Edward T. Wei

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors

Studies in mammalian skin have shown expression of the genes for corticotropin‐releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH‐Rs). These CRH‐Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)‐mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypo‐thalamic‐pituitary‐adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/ POMC system, the CRH‐Rs may be a central element.—Slominski, A., Wortsman, J., Pisarchik, A., Zbytek, B., Linton, E. A., Mazurkiewicz, J., Wei, E. T. Cutaneous expression of corticotropin‐releasing hormone (CRH), urocortin, and CRH receptors. FASEB J. 15, 1678–1693 (2001)

Effects of clonidine on morphine withdrawal signs in the rat

The influence of clonidine on the naloxone-induced withdrawal signs, escape attempts and precipitated shakes, was studied in morphine-dependent rats. Clonidine injected i.p. or intraventricularly (i. vent.) inhibited precipitated shakes and potentiated escape attempts induced by naloxone in morphine-dependent rats. Under pentobarbital anesthesia, precipitated shakes and ice water-induced wet shakes were inhibited by clonidine and norepinephrine. Clonidine injected i. vent. reduced body temperatures in morphine-dependent rats but not in placebo pellet-implanted rats. We suggest that clonidine modulates morphine withdrawal signs by potentiating the behavior associated with heat dissipation (escape attempts) and inhibiting the behavior associated with heat gain mechanisms (precipitated shakes). These effects may occur via stimulation of central noradrenergic mechanisms.

Direct-acting mutagens in automobile exhaust*

Particulate matter in city air contains chemicals which are mutagenic in the Ames Salmonella typhimurium assay. In residential urban areas, the principal mutagens in air do not require liver enzymes to be activated. The source of these liver independent (direct-acting) mutagens may be automobile exhaust because (1) the mutagenic activities were correlated to the lead content or air, (2) the mutagens were found exhaust samples from automobiles and from an experimental CFR single-cylinder gasoline engine, and (3) these mutagens were not found in fuel or unused motor oil, but were found in used motor oil. The strain specificity and the fact that liver enzymes were not required for activation indicated that the exhaust and airborne mutagens were not unsubstituted polycyclic aromatic hydrocarbons (PAH), aromatic amines, alkylnitrosamines or aliphatic epoxides, peroxides and hydroepoxides. A number of nitro-substituted aromatic compounds are direct-acting mutagens in the Ames test, and it is possible that nitration of PAH in exhaust may form the compounds observed here. We synthesized 6-nitrobenzo [a] pyrene and found it to be a potent, direct-acting mutagen with activity comparable to that of benzo-[a] pyrene.

Thyrotrophin-releasing hormone and shaking behaviour in rat.

THYROTROPHIN-RELEASING hormone (TRH) produces behavioural effects in experimental animals1 and may have psychoactive properties in man2–5. Winokur and Utiger6 and Brownstein et al.7 have described the distribution of TRH in rat brain, suggesting that TRH has a modifying role in synaptic functions in addition to its effect on the pituitary. The behavioural effects of endogenous TRH release in the brain are not known; however, Prange et al.8 noted that the systemic administration of TRH to pentobarbital-anaesthetised rats resulted in lacrimation, paw tremor and a peculiar shaking movement of the head and trunk. These behavioural effects were also obtained in partially anaesthetised rats following intracisternal injection of 10 µg TRH per animal. The TRH-induced shaking was particularly interesting because we have observed this behaviour as a characteristic sign of morphine abstinence in the anaesthetised rat9. Here, we have studied the central sites of TRH-induced shaking to determine if these sites parallel the endogenous distribution of TRH in the rat brain and also to determine if these sites correspond to brain areas where morphine withdrawal shakes are obtained.

Assessment of precipitated abstinence in morphine- dependent rats

An experimental model is described for quantifying the precipitated abstinence syndrome in morphine-dependent rats. Male rats were made dependent on morphine by subcutaneous implantation of a morphine pellet and the abstinence syndrome precipitated by intraperitoneal injection of naloxone hydrochloride. A ranking system, based on the median effective dose of naloxone for abstinence signs, quantitatively related the incidence of certain precipitated signs to the dose of naloxone. The time course for the development of dependence was shown to be maximal 70–74 h after pellet implantation. Food or water deprivation for 48 h dissociated the body weight loss during abstinence from the behavioral signs of precipitated withdrawal. Ganglionic blockade did not significantly modify abstinence behavior. An evaluative procedure which ranks abstinence signs is proposed for quantifying physical dependence on morphine.

Cutaneous Expression of CRH and CRH-R: Is There a “Skin Stress Response System?”

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca2+. The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system.

Isolation and identification of mutagenic nitro-pah in diesel-exhaust particulates

Abstract More than 50 nitro-PAH have been tentatively identified in an extract of diesel-exhaust particulates. Identifications are based on high-resolution mass spectrometry of directly mutagenic fractions derived from sequential fractionation of the extract by both low- and high-resolution liquid chromatography. The diversity of nitro-PAH tentatively identified suggests that large numbers of such compounds are formed during or following the combustion process.

