We have conducted a genome screen of autism, by linkage analysis in an initial set of 90 multiplex sibships, with parents, containing 97 independent affected sib pairs (ASPs), with follow-up in 49 additional multiplex sibships, containing 50 ASPs. In total, 519 markers were genotyped, including 362 for the initial screen, and an additional 157 were genotyped in the follow-up. As a control, we also included in the analysis unaffected sibs, which provided 51 discordant sib pairs (DSPs) for the initial screen and 29 for the follow-up. In the initial phase of the work, we observed increased identity by descent (IBD) in the ASPs (sharing of 51.6%) compared with the DSPs (sharing of 50.8%). The excess sharing in the ASPs could not be attributed to the effect of a small number of loci but, rather, was due to the modest increase in the entire distribution of IBD. These results are most compatible with a model specifying a large number of loci (perhaps >/=15) and are less compatible with models specifying

The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping consensus elements for the Sp1 and Egr-1 transcription factors located at -45 bp and -165 bp in the PNMT promoter. In the present study, we show that Sp1 and Egr-1 can specifically bind to these overlapping elements, that this binding appears to be mutually exclusive, and that binding site occupancy is dependent upon the concentration of each factor and its binding affinity for each site. Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1 binds to both sites with intermediate affinities (IC50 = 210 and 140 nM, respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT promoter activity primarily through interaction with the -165 bp site, while Sp1 stimulates PNMT promoter activity by interacting with both the -45 bp and the -165 bp sites. These results show that Sp1 and Egr-1 are capable of differentially activating PNMT gene expression, thereby suggesting that different stimuli may control the activity of the PNMT gene by selectively regulating Sp1 and/or Egr-1.

Physical challenges, emotional arousal, increased physical activity, or changes in the environment can evoke stress, requiring altered activity of visceral organs, glands, and smooth muscles. These alterations are necessary for the organism to function appropriately under these abnormal conditions and to restore homeostasis. These changes in activity comprise the "fight-or-flight" response and must occur rapidly or the organism may not survive. The rapid responses are mediated primarily via the catecholamines, epinephrine, and norepinephrine, secreted from the adrenal medulla. The catecholamine neurohormones interact with adrenergic receptors present on cell membranes of all visceral organs and smooth muscles, leading to activation of signaling pathways and consequent alterations in organ function and smooth muscle tone. During the "fight-or-flight response," the rise in circulating epinephrine and norepinephrine from the adrenal medulla and norepinephrine secreted from sympathetic nerve terminals cause increased blood pressure and cardiac output, relaxation of bronchial, intestinal and many other smooth muscles, mydriasis, and metabolic changes that increase levels of blood glucose and free fatty acids. Circulating catecholamines can also alter memory via effects on afferent sensory nerves impacting central nervous system function. While these rapid responses may be necessary for survival, sustained elevation of circulating catecholamines for prolonged periods of time can also produce pathological conditions, such as cardiac hypertrophy and heart failure, hypertension, and posttraumatic stress disorder. In this review, we discuss the present knowledge of the effects of circulating catecholamines on peripheral organs and tissues, as well as on memory in the brain.

This review summarizes knowledge on the effects of stress on two catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). Information is presented on differential responses of the enzymes to a variety of stressors as well as differential responses of the enzymes localized to the central nervous system vs. peripheral nervous system and tissues. Changes in mRNA and protein or activity are described, including species- and stressor-specific effects. While temporal changes in these parameters may differ for the particular stressor or enzyme, in general, maximal changes in mRNA and protein content occur at 6-8 and 24 h after stressor exposure, respectively. Elevation of TH and PNMT transcriptional activators prior to mRNA induction and nuclear run-on assays show that stress activates the genes encoding these enzymes. Yet, extents of induction of mRNA, protein and enzyme activity are often discordant depending on the stress, its duration and repetition of exposure. The extremes are concordant changes in mRNA and protein/activity vs. highly elevated mRNA with no change in protein/activity. Post-transcriptional and/or post-translational regulatory influences that may contribute to the complex effects of stress on TH, PNMT and the stress hormone epinephrine are explored.

No abstract

Two newly identified, overlapping (1 bp) glucocorticoid response elements (GREs) at Ϫ759 and Ϫ773 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 Ϫ533-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 M). The type II glucocorticoid receptor antagonist RU38486 (10 M) fully inhibited dexamethasone (1 M) 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 Ϫ759-bp GRE has a 2-fold greater affinity for the glucocorticoid receptor than the Ϫ773-bp GRE. Site-directed mutation of the Ϫ533-, Ϫ759-, and Ϫ773-bp GREs alone or in tandem demonstrated that the Ϫ759-bp GRE was also functionally more important, but both the Ϫ759-and Ϫ773-bp GREs are required for maximum glucocorticoid responses. Moreover, the Ϫ533-bp GRE, rather than increasing glucocorticoid sensitivity of the promoter, may limit corticosteroid responsiveness mediated via the Ϫ759-and Ϫ773-bp GREs. Finally, the glucocorticoid receptor bound to the Ϫ759-and Ϫ773-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 Ϫ533-bp GRE only seem to participate in synergistic activation of the PNMT promoter through interaction with activator protein 2.Glucocorticoids are critical regulators of phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28), the final enzyme in epinephrine biosynthesis, exerting both transcriptional and post-transcriptional influences. In vivo studies in rats have shown that depletion of corticosteroids by hypophysectomy decreases PNMT mRNA and enzyme expression (Evinger et al

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 mM) 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-de®cient 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 2165 bp Egr-1 site markedly decreased forskolin activation of the PNMT promoter. These ®ndings 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.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.