Abstract 1379: Angiotensin II Inhibits the Na+-K+ pump via PKC-dependent Activation of NAD(P)H Oxidase

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gemma A Figtree ◽  
Caroline N White ◽  
Chia Chi Liu ◽  
Alvaro Garcia ◽  
Elisha J Hamilton ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Oxidase Inhibitor ◽  
Ang Ii ◽  
Inhibitory Peptide ◽  
Pipette Solution ◽  
Myocardial Oxidative Stress ◽  
Myocyte Function

Background: The sarcolemmal Na + -K + pump, essentially the only export route for Na + and pivotal for cardiac myocyte function, is inhibited by angiotensin II (Ang II). Since Ang II activates NAD(P)H oxidase we tested the hypothesis that NAD(P)H oxidase mediates Ang II-induced pump inhibition. This is important for our understanding of pathophysiology and treatment of heart failure, a condition with increases in circulating Ang II levels; intracellular myocyte Na + ; NAD(PH oxidase activity; and myocardial oxidative stress. The raised myocyte Na + is believed to contribute to the electro-mechanical phenotype of contractile abnormalities and cardiac arrhythmias. Methods and results: Exposure to 100 nmol/L Ang II increased fluorescence of isolated rabbit ventricular myocytes loaded with a superoxide-sensitive dye. The increase was abolished by pegylated superoxide dismutase (SOD), by the NAD(P)H oxidase inhibitor apocynin and by myristolated inhibitory peptide to ϵ -protein kinase C ( ϵ PKC), previously implicated in Ang II-induced Na + -K + pump inhibition. Exposure of voltage-clamped myocytes to Ang II decreased electrogenic Na + -K + pump current, I p , from 0.48 ± 0.02 pA/pF, N = 11 to 0.36 ± 0.03 pA/pF, N = 11 (P < 0.05). The decrease was abolished (P < 0.05) by inclusion in pipette solution of: SOD, the gp91ds inhibitory peptide that blocks assembly and activation of NAD(P)H oxidase and by ϵ PKC inhibitory peptide. Since co-localization should facilitate NAD(P)H-oxidase dependent regulation of the Na + -K + pump we examined if there is physical association between the pump and NAD(P)H oxidase. Cell lysate was immunoprecipitated with antibody to the α 1 pump subunit. Immunoblotting demonstrated co-immunoprecipitation of the α 1 subunit with caveolin 3 and with membrane-associated p22 phox and cytosolic p47 phox NAD(P)H oxidase subunits at baseline. Ang II had no effect on α 1 /caveolin3 or α 1 /p22 phox interaction, but increased α 1 /p47 phox co-immunoprecipitation. Conclusion: Ang II is known to induce PKC-dependent phosphorylation and translocation of p47 phox resulting in activation of NAD(P)H oxidase, and we propose this activation occurs in a caveolar microdomain and that it mediates the inhibition of the Na + -K + pump by Ang II.

Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase

2009 ◽  
Vol 296 (4) ◽  
pp. C693-C700 ◽  
Author(s):  
Caroline N. White ◽  
Gemma A. Figtree ◽  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Elisha J. Hamilton ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Oxidase Inhibitor ◽  
Ang Ii ◽  
Inhibitory Peptide ◽  
Myocyte Function ◽  
Nadph Oxidase Activation

The sarcolemmal Na+-K+ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to ε-protein kinase C (εPKC), previously implicated in ANG II-induced Na+-K+ pump inhibition. A role for εPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of εPKC with the receptor for the activated kinase and with the cytosolic p47 phox subunit of NADPH oxidase. ANG II decreased electrogenic Na+-K+ pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by εPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na+-K+ pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The α1-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22 phox and cytosolic p47 phox NADPH oxidase subunits at baseline. ANG II had no effect on α1/caveolin 3 or α1/p22 phox interaction, but it increased α1/p47 phox coimmunoprecipitation. We conclude that ANG II inhibits the Na+-K+ pump via PKC-dependent NADPH oxidase activation.

