Release of PAI-1 in response to catecholaminergic stimulation. (A) Corelease of PAI-1 and norepinephrine from catecholaminergic cells. PC12 cells were labeled with [³H]norepinephrine and incubated at 37°C for 30 minutes in release buffer with or without the presence of secretagogue (60μM nicotine, 55mM KCl, or 2mM BaCl₂). The release of PAI-1 antigen (open bars) and [³H]norepinephrine (filled bars) were determined as described in “Secretagogue-mediated release of PAI-1.” Percent release was calculated as the amount in release buffer/total (amount in release buffer + amount in cell lysate), and the results were expressed as fold stimulation compared with basal (unstimulated) values. Values are represented as the mean ± SEM of 6 independent determinations for each group. Release of PAI-1 and [³H]norepinephrine were significantly (P < .001) greater for each of the 3 secretagogues compared with corresponding values for basal secretion. (B) Regulated secretion of PAI-1 from bovine adrenal chromaffin cells in response to secretagogues. Primary bovine adrenal chromaffin cells were incubated at 37°C for 30 minutes in release buffer without (lanes 1 and 2) or with secretagogue (55mM KCl, lanes 3 and 4, or 60μM nicotine, lanes 5 and 6). The releasates from stimulated and unstimulated cells were examined by Western blotting. (C-D) Parallel increases in plasma PAI-1 and plasma catecholamine concentrations in response to acute sympathoadrenal activation in vivo. (C) Groups of aged-matched male C57Bl6/J mice were either unstressed or exposed to restraint stress as described in “In vivo sympathoadrenal activation studies.” Data are represented as the mean ± SEM for n = 6 in each group. Significant (P < .001) increases in plasma PAI-1 concentrations were observed after sympathoadrenal activation, compared with plasma concentrations in unstressed mice. In addition, the increases in plasma PAI-1 were in parallel to those of plasma catecholamines (epinephrine plus norepinephrine), approximately 2-fold increase for both (1.94 ± 0.11-fold increase for PAI-1, and 2.01 ± 0.38-fold increase for catecholamines). (D) Linear regression of plasma PAI-1 and plasma catecholamines in control (unstressed) mice (open circles) and in response to acute sympathoadrenal activation by restraint stress (closed circles; r = 0.825, n = 12, P = .001).