Larissa A. Pohorecky

Purification and properties of rat adrenal phenylethanolamine-N-methyl transferase☆

Abstract A procedure has been developed for the purification of phenylethanolamine- N methyl transferase (PNMT) (EC 2.1.1) from adrenal glands of rats. Ninety percent of the enzyme activity was in the 105,000 g supernatant fraction. After chromatography on Sephadex G-150 and DEAE-cellulose, the PNMT showed two molecular species but the same specific activity on polyacrylamide gel electrophoresis. The final product was enriched nearly 100-fold. The methylation reaction is linear with increasing enzyme concentration, and the enzyme pH optimum was 8.0. The enzyme is relatively stable at 40 °C, but activity is partially destroyed by incubation at 60 °C. Several substrates were tested: octopamine, norepinephrine, tyramine, phenylethanolamine. Greatest affinity was for octopamine. All these substrates and the methyl group donor, S -adenosylmethionine, were inhibitory at high concentrations. Preincubation of the enzyme with norepinephrine accelerated the initial rate of the methylation reaction, while preincubation with S -adenosylmethionine had no such effect. A specific antibody against this purified enzyme was prepared. This antibody inhibited the enzyme activity and also precipitated it. Various immunological studies using this antibody are described.

Induction of Epinephrine-forming Enzyme by Glucocorticoids: Steroid Hydroxylation and Inductive Effect

THE mammalian adrenal gland consists of two anatomically distinct regions; the inner medulla composed largely of catecholamine-producing chromaffin cells, and the outer cortex containing cells that secrete steroid hormones. Although these two adrenal components are derived from different primary germ layers, they come into close contact during embryonic life through an unusual process of tissue migration. Adrenaline is formed in the adrenal medulla through the N-methylation of noradrenaline1. This process is catalysed by an enzyme, phenylethanolamine-N-methyl transferase (PNMT), which is highly concentrated in adrenal chromaffin cells2 and also present in several areas of the brain3. PNMT activity is stimulated in the rat4–6 and the dog7 by hormones secreted from the anterior pituitary gland and the adrenal cortex.

Effects of Endotoxin on Monoamine Metabolism in the Rat

Summary We have examined the effects of administered endotoxin on catecholamine metabolism in rat brain, sympathetic neurons, and adrenal medulla. Intraperitoneal endotoxin (1-5 mg) produced a dosedependent depletion of splenic and cardiac norepinephrine and of adrenal epinephrine; it also accelerated the disappearance of 3H-norepinephrine taken up from circulation. Pretreatment of animals with intraperitoneal endotoxin increased initial uptake of circulating 3H-norepinephrine into heart and spleen. Endotoxin given intraperitoneally (2.5-5 mg) or intracisternally (50-200 μg) depleted brain norepinephrine; it accelerated 3H-norepinephrine turnover in the rat brains in which norepinephrine had been radioisotopically labeled by intracisternal injection. Brain serotonin content was not affected by endotoxin. The acceleration of 3H-norepinephrine turnover in brainstem and hypothalamus after endotoxin administration preceded the induction of hypothermia. This suggests that the effects of endotoxin on body temperature may be mediated in part by central noradrenergic neurons. The work was supported by grants from the U.S. Public Health Service (AM-11237 and HE-02014), and the National Aeronautics and Space Administration (NGR-22-009-627), and by a contract from the Office of the Surgeon General, United States Army.

Decreased Sensory Responses in Osteocalcin Null Mutant Mice Imply Neuropeptide Function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4–14xa0months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-typexa0>xa0knockout; paw stimulation with Von Frey fibers, knockoutxa0<xa0wild-type; balance beam, knockout mobilityxa0<xa0WT; thermal sensitivity to heat (tail flick), knockoutxa0<xa0wild-type; and cold, knockoutxa0<xa0wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.

Effects of lighting on epinephrine synthesis in the rat

Female rats were maintained in continuous light or darkness or in cyclic illumination (i. e., twelve hours of light per day) for four weeks. Their adrenal medullae and brains were then assayed for catecholamine content and for the activity of the epinephrine-forming enzyme phenylethanolamine-N-methyl-transferase (PNMT). Continuous exposure to either light or darkness was associated with decreases in the epinephrine content and PNMT activity of the adrenals. Both treatments abolished the afternoon rise in adrenal corticosterone concentration. The PNMT activity in the olfactory bulb and tubercle of the brain was increased in rat maintained under darkness. These data indicate that changes in environmental illumination affect the synthesis and content of epinephrine within the adrenal medulla and, probably, also within the brain.

Effects of olfactory bulb removal on brain norepinephrine in golden hamsters

Abstract Changes in brain norepinephrine levels after olfactory bulb removal have been found in rats. Several investigators have suggested that the dramatic behavioral effects of bilateral olfactory bulb removal in hamsters might be due to a possible similar change rather than anosmia per se. The results of this study do not support this hypothesis since no significant differences in brain norepinephrine were found between sham operated and bilaterally bulbectomized male hamsters treated identically to those used in earlier behavioral studies on olfactory bulb removal. Also, since there were no changes in the norepinephrine levels in female hamsters which were treated identically to female rats used in earlier neurochemical studies, it is probable that there is a species difference in the effects of olfactory bulb removal on brain norepinephrine.

Control of adrenal medullary protein synthesis by corticosteroids

Abstract It has previously been shown that hypophysectomy decreases the activity of the adrenal medullary enzyme, phenylethanolamine N- methyltransferase , which catalyzes the conversion of norepinephrine to epinephrine. This effect and the consequential decrease in adrenal epinephrine content and secretion can be reversed by treatment with adrenocorticotrophic hormone (ACTH) or glucocorticoids. This report examines the effect of glucocorticoids on adrenomedullary protein synthesis in general. Hypophysectomy causes a profound disaggregation of the polysomes in the rat adrenal and decreases ability of adrenal ribosomes to incorporate amino acids into peptide chains in vitro. Both effects are restored by in vivo treatment with dexamethasone, a potent synthetic glucocorticoid. The reaggregation of adrenal polysomes precedes the phenylethanolamine N- methyltransferase activity.