Angiotensin II-induced constrictions are masked by bovine retinal vessels.
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PURPOSE: To unmask the vasoconstricting effect of angiotensin II (Ang II) on retinal smooth muscle by studying its interaction with endothelium-derived paracrine substances. This study focused specifically on determining the changes in vascular diameter and the release of endothelial-derived vasodilators, nitric oxide (NO) and prostaglandin (PG) I2, from isolated retinal microvessels. METHODS: Bovine retinal central artery and vein were cannulated, and arterioles and venules were perfused with oxygenated/heparinized physiological salt solution at 37 degrees C. This ex vivo perfused retinal microcirculation model was used to observe the contractile effects of Ang II on arterioles and venules of different diameters. The NO and PGI2 synthase inhibitors, 1-NOARG and flurbiprofen, respectively, were used to unmask Ang II vasoconstriction; the changes in vascular diameters were then measured. Enzyme immunoassays were used to measure the release of cGMP (an index of NO release) and 6-keto-PG-F1alpha (a stable metabolite of PGI2) from isolated bovine retinal vessels. RESULTS: Topically applied Ang II (10(-10) M to 10(-4) M) caused significant (P < 0.05) arteriolar and venular constrictions in a dose-dependent manner, with the smallest retinal arterioles (7+/-0.2 microm luminal diameter) and venules (12+/-2 microm luminal diameter) significantly more sensitive than larger vessels. After the inhibition of endogenous NO and PGI2 synthesis by 1-NOARG and flurbiprofen, respectively, the vasoconstriction effects of Ang II became more pronounced. Again, the smallest vessels tested were significantly more sensitive, and synthesis of endothelial-derived relaxing factor (EDRF), therefore, may be most important in these vessels. Vasoactive doses of Ang II (10(-10) M to 10(-4) M) caused a dose-dependent increase in the release of NO and PGI2 from isolated bovine retinal vessels, indicating that the increase in EDRF may nullify direct Ang II-induced vasoconstriction. Interestingly, intraluminal administration of Ang II caused only vasodilation. CONCLUSIONS: This study demonstrates that the retinal vascular endothelium acts as a buffer against the vasoconstricting agent Ang II via release of vasodilators NO and PGI2, and the vasoconstriction effects due to Ang II are most prominent in the smallest diameter vessels. (+info)
Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta.
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1. We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. 2. Compounds were delivered into the cytoplasm of de-endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10(-5) M). 3. Intracellular administration of Ang II (incorporation range: 0.01-300 nmol mg(-1)) resulted in a dose-dependent contraction, insensitive to extracellular administration (10(-6) M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non-selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]-Ang II (P<0.05). 4. Intracellular administration of CV11947 or PD 123319 right shifted the dose-response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30 nmol mg(-1) Ang II was incorporated. 5. Contraction was partially desensitized to a second intracellular Ang II addition after 45 min (P<0.05). 6. Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. 7. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre-treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction. 8. Intracellular Ang II induced contraction was not affected by pre-treatment with heparin filled liposomes, but completely abolished in Ca2+-free external medium. 9. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+-influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+-stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue. (+info)
Angiotensin II induces apoptosis in human and rat alveolar epithelial cells.
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Recent work from this laboratory demonstrated potent inhibition of apoptosis in human alveolar epithelial cells (AECs) by the angiotensin-converting enzyme inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998]. On this basis, we hypothesized that apoptosis in this cell type might be induced by angiotensin II (ANG II) through its interaction with the ANG II receptor. Purified ANG II induced dose-dependent apoptosis in both the human AEC-derived A549 cell line and in primary type II pneumocytes isolated from adult Wistar rats as detected by nuclear and chromatin morphology, caspase-3 activity, and increased binding of annexin V. Apoptosis also was induced in primary rat AECs by purified angiotensinogen. The nonselective ANG II-receptor antagonist saralasin completely abrogated both ANG II- and angiotensinogen-induced apoptosis at a concentration of 50 microgram/ml. With RT-PCR, both cell types expressed the ANG II-receptor subtypes 1 and 2 and angiotensin-converting enzyme (ACE). The nonthiol ACE inhibitor lisinopril blocked apoptosis induced by angiotensinogen, but not apoptosis induced by purified ANG II. These data demonstrate the presence of a functional ANG II-dependent pathway for apoptosis in human and rat AECs and suggest a role for the ANG II receptor and ACE in the induction of AEC apoptosis in vivo. (+info)
Proliferation of aortic smooth muscle cells and renin-angiotensin system in SHR rats.
