Thrombin‐induced prostacyclin biosynthesis in human endothelium: Role of guanine nucleotide regulatory proteins in stimulus/coupling responses

Abstract

The regulation of prostacyclin (PGl₂) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGl₂ was monitored by RIA of 6-keto PGF_1α and dose-dependent increases observed with human α-and γ-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1α approximating responses with 1 μM γ-thrombin, 5μM arachidonate, or 10 μM histamine. Diisopropyl phosphorofluo-ridate-inactivated α-thrombin did not stimulate PGl₂ release, demonstrating that catalytic activity was required for thrombin-stimulated PGl₂ release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGl₂ synthesis in a dose-dependent and time-dependent manner (20 mM NaF, 4.4 ± 0.5-fold increase at 10 min, 11.9 ± 1.5-fold increase at 30 min). Neither α-thrombin nor NaF-stimulated PGl₂ release was dependent upon the availability of extracellular Ca^{+ +}. The hypothesis that G proteins are involved in agonist-stimulated PGl₂ synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5′-0-3-thiotrisphosphate (GTP γ S), which effected significant dose-dependent increases in PGl₂ synthesis compared with control levels of 6-keto PGFα. In contrast, the G-protein inhibitor GDP β S, (guanosine 5′-0-2-thiodiphosphate), attenuated α-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 μg/ml) did not inhibit the PGl₂ synthesis stimulated by either α-thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross-reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the α-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with α-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGl₂ synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin-occupied receptor is coupled to phospholipase activities by a pertussis toxin-insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.