T. C. Tai

Epinephrine: A Short- and Long-Term Regulator of Stress and Development of Illness

Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood–brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis.

Stress-induced changes in epinephrine expression in the adrenal medulla in vivo

Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the ‘flight or fight’ response. In part, stress activates EPI via the phenylethanolamine N‐methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr‐1, with increases in Egr‐1 mRNA, protein, and protein–DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1–DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP‐2 mRNA, protein, and protein–DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post‐transcriptional regulatory mechanisms.

Genetic Mechanisms for Adrenergic Control during Stress

Abstract: 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.

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 μm) 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 ≥ 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP‐responsive element(s) lay between < 392 bp and ≥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.

Cholinergic and peptidergic regulation of phenylethanolamine N-methyltransferase gene expression.

Abstract: The splanchnic nerve, innervating the adrenal medulla, releases a variety of neurotransmitters that stimulate genes involved in catecholamine biosynthesis. In particular, cholinergic agonists have been shown to induce phenylethanolamine N‐methyltransferase (PNMT) gene expression through activation of both nicotinic and muscarinic receptors in vivo and in vitro. By contrast, the role of peptidergic neurotransmitters in adrenal medullary PNMT gene expression remains unclear. Using transient transfection assays, we demonstrate that rat PNMT promoter‐luciferase reporter gene constructs are markedly activated by 10 nM PACAP when expressed in PC12 cells. PACAP appears to mediate its effects primarily through PAC1 receptors and, subsequently, cAMP‐protein kinase A (PKA) and extracellular Ca2+ signaling mechanisms. Activation of these signal transduction pathways markedly increases nuclear levels of the immediately early gene transcription factor Egr‐1 and the developmental factor AP2. A slight decrease in Sp1 expression may also occur, whereas MAZ and glucocorticoid receptor expression remains unaltered. Although PACAP stimulates rapid changes in transcription factor expression and PNMT promoter activity, its effects are long lasting. PNMT promoter induction continues to rise and is sustained for ≥48 hours. By contrast, while muscarine, nicotine, or carbachol (100 μM) also evoke rapid increases in rat PNMT promoter activity, peak activity is observed at 6 hours, followed by a decline and restoration to basal levels by 24 hours. Cholinergic activation of the PNMT promoter also seems to involve the cAMP‐PKA signaling mechanism. However, the magnitude of stimulation and antagonist blockade with H‐89 or the polypeptide inhibitor PKI suggests that the extent of activation is much less than that with PACAP.

Hypoxic stress-induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O2) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl2 or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal −893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (−282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.

Hypoxia and adrenergic function: molecular mechanisms related to Egr-1 and Sp1 activation.

Hypoxia is shown to regulate the stress hormone epinephrine through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT) via PNMT gene activation and transcription factors Egr-1 and Sp1 in adrenal medulla-derived PC12 cells. Moderate hypoxia (5% oxygen) markedly stimulates PNMT promoter-driven luciferase activity in the cells. Hypoxia increases Egr-1 and Sp1 mRNA and nuclear protein content and Egr-1 and Sp1 protein-DNA binding complex formation. Subsequent to transcription factor induction, endogenous PNMT mRNA and protein also increase. Egr-1 and Sp1 binding site inactivation or Egr-1 and Sp1 siRNA inhibit PNMT promoter stimulation by hypoxia. Hypoxia elevates protein kinase A (PKA), phospholipase C (PLC), phosphoinositide 3-kinase, protein kinase C, ERK1/2 mitogen-activated protein kinase and p38 mitogen-activated protein kinase expression while selective inhibitors of these signaling enzymes abrogate hypoxic induction of the PNMT promoter and the rise in Egr-1, Sp1 and PNMT mRNA and protein. PC12 cells lacking PKA or PLCgamma-1 show significant reduction in PNMT promoter activation by hypoxia. Signaling inhibitors do not affect these responses or reduce hypoxic induction of the PNMT promoter to a lesser extent. Findings suggest that Egr-1 and Sp1 through synergistic interaction are critical transcriptional activators for hypoxic stress-regulated adrenergic function controlled via cAMP/PKA and PLC signaling. Identification of Sp1 as a mediator of hypoxia-induced transcriptional activation of PNMT has not been previously been shown. The effects of hypoxia on PNMT and thereby epinephrine may have important ramifications for the stress hormone epinephrine, its ability to regulate behavioral and physiological processes associated with stress and stress-elicited illness.

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 (∼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.

Neural Mechanisms Regulating Phenylethanolamine N-Methyltransferase Gene Expression

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis to stimulate hormonal and neural regulatory pathways leading to the release of glucocorticoids and epinephrine into the bloodstream. This response is the classic fight or flight mechanism originally described by Walter B. Cannon in the 1900s.1 It permits the organism to meet the challenge of a stressor, whether environmental, physiological or psychological. Eventually, however, homeostasis must be restored to ensure the organism’s survival. The latter occurs through the activation of inhibitory hormonal and neural feedback pathways impacting each level of the axis to suppress further glucocorticoid and epinephrine production and release.