Kenneth M. Halprin

Further studies on adenyl cyclase in psoriasis

Slices of human skin obtained with‐a keratome were pre‐incubated with [3H]adenine to label the ATP pool from which cyclic AMP was subsequently formed. The accumulation of radioactive cyclic AMP was measured as an index of adenyl cyclase activity. The data showed that both the ability to incorporate [3H] into ATP and adenyl cyclase activity were significantly lower in psoriatic plaques than in uninvolved skin of the psoriatic patients, or in normal skin of control subjects. The response of adenyl cyclase to the stimulation of 3.3 μM adrenaline was less than five fold in psoriatic plaques as compared to twelve to thirty‐two fold in the uninvolved skin. The response to the stimulation of prostaglandin E2 (5 μg/ml) showed no significant difference between the plaque and normal skin. The adenyl cyclase activity in uninvolved skin of psoriatic patients appeared normal. Propranolol (10 μM) blocked the stimulatory effect of adrenaline but not that of PGE2 in normal skin. These resulsuggest that the adenyl cyclase system of the skin has different regulatory sites for adrenaline and PGE2, and that the enzyme is defective in the epidermis of the psoriatic plaque, especially at the adrenaline regulatory site. A preliminary report on this study was given at the Second International Conference on Cyclic AMP at Vancouver, Canada, 8‐11 July 1974.

Multiple forms of cyclic nucleotide phosphodiesterase in pig epidermis.

Pig epidermal cyclic nucleotide phosphodiesterases (EC 3.1.4.16) have been partially purified by DEAE-cellulose column chromatography. At least three different forms of the epidermal phosphodiesterases were identified. They were cyclic GMP-specific, cyclic GMP- and cyclic AMP-hydrolyzing and apparently a cyclic AMP-specific enzyme: the first two forms were soluble and the last was the particulate enzyme. The cyclic GMP-specific soluble fraction had a relatively low Km, the cyclic GMP- and cyclic AMP-hydrolyzing fraction had a high Km for the respective substrates and the third particulate enzyme had both high and low Km values for cyclic AMP. The cyclic GMP-hydrolyzing enzyme was localized almost entirely in the soluble fraction, whereas cyclic AMP-hydrolyzing enzyme was distributed to both soluble and particulate fractions. Thus, our studies show that the multiple forms of pig epidermal enzyme differ distinctly in their substrate affinity, specificity and subcellular distribution.

Cyclosporin A induced augmentation of the beta-adrenergic adenylate cyclase response of pig epidermis

SummaryIt has been known that beta-adrenergic adenylate cyclase response is decreased in psoriatic-involved epidermis. Since the immunosuppressive agent, cyclosporin A, is reported to be effective on psoriasis clinically, the effect of cyclosporin A on beta-adrenergic adenylate cyclase response in pig skin was examined in vitro. Therapeutic serum levels of cyclosporin A (100–400 ng/ml) augmented the beta-adrenergic adenylate cyclase response of the epidermis. Highest levels of cyclosporin A (2–20 μg/ml) did not have any effect on its response. Both low Km and high Km cyclic AMP phosphodiesterases were not affected by cyclosporin A. Therefore, it is suggested that the clinical efficacy of cyclosporin A on psoriasis can be explained partially by its direct effect on the keratinocyte itself.

Monoclonal antibodies to the ?-andrenergic receptor: Modulation of catecholamine-sensitive adenylate cyclase by the antibody

SummaryWe have developed two types of hybridomas producing monoclonal antibodies to the turkey erythrocyte β1-adrenergic receptor in order to study the β-adrenergic-cAMP system of epidermis. Splenic cells from BALB/c mice immunized with partially purified turkey erythrocyte β1-adrenergic were fused with mouse myeloma cell line SP2/0-Ag14. Five hybridomas of 17 positive cells producing antibodies which could precipitate soluble turkey erythrocyte β1-receptors were cloned by the limiting dilution method. The antibodies cross-reacted with β- and β2-adrenergic receptors and stained epidermal basal cells with immunocytochemical techniques. Neither type of antibody interfered with the antagonist binding, i.e., all antibodies bound to sites other than the ligand binding site on the surface. One type of antibody inhibited epinephrine-stimulated adenylate cyclase activity in our “leaky” epidermal cell system. The data suggest that the antibody interferes with the coupling of the receptor to the regulatory protein.

Monoclonal antibodies against epidermal cell surface and dermal-epidermal junction: antigens which may be epidermal cell spreading factors

Two monoclonal antibodies which reacted with the epidermal cell surface (SF‐1) and the dermal‐epidermal‐junction (SF‐2), respectively, were obtained by immunizing mice with partially‐purified human epibolin. The corresponding antigens were partially purified from fetal bovine serum by affinity chromatography using these antibodies. SDS‐polyacrylamide gel electrophoresis showed that these antigens contained polypeptide components with molecular weights different from that of epibolin (mol. wt. 65000 daltons); SF‐1 antigen had a 68000 dalton main component, and SF‐2 antigen a broad 58000–61000 dalton main component. Both of these partially‐purified antigens promoted the spreading of dissociated pig epidermal cells. SF‐2 antigen also promoted the spreading of Pam cells (a murine keratinocyte line). The results suggest that proteins capable of promoting epidermal cell spreading may be present on the epidermal cell surface and at the dermal‐epidermal junction. However, their physiological role in keratinization remains to be elucidated.