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Title: Multiscale Spatio-Mechanical Regulation of Eph Receptors.

Abstract

Abstract not provided.

Authors:
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
USDOD
OSTI Identifier:
1375568
Report Number(s):
SAND2016-7753C
646512
DOE Contract Number:
AC04-94AL85000
Resource Type:
Conference
Resource Relation:
Conference: Proposed for presentation at the MechBio Symposium held August 4-5, 2016 in San Diego, CA, US.
Country of Publication:
United States
Language:
English

Citation Formats

Greene, Adrienne Celeste. Multiscale Spatio-Mechanical Regulation of Eph Receptors.. United States: N. p., 2016. Web.
Greene, Adrienne Celeste. Multiscale Spatio-Mechanical Regulation of Eph Receptors.. United States.
Greene, Adrienne Celeste. Mon . "Multiscale Spatio-Mechanical Regulation of Eph Receptors.". United States. doi:. https://www.osti.gov/servlets/purl/1375568.
@article{osti_1375568,
title = {Multiscale Spatio-Mechanical Regulation of Eph Receptors.},
author = {Greene, Adrienne Celeste},
abstractNote = {Abstract not provided.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Aug 01 00:00:00 EDT 2016},
month = {Mon Aug 01 00:00:00 EDT 2016}
}

Conference:
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  • The authors have studied the regulation of the coupling of alpha/sub 1/ receptor binding sites to inositide hydrolysis in primary neuronal cultures prepared from the brain stem and hypothalamus of neonatal Wistar-Kyoto rats. Cultures were exposed to norepinephrine (NE) and the NE stimulated phosphatidylinositol (PI) response or binding of (/sup 125/I)HEAT to alpha/sub 1/ recognition sites was determined. Exposure of neurons to 10 ..mu..M NE for 2 hr decreases stimulated PI hydrolysis by 70-80%, whereas alpha/sub 1/ receptor number and affinity are unchanged at this time. Further stimulation of the cultures results in a down-regulation of receptor number with amore » maximal loss of 50% binding sites after a 24 hr pretreatment. The uncoupled NE stimulated PI response after a 2 hr pretreatment with NE returns to 80-90% of control 4 hr after removal of the NE. Down-regulated alpha/sub 1/receptor binding sites after 24 hr of pretreatment with NE return after a 72 hr washout period, and the effect is blocked by cycloheximide (3.6 ..mu..M). These results suggest that stimulation of alpha/sub 1/ receptors causes a rapid uncoupling of the receptor from PI hydrolysis and that the response returns to normal in a matter of hours. In contrast, the return of receptors following down-regulation requires much longer times and appears to involve other mechanisms.« less
  • The authors have examined the effect of guanine nucleotides on the interaction of adrenergic agents with ..cap alpha../sup 1/-adrenergic receptors of two cell lines, the Madin-Darby Canine Kidney (MDCK) and BC3H-1 muscle cells. While gaunylylimidodiphosphoate (Gpp(NH)p) had no effect on the affinity or the total number of (/sup -3/H)prazosin binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonist epinephrine in competing for (/sup 3/H)prazosin binding sites in both cell types. The EC/sub 50/ of Gpp(NH)p was approx.100 ..mu..M, and a maximal effect was seen at 500 ..mu..M. In contrast, 100 ..mu..M Gpp(NH)pmore » yielding maximal shifts in binding of epinephrine to ..beta..-adrenergic receptors in BC3H-1 cell membranes. Guanine nucleotides were significantly more effective than adenine nucleotides in shifting agonist affinity for the ..cap alpha../sub 1/-receptor and Mg/sup + +/ was required to observe a maximal effect. ..cap alpha../sub 1/-receptor agonists activated phosphatidylinositol (PI) hydrolysis in both cell types, but have no direct effect on membrane adenylate cyclase activity. In intact BC3H-1 cells, ..cap alpha../sub 1/-agonists inhibited ..beta..-adrenergic cAMP production, an effect which appears in preliminary studies not to result from enhanced phosphodieterase activity. These results show that agonist binding to ..cap alpha../sup 1/-adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides. This regulation and inturn transmembrane signalling (PI hydrolysis) by these receptors appear to involve a guanine nucleotide binding (G) protein, which may be different than G/sub s/ and G/sub i/.« less
