Histamine and inositol phosphate accumulation in endothelium: cAMP and a G protein

1989 ◽  
Vol 257 (4) ◽  
pp. L259-L264 ◽  
Author(s):  
M. R. Carson ◽  
S. S. Shasby ◽  
D. M. Shasby
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
G Protein ◽  
Inositol Phosphate ◽  
Umbilical Vein ◽  
Microvascular Permeability ◽  
Alpha Toxin ◽  
H1 Receptor ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Histamine increases microvascular permeability through a calcium-dependent process, and histamine occupancy of the H1-receptor increases calcium in cultured endothelial cells. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) in endothelial cells prevent the in vivo increase in microvascular permeability that follows histamine exposure. In the current experiments, histamine occupancy of the H1-receptor increased the flux of albumin across monolayers of cultured human umbilical vein endothelial cells (HUVEC). This was prevented by pretreating the cells with theophylline, forskolin, and 8-bromo-cAMP (BrcAMP), which also decreased the flux of albumin across control monolayers. Exposing the cells to histamine increased inositol phosphate accumulation in the cells, and this was prevented by the H1-antagonist pyrilamine but not by theophylline, forskolin, and BrcAMP. Exposing the cells to histamine increased intracellular calcium measured with fura-2. The increase in cell calcium was prevented by pyrilamine but not by pretreatment with theophylline, forskolin, and BrcAMP. When endogenous cell GTP was depleted by permeabilizing the membranes of the endothelial cells with Staphylococcus aureus alpha-toxin, histamine-stimulated inositol phosphate accumulation was enhanced with addition of GTP but not with addition of GDP to the buffer. Addition of GTP alone to the buffer did not increase inositol phosphate accumulation in alpha-toxin-treated cells. Histamine stimulates inositol phosphate accumulation in HUVEC via a G protein. Inhibition of the edemagenic effects of histamine by cAMP does not occur by interrupting this signal transduction pathway between the binding of histamine to its receptor and the increase in intracellular calcium.

Thrombin Stimulates Inositol Phosphate Accumulation and Prostacyclin Synthesis in Human Endothelial Cells from Umbilical Vein but Not from Omentum

1989 ◽  
Vol 61 (01) ◽  
pp. 122-126 ◽  
Author(s):  
A J Carter ◽  
W G Eisert ◽  
T H Müller
Keyword(s):  
Endothelial Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Human Endothelial Cells ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Microvascular Endothelial ◽  
Prostacyclin Synthesis

SummaryWe have compared the effects of thrombin on the accumulation of inositol phosphates and the synthesis of prostacyclin in cultured human endothelial cells from umbilical vein and the microvasculature of omentum. Active human thrombin induced a dose-dependent accumulation of inositol phosphates and a concomitant synthesis of prostacyclin in endothelial cells from human umbilical vein. However, thrombin at all concentrations tested was unable to stimulate inositol phosphate accumulation and prostacyclin synthesis in microvascular endothelial cells from human omentum. Bradykinin was able to stimulate these effects in both types of cell. These results demonstrate that although inositol phosphate turnover is an initial event associated with prostacyclin synthesis in endothelial cells, there are differences in the way microvascular endothelial cells respond to thrombin.

G protein in stimulation of PI hydrolysis by CCK in isolated rat pancreatic acinar cells

1988 ◽  
Vol 255 (5) ◽  
pp. E652-E659 ◽  
Author(s):  
T. Matozaki ◽  
C. Sakamoto ◽  
M. Nagao ◽  
H. Nishizaki ◽  
S. Baba
Keyword(s):  
G Protein ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Secretory Rate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Phosphate Accumulation ◽  
Maximal Stimulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To clarify the possible role of a guanine nucleotide-binding protein (G protein) in the signal transducing system activated by cholecystokinin (CCK), actions of CCK on rat pancreatic acini were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. When acini were incubated with increasing concentrations of either CCK-octapeptide (CCK8) or NaF, a maximal stimulation of amylase release from acini occurred at 100 pM CCK8 or 10 mM NaF, respectively; this secretory rate decreased as CCK8 or NaF concentration was increased. NaF caused an increased in cytoplasmic Ca2+ concentration from the internal Ca2+ store and stimulated accumulation of inositol phosphates in acini, as observed with CCK. However, NaF-stimulated Ca2+ mobilization had a lag period before detectable stimulation and was potentiated by AlCl3. These stimulatory effects of NaF appeared to be independent of cellular adenosine 3',5'-cyclic monophosphate (cAMP). Pretreatment with cholera toxin or pertussis toxin did not affect CCK8- or NaF-induced inositol phosphate accumulation or Ca2+ mobilization. 5'-Guanylimidodiphosphate activated the generation of inositol phosphates in the [3H]inositol-labeled pancreatic acinar cell membrane preparation, with half-maximal and maximal stimulation at 1 and 10 microM, respectively. Furthermore, the effects of submaximal CCK concentrations on inositol phosphate accumulation in membranes were markedly potentiated in the presence of 100 microM GTP, which alone was ineffective. Combined findings of the present study strongly suggest that pancreatic CCK receptors are probably coupled to the activation of polyphosphoinositide (PI) breakdown by a G protein, which appears to be fluoride sensitive but is other than Gs- or Gi-like protein.

