17 research outputs found
Efficacy as an Intrinsic Property of the M<sub>2</sub> Muscarinic Receptor in Its Tetrameric State
Muscarinic and other G protein-coupled
receptors exhibit an agonist-specific
heterogeneity that tracks efficacy and commonly is attributed to an
effect of the G protein on an otherwise homogeneous population of
sites. To examine this notion, M<sub>2</sub> muscarinic receptors
were purified from <i>Sf</i>9 cells as monomers devoid of
G protein and reconstituted as tetramers in phospholipid vesicles.
In assays with <i>N</i>-[<sup>3</sup>H]Âmethylscopolamine,
seven agonists revealed a dispersion of affinities indicative of two
or more classes of sites. Unlabeled <i>N</i>-methylscopolamine
and the antagonist quinuclidinylbenzilate recognized one class of
sites; atropine recognized two classes with a preference that was
the opposite of that of agonists, as indicated by the effects of <i>N</i>-ethylmaleimide. The data were inconsistent with an explicit
model of constitutive asymmetry within a tetramer, and the fit improved
markedly upon the introduction of cooperative interactions (<i>P</i> < 0.00001). Purified monomers appeared to be homogeneous
or nearly so to all ligands except the partial agonists pilocarpine
and McN-A-343, where heterogeneity emerged from intramolecular cooperativity
between the orthosteric site and an allosteric site. The breadth of
each dispersion was quantified empirically as the area between the
fitted curve for two classes of sites and the theoretical curve for
a single class of lower affinity, which approximates the expected
effect of GTP if a G protein were present. The areas measured for
10 ligands at reconstituted tetramers correlated with similar measures
of heterogeneity and with intrinsic activities reported previously
for binding and response in natural membranes (<i>P</i> ≤
0.00002). The data suggest that the GTP-sensitive heterogeneity typically
revealed by agonists at M<sub>2</sub> receptors is intrinsic to the
receptor in its tetrameric state. It exists independently of the G
protein, and it appears to arise at least in part from cooperativity
between linked orthosteric sites
Heterotropic Cooperativity within and between Protomers of an Oligomeric M<sub>2</sub> Muscarinic Receptor
At least four allosteric sites have been found to mediate
the dose-dependent
effects of gallamine on the binding of [<sup>3</sup>H]Âquinuclidinylbenzilate
(QNB) and <i>N</i>-[<sup>3</sup>H]Âmethylscopolamine (NMS)
to M<sub>2</sub> muscarinic receptors in membranes and solubilized
preparations from porcine atria, CHO cells, and <i>Sf</i>9 cells. The rate of dissociation of [<sup>3</sup>H]ÂQNB was affected
in a bell-shaped manner with at least one Hill coefficient (<i>n</i><sub>H</sub>) greater than 1, indicating that at least
three allosteric sites are involved. The level of binding of [<sup>3</sup>H]ÂQNB was decreased in a biphasic manner, revealing at least
two allosteric sites; binding of [<sup>3</sup>H]ÂNMS was affected in
a triphasic, serpentine manner, revealing at least three sites, and
values of <i>n</i><sub>H</sub> >1 pointed to at least
four
sites. Several lines of evidence indicate that all effects of gallamine
were allosteric in nature and could be observed at equilibrium. The
rates of equilibration and dissociation suggest that the receptor
was predominately oligomeric, and the heterogeneity revealed by gallamine
can be attributed to differences in its affinity for the constituent
protomers of a tetramer. Those differences appear to arise from inter-
and intramolecular cooperativity between gallamine and the radioligand
MCs migrate towards CCL2 and CCL5 and are recruited to E7 ear skin.
<p>(<b>A</b>) 5 µm transwell assays were used to determine the mast cells chemotaxis towards chemokines. Graphs represent the number of migrated FcεRIα<sup>+</sup> cKit<sup>+</sup> BMCMCs towards C57 skin explant supernatants (left) or E7 explant supernatants (right) in the presence of anti-CCL2 and/or anti-CCL5 blocking antibodies used at 10 µg/ml. 3 independent experiments (<b>B,C</b>) <i>Kit</i><sup>W-sh/W-sh</sup> (Wsh) (n = 3) and E7.<i>Kit</i><sup>W-sh/W-sh</sup> (E7.Wsh) (n = 3) mice were reconstituted with 1.4×10<sup>7</sup> BMCMCs <i>i.v</i>. or left untreated (E7.Wsh (n = 3) and Wsh (n = 2)). 12 weeks later, mice were culled and MCs were identified in ear skin by (<b>B</b>) toluidine blue staining (<b>top</b>, scale bar  = 50 µm and <b>bottom</b>, scale bar  = 20 µm) and expressed as (<b>C</b>) MC number per mm cartilage length. *p<0.01, by unpaired t-test for all indicated comparisons.</p
MCs are immunosuppressive in E7 environment.
