α2-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption

2001 ◽  
Vol 281 (4) ◽  
pp. F679-F686 ◽  
Author(s):  
Craig F. Plato ◽  
Jeffrey L. Garvin
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Chloride Transport ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
No Production ◽  
Thick Ascending Limb ◽  
Chloride Absorption ◽  
Ly 83583

Stimulation of α2-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses α2-receptors. We hypothesized that selective α2-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the α2-receptor agonist clonidine (10 nM) decreased chloride flux ( J Cl) from 119.5 ± 15.9 to 67.4 ± 13.8 pmol · mm−1 · min−1 (43% reduction; P < 0.02), whereas removal of clonidine from the bath increased J Cl by 20%. When NOS activity was inhibited by pretreatment with 5 mM N G-nitro-l-arginine methyl ester, the inhibitory effects of clonidine on THAL J Clwere prevented (81.7 ± 10.8 vs. 71.6 ± 6.9 pmol · mm−1 · min−1). Similarly, when the NOS substrate l-arginine was deleted from the bath, addition of clonidine did not decrease THAL J Cl from control (106.9 ± 11.6 vs. 132.2 ± 21.3 pmol · mm−1 · min−1). When we blocked the α2-receptors with rauwolscine (1 μM), we found that the inhibitory effect of 10 nM clonidine on THAL J Cl was abolished, verifying that α2, rather than I1, receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL J Cl, indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 μM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J Cl. In conclusion, α2-receptor stimulation decreases THAL J Cl by increasing NO release and stimulating guanylate cyclase. These data suggest that α2-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.

Endothelin inhibits thick ascending limb chloride flux via ETB receptor-mediated NO release

2000 ◽  
Vol 279 (2) ◽  
pp. F326-F333 ◽  
Author(s):  
Craig F. Plato ◽  
David M. Pollock ◽  
Jeffrey L. Garvin
Keyword(s):  
Intracellular Calcium ◽  
Chloride Transport ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Inhibitory Effects ◽  
Chloride Flux ◽  
Nacl Transport ◽  
No Release ◽  
Bq 610

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ETB receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux ( J Cl). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ETBreceptors and release of NO. Exogenous ET-1 (1 nM) decreased J Cl from 118.3 ± 15.0 to 62.7 ± 13.6 pmol · mm−1 · min−1 (48.3 ± 8.2% reduction), whereas removal of ET-1 increased J Cl in a separate group of tubules from 87.6 ± 10.7 to 115.2 ± 10.3 pmol · mm−1 · min−1 (34.5 ± 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J Cl, we examined the effect of inhibiting of NO synthase (NOS) with N G-nitro-l-arginine methyl ester (l-NAME) on ET-1-induced changes in J Cl. l-NAME (5 mM) completely prevented the ET-1-induced reduction in J Cl, whereas d-NAME did not. l-NAME alone had no effect on J Cl. These data suggest that the effects of ET-1 are mediated by NO. Blockade of ETBreceptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ETB receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J Cl (from 120.7 ± 12.6 to 75.4 ± 13.3 pmol · mm−1 · min−1). In contrast, ETA receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J Cl (from 96.5 ± 10.4 to 69.5 ± 8.6 pmol · mm−1 · min−1). Endothelin increased intracellular calcium from 96.9 ± 14.0 to 191.4 ± 11.9 nM, an increase of 110.8 ± 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J Cl by activating ETB receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.

scholarly journals The G-protein regulator LGN modulates the activity of the NO receptor soluble guanylate cyclase

