No abstract

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 apr 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'-[y-thio]triphosphate (GTP[v-S]). Both light and GTP[y-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 Py subunits. This is in contrast to the transducinmediated increase in cGMP phosphodiesterase activity, where activation occurs by action of the a subunit. The a subunit, which itself slightly stimulated phospholipase A2 activity, inhibited the P8y-induced stimulation of phospholipase A2. This inhibition appears to be the result of subunit reassociation since addition of GTP[y-S] abolished the inhibitory effect of the a subunit on the Pr-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 subunits is, therefore, a mode of action for these subunits in signal transduction.Guanine nucleotide-binding regulatory proteins, G (or N) proteins, play a major role in receptor-mediated signal transduction (1-3). These G proteins are heterotrimers, which, upon ligand-induced stimulation, dissociate into a and P8y subunits (1, 3). The a subunits of these G proteins serve as substrates for specific bacterial toxins (4-7), have high affinity GTP binding sites (1, 2), and an intrinsic GTPase activity that plays a role in cycling the G proteins through their active (dissociated) and inactive (associated) states (1-3). The dissociated GTP-bound forms of the G protein a subunits are the active effectors of intracellular regulatory proteins such as adenylate cyclase and cGMP phosphodiesterase (1-3). The 8y subunits, in contrast, function primarily to facilitate the reassociation of the heterotrimeric complex, thereby deactivating the dissociated a subunits of those G proteins that share common Py subunits (1, 8). The f3y complex also appears to be required for or to enhance the interaction of specific G proteins with activated receptors (9, 10).In the rod outer segments (ROS) of the retina, the major G protein is transducin (2). The dissociated a subunit of this G protein couples the light activation of rhodopsin to stimulation of cGM...

Fibroblast growth factor (FGF)-l 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. (J. Clin. Invest. 1993. 91:553-565.)

A s bstract. The interaction of bradykinin (BK) with its specific receptors on intact cultured human fibroblasts results in production ofprostaglandins, including prostacyclin (PG12), and accumulation of cyclic AMP. Incubation of cells with 1 ,tM BK for 5 min at 37°C led to a marked reduction (75-90%) in BK-induced PGI2 release and in total number of [3H]BK-binding sites with no change in dissociation constant (6.1 and 7.6 nM for control and BK-treated cells, respectively). The decrease in receptor number did not result from BK transferred from the first incubation into the binding assay. BK-induced receptor loss was temperature dependent; exposure of cells to BK at 40C had little or no effect on receptor number. After incubation with BK for _ 15 min, further incubation in the absence of BK for 30 min at 370C almost completely restored both receptor number and BK-induced PGI2 release. With more prolonged exposure to BK (>1 h), restoration of receptors was inversely related to the length of exposure and the concentration ofBK. Recovery was unaffected by cycloheximide. During prolonged incubation without removal of BK, cells began to recover receptors by 5 h; >99% of the bradykinin initially present disappeared by 3 h. Bacitracin greatly retarded BK disappearance and totally prevented recovery. These observations provide direct evidence that the num-

No abstract