scholarly journals Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

2012 ◽  
Vol 443 (1) ◽  
pp. 249-258 ◽  
Author(s):  
Katherine J. Massey ◽  
Quanwen Li ◽  
Noreen F. Rossi ◽  
Raymond R. Mattingly ◽  
Douglas R. Yingst
Keyword(s):  
Angiotensin Ii ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Second Phase ◽  
Ang Ii ◽  
Wild Type ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Two Phases ◽  
At1a Receptor

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.

scholarly journals Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells

2016 ◽  
Vol 310 (3) ◽  
pp. C227-C232 ◽  
Author(s):  
Katherine J. Massey ◽  
Quanwen Li ◽  
Noreen F. Rossi ◽  
Susan M. Keezer ◽  
Raymond R. Mattingly ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cells ◽  
Ang Ii ◽  
Wild Type ◽  
Stimulate Activity ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Stimulation Of

How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser938 were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K·mg protein−1·min−1 and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser938 is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.

scholarly journals Angiotensin II stimulates elution of Na-K-ATPase from a digoxin-affinity column by increasing the kinetic response to ligands that trigger the decay of E2-P

2008 ◽  
Vol 294 (4) ◽  
pp. F990-F1000 ◽  
Author(s):  
Douglas R. Yingst ◽  
Tabitha M. Doci ◽  
Katherine J. Massey ◽  
Noreen F. Rossi ◽  
Ebony Rucker ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Rat Kidney ◽  
Affinity Column ◽  
Kinetic Properties ◽  
Ang Ii ◽  
Wild Type ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Truncation Mutant ◽  
Kidney Microsomes

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E2-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT1a receptor and either the wild-type rat α1-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH2 terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E2-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat α1-subunit by increasing the kinetic response to ligands that cause a decay of E2-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH2 terminus.

scholarly journals Ascospore Formation in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 69 (4) ◽  
pp. 565-584 ◽  
Author(s):  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Diploid Cell ◽  
Spore Wall ◽  
Second Phase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Membrane Compartments ◽  
Two Phases

SUMMARY Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.

Modulation of Na-H exchange activity by angiotensin II in opossum kidney cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1141-C1146 ◽  
Author(s):  
M. Jourdain ◽  
C. Amiel ◽  
G. Friedlander
Keyword(s):  
Angiotensin Ii ◽  
Ang Ii ◽  
Camp Content ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Camp Generation ◽  
Opossum Kidney Cells ◽  
Different Sources ◽  
Pkc Activation ◽  
Exchange Activity

Angiotensin II (ANG II) was shown to modulate transport in the renal proximal tubule through both inhibition of adenylate cyclase and protein kinase C (PKC) activation. We evaluated the effects of ANG II on adenosine 3',5'-cyclic monophosphate (cAMP) content and Na-H exchange activity (amiloride-sensitive Na influx) in two strains of opossum kidney (OK) cells originating from different sources, OK-VD and OK-RR cells. In OK-VD cells, ANG II inhibited basal and parathyroid hormone (PTH)-induced cAMP generation in a pertussis toxin-sensitive manner and reversed PTH inhibition of Na-H exchange. These effects of ANG II were prevented by PD 123319, a selective nonpeptide antagonist of AT2 receptors. In contrast, DuP 753, which antagonizes selectively AT1 receptors, had no effect. In OK-RR cells, ANG II had no effect on cAMP content and decreased Na-H exchange activity. The effect of ANG II persisted in the presence of PTH but was abolished by PKC downregulation and by DuP 753, but not by PD 123319. In conclusion, two types of ANG II receptors, coupled to distinct signaling pathways, were expressed independently in OK cells originating from two different sources and mediated opposite effects of ANG II on Na-H exchange activity. Those models provide a powerful tool for studying the intracellular steps involved in the tubular effects of ANG II and to evaluate the effect of pharmacological inhibitors of ANG II binding to its receptors.

