Does atrial natriuretic factor protect against right ventricular overload? I. Hemodynamic study

We studied the effects of synthetic atrial natriuretic factor (ANF, 28-amino acid peptide) on base-line perfusion pressures and pressor responses to hypoxia and angiotensin II (ANG II) in isolated rat lungs and on the following hemodynamic and renal parameters in awake, chronically instrumented rats: cardiac output (CO), systemic (Rsa) and pulmonary (Rpa) vascular resistances, ANG II- and hypoxia (10.5% O2)-induced changes in Rsa and Rpa, and urine output. Intra-arterial ANF injections lowered base-line perfusion pressures and blunted hypoxia- and ANG II-induced pressor responses in the isolated lungs. Bolus intravenous injection of ANF (10 micrograms/kg) into intact rats decreased CO and arterial blood pressures of both systemic and pulmonary circulations and increased Rsa. ANG II (0.4 micrograms/kg) increased both Rsa and Rpa, and hypoxia increased Rpa alone in the intact rats. ANF (10 micrograms/kg) inhibited both ANG II- and hypoxia-induced increases in Rpa but did not significantly affect the ANG II-induced increase in Rsa. The antagonistic effect of ANF on pulmonary vasoconstriction was reversible and dose-dependent. The threshold doses of ANF required to inhibit pulmonary vasoconstriction were in the same range as those required to elicit diuresis and natriuresis. The data demonstrate that ANF has a preferential relaxant effect on pulmonary vessels constricted by hypoxia or ANG II. Both the renal and the pulmonary vascular effects of ANF may represent fundamental physiological actions of ANF. These actions may serve as a negative feedback control system that protects the right ventricle from excessive mechanical loads.     

Angiotensin II utilizes Janus kinase 2 in hypertension, but not in the physiological control of blood pressure, during low-salt intake

Janus kinase (JAK) 2 is activated by ANG II in vitro and in vivo, and chronic blockade of JAK2 by the JAK2 inhibitor AG-490 has been shown recently to attenuate ANG II hypertension in mice. In this study, AG-490 was infused intravenously in chronically instrumented rats to determine if the blunted hypertension was linked to attenuation of the renal actions of ANG II. In male Sprague-Dawley rats, after a control period, ANG II at 10 ng·kg(-1)·min(-1) was infused intravenously with or without AG-490 at 10 ng·kg(-1)·min(-1) iv for 11 days. ANG II infusion (18 h/day) increased mean arterial pressure from 91 ± 3 to 168 ± 7 mmHg by day 11. That response was attenuated significantly in the ANG II + AG-490 group, with mean arterial pressure increasing only from 92 ± 5 to 127 ± 3 mmHg. ANG II infusion markedly decreased urinary sodium excretion, caused a rapid and sustained decrease in glomerular filtration rate to ～60% of control, and increased renal JAK2 phosphorylation; all these responses were blocked by AG-490. However, chronic AG-490 treatment had no effect on the ability of a separate group of normal rats to maintain normal blood pressure when they were switched rapidly to a low-sodium diet, whereas blood pressure fell dramatically in losartan-treated rats on a low-sodium diet. These data suggest that activation of the JAK/STAT pathway is critical for the development of ANG II-induced hypertension by mediating its effects on renal sodium excretory capability, but the physiological control of blood pressure by ANG II with a low-salt diet does not require JAK2 activation.     

Enhanced AT1 receptor-mediated vasocontractile response to ANG II in endothelium-denuded aorta of obese Zucker rats

In the present study, we tested the hypothesis that ANG II causes a greater vasoconstriction in obese Zucker rats, a model of type 2 diabetes, with mild hypertension. Measurement of isometric tension in isolated aortic rings with intact endothelium revealed a modest but not significantly greater ANG II-induced contraction in obese than lean rats. Removal of endothelium or inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester (L-NAME) enhanced 1) ANG II-induced contraction in both lean and obese rats, being significantly greater in obese rats (E(max) g/g tissue, denuded: lean 572 +/- 40 vs. obese 664 +/- 16; L-NAME: lean 535 +/- 14 vs. obese 818 +/- 23) and 2) ANG II sensitivity in obese compared with lean rats, as revealed by the pD(2) values. Endothelin-1 and KCl elicited similar contractions in the aortic rings of lean and obese rats. ACh, a NO-dependent relaxing hormone, produced greater relaxation in the aortic rings of obese than lean rats, whereas sodium nitroprusside, an NO donor, elicited similar relaxations in both rat strains. The expression of the ANG type 1 (AT(1)) receptor protein and mRNA in the endothelium-intact aorta was significantly greater in obese than lean rats, whereas the endothelium-denuded rings expressed modest but not significantly greater levels of AT(1) receptors in obese than lean rats. The endothelial NO synthase protein and mRNA expression levels were higher in the aorta of obese than lean animals. We conclude that, although ANG II produces greater vasoconstriction in obese rat aortic rings, enhanced endothelial AT(1) receptor-mediated NO production appears to counteract the increased ANG II-induced vasoconstriction, suggesting that arterial AT(1) receptor may not be a contributing factor to hypertension in this model of obesity.     

