Identification of a beta2-adrenergic receptor in mammalian epidermis

The presence of a beta2-adrenergic receptor in the epidermis has been demonstrated, based on the following pieces of information: (1) the addition of salbutamol, a beta2-agonist, to slices of epidermal tissue increased the levels of cyclic AMP in the tissue above control levels in a dose-dependent manner with a maximum response after 5 min of incubation in 5 x 10-5 M salbutamol, (2) the addition of butoxamine, a beta2-antagonist, in conjunction with isoproterenol or salbutamol reduced the epidermal cyclic AMP levels when compared to levels obtained with agonist alone, (3) practolol, a beta1-antagonist. had little effect on the salbutamol-induced increases in the cyclic AMP levels and further elevated the levels of cyclic AMP obtained by the addition of isoproterenol, (4) the addition of propranolol to the tissue in conjunction with isoproterenol or salbutamol reduced the levels of cyclic AMP to near control values, and (5) Ro 20-1724, a cyclic nucleotide phosphodiesterase inhibitor, maintained the salbutamolelevated cyclic AMP levels for a longer period of time.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23366/1/0000310.pd

Unoccluded Retinol Penetrates Human Skin In Vivo More Effectively Than Unoccluded Retinyl Palmitate or Retinoic Acid

The formation of all-trans retinoic acid is an oxidative process whereby retinol is converted to retinaldehyde and then to retinoic acid. Because retinol causes qualitative molecular changes similar to those produced by retinoic acid, we compared potency of retinol, retinaldehyde, and retinyl palmitate to retinoic acid and assessed the effects of occlusion. Retinoids were prepared in an experimental vehicle of 95% ethanol:propylene glycol (7:3) with anti-oxidant. Induction of retinoic acid 4-hydroxylase activity was the end point for comparison. Retinoic acid concentrations from 0.001% to 0.05% under occlusion produced a linear dose-response induction of 4-hydroxylase activity. The concentrations of the other retinoids under occlusion required to achieve significant induction of enzyme activity were 0.6% retinyl palmitate, 0.025% retinol, and 0.01% retinaldehyde. The linear dose-response was lost with retinoid concentrations in excess of 0.25% retinol or 0.5% retinaldehyde. Statistical analyses showed no difference in 4-hydroxylase activity between unocciuded and occluded retinol treated sites. By contrast, however, unoccluded sites treated with retinoic acid or retinyl palmitate had less induction of 4-hydroxylase activity than occluded sites. Retinol, retinaldehyde, and retinyl palmitate did not produce erythema but did increase epidermal thickness. Although retinol is a weaker retinoid than retinoic acid, the increased penetration of unoccluded retinol in comparison to unoccluded retinoic acid with this prototypic vehicle confers on retinol a more effective delivery of a retinoidal effect than unocciuded retinoic acid. Retinol at 0.25% may be a useful retinoid for application without occlusion because it does not irritate but does induce cellular and molecular changes similar to those observed with application of 0.025% retinoic acid

Retinoic Acid Isomers Applied to Human Skin in Vivo Each Induce a 4-Hydroxylase That Inactivates Only Trans Retinoic Acid

Application of all-trans retinoic acid to human skin for 4 d under occlusion produces a marked increase in retinoic acid 4-hydroxylase activity. In this study, the possible induction of other hydroxylases in response to 9-cis and 13-cis retinoic acid applications to adult human skin in vivo was determined. Application of 0.1% all-trans, 0.1% 9-cis, and 0.1% 13-cis retinoic acid to human skin for 2 d resulted in induction of only all-trans retinoic acid 4-hydroxylase activity. The 4-hydroxylase activity in microsomes from the treated tissue ranged from 383 ± 46 to 531 ± 59pg of 4-hydroxy all-trans retinoic acid formed/min/mg protein (n = 6). These same preparations were unable to use 9-cis or 13-cis retinoic acid as substrate for the hydroxylation reaction. Extraction of the retinoic acid isomers from epidermis 48h after application of 0.1% solution of each isomer yielded significant amounts of all-trans retinoic acid (36-72%) regardless of the isomer applied. The all-trans isomer produced by isomerization of both 9-cis and 13-cis retinoic acids is the likely inducer of the 4-hydroxylase. All-trans retinol and all-trans retinal were unable to compete with all-trans retinoic acid as substrate for 4-hydroxylase enzyme. The 4-hydroxylase induced in response to pharmacological doses of retinoic acids is specific for the all-trans isomer. The inability of 9-cis or 13-cis retinoic acid to induce their own hydroxylation and inactivation or act as substrate for the 4-hydroxylase in skin may have considerable implications in light of the clinical use of retinoids in the treatment of various diseases including cancers

Molecular and clinical pharmacology of psoriasis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/136054/1/cpt1974165part2919.pd

In Vitro Model of Essential Fatty Acid Deficiency

The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA) – deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty adds 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85–90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid – deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure

