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Although calcium ions have been shown to regulate the differentiation of keratinocytes in vitro, the role of divalent cations in vivo is not known. Prior attempts to localize divalent cations in epithelial tissues have been impeded by a lack of specificity of ultrastructural techniques, as well as translocation of precipitates within tissues. The availability of an improved cytochemical method (oxalate-pyroantimonate technique) has facilitated more precise, reliable localization of calcium. When this technique (+/- 10 mM EGTA) was applied to neonatal mouse epidermis, Ca++-containing precipitates localized primarily within the cytosol, mitochondria, and nuclear chromatin of some basal and spinous cells, suggesting a possible relationship of Ca++ with the cell cycle. In the lower granular layer, progressively more Ca++ precipitates appeared intercellularly, with the only intracellular Ca++ localized within mitochondria and lamellar bodies (limiting membranes and discs). The most apical granular cells always demonstrated dense extracellular deposits, and high intracellular Ca++, free in the cytosol. The extruded contents of lamellar bodies, at the granular-cornified layer interface, also demonstrated significant amounts of Ca++-containing precipitates between the lamellar discs. Although some corneocytes in the lower stratum corneum demonstrated intracellular precipitates, most were deviod of Ca++. The striking intercellular Ca++ accumulation in the mid granular layer, coupled with Ca++ influx in the upper granular layer, supports the view that changes in intracellular Ca++ may regulate epidermal differentiation. Finally, the association of Ca++ with lamellar body disc membranes and contents suggests that divalent cations may contribute to both lamellar body secretion and to the formation of intercorneocyte membrane bilayers.

Hydrolysis of glucosylceramide by j-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gaucher's disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2±0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5-to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function. (J.

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The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids, including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited fl-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function. (J. Clin. Invest. 1993.91:1656-1664

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