The PPARgamma agonist pioglitazone modifies the vascular sodium-angiotensin II relationship in insulin-resistant rats

Glitazones are efficient insulin sensitizers that blunt the effects of angiotensin II (ANG II) in the rat. Sodium chloride is another important modulator of the systemic and renal effects of ANG II. Whether glitazones interfere with the interaction between sodium and the response to ANG II is not known. Therefore, we investigated the effects of pioglitazone on the relationship between sodium and the systemic and renal effects of ANG II in rats. Pioglitazone, or vehicle, was administered for 4 wk to 8-wk-old obese Zucker rats. Animals were fed a normal-sodium (NS) or a high-sodium (HS) diet. Intravenous glucose tolerance tests, systemic and renal hemodynamic responses to ANG II, and the renal ANG II binding and expression of ANG II type 1 (AT(1)) receptors were measured. The results of our study were that food intake and body weight increased, whereas blood pressure, heart rate, filtration fraction, and insulin levels decreased significantly with pioglitazone in obese rats on both diets. Pioglitazone blunted the systemic response to ANG II and abolished the increased responsiveness to ANG II induced by a HS diet. Pioglitazone modified the renal hemodynamic response to changes in salt intake while maintaining a lower filtration fraction with ANG II perfusion. These effects were associated with a decrease in the number and expression of the AT(1) receptor in the kidney. In conclusion, these data demonstrate that the peroxisome proliferator-activated receptor-gamma agonist pioglitazone modifies the physiological relationship between sodium chloride and the response to ANG II in insulin-resistant rats.     

Enhanced renal expression of preproendothelin mRNA during chronic angiotensin II hypertension

The purpose of this study was to determine the role of endothelin in mediating the renal hemodynamic and arterial pressure changes observed during chronic ANG II-induced hypertension. ANG II (50 ng x kg(-1) x min(-1)) was chronically infused into the jugular vein by miniosmotic pump for 2 wk in male Sprague-Dawley rats with and without endothelin type A (ET(A))-receptor antagonist ABT-627 (5 mg x kg(-1) x day(-1)) pretreatment. Arterial pressure increased in ANG II rats compared with control rats (149 +/- 5 vs. 121 +/- 6 mmHg, P < 0.05, respectively). Renal expression of preproendothelin mRNA was increased by approximately 50% in both the medulla and cortex of ANG II rats. The hypertensive effect of ANG II was completely abolished in rats pretreated with the ET(A)-receptor antagonist (114 +/- 5 mmHg, P < 0.05). Glomerular filtration rate was decreased by 33% in ANG II rats, and this response was attenuated in rats pretreated with ET(A)-receptor antagonist. These data indicate that activation of the renal endothelin system by ANG II may play an important role in mediating chronic renal and hypertensive actions of ANG II.     

Mineralocorticoid treatment attenuates activation of oxytocinergic and vasopressinergic neurons by icv ANG II

Central oxytocin (OT) neurons limit intracerebroventricular (icv) ANG II-induced NaCl intake. Because mineralocorticoids synergistically increase ANG II-induced NaCl intake, we hypothesized that mineralocorticoids may attenuate ANG II-induced activation of inhibitory OT neurons. To test this hypothesis, we determined the effect of deoxycorticosterone (DOCA; 2 mg/day) on icv ANG II-induced c-Fos immunoreactivity in OT and vasopressin (VP) neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also on pituitary OT and VP secretion in male rats. DOCA significantly decreased the percentage of c-Fos-positive (%c-Fos+) OT neurons in the SON and PVN, both in the magnocellular and parvocellular subdivisions, and the %c-Fos+ VP neurons in the SON after a 5-ng icv injection of ANG II. DOCA also significantly reduced the %c-Fos+ OT neurons in the SON after 10 ng ANG II and tended to attenuate 10 ng ANG II-induced OT secretion. However, the %c-Fos+ OT neurons in DOCA-treated rats was greater after 10 ng ANG II, and DOCA did not affect the %c-Fos+ OT neurons in the PVN nor VP secretion or c-Fos immunoreactivity in either the SON or PVN after 10 ng ANG II. DOCA also did not significantly alter the effect of intraperitoneal (ip) cholecystokinin (62 microg) on %c-Fos+ OT neurons or of ip NaCl (2 ml of 2 M NaCl) on the %c-Fos+ OT and VP neurons. These findings indicate that DOCA attenuates the responsiveness of OT and VP neurons to ANG II without completely suppressing the activity of these neurons and, therefore, support the hypothesis that attenuation of OT neuronal activity is one mechanism by which mineralocorticoids enhance NaCl intake.     