Proopiomelanocortin (POMC), the ACTH/ melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin‐1 receptor (MC‐1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melano‐cytes in culture. Much lower levels of α‐MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and α‐MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone conver‐tases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and α‐MSH, and this was enhanced in response to corti‐cotrophin‐releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably trans‐fected with the MC‐1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, α‐MSH, and β‐MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC‐1R highlight the importance of POMC processing as a key regulatory event in the skin.—Rousseau, K., Kauser, S., Pritchard, L. E., Warhurst, A., Oliver, R. L., Slominski, A., Wei, E. T., Thody, A. J., Tobin, D. J., White, A. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and mela‐nocytes and stimulates melanogenesis. FASEB J. 21, 1844–1856 (2007)

AG-3-5: a chemical producing sensations of cold.

Menthol (Fig. 1) is widely used because it produces sensations of coolness and because it has a pleasing mint flavour and odour (USDHEW Publication 1979). The cold sensations elicited by menthol are a fact of human experience. In animal studies, it has been shown that menthol exerts a selective stimulation of peripheral cold receptors in the tongue, as measured by action potentials in the lingual nerve of the cat (Hensel& Zotterman 1951). We describe here a chemical, AG-3-5, which, like menthol, has the unusual property of producing cold sensations. AG-3-5 represents a class of compounds known as tetrahydropyrimidine-2-one derivatives (Fig. 1). In 1972, Podesva & DoNascimento of Delmar Chemicals, Ltd discovered that such compounds affect behaviour in animals. Processes for making these compounds were patented by these investigators. The effects of AG-3-5 were first described by Burford & Chappel (1972). Pharmacologists working at Delmar Chemicals, Ltd stated that AG-3-5 produced in rats and monkeys, among other effects, a behavioural change known as ‘wet shakes’ which are rotational body movements of animals similar to those made by a dog when wet. We were interested in AG-3-5 because we had been studying narcotic withdrawal and one prominent feature of withdrawal in rats is repetitive shaking movements of the body (Wei 1981). A sample of AG-3-5 was obtained from Delmar Chemicals, Ltd and, when tested in rodents, the shaking behaviour was observed (Wei 1976). Low doses of AG-3-5, usually less than 1 mg kg-1 , were sufficient to produce a high frequency of shaking. For example, 1 mg k g l of AG-3-5 administered intrapentoneally to rats elicited over 300 shakes in a 2 h observation period. AG-3-5 was effective in all laboratory species examined, namely, mice, rats, hamsters, gerbils, guinea-pigs, rabbits, cats and dogs (Wei 1976). The reasons for the pharmacological activity of AG-3-5 gradually became apparent as these experiments were conducted. In order to administer AG-3-5 to animals, this compound was dissolved in propylene glycol, a relatively non-toxic solvent frequently used in drug formulations. When the drug solution accidentally came into contact with our mucous membranes-nostrils, lips and eyelids-we noticed sensations of coolness which lasted for approximately 15 min. These sensations of coolness were also experienced by six other individuals in our laboratory who have had occasion to work with AG-3-5. Propylene glycol by itself was not active. On the basis of these preliminary observations, we decided to compare AG-3-5 to menthol, to examine its toxicity, and to taste and ingest a small quantity of the drug so that the sensations produced by this chemical might be characterized. Experiments were conducted on male albino rats, 250-450 g. In confirmation of previous results (Wei 1976), AG-3-5, dissolved in propylene glycol 1 mg k g l , produced in rats numerous and vigorous ‘wet shakes’ within 2 min of intraperitoneal (i.p.) injection. The median effective dose (ED50) for shaking, with a positive response defined as an animal which shook more than 10 times in the 10 min interval after injection, was estimated (Weill952) to be 0-18 mg kg1 (Table 1). Injections of menthol, dissolved 125 mg ml-l in 50% v/v ethanol-water, also produced shaking, the ED50 being 35 mg k g l i.p. The duration of shaking observed with menthol was shorter than for AG-3-5 possibly because at menthol doses >125 mg kg-1, animals became ataxic and lost their righting reflex. These high menthol doses also produced stretching movements of the abdomen (writhing) which may be indicative of local irritation. To determine the short-term toxic effects of AG-3-5, doses of up to 1.5 g k g l of Ag-3-5 were administered either orally or injected i.p. Surprisingly, none of the animals tested at these doses died or showed signs of debilitation or body weight loss in the one-month observation period following drug administration. This low acute toxicity of AG-3-5 was also mentioned in the patent disclosure for AG-3-5 but no data were presented (Podesva & DoNascimento 1974). By contrast, we confirmed the value for the median lethal dose (LD50)

Depressor and natriuretic activities of several atrial peptides

Mammalian atria contain peptides that have depressor and natriuretic activities. Four peptides, atriopeptin I to III (AP I to III) and alpha-human atrial natriuretic factor (alpha-hANP), were synthesized and assayed in the urethane-anesthetized rat for cardiovascular changes and natriuretic activities. All four peptides produced depressor responses and natriuresis. The relative activities were: alpha-hANP = AP III greater than AP II greater than AP I. The disappearance of iodinated AP III from plasma was rapid, with an estimated half-life of 2.5 min. Atriopeptin III was degraded by tissue homogenates, the relative activities being: kidney greater than liver greater than lung greater than plasma greater than heart. The HPLC profile of AP III suggested that smaller peptide fragments were formed after incubation with kidney homogenates. The degradation of AP III was inhibited by bestatin, an aminopeptidase inhibitor, and SQ 20881, a carboxypeptidase inhibitor.