ISSN:
0021-9541
Keywords:
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
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.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcp.1041420123
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