Role of endothelin ETB receptor activation in angiotensin II-induced hypertension: effects of salt intake

We showed recently that endothelin (ET)A receptors are involved in the salt sensitivity of ANG II-induced hypertension. The objective of this current study was to characterize the role of endothelin ETB receptor activation in the same model. Male rats on fixed normal (2 meq/day) or high (6 meq/day) salt intake received a continuous intravenous infusion of ANG II or salt only for 15 days. During the middle 5 days of the infusion period, rats were given either the selective ETB receptor antagonist A-192621 or the nonselective endothelin receptor antagonist A-182086 (both at 24 mg x kg(-1) x day(-1) intra-arterially). Infusion of ANG II caused a greater rise in arterial pressure in rats on high-salt intake. The administration of A-192621 increased arterial pressure further in all rats. The chronic hypertensive effect of A-192621 was not significantly affected by salt intake or ANG II. The administration of A-182086 lowered arterial pressure chronically only in rats on normal salt intake receiving ANG II. Thus the salt sensitivity of ANG II-induced hypertension is not caused by changes in ETB receptor function.     

Central estrogen inhibition of angiotensin II-induced hypertension in male mice and the role of reactive oxygen species

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP (Delta30.1 +/- 2.5 mmHg). Either central infusion of Tempol or 17beta-estradiol (E2) attenuated the pressor effect of ANG II (Delta10.9 +/- 2.3 and Delta4.5 +/- 1.4 mmHg), and the protective effect of E2 was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 (Delta23.6 +/- 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central E2-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 +/- 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (-1.8 +/- 1.6% and -1.0 +/- 1.8%). The ROS response to ANG II was also blocked by E2 (-3.2 +/- 2.4%). The results suggest that the central actions of E2 are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production.     

Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).     

Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-kappaB-dependent pathway in rat preadipocytes

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time- and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-kappaB inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1kappaB-alpha phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-kappaB p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-kappaB binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-kappaB-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease.     

Role of ET(A) receptors in experimental ANG II-induced hypertension in rats

The objectives were to determine if ANG II-induced hypertension is maintained by activation of endothelin type A (ET(A)) receptors by endogenous ET-1 and if this effect is influenced by salt intake. Male rats were maintained on high sodium intake (HS; 6 meq/day) or on normal sodium intake (NS; 2 meq/day). Hypertension was produced by intravenous infusion of ANG II (5 ng/min) for 15 days. Five-day oral dosing with the selective ET(A)-receptor antagonist ABT-627 (~2 mg. kg(-1). day(-1)) reduced mean arterial pressure (MAP) to baseline levels in rats on HS receiving ANG II infusion, but it did not affect MAP in normotensive HS controls. In rats on NS, ABT-627 only transiently decreased MAP in rats receiving ANG II and slightly reduced MAP in normotensive controls. ABT-627 produced mild retention of sodium and water in NS rats receiving ANG II, but not in any other group. These results indicate that ET-1 plays a role in ANG II-induced hypertension via activation of ET(A) receptors and that this role is more prominent in rats on HS.     

Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles

We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.     

Role of myofibrillogenesis regulator-1 in myocardial hypertrophy

Myofibrillogenesis regulator-1 (MR-1) is a novel homologous gene, identified from a human skeletal muscle cDNA library, that interacts with contractile proteins and exists in human myocardial myofibrils. The present study investigated MR-1 protein expression in hypertrophied myocardium and MR-1 involvement in cardiac hypertrophy. Cardiac hypertrophy was induced by abdominal aortic stenosis (AAS) in Sprague-Dawley rats. Left ventricular (LV) hypertrophy was assessed by the ratio of LV wet weight to whole heart weight (LV/HW) or LV weight to body weight (LV/BW). Rat MR-1 (rMR-1) expression in the myocardium was detected by immunohistochemical and Western blotting analysis. Hypertrophy was induced by ANG II incubation in cultured neonatal rat cardiomyocytes. The effect of rMR-1 RNA interference on ANG II-induced hypertrophy was studied by transfection of cardiomyocytes with an RNA interference plasmid, pSi-1, which targets rMR-1. Hypertrophy in cardiomyocytes was assessed by [3H]Leu incorporation and myocyte size. rMR-1 protein expression in cardiomyocytes was detected by Western blotting. We found that AAS resulted in a significant increase in LV/HW and LV/BW: 89% and 86%, respectively (P < 0.01). Immunohistochemistry and Western blot analysis demonstrated upregulated rMR-1 protein expression in hypertrophic myocardium. ANG II induced a 24% increase in [3H]Leu incorporation and a 65.8% increase in cell size compared with control cardiomyocytes (P < 0.01), which was prevented by treatment with losartan, an angiotensin (AT1) receptor inhibitor, or transfection with pSi-1. rMR-1 expression increased in ANG II-induced hypertrophied cardiomyocytes, and pSi-1 transfection abolished the upregulation. These findings suggest that MR-1 is associated with cardiac hypertrophy in rats in vivo and in vitro.     

