Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.

Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand. During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded. At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II. Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 4.6 +/- 0.5 nM (mean +/- SE, n = 4). Linear Scatchard plots, Hill coefficients close to unity (0.95-1.06), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity. Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25%. The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor. Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha. This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes.     

Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit.

In the rod outer segments (ROS) of bovine retina, light activation of phospholipase A2 has been shown to occur by a transducin-dependent mechanism. In this report, the transducin-mediated stimulation of phospholipase A2 is shown to require dissociation of the alpha beta gamma heterotrimer. Addition of transducin to dark-adapted transducin-poor ROS stimulated phospholipase A2 activity only with coincident exposure to white light or, in the dark, with addition of the hydrolysis-resistant GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]). Both light and GTP[gamma-S] induced dissociation of the transducin subunits and led to severalfold increases in the phospholipase A2 activity of transducin-rich, but not transducin-poor, ROS. In contrast, pertussis toxin treatment of transducin, which stabilizes the associated state of this G protein, prevented the stimulation of phospholipase A2 by exogenous transducin in the presence of light. Addition of purified transducin subunits to dark-adapted transducin-poor ROS revealed that phospholipase A2 stimulation occurred by action of the beta gamma subunits. This is in contrast to the transducin-mediated increase in cGMP phosphodiesterase activity, where activation occurs by action of the alpha subunit. The alpha subunit, which itself slightly stimulated phospholipase A2 activity, inhibited the beta gamma-induced stimulation of phospholipase A2. This inhibition appears to be the result of subunit reassociation since addition of GTP[gamma-S] abolished the inhibitory effect of the alpha subunit on the beta gamma-induced increase in phospholipase A2, while pertussis toxin treatment of the subunits further inhibited phospholipase A2 activity. Modulation of phospholipase A2 activity by the transducin subunit is, therefore, a mode of action for these subunits in signal transduction.     

Is placenta previa a determinant of preeclampsia?

Low implantation of the placenta has been reported to be associated with a decreased risk for preeclampsia and this has been attributed to increased placental blood flow. However, placenta previa is known to be associated with separation and bleeding, intrauterine growth retardation, and elevated umbilical blood flow resistance by Doppler studies, suggesting decreased umbilical blood flow. To better evaluate the relationship of placenta previa and preeclampsia, 6576 consecutive patients who had ultrasound examination after 28 weeks gestation and delivery at our institution were studied. The placental location, parity, maternal weight, development of preeclampsia, and gestational age were evaluated by using frequency tables and stepwise discriminant analysis. Results showed that placenta previa is not a significant determinant of the development of preeclampsia, but parity, maternal weight, and gestational age contributed significantly to the development of preeclampsia. The finding of decreased incidence of preeclampsia with previa is explained not by increased placental blood flow but by associated maternal characteristics, and particularly by the strong association of previa with premature delivery.     

Coexpression of phosphotyrosine-containing proteins, platelet-derived growth factor-B, and fibroblast growth factor-1 in situ in synovial tissues of patients with rheumatoid arthritis and Lewis rats with adjuvant or streptococcal cell wall arthritis.

Fibroblast growth factor (FGF)-1 and PDGF-B-like factors have been implicated in the pathobiology of RA and animal models of this disease. Since the receptors for FGF-1 and PDGF are tyrosine kinases, we examined the expression of tyrosine phosphorylated proteins (phosphotyrosine, P-Tyr) in synovial tissues from patients with RA and osteoarthritis (OA), and rats with streptococcal cell wall (SCW) and adjuvant arthritis (AA). Synovia from patients with RA and LEW/N rats with SCW and AA arthritis, in contrast to controls, stained intensely with anti-P-Tyr antibody. The staining colocalized with PDGF-B and FGF-1 staining. Comparative immunoblot analysis showed markedly enhanced expression of a 45-kD P-Tyr protein in the inflamed synovia. Treatment with physiological concentrations of dexamethasone suppressed both arthritis and P-Tyr expression in AA. P-Tyr was only transiently expressed in athymic nude Lewis rats and was not detected in relatively arthritis-resistant F344/N rats. These data suggest that (a) FGF-1 and PDGF-B-like factors are upregulated and may induce tyrosine phosphorylation of proteins in vivo in inflammatory joint diseases, (b) persistent high level P-Tyr expression is T lymphocyte dependent, correlates with disease severity, and is strain dependent in rats, (c) corticosteroids, in physiological concentrations, downregulate P-Tyr expression in these lesions.     

Thyrotropin induces growth and iodothyronine production in a human thyroid cell line without affecting adenosine 3',5'-monophosphate production

Using a recently described cell line derived from the fusion of human thyroid and human myeloma cells, we studied the effects of TSH on cell growth, iodide organification, and cAMP production. Although these cells grow in the absence of TSH, when incubated for 2 days in serum-free medium containing purified human or bovine TSH, there was a significant increase in cellular DNA content. The stimulatory effect was observed at concentrations as low as 0.5 microU/ml, which produced a significant increase in DNA content, and was maximal with 5 microU/ml. This effect was still present after 6 days of incubation. Electron microscopy performed by an unbiased observer on cells incubated in the presence of TSH showed an increase in the calculated size of the cells and the nucleus which was significant at 0.1 microU/ml and maximal at 1 microU/ml. Stimulation of 125I organification and hormone production was observed at TSH concentrations as low as 1 microU/ml and was maximal at 10 microU/ml. However, neither TSH (1-50 microU/ml) nor forskolin (10(-6) M) stimulated cAMP production. These data suggest that these cells lack a functional adenylate cyclase pathway and that growth and iodide organification are mediated by other second messenger systems. Such a cell line could yield new insights into the mechanisms of TSH action and may provide a sensitive bioassay for TSH and other thyrotropic factors.