synthetic pathways of Long Evans cinnamon (LEC) rats mutant for PINA, a pineal night-specific ATPase defective in Wilson disease, we discovered that NAT activity and protein levels are greatly reduced in LEC rats, and that the highly conserved histidine 28 is mutated to tyrosine. To study the effect of H28Y, we isolated a new strain of rat termed LPN that is mutant for NAT but wildtype for both PINA and coat color. Compared with control rats, the LPN rats displayed low NAT protein levels and enzyme activities. These results suggest that the H28Y mutation in NAT is the cause of reduced NAT levels in vivo. The identical H28Y mutation was also found in Sprague-Dawley rats from Zivic-Miller, suggesting it may be a common mutation in rodents. When analyzed in bacterial cells and HEK293 cells, the mutation resulted in reduction of both NAT protein stability and catalytic activity, confirming that the in vivo NAT phenotype in LPN rats was due to the H28Y mutation. Further analysis of the NAT-H28Y will focus on the mechanisms of the increased degradation both in vitro and in vivo, which will facilitate our understanding of how melatonin synthesis is controlled at the molecular level.
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PMID:A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro. 1597 62