2012 ◽  
Vol 446 (3) ◽  
pp. 445-453 ◽  
Author(s):  
Swati Chauhan ◽  
Filip Jelen ◽  
Iraida Sharina ◽  
Emil Martin
Keyword(s):  
G Protein ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Activated State ◽  
Ic50 Values

sGC (soluble guanylate cyclase) is the main mediator of NO signalling. Biochemical and physiological studies suggest that, besides NO, in vivo regulation of sGC involves direct interaction with other proteins. Using yeast two-hybrid screening, we identified that the multidomain LGN (Leu-Gly-Asn repeat-enriched protein) interacts with both α1 and β1 sGC subunits. LGN and sGC co-localized in the cell cytoplasm, and the LGN–sGC complex was co-immunoprecipitated from cells expressing both proteins and from native tissues. Their interaction requires the N-terminal tetratricopeptide repeats of LGN, but does not require the N-terminal portions of α1 or β1 sGC subunits. Overexpression of LGN decreases the activity of cellular sGC, whereas knockdown of LGN mRNA and protein correlated with increased sGC activity. Although purified LGN interacts directly with purified sGC, the inhibitory effect in vitro is observed only after supplementation of cell lysate to the reaction. Although resting sGC and sGC activated by the stimulator BAY41-2272 have very similar LGN-IC50 values to the NO-stimulated sGC, they have a much higher Hill coefficient, suggesting co-operative binding with respect to LGN in the low-activated state of sGC. AGS3 (activator of G-protein signalling 3), the closest LGN homologue, also inhibits sGC. The interaction of sGC with these scaffolding proteins may expand the cross-talk between NO/cGMP signalling and other cellular pathways and tailor sGC function to specific tissues or signals.

scholarly journals Endogenous flow-induced nitric oxide reduces superoxide-stimulated Na/H exchange activity via PKG in thick ascending limbs

2015 ◽  
Vol 308 (5) ◽  
pp. F444-F449 ◽  
Author(s):  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Dependent Protein Kinase ◽  
Two Factors ◽  
Endogenous Nitric Oxide ◽  
Dependent Protein ◽  
Acid Load ◽  
Luminal Flow ◽  
Ly 83583

Luminal flow stimulates endogenous nitric oxide (NO) and superoxide (O2−) production by renal thick ascending limbs (TALs). The delicate balance between these two factors regulates Na transport in TALs; NO enhances natriuresis, whereas O2− augments Na absorption. Endogenous, flow-stimulated O2− enhances Na/H exchange (NHE). Flow-stimulated NO reduces flow-induced O2−, a process mediated by cGMP-dependent protein kinase (PKG). However, whether flow-stimulated, endogenously-produced NO diminishes O2−-stimulated NHE activity and the signaling pathway involved are unknown. We hypothesized that flow-induced NO reduces the stimulation of NHE activity caused by flow-induced O2− via PKG in TALs. Intracellular pH recovery after an acid load was measured as an indicator of NHE activity in isolated, perfused rat TALs. l-Arginine, the NO synthase substrate, decreased NHE activity by 34 ± 5% ( n = 5; P < 0.04). The O2− scavenger tempol decreased NHE activity by 46 ± 8% ( n = 6; P < 0.004) in the absence of NO. In the presence of l-arginine, the inhibitory effect of tempol on NHE activity was reduced to −19 ± 6% ( n = 6; P < 0.03). The soluble guanylate cyclase inhibitor LY-83583 blocked the effect of l-arginine thus restoring tempol's effect on NHE activity to −42 ± 4% ( n = 6; P < 0.0005). The PKG inhibitor KT-5823 also inhibited l-arginine's effect on tempol-reduced NHE activity (−43 ± 5%; n = 5; P < 0.03). We conclude that flow-induced NO reduces the stimulatory effect of endogenous, flow-induced O2− on NHE activity in TALs via an increase in cGMP and PKG activation.

scholarly journals Acute stimulation of the soluble guanylate cyclase does not impact on left ventricular capacitance in normal and hypertrophied porcine hearts in vivo

2018 ◽  
Vol 315 (3) ◽  
pp. H669-H680 ◽  
Author(s):  
Alessio Alogna ◽  
Michael Schwarzl ◽  
Martin Manninger ◽  
Nazha Hamdani ◽  
Birgit Zirngast ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Diastolic Pressure ◽  
Aortic Pressure ◽  
Left Ventricular ◽  
Concentric Hypertrophy ◽  
Ventricular Compliance ◽  
Left Ventricular Compliance ◽  
Stimulation Of

