Calcium-mobilizing agonists stimulate anion fluxes in cultured endothelial cells from human umbilical vein

Abstract

The goal of the present studies was to determine whether anion fluxes are involved in thrombin-and histamine-activated signal transduction pathways in human umbilical vein endothelial cells (HUVECs). ¹²⁵Iodine (¹²⁵I) efflux techniques were used to test the sensitivity of anion fluxes to increases in [Ca²⁺] _i and activation of protein kinase C. HUVECs exhibited constant ¹²⁵I efflux rates under basal conditions. Administration of thrombin or histamine stimulated an increase in ¹²⁵I efflux rates which returned to control values after approximately 1–2 min. Since both agonists stimulate increases in [Ca²⁺] _i , we tested the hypothesis that ¹²⁵I efflux was sensitive to changes in [Ca²⁺] _i . When HUVECs were exposed to ionomycin or thapsigargin, the ¹²⁵I efflux rate increased and remained elevated for several minutes. In subsequent experiments, HUVECs were incubated with the cell permanent Ca²⁺ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-AM, to buffer changes in [Ca²⁺] _i . This treatment reduced both basal and thrombin-stimulated ¹²⁵I efflux. However, when Ca²⁺ was removed from the efflux buffer and replaced with EGTA, peak thrombin-stimulated ¹²⁵I efflux remained unchanged. This anion efflux was also sensitive to activation of protein kinase C since phorbol 12-myristate 13-acetate and phorbol, 12,13-dibutyrate blunted thrombin-mediated increases in ¹²⁵I efflux. Preincubation of HUVECs with protein kinase C inhibitor peptide [19–36] antagonized the phorbol ester-mediated decrease in thrombin-stimulated ¹²⁵I efflux. We suggest that ¹²⁵I efflux in HUVECs represents a Ca²⁺-sensitive anion conductance and that intracellular Ca²⁺ release, but not extracellular Ca²⁺ influx, is sufficient to initiate channel activity.