Epidermal Aminopeptidase Activity and Metabolism as Observed in an Organized HaCaT Cell Sheet Model

Abstract

Metabolism studies on organized HaCaT keratinocyte cell sheets are reported. Cells were grown on porous membranes to form organized cell sheets of several cell layers, which were considered as a model of viable epidermis. Metabolism was studied by reflection kinetics, with the top side of the cell sheets in contact with a donor solution and the basal side closed by an impermeable backing layer. Metabolite formation was followed by HPLC of substrate and metabolite in the donor. For comparison, studies with homogenized HaCaT cells were also performed. Model substrates were amino acid amides of 4-methoxy-2-naphthylamine (MNA) (i.e., Ala-MNA, Arg-MNA, Glu-MNA, and Leu-MNA). Also Leu-enkephalin was studied as a model peptide. Except for Glu-MNA, all substrates were metabolized in both the organized cell sheet and in the homogenates. In homogenate studies, saturation of the metabolic reaction was reached at <100 nmol mL(-1) substrate, whereas metabolism in organized cell sheets was below saturation (up to 500 nmol mL(-1)) except for Leu-enkephalin that showed saturation at >100 nmol mL(-1). In homogenates, substrate inhibition was found with Leu-MNA (> approximately 20 nmol mL(-1)) but not with Ala-MNA and Arg-MNA, both of which showed saturation. Differences of homogenates versus organized cell sheets are due to the intact organization and enzyme compartmentation of the cell sheets as opposed to the loss of organization and compartmentation in homogenates. Also, diffusion of substrate into cell sheets may be rate limiting.

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

“…In organotypic co-cultures with fibroblasts, conditions that induce normal keratinocytes to form stratified epithelia with all features of a normal epidermis (Smola et al, 1998;Stark et al, 1999), HaCaT cells have been described to be deficient in this complex function of tissue regeneration (Haake and Polakowska, 1993;Syrjänen et al, 1996;Steinsträsser et al, 1997;Boelsma et al, 1999). We have been able to improve their epidermal tissue formation and differentiation in this model by intensifying stromal influences by elevating the number of supporting fibroblasts (Schoop et al, 1999).…”

“…This is best demonstrated with the HaCaT cell line, a non-tumorigenic keratinocyte population derived from adult trunk skin exhibiting a rather normal differentiation capacity despite multiple chromosomal alterations (Boukamp et al, 1988;Boukamp et al, 1997;Breitkreutz et al, 1997;Breitkreutz et al, 1998;Ryle et al, 1989). However, organotypic co-cultures of HaCaT cells with skin fibroblasts exhibit some stratification but usually lack typical criteria of an ordered structure and regular keratinization (Haake and Polakowska, 1993;Syrjänen et al, 1996;Steinsträsser et al, 1997;Boelsma et al, 1999). This deficiency, however, was not due to the permanent loss of essential differentiation functions.…”

Modeling of Diffusion and Concurrent Metabolism in Cutaneous Tissue

“…Samples were analyzed by HPLC for substrate and metabolite as previously described (Steinsträsser et al, 1997). All experiments were run at least in triplicate.…”

“…Values were taken from previously performed independent experiments (Steinsträsser, 1994;Steinsträsser et al, 1997):…”

Stability of a Transdermal Salmon Calcitonin Formulation

“…The drug does not encounter the enzymes inside the cells as it penetrates the skin so that homogenates were not used for this study. It has been shown that homogenates of cells may overestimate the enzymatic activity of the intact cells due to the loss of the organization and compartmentation in homogenates (Steinstrasser et al 1997).…”