Biomedical subjects

T C Tai

Publications and source records attributed to T C Tai.

10 recordsLinked to original sources

PubMed2006

Nerve growth factor regulates adrenergic expression.

The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC-12 cells transfected with a rat phenylethanolamine N-methyl-transferase (PNMT) promoter-luciferase reporter gene construct pGL3RP893. NGF treatment increased PNMT promoter-driven luciferase activity in a dose- and time-dependent manner. Induction was attenuated by inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway ( approximately 60%) but not by inhibition of the protein kinase A (PKA), protein kinase C, phosphoinositol kinase, or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF-responsive sequences lay within the proximal -392 base pairs (bp) of PNMT promoter, wherein binding elements for Egr-1 (-165 bp) and Sp1 (-48 bp) reside. Western analysis further showed that NGF increased nuclear levels of Egr-1, but not Sp1 or the catalytic subunit of PKA. Gel mobility shift assays showed increased potential for Egr-1, but not Sp1, protein-DNA binding complex formation. Mutation of either the Egr-1 or Sp1 binding sites in the PNMT promoter attenuated NGF activation. NGF, combined with pituitary adenylyl cyclase-activating protein (PACAP), another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control seems to be another important mechanism by which neurotrophins regulate the adrenergic phenotype. NGF, PACAP, and a combination of the two stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein, but to different extents.

Adrenal Glands↗

PubMed2004

Genetic mechanisms for adrenergic control during stress.

Cortisol and epinephrine released in response to stress are replenished via activation of the hypothalamic-pituitary-adrenal (HPA or stress) axis. Immobilization (IMMO) stress in rats stimulates epinephrine production in part via the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT mRNA rose up to 7.0-fold with acute or chronic stress. Two transcription factors mediating stress induction of the PNMT gene are the glucocorticoid receptor (GR) and Egr-1, which interact with -533, -759, and -773 bp, and -165 bp binding sites in the rat PNMT promoter, respectively. To identify molecular mechanisms involved, effects of hypoxic stress on PNMT promoter activity were examined in PC12 cells transfected with the PNMT promoter-luciferase reporter gene construct pGL3RP893. Oxygen reduction to 5% increased PNMT promoter-driven luciferase expression, with maximum activity at 6 h. Pretreatment of the cells with protein kinase A (PKA) and protein kinase C (PKC) inhibitors, H-89 and GF109203X, respectively, attenuated the rise in luciferase. Similarly, PKA-deficient PC12 cells transfected with pGL3RP893 and exposed to hypoxia also showed attenuated PNMT promoter-driven luciferase expression. Mutation of the Egr-1 binding site completely prevented PNMT promoter activation, indicating that Egr-1 is essential to the stress response. Consistent with this result, hypoxia increased Egr-1 protein. Hypoxia also increased endogenous PNMT mRNA. However, a shift to intron-retaining mRNA from which truncated, nonfunctional protein is produced, occurred, suggesting that posttranscriptional regulation may be an important genetic mechanism controlling adrenergic expression and hence, epinephrine, during stress.

Animals↗

PubMed2003

Protein kinase A and protein kinase C signaling pathway interaction in phenylethanolamine N-methyltransferase gene regulation.

The protein kinase A (PKA) and protein kinase C (PKC) signaling pathways appear to interact in regulating phenylethanolamine N-methyltransferase (PNMT) promoter-driven gene transcription in PC12 cells. Forskolin treatment of cells transfected with the rat PNMT promoter-luciferase reporter gene construct pGL3RP893 increased promoter activity approximately two-fold whereas phorbol-12-myristate-13 acetate (PMA) treatment had no effect. However, simultaneous forskolin and PMA treatment synergistically activated the PNMT promoter approximately four-fold, suggesting that PKC stimulation requires prior induction of the PKA pathway. Consistent with this possibility the adenylate cyclase inhibitor MDL12,330A, and the PKA inhibitor H-89 prevented PNMT promoter stimulation by the combination of forskolin and PMA. PKA and PKC regulation seems to be mediated in part by Egr-1 and Sp1 through their consensus elements in the PNMT promoter. Forskolin and PMA treatment of PC12 cells increased Egr-1 protein and phosphorylated Egr-1/DNA-binding complex formation to the same extent but only increased phosphorylated Sp1/DNA binding complex formation without altering Sp1 protein levels. Mutation of the - 165 bp Egr-1 and - 48 bp Sp1 sites, respectively, attenuated and abolished combined forskolin and PMA-mediated promoter activation. PNMT promoter analysis further showed that synergistic stimulation by PKA and PKC involves DNA sequences between - 442 and - 392 bp, and potentially a GCM binding element lying within this region.

Animals↗

PubMed2003

Regulation of the rat phenylethanolamine N-methyltransferase gene by transcription factors Sp1 and MAZ.