Experimental data indicate that stimulation of the nitric oxide-soluble guanylate cyclase(sGC)-cGMP-PKG pathway can increase left ventricular (LV) capacitance via phosphorylation of the myofilamental protein titin. We aimed to test whether acute pharmacological sGC stimulation with BAY 41-8543 would increase LV capacitance via titin phosphorylation in healthy and deoxycorticosteroneacetate (DOCA)-induced hypertensive pigs. Nine healthy Landrace pigs and 7 pigs with DOCA-induced hypertension and LV concentric hypertrophy were acutely instrumented to measure LV end-diastolic pressure-volume relationships (EDPVRs) at baseline and during intravenous infusion of BAY 41-8543 (1 and 3 μg·kg−1·min−1 for 30 min, respectively). Separately, in seven healthy and six DOCA pigs, transmural LV biopsies were harvested from the beating heart to measure titin phosphorylation during BAY 41-8543 infusion. LV EDPVRs before and during BAY 41-8543 infusion were superimposable in both healthy and DOCA-treated pigs, whereas mean aortic pressure decreased by 20–30 mmHg in both groups. Myocardial titin phosphorylation was unchanged in healthy pigs, but total and site-specific (Pro-Glu-Val-Lys and N2-Bus domains) titin phosphorylation was increased in DOCA-treated pigs. Bicoronary nitroglycerin infusion in healthy pigs ( n = 5) induced a rightward shift of the LV EDPVR, demonstrating the responsiveness of the pathway in this model. Acute systemic sGC stimulation with the sGC stimulator BAY 41-8543 did not recruit an LV preload reserve in both healthy and hypertrophied LV porcine myocardium, although it increased titin phosphorylation in the latter group. Thus, increased titin phosphorylation is not indicative of increased in vivo LV capacitance. NEW & NOTEWORTHY We demonstrate that acute pharmacological stimulation of soluble guanylate cyclase does not increase left ventricular compliance in normal and hypertrophied porcine hearts. Effects of long-term soluble guanylate cyclase stimulation with oral compounds in disease conditions associated with lowered myocardial cGMP levels, i.e., heart failure with preserved ejection fraction, remain to be investigated.

Abstract 1415: Activation of Soluble Guanylate Cyclase Regulates Phosphodiesterase 5A Localization and Restores Anti-adrenergic Action of Sildenafil despite Nitric Oxide Synthase Inhibition

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Takahiro Nagayama ◽  
Manling Zhang ◽  
Eiki Takimoto ◽  
David A Kass
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Soluble Guanylate Cyclase ◽  
Synergistic Effects ◽  
Adrenergic Stimulation ◽  
Adrenergic Activity ◽  
Oxide Synthase

Background: We have shown that inhibition of cyclic GMP-phosphodiesterase 5A (PDE5A) by sildenafil (SIL) blunts cardiomyocyte β-adrenergic stimulation, but this effect depends on the activity of endothelial nitric oxide synthase (eNOS) to generate a specific pool of cyclic GMP. PDE5A normally localizes at Z-bands in myocytes, but localization is more diffuse in cells with eNOS chronically inhibited. Here, we tested whether the influence of eNOS on PDE5A localization and anti-adrenergic action depends upon cyclic GMP. Methods and Results: Mouse in vivo hemodynamics were assessed by pressure-volume analysis. Isoproterenol (ISO: 20 ng/kg/min, iv ) stimulated contractility was inhibited by SIL (100 μg/kg/min, iv ), however this did not occur in mice given N w -nitro-L-arginine methyl ester (L-NAME: 1 mg/mL in drinking water for 1 week) to inhibit NOS. Myocytes transfected with an adenoviral vector encoding a fusion protein (PDE5A-DSred) in vivo were subsequently isolated and examined for PDE5A/α-actinin localization. Normal cells showed strong co-localization, whereas L-NAME-treated cells had diffuse PDE5A distribution. If L-NAME was stopped for 1-wk washout, SIL regained anti-adrenergic activity, and PDE5A z-band localization was restored. If L-NAME was continued but combined with Bay 41– 8543 (BAY: 30 mg/kg/day, po ), a soluble guanylate cyclase (sGC) activator, both PDE5A localization and SIL anti-adrenergic action were also restored. Chronic L-NAME suppressed phosphorylation of vasodilator-stimulated protein (VASP), a marker of protein kinase G (PKG) activity, in hearts acutely exposed to ISO+SIL. After L-NAME washout or L-NAME+BAY, VASP phosphorylation with ISO+SIL was restored. Conclusion: NOS-dependent modulation of both PDE5A sarcomere localization and anti-adrenergic activity depends upon sGC-derived cyclic GMP, and is linked to PKG activation. This suggests sGC activators may have synergistic effects with PDE5A inhibitors.

