TY - JOUR
T1 - Thrombin‐induced prostacyclin biosynthesis in human endothelium
T2 - Role of guanine nucleotide regulatory proteins in stimulus/coupling responses
AU - Garcia, Joe G.N.
AU - Painter, Richard G.
AU - Fenton, John W.
AU - English, Denis
AU - Callahan, Karleen S.
PY - 1990/1
Y1 - 1990/1
N2 - 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 PGF1α 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.
AB - 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 PGF1α 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.
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U2 - 10.1002/jcp.1041420123
DO - 10.1002/jcp.1041420123
M3 - Article
C2 - 2105325
AN - SCOPUS:0025062278
SN - 0021-9541
VL - 142
SP - 186
EP - 193
JO - Journal of Cellular Physiology
JF - Journal of Cellular Physiology
IS - 1
ER -