  • Activation of protein kinase C (PKC) with suboptimal does of phorbol myristyl acetate (PMA) will increase fMP receptor expression with parallel potentiation of superoxide generation. PMA-induced changes in leukotriene B{sub 4} (LTB{sub 4}) receptor expression were assessed in parallel with fMP receptor expression to determine if these two independent receptor classes are regulated in a similar manner by PKC. The relative density of fMP receptors was assessed by flow cytometry. The relative density of receptors for LTB{sub 4} was quantitated by incubating 2 {times} 10{sup 6} Ns with 10nM({sup 3}H)-LTB{sub 4} and determining the amount of radioactivity bound after filtrationmore » on glass fiber filters. Incubation of N with 10ng/mL PMA induced a 3.2-fold increase in fMP receptor expression by 5 min which was sustained for up to 15 min. In contrast, LTB{sub 4} receptor density decreased by 36% within 5 min. in response to 10 ng/mL PMA. Staurosporine, a potent antagonist of PKC, had no effect of fMP receptor expression but markedly enhanced LTB{sub 4} receptor expression by 1.7-fold at 200nM. PKC acts to decrease the surface expression of LTB{sub 4} receptors in contrast to the enhancement of fMP receptor expression, suggesting in contrast to the enhancement of fMP receptor expression, suggesting that potentiation of N function by PMA may be stimulus-specific.« less
  • Pulmonary surfactant, of which phosphatidylcholine (PC) is a major component, is produced by the type II pneumocyte. PC secretion in type II cells is stimulated by beta-agonists, leukotrienes and agents that activate protein kinase C. Since the influence of purinergic agonists on surfactant secretion has not been reported previously they examined the effects of adenosine and its phosphorylated derivatives on PC secretion in type II cells. The cells were isolated from adult rats and cultured for 20 h in medium containing serum, antibiotics and /sup 3/H-choline. They were then transferred to fresh serum-free medium and incubated with the test agentsmore » for 90 min after which /sup 3/H-PC in the cells and medium was measured. Adenosine, AMP, ADP and ATP all stimulated PC secretion. The effects were dose-dependent in the range 10/sup -8/ to 10/sup -3/ M. At 10/sup -3/M, adenosine stimulated secretion by 140% from a basal rate of 0.91 +/- 0.06 (/sup 3/H-PC in the medium as % of that in cells + medium) to 2.19 +/- 0.26 (n = 6, P < 0.005, paired t test), AMP stimulated by 140% from 0.87 +/- 0.07 to 2.09 +/- 0.09 (n = 4, P < 0.002), ADP by 570% from 0.84 +/- 0.05 to 5.65 +/- 0.34 (n = 4, P < 0.002) and ATP by 410% from 0.84 +/- 0.04 to 4.32 +/- 0.35 (n = 5, P < 0.001). The EC/sub 50/ values for adenosine, AMP, ADP and TP were 3.0, 0.6, 20.0 and 5.0 x 10/sup -6/ M, respectively. None of the agents increased release of lactate dehydrogenase or PC synthesis as determined by choline incorporation. The effects are therefore likely to be directly on secretion rather than secondary to PC synthesis or cell injury.« less
  • The purpose of these experiments was to characterize Ca/sup 2 +/ regulation of phorbol dibutyrate (PDBu) binding to both protein kinase C (PKC) and a previously undescribed PDBu receptor. PKC from rabbit brain cytosol was prepared by anion exchange chromatography. Subsequent chromatography on hydroxylapatite revealed 2 peaks of PDBu binding. The 2nd peak eluted with PKC with approximately 190 mM phosphate; the first peak eluted with an independent kinase activity (OINK) with approximately 100 mM phosphate. Scatchard analysis of binding in the presence of excess EGTA or Ca/sup 2 +/ showed that Ca/sup 2 +/ increased PDBu receptor affinity ofmore » the PKC peak from Kd = 46 +/- 10 nM to 1.2 +/- 0.4 nM (all values are means +/- S.E., n = 4). The total amount bound in the presence of excess EGTA was slightly greater (126 +/- 11%). The effect of Ca/sup 2 +/ on OINK affinity was smaller with Kd = 4.4 +/- 0.8 nM in EGTA vs 1.8 +/- 0.6 nM in Ca/sup 2 +/. Binding capacity was increased in the presence of Ca/sup 2 +/ by 127 +/- 21%. The amount of Ca/sup 2 +/ required for these effects on both receptors was in the range of an EGTA:Ca/sup 2 +/ ratio of 1.5. These results may help to define the relationship between PDBu receptor occupancy and cellular responses to phorbol esters, including activation of PKC.« less