In Vitro and In Vivo Anti-Inflammatory Activity of the Rhizomes of Globba pendula Roxb

2021 ◽  
Vol 16 (10) ◽  
pp. 1934578X2110559
Author(s):  
Le Minh Ha ◽  
Ngo Thi Phuong ◽  
Nguyen Thi Thu Hien ◽  
Pham Thi Tam ◽  
Do Thi Thao ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Ethyl Acetate Extract ◽  
Inhibitory Effect ◽  
Potential Effect ◽  
Inflammatory Activity ◽  
No Production ◽  
Anti Inflammatory ◽  
Inflammatory Effect

In this study, we aimed at evaluating in vitro and in vivo anti-inflammatory activity of various extracts of the rhizomes of Globba pendula Roxb. Three extracts ( n-hexane, ethyl acetate, and water) were screened for their inhibitory effect on NO production by lipopolysaccharide-stimulated RAW 264.7 macrophages. The ethyl acetate extract of G. pendula rhizomes (EGP) showed a potential effect with an IC50 value of 32.45 µg/mL. For in vivo study, the ethyl acetate extract was further investigated for its anti-inflammatory effect using collagen antibody-induced arthritic mice (CAIA). The level of arthritis in experimental mice significantly reduced ( P < .05) after treatment with EGP at a dose of 500 mg/kg body weight (b.w.). This study also revealed that EGP is orally non-toxic. Ethyl p-methoxy cinamate was identified as the main constituent of EGP, which may result in its anti-inflammatory effect.

scholarly journals A theoretical model of nitric oxide transport in arterioles: frequency- vs. amplitude-dependent control of cGMP formation

2004 ◽  
Vol 286 (3) ◽  
pp. H1043-H1056 ◽  
Author(s):  
Nikolaos M. Tsoukias ◽  
Mahendra Kavdia ◽  
Aleksander S. Popel
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Soluble Guanylate Cyclase ◽  
Muscle Tone ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
No Production ◽  
Cgmp Formation

Nitric oxide (NO) plays many important physiological roles, including the regulation of vascular smooth muscle tone. In response to hemodynamic or agonist stimuli, endothelial cells produce NO, which can diffuse to smooth muscle where it activates soluble guanylate cyclase (sGC), leading to cGMP formation and smooth muscle relaxation. The close proximity of red blood cells suggests, however, that a significant amount of NO released will be scavenged by blood, and thus the issue of bioavailability of endothelium-derived NO to smooth muscle has been investigated experimentally and theoretically. We formulated a mathematical model for NO transport in an arteriole to test the hypothesis that transient, burst-like NO production can facilitate efficient NO delivery to smooth muscle and reduce NO scavenging by blood. The model simulations predict that 1) the endothelium can maintain a physiologically significant amount of NO in smooth muscle despite the presence of NO scavengers such as hemoglobin and myoglobin; 2) under certain conditions, transient NO release presents a more efficient way for activating sGC and it can increase cGMP formation severalfold; and 3) frequency-rather than amplitude-dependent control of cGMP formation is possible. This suggests that it is the frequency of NO bursts and perhaps the frequency of Ca2+ oscillations in endothelial cells that may limit cGMP formation and regulate vascular tone. The proposed hypothesis suggests a new functional role for Ca2+ oscillations in endothelial cells. Further experimentation is needed to test whether and under what conditions in silico predictions occur in vivo.

scholarly journals Angiotensin II inhibits NaCl absorption in the rat medullary thick ascending limb