scholarly journals Phosphorylation of the Angiotensin II (AT1A) Receptor Carboxyl Terminus: A Role in Receptor Endocytosis

1998 ◽  
Vol 12 (10) ◽  
pp. 1513-1524 ◽  
Author(s):  
Walter G. Thomas ◽  
Thomas J. Motel ◽  
Christopher E. Kule ◽  
Vijay Karoor ◽  
Kenneth M. Baker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Chinese Hamster ◽  
Carboxyl Terminus ◽  
Amino Acid Substitutions ◽  
Ang Ii ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
At1a Receptor

Abstract The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar1Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.

Membrane potential measurements in renal afferent and efferent arterioles: actions of angiotensin II

1997 ◽  
Vol 273 (2) ◽  
pp. F307-F314 ◽  
Author(s):  
R. Loutzenhiser ◽  
L. Chilton ◽  
G. Trottier
Keyword(s):  
Angiotensin Ii ◽  
Perfusion Pressure ◽  
Rat Kidney ◽  
Renal Perfusion ◽  
Membrane Potentials ◽  
Ang Ii ◽  
Ca Channels ◽  
Renal Perfusion Pressure

An adaptation of the in vitro perfused hydronephrotic rat kidney model allowing in situ measurement of arteriolar membrane potentials is described. At a renal perfusion pressure of 80 mmHg, resting membrane potentials of interlobular arteries (22 +/- 2 microns) and afferent (14 +/- 1 microns) and efferent arterioles (12 +/- 1 microns) were -40 +/- 2 (n = 8), -40 +/- 1 (n = 45), and -38 +/- 2 mV (n = 22), respectively (P = 0.75). Using a dual-pipette system to stabilize the impalement site, we measured afferent and efferent arteriolar membrane potentials during angiotensin II (ANG II)-induced vasoconstriction. ANG II (0.1 nM) reduced afferent arteriolar diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.005) and membrane potentials from -40 +/- 2 to -29 +/- mV (P = 0.012). ANG II elicited a similar vasoconstriction in efferent arterioles, decreasing diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.004), but failed to elicit a significant depolarization (-39 +/- 2 for control; -36 +/- 3 mV for ANG II; P = 0.27). Our findings thus indicate that resting membrane potentials of pre- and postglomerular arterioles are similar and lie near the threshold activation potential for L-type Ca channels. ANG II-induced vasoconstriction appears to be closely coupled to membrane depolarization in the afferent arteriole, whereas mechanical and electrical responses appear to be dissociated in the efferent arteriole.

scholarly journals Proximal Tubule-Specific Deletion of Angiotensin II Type 1a Receptors in the Kidney Attenuates Circulating and Intratubular Angiotensin II–Induced Hypertension in PT- Agtr1a −/− Mice

Hypertension ◽  
2021 ◽  
Author(s):  
Xiao Chun Li ◽  
Ana Paula Oliveira Leite ◽  
Xiaowen Zheng ◽  
Chunling Zhao ◽  
Xu Chen ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Angiotensin Ii ◽  
Fusion Protein ◽  
Proximal Tubule ◽  
Proximal Tubules ◽  
Normal Blood ◽  
Ang Ii ◽  
Normal Blood Pressure ◽  
Wild Type ◽  
Induced Hypertension

