Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-kappa B transcription factors.
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Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar1 Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-kappaB (NF-kappaB) motif. Sar1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-kappaB subunits Rel A and NF-kappaB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-kappaB translocation was dependent on weak simultaneous proteolysis of the IkappaBalpha and beta inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-kappaB1 form. Pretreatment with a potent inhibitor of IkappaBalpha proteolysis, TPCK, completely blocked Sar1 Ang IIAng II-induced NF-kappaB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-kappaB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-kappaB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs. (+info)
N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane.
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The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn-->Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies. (+info)
Recent insight into therapy of congestive heart failure: focus on ACE inhibition and angiotensin-II antagonism.
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One possible intervention to interrupt the deleterious effects of the renin-angiotensin system is suppression of angiotensin II (Ang II) formation by inhibition of angiotensin-converting enzyme (ACE). However, ACE inhibition incompletely suppresses Ang II formation and also leads to accumulation of bradykinin. Angiotensin II type 1 (AT1) receptors are believed to promote the known deleterious effects of Ang II. Therefore, AT1 receptor antagonists have been recently introduced into therapy for hypertension and congestive heart failure (CHF). Although there are significant differences between the effects of AT1 receptor antagonists and ACE inhibitors including the unopposed stimulation of angiotensin II type 2 (AT2) receptors by AT1 receptor antagonists, the discussion of whether ACE inhibitors, AT1 receptor antagonists or the combination of both are superior in the pharmacotherapy of CHF is still largely theoretical. Accordingly, AT1 receptor antagonists are still investigational. Angiotensin-converting enzyme inhibitors remain first line therapy in patients with CHF due to systolic dysfunction. However, in patients not able to tolerate ACE inhibitor induced side effects, in particular cough, AT1 receptor antagonism is a good alternative. In clinical practice, emphasis should be placed on increasing the utilization of ACE inhibitors, as more than 50% of patients with CHF do not receive ACE inhibitors. In addition, the majority of those on ACE inhibitors receive doses lower than the dosage used in the large clinical trials. Although not yet completely proved, it is likely that high doses of ACE inhibition are superior to low doses with respect to prognosis and symptoms. (+info)
Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors.
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1. CHO-K1 cells that were stably transfected with the gene for the human AT1 receptor (CHO-AT1 cells) were used for pharmacological studies of non-peptide AT1 receptor antagonists. 2. In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 3. [3H]-Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT1 antagonists inhibited the angiotensin II (0.1 microM) induced IP accumulation and the binding of [3H]-angiotensin II (1 nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. 5. Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half-maximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 microM losartan indicating a syntopic action of both antagonists. 6. Losartan caused a parallel rightward shift of the angiotensin II concentration-response curves and did not affect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. 7. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT1 receptor antagonists in functional studies was related to their long-lasting inhibition. (+info)
The systemic and renal response to NO inhibition is not modified by angiotensin-II-receptor blockade in healthy humans.
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BACKGROUND: The role of angiotensin II (Ang II) in the systemic and renal responses to acute nitric oxide (NO) synthesis inhibition has not been studied in detail in healthy humans. The purpose of the present study was to investigate the effects of Ang II receptor blockade on the systemic and renal response to acute treat ment with Ng-monomethyl-L-arginine (L-NMMA) in healthy subjects. METHODS: Mean arterial blood pressure (MAP), renal plasma flow (RPF), glomerular filtration rate (GFR), sodium excretion (UNa*V), and plasma levels of renin, Ang II, ANP, BNP, and cGMP were assessed in 15 healthy sodium replete humans before and after acute L-NMMA treatment (3 mg/kg) on two occasions, i.e. after pretreatment with the Ang II type 1 receptor (AT-1) antagonist candesartan cilexetil (CAND; 8 mg) or placebo in a double blind, randomized fashion. Renal haemodynamics were measured during water diuresis using renal clearances of [125I]hippuran and [51Cr]EDTA. Plasma hormones were measured by radioimmunoassays. RESULTS: On both study days L-NMMA treatment induced a significant increase in MAP and a significant decrease in GFR, RPF, and UNa*V. These effects of L-NMMA were not affected significantly by pretreatment with CAND. The effects of L-NMMA on hormones were roughly similar on both occasions with a drop in P-cGMP and U-cGMP. However, a fall in renin was observed only during CAND pretreatment. CONCLUSIONS: We conclude that Ang II is not a major mediator of acute vasoconstriction and sodium retention during acute lowering of NO activity in healthy man. (+info)
Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor.
