Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes.
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The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. We have cloned an IL-2-induced gene from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation. (+info)
Effect of sodium glycyrrhetinate on chemical peritonitis in rats.
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AIM: To study the anti-inflammatory mechanisms of sodium glycyrrhetinate (SG). METHODS: Rat chemical peritonitis was used. The protein content and prostaglandin E2 (PGE2) content in exudate were measured by Folin-phenol assay and RIA, respectively. SOD activity in neutrophils (Neu) was determined by pyrogallol-NBT colorimetry. cAMP content in Neu was detected by competitive protein binding assay. RESULTS: In peritonitis caused by histamine, SG 10-20 mg.kg-1 i.m. reduced exudate volume and Neu counts, and 5-20 mg.kg-1 i.m. lowered the protein content in exudate. In peritonitis induced by carrageenan, SG 20 mg.kg-1 i.m. reduced exudate volume, Neu counts, protein content and PGE2 content in exudate, increased SOD activity in Neu, but did not affect beta-glucuronidase release from Neu. In peritonitis induced by arachidonic acid, SG 20 mg.kg-1 i.m. reduced Neu counts, protein content, and PGE2 content in exudate, and attenuated the reduction of cAMP level in Neu. CONCLUSION: SG exerts its anti-inflammatory action by lowering permeability of capillaries in inflammatory site, inhibiting Neu emigration and PGE2 biosynthesis, and scavenging oxygen free radicals. (+info)
Effects of clonidine on myocardial beta-adrenergic receptor-adenyl cyclase-cAMP system after scalds in rats.
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AIM: To study the role of clonidine (Clo) on the myocardial beta-adrenergic receptor (beta-AR)-adenyl cyclase (AC)-cAMP system after the scalds in rats. METHODS: A 30% skin-full-thickness scald was produced by immersing rats in 95 degrees C water for 9 s. Clo 0.1-3.0 mg.kg-1 was injected i.p. to rats at 30 min before scalds, yohimbine (Yoh) 0.05 mg.kg-1 or prazosin (Pra) 0.03 mg.kg-1 to rats at 30 min before i.p. Clo. beta-AR density and affinity, AC activity, phosphoric diester hydrolases (PDH) activity, and cAMP content were determined with radioreceptor assay, indirect method, enzymeradiochemical assay, and radioimmunoassay, respectively. RESULTS: Clo inhibited the decrease of the myocardial beta-AR density, the attenuation of AC activity, and the reduction of cAMP content at 12 h after the scalds. Yoh partially reversed the effects of Clo on the three parameters. But Pra did not. CONCLUSION: Clo reversed the changes of the myocardial beta-AR-AC-cAMP system resulted from the scalds in rats. (+info)
cGMP-dependent and -independent inhibition of a K+ conductance by natriuretic peptides: molecular and functional studies in human proximal tubule cells.
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In immortalized human kidney epithelial (IHKE-1) cells derived from proximal tubules, two natriuretic peptide receptors (NPR) were identified. In addition to NPR-A, which is bound by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and urodilatin (URO), a novel form of NPR-B that might be bound by C-type natriuretic peptide (CNP) was identified using PCR. This novel splice variant of NPR-B (NPR-Bi) was also found in human kidney. Whereas ANP, BNP, and URO increased intracellular cGMP levels in IHKE-1 cells in a concentration-dependent manner, CNP had no effect on cGMP levels. To determine the physiologic responses to these agonists in IHKE-1 cells, the membrane voltage (Vm) was monitored using the slow whole-cell patch-clamp technique. ANP (10 nM), BNP (10 nM), and URO (16 nM) depolarized these cells by 3 to 4 mV (n = 47, 7, and 16, respectively), an effect that could be mimicked by 0.1 mM 8-Br-cGMP (n = 15). The effects of ANP and 8-Br-cGMP were not additive (n = 4). CNP (10 nM) also depolarized these cells, by 3+/-1 mV (n = 28), despite the absence of an increase in cellular cGMP levels, indicating a cGMP-independent mechanism. In the presence of CNP, 8-Br-cGMP further depolarized Vm significantly, by 1.6+/-0.3 mV (n = 5). The depolarizations by ANP were completely abolished in the presence of Ba2+ (1 mM, n = 4) and thus can be related to inhibition of a K+ conductance in the luminal membrane of IHKE-1 cells. The depolarizations attributable to CNP were completely blocked when genistein (10 microM, n = 6), an inhibitor of tyrosine kinases, was present. These findings indicate that natriuretic peptides regulate electrogenic transport processes via cGMP-dependent and -independent pathways that influence the Vm of IHKE-1 cells. (+info)
"The FSGS factor:" enrichment and in vivo effect of activity from focal segmental glomerulosclerosis plasma.
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A circulating causative factor has been postulated in focal segmental glomerulosclerosis (FSGS). It has been shown that serum or plasma from some FSGS increases glomerular albumin permeability (Palb) in vitro. Palb greater than 0.5 (i.e., FS activity) is associated with recurrence after transplantation. Specimens from 15 FSGS patients were studied to document the presence of a permeability factor, to isolate this factor, to characterize its biochemical properties, and to show its effect in vivo. Total lipids were extracted by chloroform/methanol (2: 1); FS activity was absent from total lipid extract. Chylomicrons and lipoproteins were removed from the plasma with dextran sulfate, followed by sequential precipitation of proteins at 50 and 70% ammonium sulfate saturation. FS activity was retained in the 70% ammonium sulfate supernatant and exhibited a 100-fold purification. FS activity was lost after heating at 100 degrees C for 10 min or after protease digestion. Under nondenaturing conditions, electrophoresis of the FSGS 70% supernatant showed a prominent low molecular weight band that was not evident in the 70% supernatant from normal plasma. Dialysis and centrifugation-based membrane ultrafiltration of the FSGS factor indicated a molecular size between 30 and 50 kD. Injection of the 70% FSGS supernatant into rats caused a threefold increase in urine protein in collections from 6 to 24 h after injection. No increase in proteinuria occurred in rats injected with 70% supernatant from normal individuals. It is concluded that the FSGS factor is a low molecular weight protein with the potential to increase Palb in vitro and to cause proteinuria in vivo. (+info)
Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites.
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Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC. (+info)
Catabolic repression of secB expression is positively controlled by cyclic AMP (cAMP) receptor protein-cAMP complexes at the transcriptional level.
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SecB, a protein export-specific chaperone, enhances the export of a subset of proteins across cytoplasmic membranes of Escherichia coli. Previous studies showed that the synthesis of SecB is repressed by the presence of glucose in the medium. The derepression of SecB requires the products of both the cya and crp genes, indicating that secB expression is under the control of catabolic repression. In this study, two secB-specific promoters were identified. In addition, 5' transcription initiation sites from these two promoters were determined by means of secB-lacZ fusions and primer extension. The distal P1 promoter appeared to be independent of carbon sources, whereas the proximal P2 promoter was shown to be subject to control by the cyclic AMP (cAMP) receptor protein (CRP)-cAMP complexes. Gel-mobility shift studies showed that this regulation results from direct interaction between the secB P2 promoter region and the CRP-cAMP complex. Moreover, the CRP binding site on the secB gene was determined by DNase I footprinting and further substantiated by mutational analysis. The identified secB CRP binding region is centered at the -61.5 region of the secB gene and differed from the putative binding sites predicted by computer analysis. (+info)
Calmodulin-binding sites on adenylyl cyclase type VIII.
(48/18083)
Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin. (+info)