ATP-induced cell contraction with epidermolysis bullosa dystrophica recessive and normal dermal fibroblasts

Abstract

Human dermal fibroblasts cultured on glass coverslips and permeabilized by glycerol can be induced to undergo cell shrinkage by the addition of ATP in buffer containing calcium and magnesium. They reduce in size by 72% in 10 min. ATP-induced cell contraction is linked to an aggregation of cytoplasmic filaments as demonstrated by rhodamine-phalloidin F-actin staining. Before the addition of ATP, glycerinated cells have parallel arrangements of staining cytoplasmic filaments. Afterward, dermal fibroblasts from patients with epidermolysis bullosa dystrophica recessive (EBdr) show only a 10-20% reduction in cell size, and little F-actin aggregation staining can be demonstrated. Epidermolysis bullosa dystrophica recessive fibroblasts have been reported to produce excess prostaglandin E2 (PGE2) and cAMP. The preincubation of normal dermal fibroblasts for 24-30 h with 10 micrograms/ml PGE2, 10 micrograms/ml cholera toxin, or 1 mM dibutyl cAMP will reduce ATP-induced cell contraction to less than 20%. Treated cells showed little disruption of cytoplasmic F-actin. Epidermolysis bullosa dystrophica recessive fibroblasts preincubated with the cyclooxygenase inhibitor indomethacin at 10 micrograms/ml restored cell contraction to 74%. These treated cells also show aggregation of F-actin filaments. The process of ATP-induced cell contraction can be altered by the intracellular concentrations of cAMP, the levels of which are elevated in the fibroblasts in EBdr patients. A mechanism for cAMP inhibition of cell contraction is discussed.