Intact epidermal cell assay for ornithine decarboxylase activity

A procedure measuring the ornithine decarboxylase (ODC) activity and polyamine formation of intact neonatal mouse epidermal cells in culture has been developed and tested. Basal cells prepared from neonatal mouse epidermis were plated on round 15-mm Lux coverslips, placed in Costar 24 well culture clusters and grown at 32°C in M-199 + 13% fetal bovine serum. Before assay the cells were rendered permeable to ornithine 14 C and ODC inhibitors using the buffer described by Berger et al. [3]. The slides, covered with adhering cell layers, were then placed in vials, covered with assay buffer and assayed intact for ODC activity. The ODC reaction was terminated by addition of citric acid to the buffer and the amount of 14 CO 2 released was determined by scintillation counting of a center well filled with trapping agent. The baseline ODC activity of the intact cells was 500–1,000 pmol 14 CO 2 /mg protein/45 min. The validity of this ODC assay procedure using intact neonatal mouse keratinocytes was tested by use of three specific ODC inhibitors and by measuring the formation of polyamines from uniform labeled ornithine. The results indicated that authentic ODC activity was measured and preserved in this intact neonatal mouse epidermal cell assay. This technique holds promise for future studies of epidermal cell regulation of ODC and polyamine synthesis and studies of the multiple ornithine metabolites and conjugates formed, using a highly manipulable in vitro system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47233/1/403_2004_Article_BF00509038.pd

A retinoic acid-inducible skin-specific gene (RIS-1/psoriasin): molecular cloning and analysis of gene expression in human skin in vivo and cultured skin cells in vitro

A retinoic acid (RA) inducible skin-specific gene transcript (RIS-1) was isolated by differential hybridization screening of a RA-treated human skin cDNA library. The library was constructed from pooled RNA derived from normal adult human skin treated with all trans -RA for 4 h (n=6) and 12 h (n=6) in vivo . RIS-1 cDNA corresponded to a 0.6 kb transcript that was barely detectable in normal adult human skin but was significantly induced by 8 h in RA-treated compared to vehicle-treated skin (range 1.1–3.6 fold). Prolonged RA treatment for up to 24 h further increased relative RIS-1 mRNA levels by 1.3–5.5 fold. HPLC analysis of the RA content of 0.1% RA-treated skin in vivo revealed significant levels at 6 h (18.8–120.6 ng RA/g wet weight tissue; approximately 240 nM), immediately preceding the time point at which the increased RIS-1 mRNA level was first seen. This concentration of RA also induced the mRNA levels for cellular RA binding protein II (1.6–19 fold), a marker of RA activity in human skin. RIS-1 mRNA was detected by Northern and dot blotting only in normal skin but not in any other normal human tissues examined, indicating a tissue-specific pattern of gene expression. RIS-1 transcripts were detected at very low levels in untreated cultured human epidermal keratinocytes, while no expression was seen in dermal fibroblasts and melanocytes, the other major cell types in skin. Southern analysis of human and mouse DNA indicated the existence of evolutionarily conserved sequences for RIS-1 between these two species. The polypeptide sequence derived from the partial RIS-1 cDNA was found to be identical to the calcium binding domain found in ‘psoriasin’, a gene whose expression appears to be increased in the skin of psoriasis patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43250/1/11033_2004_Article_BF00996356.pd

Increased cyclic GMP and decreased cyclic AMP levels in the hyperplastic, abnormally differentiated epidermis of psoriasis

The genetic skin disease psoriasis has been examined as a model system that may provide an understanding of the control of normal epidermal specialization (differentiation) and the perturbed regulatory processes in proliferative diseases. The excessive glycogen accumulation, increased proliferation and decreased tissue specialization characteristic of psoriasis involve cellular processes that have been shown to be regulated by cyclic AMP in other cells and tissues. It has also been suggested that cyclic GMP is a cellular effector that may be involved in promoting cell proliferation and other events that oppose those believed to be mediated by cyclic AMP. It was postulated, therefore, that the epidermis of the psoriasis lesion might exhibit an imbalance in the cellular concentrations of these two cyclic nucleotides. In this study the levels of cyclic AMP were measured in the involved epidermis (IE) and uninvolved epidermis (UE) from 25 psoriasis patients. The concentrations of cyclic AMP were found as reported previously using a different analytical procedure, to be significantly lower in IE based on protein and DNA. A comparison of the levels of cyclic GMP in IE versus UE of 12 other psoriasis patients showed the levels of this cyclic nucleotide to be significantly increased in IE based on protein, DNA and wet weight. We suggest that this imbalance in the ratio of these two cyclic nucleotides may have pathophysiological relevance to the initiation and/or the maintenance of the psoriasis lesion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33811/1/0000067.pd

Arachidonic acid metabolites: Effects on inflammation of fetal rabbit excisional wounds

Uncovered fetal rabbit excisional wounds do not exhibit any classic signs of healing; wounds covered with an impermeable cover do contract, reepithelialize, and exhibit inflammation. Prostaglandin E 2 (PGE 2 ) is elevated in amniotic fluid, acting as an immunosuppressant at the maternal-fetal interface. Full-thickness excisional wounds were made on 25-day gestational age rabbit fetuses. Half the wounds were covered with an impermeable cover. Tissue from covered, uncovered, and nonwounded fetuses was examined 72 h after wounding for arachidonic acid metabolites. Uncovered wounds had significantly ( P ≤0.05) elevated levels of PGE 2 , PGE 2α , and 12-HETE versus covered wounds and control tissue. Covered wounds had significantly elevated levels of 15-HETE compared to uncovered and control tissue. The elevated PGE 2 in uncovered wounds may act as a fetal immunosuppressant; covered wounds (lower PGE 2 ) developed cellular inflammation. Further investigations of these interactions may permit modulation of adult inflammation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44506/1/10753_2004_Article_BF00918814.pd