Barrier recovery is impeded at neutral pH, independent of ionic effects: implications for extracellular lipid processing

Abstract

Epidermal permeability barrier homeostasis requires the postsecretory processing of polar lipid precursors into nonpolar lipid products within the stratum corneum (SC) interstices by a family of lipid hydrolases. A specific requirement for beta-glucocerebrosidase (beta-GlcCer'ase), which exhibits a distinct acidic pH optimum, is particularly well documented. Therefore, we sought to determine whether the recovery of the barrier after acute insults requires acidification of the SC. We examined permeability barrier recovery by assessing changes in transepidermal water loss (TEWL), SC membrane ultrastructure utilizing ruthenium tetroxide (RuO4) postfixation, and beta-GlcCer'ase activity by in situ zymography at an acidic vs neutral pH. Barrier recovery proceeded normally when acetone-treated skin was exposed to solutions buffered to an acidic pH. In contrast, the initiation of barrier recovery was slowed when treated skin was exposed to neutral or alkaline pH, regardless of buffer composition. In addition, enhancement of the alkaline buffer-induced delay in barrier recovery occurred with Ca2+ and K+ inclusion in the buffer. Moreover, the pH-dependent alteration in barrier recovery appeared to occur through a mechanism that was independent of Ca(2+)- or K(+)-controlled lamellar body secretion, since both the formation and secretion of lamellar bodies proceeded comparably at pH 5.5 and pH 7.4. In contrast, exposure to pH 7.4 (but not pH 5.5) resulted in both the persistence of immature, extracellular lamellar membrane structures, and a marked decrease in the in situ activity of beta-GlcCer'ase. These results suggest first that an acidic extracellular pH is necessary for the initiation of barrier recovery, and second that the delay in barrier recovery is a consequence of inhibition of postsecretory lipid processing.

Neonatal Development of the Stratum Corneum pH Gradient: Localization and Mechanisms Leading to Emergence of Optimal Barrier Function

“…All diagrams in comparison show acidity increasing initially at the surface, which peaks at day 4, and in parallel, but separately increasing acidity at the SC/SG interface, which by day 4 forms a continuously spreading extracellular acidic pH domain (panels C), while pH distribution inside-out dominates thereafter (panels D). less active when SC is exposed to a neutral pH (Mauro et al, 1998), consistent with their known acidic pH optima (Jensen et al, 1999;Takagi et al, 1999;Schmuth et al 2000). Clearly, such correlations within localized domains are not possible with standard flat electrodes, which measure only surface pH.…”

“…The novel FLIM technique recently developed for epidermal use by our group reveals heretofore undetectable, spatiotemporal details about the development of the epidermal pH gradient, and provides a unique opportunity to gain insight from pH localization as correlated with SC structural/functional alterations. Since the SC acid mantle regulates not only epidermal barrier homeostasis (Thune et al, 1988;Berg et al, 1994;Mauro et al, 1998), but also key SC functions such as desquamation (Ohman and Vahlquist, 1998), and antimicrobial capacity (Aly et al, 1978;Korting et al, 1990), FLIM results also will provide insights into the role of pH in regulating these and other SC functions.…”

“…In adults and neonates, formation and maintenance of the cutaneous permeability barrier requires hydrolytic processing of the relatively polar, secreted lipid mixture of lamellar bodies into their less polar lipid products, a process that is controlled by SC pH (Mauro et al, 1998). Two of the ''lysosomal-type'' enzymes that are required for this lipid processing, b-glucocerebrosidase (b-GlcCer'-ase) and acidic sphingomyelinase (aSM'ase), are cosecrcted with the lipids to the extracellular domains of the lower SC, but both require an acidic milieu for optimal activity (Holleran et al, 1992;Jensen et al, 1999).…”

Acute Modulations in Permeability Barrier Function Regulate Epidermal Cornification

“…Baseline control groups consisted of nontreated animals (n ϭ 4 to 5). Harvesting of tissue samples was performed at 30 minutes, 1,3,6,8,18, and 24 hours after the treatment.…”

“…9 Two signaling mechanisms are known to regulate the lamellar body secretory response in the epidermis underlying disrupted skin sites: an abrupt decline in epidermal calcium levels stimulates secretion of preformed lamellar bodies from the outer granular layer; 10 -14 and acute barrier disruption also raises the ambient pH of normal SC transiently from its usual acidic levels (ϳ5.0) toward neutrality, which, in turn, activates serine proteases (SPs) in the outer epidermis, a sequence that retards barrier recovery kinetics. [15][16][17] Conversely, either immediate reacidification of SC, 18 or applications of SP inhibitors accelerates barrier recovery. 15,19 The increase in SP activity that results from either barrier disruption or a discrete increase in the pH of SC, in turn, activates the proteaseactivated receptor type 2 (PAR2), which is expressed on the plasma membranes of cells of the stratum granulosum.…”