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Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. We investigated 25 preeclamptic patients and compared them with 12 normotensive pregnant women and 10 pregnant patients with essential hypertension. Antibodies were detected by the chronotropic responses to AT1 receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Immunoglobulin from all preeclamptic patients stimulated the AT1 receptor, whereas immunoglobulin from controls had no effect. The increased autoimmune activity decreased after delivery. Affinity-column purification and anti-human IgG and IgM antibody exposure implicated an IgG antibody directed at the AT1 receptor. Peptides corresponding to sites on the AT1 receptor's second extracellular loop abolished the stimulatory effect. Western blotting with purified patient IgG and a commercially obtained AT1 receptor antibody produced bands of identical molecular weight. Furthermore, confocal microscopy of vascular smooth muscle cells showed colocalization of purified patient IgG and AT1 receptor antibody. The protein kinase C (PKC) inhibitor calphostin C prevented the stimulatory effect. Our results suggest that preeclamptic patients develop stimulatory autoantibodies against the second extracellular AT1 receptor loop. The effect appears to be PKC-mediated. These novel autoantibodies may participate in the angiotensin II-induced vascular lesions in these patients. (+info)
Angiotensin II attenuates renal cortical cyclooxygenase-2 expression.
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We have previously shown that in rat renal cortex, cyclooxygenase-2 (COX-2) expression is localized to cTALH cells in the region of the macula densa, and that dietary salt restriction increases COX-2 expression. Administration of the angiotensin converting inhibitor, captopril, further increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the most pronounced expression in the macula densa. Administration of an AT1 receptor antagonist, losartan, also significantly increased cortical COX-2 mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for both Agtr1a and Agtr1b null mutations (Agtr1a-/-,Agtr1b-/-) demonstrated large increases in immunoreactive COX-2 expression inthe cTALH/macula densa. To determine whether increased COX-2expression in response to ACE inhibition mediated increases in renin production, rats were treated with captopril for one week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the captropril group, and this increase was significantly inhibited by simultaneous treatment with SC58236. Thus, these studies indicated that angiotensin II inhibitors augment upregulation of renal cortical COX-2 in states of volume depletion, suggesting that negative feedback by the renin-angiotensin system modulates renal cortical COX-2 expression and that COX-2 is a mediator of increased renin production in response to inhibition of angiotension II production. (+info)
Bradykinin-stimulated protein synthesis by myocytes is dependent on the MAP kinase pathway and p70(S6K).
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Bradykinin (BK) has a direct hypertrophic effect on rat ventricular cardiomyocytes (VCM) as defined by an increase in protein synthesis and an increase in atrial natriuretic peptide mRNA and secretion. In the current study, we have examined the dependence of BK-induced protein synthesis on activation of 90-kDa ribosomal S6 kinase (p90(rsk)) and 70-kDa S6 kinase (p70(S6K)). Both of these kinases possess the ability to phosphorylate the ribosomal protein S6, which plays an important role in initiating mRNA translation. Stimulation of adult VCM with 10 microM BK increased p90(rsk) activity by 2.5 +/- 0.3-fold and increased p70(S6K) activity by 2.0 +/- 0.3-fold. p90(rsk) is a terminal kinase in the mitogen-activated protein (MAP) kinase pathway. Inhibition of MAP kinase kinase activation by Raf in the MAP kinase pathway with PD-098059 (25 microM) blocked BK-stimulated activation of p90(rsk) by 70% and unexpectedly blocked p70(S6K) by 72%. Rapamycin inhibited BK-stimulated p70(S6K) activity by 93% but had no effect on p90(rsk) activation by BK. Inhibition of the MAP kinase pathway and p70(S6K) with PD-098059 was paralleled by changes in protein synthesis. BK (10 microM) increased [3H]phenylalanine incorporation by 27 +/- 3 and 39 +/- 6% in cultured adult and neonatal VCM, respectively. Treatment with PD-098059 or rapamycin abolished the increase in protein synthesis stimulated by BK. These results suggest that 1) BK activates p70(S6K) and p90(rsk); 2) although both p70(S6K) and p90(rsk) have the potential to phosphorylate the ribosomal S6 protein, p70(S6K) and not p90(rsk) is the predominant kinase involved in increasing protein synthesis by BK; and 3) p70(S6K) activation is dependent on stimulation of the MAP kinase pathway at a point distal to Raf. (+info)