Susan Forster

Characterization and activity of phospholipase A2 in normal human epidermis and in lesion-free epidermis of patients with psoriasis or eczema

The kinetic properties of phospholipase A2 isolated from single large specimens of normal human epidermis and‘uninvolved’(lesion‐free) psoriatic epidermis were determined. The enzymes from the two sources behaved identically with respect to changes in protein concentration, Ca2+ concentration and pH, but the enzymes responded differently to changes in substrate concentration. Furthermore, the specific activity of the enzyme derived from lesion‐free psoriatic epidermis was higher than that from normal epidermis under all conditions used. Increased specific activity of the enzyme in the lesion‐free epidermis was also found when biopsy specimens taken from thirty‐five patients with psoriasis vulgaris at varying severity were compared with biopsies of normal epidermis from thirty‐one control volunteers (P < 0.001). Mixing experiments, in which homogenates of lesion‐free psoriatic epidermis and control epidermis were combined, suggested that the relatively low activity of the enzyme in normal epidermis was due to the presence of an inhibitor. As the activity of the enzyme was not elevated in the lesion‐free epidermis from twelve cases of eczema, which is also an inflammatory condition of the epidermis and superficial dermis, it is suggested that the raised phospholipase A2 activity demonstrated in the lesion‐free epidermis of psoriasis may play an important role in the pathogenesis of the disease.

The level of phospholipase A2 activity is raised in the uninvolved epidermis of psoriasis

The activity of phospholipase A2 was measured in uninvolved psoriatic epidermis and normal epidermis. The mean phospholipase A2 activity in uninvolved psoriatic epidermis, expressed as nmol fatty acid released per mg protein per h, was raised compared with normal epidermis (7.88±s.e. 1·20 vs 2.18±s.e. 0·38; P<0·01). This observation is, we believe, the first demonstration of an abnormality in the uninvolved untreated psoriatic epidermis which could account for the pathology of the disease.

The lysosome membrane

Abstract The membrane around the lysosome is more than a cordon sanitaire , protecting the cytoplasm from the dangerous brew of hydrolytic enzymes in these organelles. Its permeability properties are tailor-made to enhance the efficiency of lysosome function. Specific transporters exist in the membrane for certain metabolites.

Epidermal phospholipase A2 activity is raised in the uninvolved skin of psoriasis

In uninvolved epidermis from nine psoriatic subjects, the mean phospholipase A2 activity was significantly greater (P <0·01) than in nine controls. The increased activity of this enzyme could lead to an increase in the concentration of free arachidonic acid which, in turn, could lead to increased concentrations of inflammatory mediators.

Utilization of epidermal phospholipase A2 inhibition to monitor topical steroid action

The effect of several steroid creams on epidermal phospholipase A2 (PLA2) activity in symptomless psoriatic and normal epidermis was studied. The magnitude of PLA2 inhibition produced by the steroids was directly proportional to the initial level of the enzyme activity. This differential inhibition resulted in PLA2 activity approaching or attaining the normal range regardless of its initial level. Clobetasol propionate 0·05% (Dermovate®) produced more enzyme inhibition than betamethasone valerate 0·1% (Betnovate®) but there was no difference in inhibition between this latter steroid and clobetasone butyrate 0 ·05% (Eumovate®). All were more inhibitory than hydrocortisone 1 % (Efcortelan®).

The effect of lysosomotropic detergents on the permeability properties of the lysosome membrane

Compounds such as N-dodecylimidazole and N-dodecylmorpholine kill cells in culture. Their cytotoxicity has been attributed to accumulation in lysosomes where protonation confers detergent properties resulting in membrane destabilization. This hypothesis has been tested by examining the ability of N-dodecylimidazole and N-dodecylmorpholine to decrease the latency of alpha-glucosidase in isolated rat liver lysosomes. No effect was observed. Nor was N-dodecylimidazole apparently able to increase the permeability of isolated rat liver lysosomes to L-alanine, as no diminution of the disruptive effect of L-alanine methyl ester was seen. N-Dodecylimidazole (10-20 micrograms per ml) caused lactate dehydrogenase release from cystinotic fibroblasts, but marginally toxic concentrations failed to induce cystine release, as might have been expected if lysosome membrane damage had occurred. It is concluded that the cytotoxic effects of lysosomotropic detergents may be mediated by a non-lysosomal mechanism.

The role of pinocytosis in the cellular uptake of an amino acid.

Uptake of L- and D-alanine by the rat visceral yolk sac has been studied in vitro in incubations of short duration. It is concluded that much of the uptake of D-alanine is due to fluid-phase pinocytosis and that the plasma membrane L-alanine porter is more stereospecific than has hitherto been supposed.

pH-profile of cystine and glutamate transport in normal and cystinotic human fibroblasts

In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porters ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct.

The effects of decreased growth temperature on the cystine content of cystinotic fibroblasts

Cystinotic fibroblasts transferred from 37 degrees C to 28 degrees C accumulated additional cystine over the period from 4 to 7 days of incubation at 28 degrees C, after which the additional cystine was lost; warming (to 37 degrees C) of cells with elevated cystine stores led to rapid cystine loss. These results, taken together with previously published data showing cystine release from cystinotic fibroblasts incubated at above-normal temperature, are interpreted as indicating the presence in the cystinotic fibroblast lysosome membrane of a cystine-porter whose efficacy is increased by an increase in membrane fluidity. This porter may be the residual activity of the cystine porter that is known to be deficient in cystinosis, or it may be a second as yet unrecognized porter. It is further proposed that this porter is responsible for the presumed efflux of cystine from cystinotic lysosomes.

Mechanism of cystine reaccumulation by cystinotic fibroblastsin vitro

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.