The rat phenylethanolamine N-methyltransferase (PNMT) gene promoter contains 1-base pair (bp) overlapping consensus sequences for Sp1 and MAZ transcription factors at -48 and -38 bp, respectively. Gel mobility assays using PC-12-derived RS1 cell nuclear extracts or in vitro translated proteins showed that Sp1 and MAZ specifically bind to these elements, that MAZ displaces/prevents Sp1 binding, and that Sp1 and MAZ binding is mutually exclusive, with occupancy dependent on each factor's concentration and affinity for its consensus element. In transfection assays, PNMT promoter activation by Sp1 and MAZ depends on promoter length, with -893 bp of sequence yielding greatest activation. Although MAZ has higher affinity for its binding element, it is a less effective activator. Changes in PNMT promoter activity for the constructs pGL3RP60 or pGL3RP893 using a fixed amount of MAZ expression construct and a variable amount of Sp1 expression construct or vice versa confirmed the latter. Mutation of the MAZ or Sp1 sites in pGL3RP60 attenuated but did not eliminate PNMT promoter activity, even though the proteins no longer bind to their consensus elements. Phosphatase treatment of RS1 cell nuclear extracts prevented MAZ- and Sp1-DNA binding complex formation. Although MAZ and Sp1 elevate endogenous PNMT mRNA in RS1 cells, MAZ preferentially increases intron-retaining whereas Sp1 preferentially increases intronless mRNA. Thus, expression of the PNMT gene seems to be modulated through competitive binding of phosphorylated Sp1 and MAZ to their consensus elements in the promoter. In addition, post-transcriptional regulation seems to be another important mechanism controlling PNMT expression.

Animals↗

PubMed2002

Glucocorticoid responsiveness of the rat phenylethanolamine N-methyltransferase gene.

Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at -759 and -773 bp in the promoter of the rat phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) gene are primarily responsible for its glucocorticoid sensitivity, rather than the originally identified -533-bp GRE. A dose-dependent increase in PNMT promoter activity was observed in RS1 cells transfected with a wild-type PNMT promoter-luciferase reporter gene construct and treated with dexamethasone (maximum activation at 0.1 microM). The type II glucocorticoid receptor antagonist RU38486 (10 microM) fully inhibited dexamethasone (1 microM) activation of the PNMT promoter, consistent with classical glucocorticoid receptors mediating corticosteroid-stimulated transcriptional activity. Relative IC(50) values from gel mobility shift competition assays showed that the -759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the -773-bp GRE. Site-directed mutation of the -533-, -759-, and -773-bp GREs alone or in tandem demonstrated that the -759-bp GRE was also functionally more important, but both the -759- and -773-bp GREs are required for maximum glucocorticoid responses. Moreover, the -533-bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the -759- and -773-bp GREs. Finally, the glucocorticoid receptor bound to the -759- and -773-bp GREs interacts cooperatively with Egr-1 and/or AP-2 to stimulate PNMT promoter activity in RS1 cells treated with dexamethasone. In contrast, glucocorticoid receptors bound to the -533-bp GRE only seem to participate in synergistic activation of the PNMT promoter through interaction with activator protein 2.

Animals↗

PubMed2001

Role of Egr-1 in cAMP-dependent protein kinase regulation of the phenylethanolamine N-methyltransferase gene.

The molecular mechanism by which cAMP activates the rat phenylethanolamine N-methyltransferase (PNMT) gene was examined by transient transfection of the wild-type rat PNMT promoter-luciferase reporter gene construct pGL3RP893 into PC12 cells. Forskolin treatment (10 microM) of the transfected cells for 3--6 h maximally induced luciferase threefold. Induction by forskolin was mimicked by the cAMP analog, 8-Br-cAMP, and prevented in PC12 cells pretreated with the protein kinase A (PKA) inhibitor H-89 or co-transfected with an expression construct for PKI, a polypeptide inhibitor of PKA. Furthermore, forskolin did not activate the PNMT promoter when the 893 bp PNMT promoter-reporter gene construct was transfected into the PKA-deficient cell line, A126. Detailed examination of the forskolin responsiveness of PNMT constructs harboring > or = 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP-responsive element(s) lay between < 392 bp and > or =60 bp. Within this region of the promoter lies a functional binding element for Egr-1, a transcriptional activator of the PNMT gene. Forskolin treatment of PC12 cells also rapidly increased nuclear levels of Egr-1 and the catalytic subunit of PKA (PKA-C), with the rise in PKA-C preceding that of Egr-1. Mutation of the --165 bp Egr-1 site markedly decreased forskolin activation of the PNMT promoter. These findings demonstrate that the rat PNMT gene promoter can be activated via the cAMP-PKA signal transduction pathway, mediated by the immediate early gene transcription factor, Egr-1.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Developmental changes in respiratory, febrile, and cardiovascular responses to PGE(2) in newborn lambs.