The present study used a novel mouse model with proximal tubule-specific knockout of AT 1a receptors in the kidney, PT- Agtr1a −/− , to test the hypothesis that intratubular Ang II (angiotensin II) and AT 1a receptors in the proximal tubules are required for maintaining normal blood pressure and the development of Ang II–induced hypertension. Twenty-six groups (n=6–15 per group) of adult male wild-type, global Agtr1a −/− , and PT- Agtr1a −/− mice were infused with Ang II (1.5 mg/kg per day, IP), or overexpressed an intracellular Ang II fusion protein in the proximal tubules for 2 weeks. Basal telemetry blood pressure were ≈15±3 mm Hg lower in PT- Agtr1a −/− than wild-type mice and ≈13±3 mm Hg higher than Agtr1a −/− mice ( P <0.01). Basal glomerular filtration was ≈23.9% higher ( P <0.01), whereas fractional proximal tubule Na + reabsorption was lower in PT- Agtr1a −/− mice ( P <0.01). Deletion of AT 1a receptors in the proximal tubules augmented the pressure-natriuresis response ( P <0.01) and natriuretic responses to salt loading or Ang III infusion ( P <0.01). Ang II induced hypertension in wild-type, PT- Agtr1a −/− and PT- Nhe3 −/− mice, but the pressor response was ≈16±2 mm Hg lower in PT- Agtr1a −/− and PT- Nhe3 −/− mice ( P <0.01). Deletion of AT 1a receptors or NHE3 (Na + /H + exchanger 3) in the proximal tubules attenuated ≈50% of Ang II–induced hypertension in wild-type mice ( P <0.01), but blocked intracellular Ang II fusion protein-induced hypertension in PT- Agtr1a −/− mice ( P <0.01). Taken together, the results of the present study provide new insights into the critical role of intratubular Ang II/AT 1 (AT 1a )/NHE3 pathways in the proximal tubules in normal blood pressure control and the development of Ang II–induced hypertension.

scholarly journals Genetic deletion of AT1a receptors attenuates intracellular accumulation of ANG II in the kidney of AT1a receptor-deficient mice

2007 ◽  
Vol 293 (2) ◽  
pp. F586-F593 ◽  
Author(s):  
Xiao C. Li ◽  
L. Gabriel Navar ◽  
Yuan Shao ◽  
Jia L. Zhuo
Keyword(s):  
Blood Pressure ◽  
Sodium Excretion ◽  
Rat Kidney ◽  
Systolic Pressure ◽  
Weight Ratio ◽  
Urinary Sodium Excretion ◽  
Urinary Sodium ◽  
Ang Ii ◽  
Wild Type ◽  
Deficient Mice

We and others have previously shown that high levels of ANG II are accumulated in the rat kidney via a type 1 (AT1) receptor-mediated mechanism, but it is not known which AT1 receptor is involved in this process in rodents. We tested the hypothesis that AT1a receptor-deficient mice (Agtr1a−/−) are unable to accumulate ANG II intracellularly in the kidney because of the absence of AT1a receptor-mediated endocytosis. Adult male wild-type (Agtr1a+/+), heterozygous (Agtr1a+/−), and Agtr1a−/− were treated with vehicle, ANG II (40 ng/min ip via osmotic minipump), or ANG II plus the AT1 antagonist losartan (10 mg·kg−1·day−1 po) for 2 wk. In wild-type mice, ANG II induced hypertension (168 ± 4 vs. 113 ± 3 mmHg, P < 0.001), increased kidney-to-body weight ratio ( P < 0.01), caused pressure natriuresis ( P < 0.05), and elevated plasma and whole kidney ANG II levels ( P < 0.001). Concurrent administration of ANG II with losartan attenuated these responses to ANG II. In contrast, Agtr1a−/− mice had lower basal systolic pressures ( P < 0.001), smaller kidneys with much fewer AT1b receptors ( P < 0.001), higher basal 24-h urinary sodium excretion ( P < 0.01), as well as basal plasma and whole kidney ANG II levels ( P < 0.01). However, intracellular ANG II levels in the kidney were lower in Agtr1a−/− mice. In Agtr1a−/− mice, ANG II slightly increased systolic pressure ( P < 0.05) but had no effect on the kidney weight, urinary sodium excretion, and whole kidney ANG II levels. Losartan restored systolic pressure to basal levels and decreased whole kidney ANG II levels by ∼20% ( P < 0.05). These results demonstrate a predominant role of AT1a receptors in blood pressure regulation and in the renal responses to long-term ANG II administration, that AT1b receptors may play a limited role in blood pressure control and mediating intrarenal ANG II accumulation in the absence of AT1a receptors.

