Thrombin-induced prostacyclin biosynthesis in human endothelium: role of guanine nucleotide regulatory proteins in stimulus/coupling responses.
The regulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGI2 was monitored by RIA of 6-keto PGF1 alpha and dose-dependent increases observed with human alpha- and gamma-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1 alpha approximating responses with 1 microM gamma-thrombin, 5 microM arachidonate, or 10 microM histamine. Diisopropyl phosphorofluoridate-inactivated alpha-thrombin did not stimulate PGI2 release, demonstrating that catalytic activity was required for thrombin-stimulated PGI2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGI2 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 alpha-thrombin nor NaF-stimulated PGI2 release was dependent upon the availability of extracellular Ca++). The hypothesis that G proteins are involved in agonist-stimulated PGI2 synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5'-0-3-thiotrisphosphate (GTP gamma S), which effected significant dose-dependent increases in PGI2 synthesis compared with control levels of 6-keto PGF1 alpha. In contrast, the G-protein inhibitor GDP beta S, (guanosine 5'-0-2-thiodiphosphate), attenuated alpha-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 microgram/ml) did not inhibit the PGI2 synthesis stimulated by either alpha-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 alpha-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with alpha-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGI2 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.
Garcia
J G
JG
Department of Medicine, Indiana University School of Medicine, Indianapolis 46208.
Painter
R G
RG
Fenton
J W
JW
English
D
D
Callahan
K S
KS
D000244:Adenosine Diphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D011464:Epoprostenol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D037342:Pertussis Toxin; D013917:Thrombin / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008565:Membrane Proteins; D010566:Virulence Factors, Bordetella; D000244:Adenosine Diphosphate; D011464:Epoprostenol; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins