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

The regulation of prostacyclin (PGl 2 ) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGl 2 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 PGl 2 release, demonstrating that catalytic activity was required for thrombin‐stimulated PGl 2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGl 2 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 PGl 2 release was dependent upon the availability of extracellular Ca + + . The hypothesis that G proteins are involved in agonist‐stimulated PGl 2 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 PGl 2 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 PGl 2 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 PGl 2 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.