Abstract 478: FGF23 Expression in Cardiac Fibroblasts Is Augmented by S100/Calgranulins-Mediated Inflammation and Associated With Cardiac Hypertrophy, but Not in Angiotensin II-Induced Cardiac Hypertrophy

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marion Hofmann Bowman ◽  
Brandon Gardner ◽  
Judy Earley ◽  
Debra L Rateri ◽  
Alan Daugherty ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Cardiac Fibroblasts ◽  
Left Ventricular ◽  
Heart Weight ◽  
Ang Ii ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Transgenic Expression ◽  
Significant Difference

Background: Serum S100A12 and fibroblast growth factor (FGF) 23 are biomarkers for cardiovascular mortality in patients with chronic kidney disease (CKD) and are associated with left ventricular hypertrophy (LVH). FGF23 is induced in cultured cardiac fibroblasts in response to cytokines including IL-6, TNF-a, LPS and S100/calgranulins. Moreover, hBAC-S100 transgenic mice with CKD had increased FGF23 in valvular interstitial cells and exhibited LVH. The present study was designed to examine cardiac FGF23 expression in other murine models of LVH in the absence of CKD. Methods: Hearts from five groups of male mice were studied: (i) C57BL6/J with transgenic expression a bacterial artificial chromosome of the human S100/calgranulins (S1008/9 and S100A12, hBAC-S100), (ii) wild type littermates, (iii) LDLR-/- infused with saline (29 days, 0.9%), (iv) LDLR-/- infused with angiotensin (Ang) II (29 days, 1000 ng/kg/min), and (v) fibroblast specific depletion of angiotensin II type 1a receptor (AT1aR) (S100A4-Cre x AT1aR-/- x LDLR-/-) infused with AngII. Results: hBAC-S100, but not wild type littermate mice, developed significant LVH at 10 months by heart weight/body weight (5.9 ±1.1 mg/g vs. 4.2 ±0.8, p<0.04), decreased E/A ratio, and increased LVPW thickness, and associated with increased expression of FGF23 mRNA and protein in cardiac tissue lysates (2-4 fold increase). Similarly, Ang II induced significant LVH compared to saline infused LDLR-/- mice (6.1±1.3 vs. 3.6 ±0.9 mg/g, p<0.01), and associated with increased mRNA for hypertrophic genes (ANP, BNP, b-MHC, CTGF and Col1a1). However, there was no significant difference in FGF23 mRNA and protein between Ang II and saline infused mice. Cardiac hypertrophy was attenuated in AngII-infused mice with deficiency of AT1aR (S100A4-Cre+/-xAT1aRxLDLR-/-). In vitro, Ang II (100nM) did not induce FGF23 in valvular interstitial fibroblasts or myocytes. Summary: Transgenic expression of S100/calgranulins is sufficient to induce LVH in aged mice with normal renal function, and this is associated with FGF23 expression in cardiac interstitial fibroblasts. Future studies are needed to determine whether cardiac FGF23 promotes LVH in a paracrine manner. However, FGF23 does not play a role in Ang II-induced LVH.

Abstract 229: TNF Receptor 1 Signaling Is Critically Involved in Mediating Angiotensin II-Induced Cardiac Fibrosis and Dysfunction

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sandra B Haudek ◽  
Jeff Crawford ◽  
Erin Reineke ◽  
Alberto A Allegre ◽  
George E Taffet ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Fibrosis ◽  
Smooth Muscle Actin ◽  
Protein A ◽  
Ang Ii ◽  
Wild Type ◽  
Monocyte Chemoattractant Protein 1 ◽  
Reactive Fibrosis

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34 + /CD45 + monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34 + /CD45 + cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.

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