Larry A. Wheeler

Transforming growth factor α : in vivo release by normal human skin following UV irradiation and abrasion

Transforming growth factor alpha (TGF alpha) is a keratinocyte-growth-stimulating factor which may have a role in epidermal hyperproliferation, psoriasis, and wound healing. Since increased epidermal proliferation occurs in response to UV radiation, we have measured the amount of TGF alpha in exudates from normal and UVB-irradiated human skin. Cutaneous exudates were obtained using the skin chamber abrasion technique from one side of the back of volunteers (n = 10) with normal skin (collected following skin contact times of 2 and 30 min). Exudates were similarly obtained from the contralateral side of the back at sites irradiated 2 h previously with 3 x the minimum erythemal dose UVB. Levels of TGF alpha were measured by radioimmunoassay. Normal human skin released TGF alpha immediately after abrasion: unirradiated, 63 +/- 18 ng/ml; irradiated, 89 +/- 15 ng/ml. Levels of TGF alpha increased within 30 min to 110 +/- 14 ng/ml in unirradiated skin and to 190 +/- 17 ng/ml in irradiated skin. Irradiated sites at 30-min time points were significantly higher (p less than 0.05) than all other samples. The presence of releasable TGF alpha in normal skin suggests a role for TGF alpha in wound repair mechanisms.

Stimulation of endogenous cyclic AMP levels in ciliary body by SK&F 82526, a novel dopamine receptor agonist

The effect of dopamine and SK&F 82526 on cyclic AMP metabolism in ocular tissues has been examined. The in vitro incubation of human and bovine ciliary body with these agonists produced a dose-dependent increase in endogenous levels of cyclic AMP. This stimulation was blocked by the selective DA1 receptor antagonist SK&F 83566, but not by the beta-receptor antagonist propranolol. The response to SK&F 82526 was stereoselective, with SK&F R-82526 being approximately 100 times more potent than SK&F S-82526 in this preparation. This stimulation of ciliary body cyclic AMP content was not observed in the rabbit or cat, nor was it seen in human, bovine or rabbit iris tissue. These data suggest that adenylate cyclase linked dopamine receptors are present in both human and bovine ciliary body.

The antiinflammatory activity of topically applied novel calcium-channel antagonists

The antiinflammatory activities of two novel calcium-channel antagonists, AGN 190742 and AGN 190744, were evaluated in murine models of cutaneous inflammation. These 2(5H)-furanone ring compounds block both depolarizationdependent Ca2+ entry and receptor-mediated responses in GH3 cells. Topical application of AGN 190742 or AGN 190744 inhibits neutrophil infiltration and epidermal hyperplasia induced by repeated treatment of mouse skin with phorbol ester. AGN 190744 also is active in an arachidonic acid model of acute inflammation. These data suggest that topical application of calcium-channel antagonists can inhibit cutaneous inflammatory responses and that AGN 190742 and/or AGN 190744 may serve as useful pharmacological probes for examining these responses in vivo.

Mutagenicity of coal tar preparations used in the treatment of psoriasis

Abstract Four therapeutic coal tar preparations used in the treatment of psoriasis (Zetar ® Emulsion, Estar ® , Lavatar, and Coal Tar Solution USP) were evaluated by the Ames Salmonella/microsome mutagenicity test. All the tar products were mutagenic when plated with liver homogenate and cofactors (S9 mix) on strain TA98. There was a five-fold difference in the mutagenicities of these products when compared on the basis of his + revertants per μg tar. Zetar ® Emulsion, which contains whole coal tar, was more mutagenic than the other preparations which contain selective extracts or distillates of coal tar. This suggests that some of the processed coal tars used in various psoriasis medications may be less mutagenic than crude coal tar itself.

INHIBITION BY MANOALIDE OF FMLP-STIMULATED ELASTASE RELEASE FROM HUMAN NEUTROPHILS

Incubation of human polymorphonuclear leukocytes (PMNLs) with the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) resulted in a concentration-dependent release of the neutral protease elastase. This response was inhibited by pretreatment of the PMNLs with manoalide (IC50 approximately 0.08 microM). To understand the mechanism of this inhibition, we examined the effect of manoalide on the signal-transduction pathway believed to mediate fMLP stimulation. We observed in fura-2 loaded cells that pretreatment with manoalide blocked fMLP-induced increases in cytosolic free-calcium (IC50 approximately 0.15 microM). However, manoalide had no effect on inositol 1,4,5-trisphosphate (IP3) production at concentrations which completely inhibited the Ca2+ signal. Furthermore, manoalide was approximately 50-fold less potent as an inhibitor of phospholipase C activity in membrane preparations of PMNLs than as an inhibitor of calcium mobilization in whole cells. These data indicate that manoalide can block stimulation of human PMNLs through inhibition of Ca2+ mobilization, but that this occurs at a site beyond phospholipase C activation and inositol phosphate turnover.

AGN 190383, a novel phospholipase inhibitor with topical anti-inflammatory activity

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activityin vivo.

PROPHAGE INDUCTION, MUTAGENESIS AND CELL SURVIVAL OF AMES' MUTAGEN TESTER STRAINS AFTER 8‐METHOXYPSORALEN PLUS ULTRAVIOLET LIGHT‐A

Abstract— The furocoumarin 8‐methoxypsoralen (8‐MOP) is not detected as a mutagen in the standard Ames test either in the presence or absence of S9‐mix and/or ultraviolet light‐A (320–400 nm). The Ames strains have recently been shown to harbor bacteriophages that are inducible by carcinogens and mutagens. Psoralen (8‐MOP) plus UVA (PUVA) was found to be a potent prophage inducing treatment. Induction was observed in TA1535, TA1538, TA98, TA100, TA1978 and TA1975 with 0.1 μg/ml of 8‐MOP and 2.5 kJ/m2 of UVA. PUVA is a potent bactericidal treatment at concentrations of 8‐MOP above 0.5 μg/ml and 2.5 kJ/m2 in tester strains TA1535, TA1538, TA98, and TA100. PUVA is known to be bactericidal, but the cytotoxicity observed in this study was unique in that the frameshift tester strains (TA1538 and TA98) were more sensitive to the lethal effects of PUVA than the base pair tester strains (TA1535 and TA100). The differential cytotoxicity in such closely related strains led to the examination of some of the strains from which the Ames strains were derived. The data suggest mutations introduced into the Ames strains to make them more sensitive to carcinogens and mutagens (ΛuvrB) have resulted in an altered response to PUVA. It is postulated that TA1535 retains a DNA repair function that is lost by TA1538 during the selection for uvrB deficient strains.

Effect of Forskolin on Beta-Adrenergic Hyporesponsiveness in Skin

beta-Adrenergic receptor hyporesponsiveness has been observed in psoriasis and after exposure of epidermis to phorbol esters. It was the purpose of our studies to determine if forskolin, which is known to act synergistically with receptor agonists in elevating endogenous levels of cyclic AMP, could return these responses to those seen under control conditions. It was observed that topical application of phorbol ester to mouse ears in vivo led to a significant reduction in isoproterenol stimulation of cyclic AMP in vitro. Low doses of forskolin (10(-7) M) were able to enhance isoproterenols effect under these conditions. Similarly, human keratinocyte cell cultures treated with phorbol esters and human psoriatic epidermis in vitro were both hyporesponsive to isoproterenol. Again, pretreatment of these samples with forskolin restored the beta-agonist stimulation to control values. These data indicate that forskolin is still able to act synergistically with beta-agonists in hyporesponsive systems and suggest that forskolin may be a useful probe in defining the mechanism of this decreased responsiveness both in phorbol-ester-treated skin and in psoriasis.