Involvement of endogenous nitric oxide in angiotensin II-induced activation of vascular mitogen-activated protein kinases

Angiotensin II (ANG II) is a powerful activator of mitogen-activated protein (MAP) kinase cascades in cardiovascular tissues through a redox-sensitive mechanism. Nitric oxide (NO) is considered to antagonize the vasoconstrictive and proarteriosclerotic actions of ANG II. However, the role of endogenous NO in ANG II-induced redox-sensitive signal transduction is not yet clear. In this study using catheterized, conscious rats, we found that acute intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg) enhanced phosphorylation of aortic MAP kinases extracellular signal regulated kinase (ERK) 1/2 and p38, which were suppressed only partially by a superoxide dismutase mimetic (Tempol), whereas ANG II-induced MAP kinase phosphorylation was markedly suppressed by Tempol. FK409, a NO donor, had little effect on vascular MAP kinase phosphorylation. On the other hand, acute exposure to a vasoconstrictor dose of ANG II (200 ng x kg(-1) x min(-1) iv) failed to enhance phosphorylation of aortic MAP kinases in the chronically L-NAME-treated rats, whereas the vasoconstrictor effect of ANG II was not affected by L-NAME treatment. Furthermore, three different inhibitors of NO synthase suppressed, in a dose-dependent manner, ANG II-induced MAP kinase phosphorylation in rat vascular smooth muscle cells, which was closely linked to superoxide generation in cells. These results indicate the involvement of endogenous NO synthase in ANG II-induced signaling pathways, leading to activation of MAP kinase, and that NO may have dual effects on the vascular MAP kinase activation associated with redox sensitivity.     

Contribution of the subfornical organ to angiotensin II-induced hypertension

Previous studies clearly demonstrated acute actions of angiotensin II (ANG II) at one of the central circumventricular organs, the subfornical organ (SFO), but studies demonstrating a role for the SFO in the chronic actions of ANG II remain uncertain. The purpose of this study was to examine the role of the SFO in the chronic hypertensive phase of ANG II-induced hypertension. We hypothesized that the SFO is necessary for the full hypertensive response observed during the chronic phase of ANG II-induced hypertension. To test this hypothesis, male Sprague-Dawley rats were subjected to sham operation (sham rats) or electrolytic lesion of the SFO (SFOx rats). After 1 wk, the rats were instrumented with venous catheters and radiotelemetric transducers for intravenous administration of ANG II and measurement of blood pressure and heart rate, respectively. Rats were then allowed 1 wk for recovery. After 3 days of saline control infusion (7 ml of 0.9% NaCl/day), sham and SFOx rats were infused with ANG II at 10 ng.kg(-1).min(-1) i.v. for 10 consecutive days and then allowed to recover for 3 days. A 0.4% NaCl diet and distilled water were provided ad libitum. At day 5 of ANG II infusion, mean arterial pressure increased 11.7 +/- 3.0 mmHg in sham rats (n = 9) but increased only 3.7 +/- 1.4 mmHg in SFOx rats (n = 9). This trend continued through day 10 of ANG II treatment. These results support the hypothesis that the SFO is necessary for the full hypertensive response to chronic ANG II administration.     