2004 ◽  
Vol 287 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
Nicolas Lerolle ◽  
Soline Bourgeois ◽  
Françoise Leviel ◽  
Gaëtan Lebrun ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Plasma Membranes ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Experimental Conditions ◽  
Nacl Absorption ◽  
Medullary Thick Ascending Limb

NaCl reabsorption in the medullary thick ascending limb of Henle (MTALH) contributes to NaCl balance and is also responsible for the creation of medullary interstitial hypertonicity. Despite the presence of angiotensin II subtype 1 (AT1) receptors in both the luminal and the basolateral plasma membranes of MTALH cells, no information is available on the effect of angiotensin II on NaCl reabsorption in MTALH and, furthermore, on angiotensin II-dependent medullary interstitial osmolality. MTALHs from male Sprague-Dawley rats were isolated and microperfused in vitro; transepithelial net chloride absorption ( JCl) as well as transepithelial voltage ( Vte) were measured. Luminal or peritubular 10−11 and 10−10 M angiotensin II had no effect on JCl or Vte. However, 10−8 M luminal or peritubular angiotensin II reversibly decreased both JCl and Vte. The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10−6 M). By contrast, PD-23319, an AT2-receptor antagonist, did not alter the inhibitory effect of 10−8 M angiotensin II. Finally, no additive effect of luminal and peritubular angiotensin II was observed. We conclude that both luminal and peritubular angiotensin II inhibit NaCl absorption in the MTALH via AT1 receptors. Because of intrarenal angiotensin II synthesis, angiotensin II concentration in medullary tubular and interstitial fluids may be similar in vivo to the concentration that displays an inhibitory effect on NaCl reabsorption under the present experimental conditions.

Coupling of M2 muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

2000 ◽  
Vol 278 (3) ◽  
pp. G429-G437 ◽  
Author(s):  
Amy K. Cook ◽  
Michael Carty ◽  
Cherie A. Singer ◽  
Ilia A. Yamboliev ◽  
William T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Muscarinic Receptors ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Subsequent Treatment ◽  
P38 Map Kinase

Coupling of M2 and M3 muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M3 receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser789), a putative downstream target of MAP kinases. Alkylation of M3 receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 μM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M2 receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M2receptor activation in smooth muscle.

Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes

1992 ◽  
Vol 262 (4) ◽  
pp. C1077-C1082 ◽  
Author(s):  
T. R. Billiar ◽  
R. D. Curran ◽  
B. G. Harbrecht ◽  
J. Stadler ◽  
D. L. Williams ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrogen Oxide ◽  
Soluble Guanylate Cyclase ◽  
Cell Damage ◽  
Interleukin 1 ◽  
Isolated Hepatocytes ◽  
No Production ◽  
Time Period ◽  
Extracellular Release

Hepatocytes are known to synthesize nitric oxide (NO) from L-arginine via an inducible NO synthase. Studies were performed to determine the relationship between hepatocyte NO production and the stimulation of hepatocyte soluble guanylate cyclase. A combination of lipopolysaccharide (LPS), interferon-gamma, tumor necrosis factor, and interleukin-1 stimulates the biosynthesis of large quantities of nitrite and nitrate (NO2- + NO3-). Hepatocyte NO2- + NO3- production was associated with only small increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels but much greater increases in extracellular cGMP release over an 18-h time period. This cGMP synthesis was dependent on the L-arginine concentration and was inhibited in a reversible manner by NG-monomethyl-L-arginine. The cytokines or LPS added alone induced small increases in nitrogen oxide production and concomitant minor elevations in cGMP release. Atrial natriuretic peptide also stimulated the release of cGMP by hepatocytes which appeared to be independent of the cytokine+LPS-induced cGMP release. The addition of probenecid reduced the cGMP release by 66%, while cell damage was excluded as a cause for the extracellular release. Addition of 3-isobutyl-1-methylxanthine, but not M&B 22948, increased hepatocyte intra- and extracellular cGMP levels after cytokine+LPS stimulation. Induction of nitrogen oxide synthesis by hepatocytes in vivo by injecting rats with killed Corynebacterium parvum resulted in increased cGMP levels in freshly isolated hepatocytes and increased cGMP release by the hepatocytes when placed in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

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