Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osteoblast-like osteosarcoma cells (ROS 17/2.8) display a rapid transmembrane influx of extracellular calcium after stimulation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] that is mediated largely by the opening of voltage-gated calcium channels. These cells also constitutively express high numbers (greater than 18,000/cell) of nuclear receptors for this seco-steroid hormone that are involved in the modulation of genomic activity in the osteoblast and in the up-regulation of transcript ion of osteoblast-specific genes such as osteocalcin. The objective of this study was to determine the structural hierarchy of vitamin D3 analogs with regard to their efficacy as molecular transducers of the genomic and nongenomic pathways that are activated upon treatment of osteoblasts with 1,25-(OH)2D3. To test the structural features of the agonist required for initiation of these distinct pathways, a series of ligand analogs and naturally occurring metabolites of 1,25-(OH)2D3 were used that contain A-ring, D-ring, and side-chain modifications. The abilities of these analogs/metabolites to 1) bind to nuclear receptors and 2) stimulate transmembrane calcium influx were measured. Several analogs (25-hydroxy-16-ene-23-yne-D3 and 25-hydroxy-23-yne D3) were found to stimulate Ca2+ channel opening, but bind only poorly to the 1,25-(OH)2D3 nuclear receptor. Conversely, other analogs (1,24-dihydroxy-22-ene-24-cyclopropyl D3 and 1,25-dihydroxy-16-ene-23-yne,26,27 F6-D3) were found to bind very well to the nuclear receptor, but displayed little or no activity in opening Ca2+ channels. Pertussis toxin, which interferes with coupling of certain ligand-gated receptors to ion channels, failed to block the activation of calcium channels by 1,25-(OH)2D3 or active agonist analogs. Our results indicate that there are likely to be distinct nuclear and plasma membrane-associated forms of the 1,25-(OH)2D3 receptor that are involved in genomic and nongenomic activation of osteoblast activity, respectively. The membrane-associated receptors do not appear to be coupled to pertussis toxin-sensitive G-proteins.
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PMID:Nongenomic actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: structure-function studies using ligand analogs. 165 87

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.
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PMID:Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH2 by substitution of PTH amino acids at positions 10 and 11. 216 25

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.
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PMID:Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists. 216 26

Preparations of rod outer segments from cattle retinas contained soluble and particulate phospholipase C activities which hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2) and the other phosphoinositides. Ca2+ was required for PIP2 hydrolysis, but high (greater than 300 microM) concentrations were inhibitory. Mg2+ and spermine at low concentrations stimulated the particulate activity but inhibited the soluble. Mn2+ inhibited both. High (greater than 100 microM) concentrations of the nonhydrolyzable GTP analogue guanylyl beta,gamma-methylenediphosphonate inhibited PIP2 hydrolysis by both the soluble and particulate activities, but guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), fluoride, and cholera and pertussis toxins were without effect. Overall phospholipase C activity in ROS was unaffected by light. Evidence was found for multiple forms of the enzyme, requiring isolation and separate characterization before ruling out regulation by light or G-protein.
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PMID:Phosphatidylinositol-4,5-bisphosphate phospholipase C in bovine rod outer segments. 216 27

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells. 246 56

We examined mechanisms of down-regulation of PTH receptors and desensitization of the PTH-stimulated increase in intracellular cAMP in clonal rat osteosarcoma cells, ROS 17/2.8. ROS cells treated with 10 nM [Nle8,Nle18,Tyr34] bovine (b) PTH-(1-34) amide (NlePTH) for 3 days showed loss of specific PTH binding and PTH-stimulated cAMP accumulation to 10% of that in vehicle-treated control cells. Treatment of these cells with both 0.5 mM 8-bromo-cAMP (8-Br-cAMP) and 1 mM methylisobutylxanthine or 100 ng/ml cholera toxin for 3 days elicited no change in either of these responses. Treatment with 10 nM NlePTH for 3 days did not modify the cAMP accumulation stimulated by 30 microM forskolin or 1 micrograms/ml cholera toxin, indicating that agonist-specific desensitization of PTH-stimulated cAMP accumulation is not due to diminished activity of either the stimulatory guanyl nucleotide regulatory subunit (Gs) or the catalytic subunit of the adenylate cyclase. Treatment of ROS cells with pertussis toxin (PT; 10 ng/ml) for 12, 24, 48, and 72 h increased specific PTH binding by 21%, 28%, 35%, and 39%. The increase in PTH binding was associated with a parallel increase in PTH-stimulated cAMP accumulation and was due to an increase in the number of PTH receptors. PTH receptor affinity remained constant (apparent Kd = 0.3 nM). PT treatment of the cells partially blocked agonist-specific PTH receptor down-regulation. PT catalyzed ADP ribosylation of 41K and 39K membrane proteins, consistent with the alpha-subunits of Gi and Go, respectively. In conclusion, agonist-induced PTH receptor down-regulation in ROS 17/2.8 cells is cAMP independent and can be reversed by PT treatment. PTH receptor expression in these cells appears to be under tonic inhibitory control by mechanisms involving a PT-sensitive G protein(s).
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PMID:Inactivation of pertussis toxin-sensitive guanyl nucleotide-binding proteins increase parathyroid hormone receptors and reverse agonist-induced receptor down-regulation in ROS 17/2.8 cells. 247 33