A novel phosphoinositide 3-kinase-dependent pathway for angiotensin II/AT-1 receptor-mediated induction of collagen synthesis in MES-13 mesangial cells

Chronic activation of the angiotensin II (ANG II) type 1 receptor (AT-1R) is critical in the development of chronic kidney disease. ANG II activates mesangial cells (MCs) and stimulates the synthesis of extracellular matrix components. To determine the molecular mechanisms underlying the induction of MC collagen, a mouse mesangial cell line MES-13 was employed. ANG II treatment induced an increase in collagen synthesis, which was abrogated by co-treatment with losartan (an AT-1R antagonist), wortmannin (a phosphoinositide 3-kinase (PI3K) inhibitor), an Akt inhibitor, and stable transfection of dominant negative-Akt1. ANG II induced a significant increase in PI3K activity, which was abolished by co-treatment with losartan or 2',5'-dideoxyadenosine (2',5'-DOA, an adenylyl cyclase inhibitor) but not by PD123319 (an AT-2R antagonist) or H89 (a protein kinase A (PKA) inhibitor). The Epac (exchange protein directly activated by cAMP)-specific cAMP analog, 8-pHPT-2'-O-Me-cAMP, significantly increased PI3K activity, whereas a PKA-specific analog, 6-benzoyladenosine-cAMP, showed no effect. The ANG II-induced increase in PI3K activity was also blocked by co-treatment with PP2, an Src inhibitor, or AG1478, an epidermal growth factor receptor (EGFR) antagonist. ANG II induced phosphorylation of Akt and p70S6K and EGFR, which was abrogated by knockdown of c-Src by small interference RNA. Knockdown of Src also effectively abolished ANG II-induced collagen synthesis. Conversely, stable transfection of a constitutively active Src mutant enhanced basal PI3K activity and collagen production, which was abrogated by AG1478 but not by 2',5'-DOA. Moreover, acute treatment with ANG II significantly increased Src activity, which was abrogated with co-treatment of 2',5'-DOA. Taken together, these results suggest that ANG II induces collagen synthesis in MCs by activating the ANG II/AT-1R-EGFR-PI3K pathway. This transactivation is dependent on cAMP/Epac but not on PKA. Src kinase plays a pivotal role in this signaling pathway between cAMP and EGFR. This is the first demonstration that an AT1R-PI3K/Akt crosstalk, along with transactivation of EGFR, mediates ANG II-induced collagen synthesis in MCs.     

Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).     

Adenosine enhances long term the contractile response to angiotensin II in afferent arterioles

Adenosine (Ado) enhances ANG II-induced constrictions of afferent arterioles (Af) by receptor-dependent and -independent pathways. Here, we test the hypothesis that transient Ado treatment has a sustained effect on Af contractility, resulting in increased ANG II responses after longer absence of Ado. Treatment with Ado (cumulative from 10(-11) to 10(-4) mol/l) and consecutive washout for 10 or 30 min increased constrictions on ANG II in isolated, perfused Af. Cytosolic calcium transients on ANG II were not enhanced in Ado-treated vessels. Selective or global inhibition of A(1)- and A(2)-adenosine receptors did not inhibit the Ado effect. Nitrobenzylthioinosine (an Ado transport inhibitor) clearly reduced the Ado-mediated responses. Selective inhibition of p38 MAPK with SB-203580 also prevented the Ado effect. Inosine treatment did not influence arteriolar reactivity to ANG II. Contractile responses of Af on norepinephrine and endothelin-1 were not influenced by Ado. Phosphorylation of the p38 MAPK and of the regulatory unit of 20-kDa myosin light chain was enhanced after Ado treatment and ANG II in Af. However, phosphorylation of p38 MAPK induced by norepinephrine or endothelin-1 was reduced in vessels treated with Ado, whereas 20-kDa myosin light chain was unchanged. The results suggest an intracellular, long-lasting mechanism including p38 MAPK activation responsible for the increase of ANG II-induced contractions by Ado. The effect is not calcium dependent and specific for ANG II. The prolonged enhancement of the ANG II sensitivity of Af may be important for tubuloglomerular feedback.     

Effect of cicletanine on renal cGMP production.

The aim of the present study was to assess the effect of cicletanine on renal cGMP production. To do so we measured mean arterial pressure (MAP), creatinine clearance (CC), and urinary excretion of electrolytes and cGMP under basal conditions and after 6 h of cicletanine administration (10 and 15 mg/kg body weight by oral gavage) in conscious Wistar rats. Also, the in vitro effect of cicletanine was assessed by incubating renal slices and isolated rat glomeruli with two concentrations of cicletanine (0.1 and 1 mM) for different times (1, 2, 5, and 30 min) in the presence of 3-isobutyl-1-methylxanthine. Oral administration of cicletanine induced an increase in urinary flow (V) and the urinary excretion of electrolytes and cGMP, with no changes in CC. In addition, a significant decrease in MAP was observed, but only with the lower dose. Incubation with cicletanine did not induce significant changes in cGMP production in glomeruli or renal slices. These results show that cicletanine, administered in vivo at diuretic and antihypertensive doses, induces an increase in urinary cGMP excretion.