Natriuretic peptides stimulate the cardiac sodium pump via NPR-C-coupled NOS activation

2008 ◽  
Vol 294 (4) ◽  
pp. C1067-C1073 ◽  
Author(s):  
M. William ◽  
E. J. Hamilton ◽  
A. Garcia ◽  
H. Bundgaard ◽  
K. K. M. Chia ◽  
...  
Keyword(s):  
Natriuretic Peptides ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
No Production ◽  
Pipette Solution ◽  
Selective Ligand ◽  
Myocyte Function ◽  
Clearance Receptor ◽  
Rabbit Ventricular Myocytes ◽  
Dependent Pathway

Natriuretic peptides (NPs) and their receptors (NPRs) are expressed in the heart, but their effects on myocyte function are poorly understood. Because NPRs are coupled to synthesis of cGMP, an activator of the sarcolemmal Na+-K+ pump, we examined whether atrial natriuretic peptide (ANP) regulates the pump. We voltage clamped rabbit ventricular myocytes and identified electrogenic Na+-K+ pump current (arising from the 3:2 Na+:K+ exchange and normalized for membrane capacitance) as the shift in membrane current induced by 100 μmol/l ouabain. Ten nanomoles per liter ANP stimulated the Na+-K+ pump when the intracellular compartment was perfused with pipette solutions containing 10 mmol/l Na+ but had no effect when the pump was at near maximal activation with 80 mmol/l Na+ in the pipette solution. Stimulation was abolished by inhibition of cGMP-activated protein kinase with KT-5823, nitric oxide (NO)-activated guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), or NO synthase with NG-nitro-l-arginine methyl ester (l-NAME). Since synthesis of cGMP by NPR-A and NPR-B is not NO dependent or ODQ sensitive, we exposed myocytes to AP-811, a highly selective ligand for the NPR-C “clearance” receptor. It abolished ANP-induced pump stimulation. Conversely, the selective NPR-C agonist ANP(4-23) reproduced stimulation. The stimulation was blocked by l-NAME. To examine NO production in response to ANP(4-23), we loaded myocytes with the NO-sensitive fluorescent dye diacetylated diaminofluorescein-2 and examined them by confocal microscopy. ANP(4-23) induced a significant increase in fluorescence, which was abolished by l-NAME. We conclude that NPs stimulate the Na+-K+ pump via an NPR-C and NO-dependent pathway.

scholarly journals Central interactions of aldosterone and angiotensin II in aldosterone- and angiotensin II-induced hypertension

2011 ◽  
Vol 300 (2) ◽  
pp. H555-H564 ◽  
Author(s):  
Baojian Xue ◽  
Terry G. Beltz ◽  
Yang Yu ◽  
Fang Guo ◽  
Celso E. Gomez-Sanchez ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Oxidase Inhibitor ◽  
Male Rats ◽  
Ang Ii ◽  
Adeno Associated Virus ◽  
Synergistic Interactions ◽  
Induced Hypertension ◽  
Ros Scavenger ◽  
The Brain

Many studies have implicated both angiotensin II (ANG II) and aldosterone (Aldo) in the pathogenesis of hypertension, the progression of renal injury, and cardiac remodeling after myocardial infarction. In several cases, ANG II and Aldo have been shown to have synergistic interactions in the periphery. In the present studies, we tested the hypothesis that ANG II and Aldo interact centrally in Aldo- and ANG II-induced hypertension in male rats. In rats with blood pressure (BP) and heart rate (HR) measured by DSI telemetry, intracerebroventricular (icv) infusions of the mineralocorticoid receptor (MR) antagonists spironolactone and RU28318 or the angiotensin type 1 receptor (AT1R) antagonist irbesartan significantly inhibited Aldo-induced hypertension. In ANG II-induced hypertension, icv infusion of RU28318 significantly reduced the increase in BP. Moreover, icv infusions of the reactive oxygen species (ROS) scavenger tempol or the NADPH oxidase inhibitor apocynin attenuated Aldo-induced hypertension. To confirm these effects of pharmacological antagonists, icv injections of either recombinant adeno-associated virus carrying siRNA silencers of AT1aR (AT1aR-siRNA) or MR (MR-siRNA) significantly attenuated the development of Aldo-induced hypertension. The immunohistochemical and Western blot analyses of AT1aR-siRNA- or MR-siRNA-injected rats showed a marked reduction in the expression of AT1R or MR in the paraventricular nucleus compared with scrambled siRNA rats. When animals from all studies underwent ganglionic blockade with hexamethonium, there was a smaller reduction in the fall of BP in animals receiving icv AT1R or MR antagonists. These results suggest that ANG II and Aldo interact in the brain in a mutually cooperative manner such that the functional integrity of both brain AT1R and MR are necessary for hypertension to be induced by either systemic ANG II or Aldo. The pressor effects produced by systemic ANG II or Aldo involve increased central ROS and sympathetic outflow.