PGE(2) has centrally mediated respiratory, febrile, and cardiovascular effects that markedly differ between fetal and adult life. We hypothesized that the transition from fetal to adult responses to PGE(2) occurs in the newborn period. Thus effects of an intracarotid infusion of PGE(2) (3 microg/min for 60 min) were determined in unanesthetized newborn lambs at 5, 10, and 15 days after birth. At 5 days, PGE(2) reduced central CO(2) sensitivity, reduced lung ventilation due to a decrease in breathing frequency, and induced hypercapnia. By 15 days, these effects of PGE(2) had waned significantly. In contrast, phasic (expiratory) thyroarytenoid muscle electromyogram activity, number of short apneas, and incidence of Biot periodic breathing were similarly increased at all three ages. PGE(2) induced a sustained fever at 10 and 15 days. Heart rate and mean arterial blood pressure were unchanged in contrast to marked increases observed by others in adults. Results showed that the transition from fetal to adult respiratory and febrile responses to PGE(2) occurs in early postnatal life, whereas adult cardiovascular responses develop later in life in sheep.

Age Factors↗

PubMed1998

Expression of prostaglandin E2 receptor subtypes in the developing sheep brainstem.

Expression in sheep brain of EP transcripts and their ontogeny in the brainstem was determined using RT-PCR. Sheep EP subtypes showed high homology (>80%) with other species. EP1, EP2, EP3 and EP4 were expressed in spinal cord, medulla, pons, diencephalon, hippocampus, pituitary, cortex and cerebellum of the fetal, newborn and adult sheep brain. Expression of all four peaked in the newborn, whereas fetal and adult expression were similar. A similar ontogeny suggests a common regulation.

Age Factors↗

PubMed1995

Changes in regional cytochrome oxidase activity in the fetal, newborn and adult ovine brainstem.

Metabolic activity of specific brain regions (e.g. brainstem respiratory centers) may increase during the physiologic adaptations at birth. Since regional activity of cytochrome oxidase is correlated with the level of oxidative metabolism, cytochrome oxidase histochemistry was used to investigate whether there are sustained changes in metabolic activity within specific nuclei of the ovine brainstem during the perinatal period and whether further changes occur in the adult. Histochemistry was performed on 10-microns-thick frozen sections of the perinatal (130 d fetus, 140 d fetus, 8 h newborn and 10 d newborn) and adult ovine brainstem (n = 3 at each age). Computer-assisted image analysis was performed on 20 brainstem regions. A general decreasing trend, interrupted by a tendency for a transient increase at 8 h after birth was observed in most regions analyzed. Statistically significant decreases (P < 0.05) in cytochrome oxidase levels between the perinatal age groups and the adult were found in 7 brainstem nuclei studied: ambiguus, cuneate, inferior olivary, reticularis lateralis, spinal trigeminal, parabrachial and superior olivary nuclei. Within the perinatal period, the nucleus gracilis was the only region to show statistically significant decreases in 140 d fetus and 8 h newborns in all nuclei analyzed, but this change was not statistically significant (P > 0.1). These results indicate that the dramatic changes in physiology and environment at birth do not result in a significant change in the metabolic capacity of brainstem nuclei in the immediate perinatal period. However, more gradual developmental changes are observed in specific brainstem nuclei suggesting a decrease in neuronal activity occurs in these areas during development in the sheep.

Aging↗

PubMed1994

Ontogenesis of prostaglandin E2 binding sites in the brainstem of the sheep.

Perinatal changes in plasma prostaglandin E2 (PGE2) act at the level of the fetal brainstem to modulate respiratory activity. In-vitro receptor autoradiography was used to determine whether PGE2 binding sites (putative receptors) are located in brainstem regions responsible for respiratory control. Frozen brainstem sections were obtained from fetal sheep at 140 d of gestation, lambs at 5 h and 10 d after birth, and adults (n = 3 at each age). In the perinatal brainstem, high density binding was localized near the following nuclei: hypoglossal, tractus solitarius, parabrachial and spinal trigeminal-oral. Moderate levels of binding were localized near the nuclei of the spinal trigeminal tract, ambiguus, inferior olivary, cuneate, gracilis, facial, medial vestibular, superior vestibular, and abducens. Binding density did not change with age within the perinatal period. However, binding in three high density regions (hypoglossal, spinal trigeminal and parabrachial nuclei) decreased significantly in adulthood, whereas the binding in the nucleus of the solitary tract remained high. We hypothesize that the PGE2 binding sites that we identified near respiratory regions of the brainstem may mediate the central respiratory effects of PGE2.

Animals↗