scholarly journals A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

2004 ◽  
Vol 9 (4) ◽  
pp. 343-353 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Sarah L. Haftl ◽  
Dimitris N. Xanthos ◽  
Gregg Crumley ◽  
Mohamed Hachicha ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Column Chromatography ◽  
Large Volume ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Assay Method ◽  
Signal To Noise ◽  
Chromatography Method ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

scholarly journals Beryllium competitively inhibits brain myo-inositol monophosphatase, but unlike lithium does not enhance agonist-induced inositol phosphate accumulation

1993 ◽  
Vol 291 (2) ◽  
pp. 369-374 ◽  
Author(s):  
W S Faraci ◽  
S H Zorn ◽  
A V Bakker ◽  
E Jackson ◽  
K Pratt
Keyword(s):  
Rat Brain ◽  
Inositol Phosphate ◽  
Bipolar Depression ◽  
Neuroblastoma Cell ◽  
Brain Slices ◽  
Human Neuroblastoma Cell ◽  
Inositol Monophosphatase ◽  
Human Neuroblastoma ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Despite limiting side-effects, lithium is the drug of choice for the treatment of bipolar depression. Its action may be due, in part, to its ability to dampen phosphatidylinositol turnover by inhibiting myo-inositol monophosphatase. Beryllium has been identified as a potent inhibitor of partially purified myo-inositol monophosphatase isolated from rat brain (Ki = 150 nM), bovine brain (Ki = 35 nM), and from the human neuroblastoma cell line SK-N-SH (Ki = 85 nM). It is over three orders of magnitude more potent than LiCl (Ki = 0.5-1.2 mM). Kinetic analysis reveals that beryllium is a competitive inhibitor of myo-inositol monophosphatase, in contrast with lithium which is an uncompetitive inhibitor. Inhibition of exogenous [3H]inositol phosphate hydrolysis by beryllium (IC50 = 250-300 nM) was observed to the same maximal extent as that seen with lithium in permeabilized SK-N-SH cells, reflecting inhibition of cellular myo-inositol monophosphatase. However, in contrast with that observed with lithium, agonist-induced accumulation of inositol phosphate was not observed with beryllium in permeabilized and non-permeabilized SK-N-SH cells and in rat brain slices. Similar results were obtained in permeabilized SK-N-SH cells when GTP-gamma-S was used as an alternative stimulator of inositol phosphate accumulation. The disparity in the actions of beryllium and lithium suggest that either (1) selective inhibition of myo-inositol monophosphatase does not completely explain the action of lithium on the phosphatidylinositol cycle, or (2) that uncompetitive inhibition of myo-inositol monophosphatase is a necessary requirement to observe functional lithium mimetic activity.

scholarly journals Functional analysis of human cytomegalovirus pUS28 mutants in infected cells

2008 ◽  
Vol 89 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Melissa P. M. Stropes ◽  
William E. Miller
Keyword(s):  
G Protein ◽  
Human Cytomegalovirus ◽  
Inositol Phosphate ◽  
Biological Effects ◽  
Artificial Chromosome ◽  
Binding Domain ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
Inositol Phosphate Accumulation ◽  
Infected Cells

The human cytomegalovirus (HCMV)-encoded viral G protein-coupled receptor pUS28 contributes to an array of biological effects, including cell migration and proliferation. Using FIX-BAC (bacterial artificial chromosome, derived from the HCMV clinical isolate VR1814) and lambda red recombination techniques, we generated HCMV recombinants expressing amino-terminally FLAG-tagged versions of wild-type pUS28 (FLAG–US28/WT), G-protein coupling deficient pUS28 (FLAG–US28/R129A) and chemokine-binding domain deficient pUS28 (FLAG–US28/ΔN). Infection with the FLAG–US28/R129A virus failed to induce inositol phosphate accumulation, indicating that G-protein coupling is essential for pUS28 signalling to phospholipase C-β (PLC-β) during HCMV infection. The FLAG–US28/ΔN virus induced about 80 % of the level of PLC-β signalling induced by the FLAG–US28/WT virus, demonstrating that the N-terminal chemokine-binding domain is not required for pUS28-induced PLC-β signalling in infected cells. The data presented here are the first to describe the functional analyses of several key pUS28 mutants in HCMV-infected cells. Elucidating the mechanisms by which pUS28 signals during infection will provide important insights into HCMV pathogenesis.

Mechanisms underlying AlCl3 inhibition of agonist-stimulated inositol phosphate accumulation

1994 ◽  
Vol 47 (8) ◽  
pp. 1417-1425 ◽  
Author(s):  
Timothy J. Shafer ◽  
Amy C. Nostrandt ◽  
Hugh A. Tilson ◽  
William R. Mundy
Keyword(s):  
Inositol Phosphate ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation
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