scholarly journals Suppressor of Cytokine Signaling 3 Is Induced by Angiotensin II in Heart and Isolated Cardiomyocytes, and Participates in Desensitization

Endocrinology ◽  
2003 ◽  
Vol 144 (10) ◽  
pp. 4586-4596 ◽  
Author(s):  
Vivian C. Calegari ◽  
Rosangela M. N. Bezerra ◽  
Márcio A. Torsoni ◽  
Adriana S. Torsoni ◽  
Kleber G. Franchini ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Rat Heart ◽  
Ventricular Myocytes ◽  
Janus Kinase ◽  
Neonatal Rat ◽  
Cytokine Signaling ◽  
Ang Ii ◽  
Suppressor Of Cytokine Signaling ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

Angiotensin II (Ang II) exerts a potent growth stimulus on the heart and vascular wall. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) intracellular signaling pathway by Ang II mediates at least some of the mitogenic responses to this hormone. In other signaling systems that use the JAK/STAT pathway, proteins of the suppressor of cytokine signaling (SOCS) family participate in signal regulation. In the present study it is demonstrated that SOCS3 is constitutively expressed at a low level in rat heart and neonatal rat ventricular myocytes. Ang II at a physiological concentration enhances the expression of SOCS3 mRNA and protein, mainly via AT1 receptors. After induction, SOCS3 associates with JAK2 and impairs further activation of the JAK2/STAT1 pathway. Pretreatment of rats with a specific phosphorthioate antisense oligonucleotide to SOCS3, reverses the desensitization to angiotensin signaling, as detected by a fall in c-Jun expression after repetitive infusions of the hormone. Thus, SOCS3 is induced by Ang II in rat heart and neonatal rat ventricular myocytes and participates in the modulation of the signal generated by this hormone.

scholarly journals Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

2015 ◽  
Vol 309 (4) ◽  
pp. C239-C250 ◽  
Author(s):  
Karin K. M. Chia ◽  
Chia-Chi Liu ◽  
Elisha J. Hamilton ◽  
Alvaro Garcia ◽  
Natasha A. Fry ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Pump Current ◽  
Reciprocal Relationship ◽  
Ang Ii ◽  
Α Subunit

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current ( Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47 phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22 phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47 phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22 phox and p47 phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo.

scholarly journals Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain

2018 ◽  
Author(s):  
Siyu Wei ◽  
Christian Cassara ◽  
Xianming Lin ◽  
Richard D Veenstra
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ventricular Myocytes ◽  
External Solution ◽  
Inhibitory Peptide ◽  
Pipette Solution ◽  
Calmodulin Binding ◽  
Whole Cell Patch Clamp ◽  
Gating Mechanism ◽  
Mimetic Peptides

AbstractIntracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction channels and the synergism or antagonism between pH and Ca2+ has been questioned for decades. In this study, we assessed whether the calcium gating mechanism occurs independently of the pH gating mechanism by utilizing the Cx43-M257 (Cx43K258stop) mutant, a carboxyl-terminal (CT) truncated version of Cx43 lacking the pH gating domain. Dual whole cell patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or Cx43-M257 proteins. Addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 macroscopic gap junction conductance (gj) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl2 from the external solution or adding 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. The ability of connexin calmodulin binding domain (Cx CaMBD) mimetic peptides and the Gap19 peptide to inhibit the Ca2+/CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had no effect. Lastly, the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions were investigated. We confirmed that the fast kinetic inactivation component was absent in Cx43-M257 gap junctions, but also observed that the previously reported facilitated recovery of gj from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.

Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes

1991 ◽  
Vol 261 (1) ◽  
pp. C77-C85 ◽  
Author(s):  
D. C. Kem ◽  
E. I. Johnson ◽  
A. M. Capponi ◽  
D. Chardonnens ◽  
U. Lang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Acute Effect ◽  
Neonatal Rat ◽  
Free Calcium ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Ventricular Myocytes

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.

Abstract 331: 20-hydroxyeicosatetraenoic Acid is Involved in Angiotensin II-mediated Apoptosis in Rat Cardiac Myocyte

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Yong Han ◽  
Xin Shen ◽  
Fanrong Yao ◽  
Chengluan Xuan ◽  
Yan Gu ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Myocyte ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Chromatin Condensation ◽  
Neonatal Rat ◽  
Spectrometric Analysis ◽  
Ang Ii ◽  
Hydroxyeicosatetraenoic Acid ◽  
Induced Apoptosis

Cardiomyocyte apoptosis has been documented involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both angiotensin II (Ang II) and 20-Hydroxyeicosatetraenoic Acid (20-HETE), a hydroxylation product of arachidonic acid catalyzed by CYP450-ω hydroxylase, induce apoptosis in cardiomyocyte. However, the crosstalk between 20-HETE and Ang II in cardiomyocytes apoptosis process is unclear. In the current study, we examined apoptosis using flow cytometry in primary cultured neonatal rat ventricular myocytes treated with control, Ang II (100nM), Ang II plus HET0016 (10μM), HET0016, or 20-HETE (10nM) along. The results demonstrated that treatment with Ang II or 20-HETE significantly increased apoptosis and that Ang II-induced apoptosis were markedly attenuated by HET0016, a 20-HETE agonist. In addition, Ang II-induced increases of caspase-3 activity were significantly attenuated by 20.9±3.4% after co-treatment with HET0016. Our results also demonstrated that HET0016 significantly suppressed Ang II-induced increases of superoxide production by 27.52.3% and mitochondrial membrane potential by 64.5±6.3%. Finally, Ang II-induced nuclei crenation, chromatin condensation and fractionation were attenuated by 73.6±8.5% with HET0016 pre-treatment. In addition, treatment cardiomyocytes with Ang II increased CYP4A1 expression and 20-HETE production, measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of Ang II related cardiac diseases.

scholarly journals NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

2010 ◽  
Vol 298 (3) ◽  
pp. R707-R712 ◽  
Author(s):  
Yiling Fu ◽  
Rui Zhang ◽  
Deyin Lu ◽  
Haifeng Liu ◽  
Kiran Chandrashekar ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Primary Source ◽  
Oxidase Inhibitor ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Ang Ii ◽  
Xanthine Oxidase Inhibitor ◽  
Si Rna ◽  
Full Activation

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O2−) and angiotensin II (ANG II). We have reported that NaCl-induced O2−in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O2−in MD is unknown. Thus we determined the pathways by which ANG II increased O2−in the MD by measuring O2−in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O2−levels to increase by more than twofold ( P < 0.01). This increase was blocked by losartan (AT1receptor blocker) but not PD-123319 (AT2receptor antagonist). Apocynin (a NOX inhibitor) decreased O2−by 86% ( P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O2−was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O2−, whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O2−production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O2−production in the MD primarily via AT1-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

Intracellular angiotensin II induces cell proliferation independent of AT1 receptor

2006 ◽  
Vol 291 (5) ◽  
pp. C995-C1001 ◽  
Author(s):  
Kenneth M. Baker ◽  
Rajesh Kumar
Keyword(s):  
Cell Proliferation ◽  
Angiotensin Ii ◽  
Cho Cells ◽  
Cardiac Myocyte ◽  
Chinese Hamster ◽  
Cell Types ◽  
Growth Effect ◽  
Ang Ii

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT1) antagonist, losartan. To further determine the role of AT1 in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT1 receptor (CHO-AT1). CHO-AT1, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT1 cells were stably transfected for iANG II expression (CHO-iA and CHO-AT1-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT1-iA cells showed a steeper growth curve compared with CHO and CHO-AT1, respectively. These observations were confirmed by Wst-1 assay. The AT1 receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT1-iA cells. eANG II showed an additional growth effect in CHO-AT1-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT1 receptors and suggest novel intracellular mechanisms of action for ANG II.

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