<p>(A) C57 mice were double grafted with C57 syngeneic ear skin (n = 9), and either <i>Kit</i><sup>W-sh/W-sh</sup> (Wsh) ear skin (n = 9) or E7. <i>Kit</i><sup>W-sh/W-sh</sup> (E7.Wsh) ear skin (n = 9). Control E7 ear skin (n = 6) are tolerated. Kaplan-Meier survival curves show the median graft survival. ****p<0.0001 using a log-rank test. Data are combined from 3 independent experiments. (<b>B</b>) Representative graft histology of C57, Wsh and one remaining E7.Wsh graft at day 90 by toluidine blue staining (scale bar  = 100 µm). (<b>C</b>) MCs per mm of graft tissue at day 28 in C57 (n = 4), Wsh non-rejected (n = 4) and E7.Wsh rejected (n = 3) grafts. (**p<0.01, by unpaired t-test).</p
MC infiltration in ear skin is dependent on E7 hyperplasia.
<p>E7.Rb<sup>mut</sup> ear skin is not hyperplasic and contains normal MC numbers. (A) Representative images of E7.Rb<sup>wt</sup> and E7.Rb<sup>mut</sup> ear skin by toluidine blue stain (original magnification ×400, scale  = 50 µm). (B) MC number per mm cartilage length (n = 3 in each group). ***p<0.001 by unpaired t-test for indicated comparison. Rb<sup>wt</sup> are same as C57.</p
Comparison of Arginase and Asparaginase supplementation on the survival of canine lymphoid cells.
<p>Canine lymphoid cells (A–E), or MAPC (F) were cultured in normal RPMI for 3 days with or without arginase or asparaginase supplementation as indicated below. Enzymes were quenched on day 3 as indicated by the arrows. Cell viability was determined by ATP quantification. Blue diamonds = normal RPMI, red triangles = +0.3 U/ml Asparaginase, green triangles = +1 U/ml Asparaginase, purple triangles = +10 U/ml Asparaginase, Orange circles = +10 U/ml Arginase. Graphs show a representative experiment from three independent experiments with similar results. Error bars represent SD.</p
Arginase activity is cell-associated.
<p>GL-1 cells were cultured for 24 hours in arginine-free (AF) or normal RPMI media plus/minus 10 U/ml Arginase. Arginase activity was then measured in filtered supernatant (A, B) or cell lysate (C, D) using a QuantiChrom Arginase Assay kit. Graphs show a representative experiment from three independent experiments with similar results. Error bars represent SD.</p
Sensitivity of canine lymphoid cells and osteosarcomas to prolonged arginine-deprivation.
<p>Canine lymphoid cells and osteosarcomas (A–L, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054464#pone-0054464-t001" target="_blank">Table 1</a>) were cultured in Arginine-free media for 6 days with the amino acids indicated below. Arginine was reintroduced into cultures on day 6 as indicated by the arrows. Cell viability was determined by ATP quantification. Blue squares = +1 mM Arginine, Red diamonds = Arginine-free, Green triangles = +1 mM Citruline, Purple crosses = +1 mM Ornithine. Graphs show a representative experiment from three independent experiments with similar results. Error bars represent SD.</p
Arginase supplementation can prevent recovery of canine lymphoid cells and osteosarcomas subjected to arginine-deprivation.
<p>Canine lymphoid cells and osteosarcomas (A–H), and MAPC (I) were cultured in Arginine-free media for 3 days with or without arginase supplementation as indicated below. Arginase was removed and arginine reintroduced into cultures on day 3 (A–E), day 6 (F, G), day 8 (H), or day 9 (I) as indicated by the arrows. Cell viability was determined by ATP quantification. Blue squares = +1 mM Arginine, Red diamonds = Arginine-free, Blue crosses = +1 U/ml Arginase, Orange circles = +10 U/ml Arginase. Graphs show a representative experiment from three independent experiments with similar results. Error bars represent SD.</p