Calcium‐independent increases in pericellular plasminogen activator activity in pemphigus vulgaris*

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Abstract
Intracellular free calcium ([Ca2+]i), an important second messenger, plays a crucial role in a variety of biochemical reactions leading to cell activation and protein secretion. This study examines the potential role of [Ca2+]i in mediating increases in pericellular plasminogen activator activity of canine keratinocytes observed upon binding of human pemphigus vulgaris IgG (hPV IgG). Using the calcium-sensitive fluorescent probe fura-2 and digital video fluorescence imaging microscopy, [Ca2+]i levels were determined in individual keratinocytes for up to 29 minutes after addition of 0.1-5 mg/ml hPV IgG to monolayers of subconfluent and confluent cultures. Extracellular ATP (a known [Ca2+]i-agonist in canine keratinocytes) and normal human IgG (nh IgG) served as positive and negative controls, respectively. HPV IgG and nh IgG failed to induce significant increases in [Ca2+]i, whereas 500 microM ATP induced a rapid, 3- to 12-fold transient increase above resting levels. Binding of hPV IgG to these keratinocyte cultures was demonstrated by immunofluorescence at the end of selected experiments. ATP stimulation of cultures previously treated with hPV IgG showed normal responsiveness and more than 90% of the cells were still viable at the end of [Ca2+]i imaging, thus demonstrating that failure to respond to hPV IgG was not due to an experimental artifact. Plasminogen activator activity in supernatants of confluent cultures incubated with 0.1-1 mg/ml hPV IgG or nh IgG and harvested at various time intervals was dependent on the IgG dose used and increased steadily over time. Increases in activity were 47-92% higher in cultures treated with hPV IgG than those incubated with the same dose of nh IgG.(ABSTRACT TRUNCATED AT 250 WORDS)