Steinsträsser I, Koopmann K, Merkle H P
Department of Pharmacy, Swiss Federal Institute of Technology Zurich (ETH), Irchel Campus, Switzerland.
J Pharm Sci. 1997 Mar;86(3):378-83. doi: 10.1021/js960201u.
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.
报道了对有组织的HaCaT角质形成细胞片层的代谢研究。细胞在多孔膜上生长，形成几层细胞的有组织的细胞片层，其被视为活表皮的模型。通过反射动力学研究代谢，细胞片层的顶面与供体溶液接触，底面由不透水的背衬层封闭。通过高效液相色谱法监测供体中底物和代谢物的代谢产物形成。为了进行比较，还对匀浆的HaCaT细胞进行了研究。模型底物是4-甲氧基-2-萘胺（MNA）的氨基酸酰胺（即丙氨酸-MNA、精氨酸-MNA、谷氨酸-MNA和亮氨酸-MNA）。还研究了亮脑啡肽作为模型肽。除了谷氨酸-MNA外，所有底物在有组织的细胞片层和匀浆中均被代谢。在匀浆研究中，代谢反应在底物浓度<100 nmol mL(-1)时达到饱和，而有组织的细胞片层中的代谢在<500 nmol mL(-1)时低于饱和，除了亮脑啡肽在>100 nmol mL(-1)时显示饱和。在匀浆中，亮氨酸-MNA（>约20 nmol mL(-1)）存在底物抑制，但丙氨酸-MNA和精氨酸-MNA不存在底物抑制，这两种底物均显示饱和。匀浆与有组织的细胞片层之间的差异是由于细胞片层完整的组织结构和酶分隔，而匀浆中组织结构和分隔丧失。此外，底物扩散到细胞片层中可能是限速因素。