In rat osteosarcoma (ROS 17/2.8) cells, which express osteoblastic features in culture, basic fibroblast growth factor (bFGF) reduces the level of alkaline phosphatase, type I collagen, and osteocalcin mRNA and increases osteopontin mRNA, independent of growth stimulation. The fibroblast growth factor (FGF) effects are dose dependent (EC50 about 6 pM) and are detected 24 h after addition of the growth factor. bFGF also reduces parathyroid hormone-stimulatable adenylate cyclase and alkaline phosphatase activity in these cells. Concomitant treatment with pertussis toxin (20 ng/ml) opposes the FGF effects. Although cyclic AMP elevating agents mimic pertussis toxin action on some parameters, they produce opposite effects on others, indicating that antagonism between pertussis toxin and bFGF is not mediated by cyclic AMP. bFGF caused a small reduction in steady state NAD-dependent ADP-ribosylation and had no detectable effects on the steady-state levels of the Gi alpha (alpha subunit of the inhibitory G protein) 1, 2, and 3, visualized with specific antibodies in these cells. Although the site of interaction of pertussis toxin and FGF remains to be determined, the findings presented here suggest separate control of growth and differentiation by bFGF and show that pertussis toxin treatment can modulate differentiation in these cells, presumably via Gi proteins.
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PMID:Opposing effects of fibroblast growth factor and pertussis toxin on alkaline phosphatase, osteopontin, osteocalcin, and type I collagen mRNA levels in ROS 17/2.8 cells. 247 40

Acidic (a) and basic (b) fibroblast growth factors (FGFs) are two related mitogenic and angiogenic factors. They are multifunctional in that they can affect proliferation and induce or delay differentiation. Both aFGF and bFGF were shown to stimulate proliferation of calvaria cells in situ as well as osteoblast-enriched calvaria-derived cells. bFGF was also found to suppress the expression of alkaline phosphatase, parathyroid hormone stimulatable adenylate cyclase, osteocalcin, and type I collagen in the osteoblastic ROS 17/2.8 cells. To explore a possible role for guanine nucleotide binding proteins we assessed the effects of pertussis toxin (PT) on FGF action. PT had opposite effects to those of bFGF on all parameters examined.
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PMID:Effects of acidic and basic fibroblast growth factors on osteoblastic cells. 261 59

Treatment of ROS 17/2.8 cells with dexamethasone (dex) increases (-)isoproterenol (ISO)-, PTH-, cholera toxin-, guanine nucleotide-, NaF-, and forskolin-stimulated adenylate cyclase activity. Enhanced hormone stimulation was first apparent 12 h after dex addition. (-)-[3H]Dihydroalprenolol binding, displaceable by ISO, increased up to 2-fold in dex-treated cells. This effect depended on protein synthesis and closely paralleled the extent and time course of the increase in adenylate cyclase stimulation. In dex-treated cells there was also an increase in the maximum velocity of guanyl-5'-yl imidodiphosphate-stimulated adenylate cyclase, a decrease in the lag time for guanyl-5'-yl imidodiphosphate enzyme activation in the presence of ISO from 3 to 1 min, increased stimulation of adenylate cyclase by cholera toxin, and increased labeling of 47,000 and 42,000 mol wt proteins by [32P]NAD in the presence of cholera toxin. [32P]NAD ribosylation in the presence of pertussis toxin resulted in the labeling of 40,000 mol wt protein, which was also increased by 20-50% in dex-treated cells. However, pertussis toxin treatment did not augment or reduce the effect on hormone stimulation, although it increased the cAMP response to PTH and (-)ISO. These findings suggest that dex increases (-)ISO stimulation of adenylate cyclase in ROS 17/2.8 cells by jointly increasing the number of hormone receptors and the abundance of Gs, the guanine nucleotide binding regulatory protein.
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PMID:Dexamethasone effects on beta-adrenergic receptors and adenylate cyclase regulatory proteins Gs and Gi in ROS 17/2.8 cells. 300 62

Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural and functional relationships of guanosine triphosphate binding proteins. 313 54


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