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AIM: To study the relationship between the enhanced proliferation and renin-angiotensin system (RAS) of aortic smooth muscle cells (ASMC) from SHR rats. METHODS: To measure the effects of angiotensin II (Ang), captopril (Cap), saralasin (Sar) on proliferation, Ang and angiotensin converting enzyme (ACE) levels in cultured ASMC from WKY and SHR rats. RESULTS: Ang was a bifunctional growth factor, which induced SHR ASMC hyperplasia in 2% FCS-RPMI 1640 medium, but not in serum free (SF)-medium. SHR ASMC had stronger proliferative ability compared with WKY while SHR ASMC RAS was activated. Enhanced proliferation of SHR ASMC and ACE activity were obviously inhibited by long-term treatment (4-wk) of both Cap and Sar, while Ang content decreased in Cap treatment group and increased in Sar treatment group. The antiproliferative effect of Cap and Sar on SHR ASMC was stronger than that on WKY. SHR, WKY ASMC RAS were not influenced by short-term (24 h) treatment of Cap. CONCLUSION: Long-term treatment of Cap and Sar suppressed SHR ASMC growth through inhibition of Ang generation or blockade of Ang binding to its receptor. (+info)
Activation of AT(2) receptors by endogenous angiotensin II is involved in flow-induced dilation in rat resistance arteries.
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Pressure-induced tone (myogenic, MT) and flow (shear stress)-induced dilation (FD) are potent modulators of resistance artery tone. We tested the hypothesis that locally produced angiotensin II interacts with MT and FD. Rat mesenteric resistance arteries were perfused in situ. Arterial diameter was measured by intravital microscopy after a bifurcation on 2 distal arterial branches equivalent in size (150 microm, n=7 per group). One was ligated distally and thus submitted to pressure only (MT, no FD). The second branch was submitted to flow and pressure (MT and FD). The difference in diameter between the 2 vessels was considered to be FD. Flow-diameter-pressure relationship was established in the absence and then in the presence of 1 of the following agents. In the nonligated segment (MT+FD), angiotensin II type 1 (AT(1)) receptor blockade (losartan) had no significant effect, whereas angiotensin II type 2 (AT(2)) receptor blockade (PD123319) or saralasin (AT(1)+AT(2) blocker) decreased the diameter significantly, by 9+/-1 and 10+/-0.8 microm, respectively. Angiotensin II in the presence of losartan increased the diameter by 18+/-0.6 microm (inhibited by PD123319). PD123319 or saralasin had no effect after NO synthesis blockade or after endothelial disruption. In the arterial segment ligated distally (MT only), AT(1) or AT(2) receptor blockade had no significant effect. AT(2)-dependent dilation represented 20% to 39% of FD (shear stress from 22 to 37 dyn/cm(2)), and AT(2)-receptor mRNA was found in mesenteric resistance arteries. Thus, resistance arterial tone was modulated in situ by locally produced angiotensin II, which might participate in flow-induced dilation through endothelial AT(2) receptor activation of NO release. (+info)
Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides.
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Earlier work from this laboratory found that fibroblasts isolated from fibrotic human lung [human interstitial pulmonary fibrosis (HIPF)] secrete a soluble inducer(s) of apoptosis in alveolar epithelial cells (AECs) in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. The cultured human fibroblast strains most active in producing the apoptotic activity contained high numbers of stellate cells expressing alpha-smooth muscle actin, a myofibroblast marker. The apoptotic activity eluted from gel-filtration columns only in fractions corresponding to proteins. Western blotting of the protein fraction identified immunoreactive angiotensinogen (ANGEN), and two-step RT-PCR revealed expression of ANGEN by HIPF fibroblasts but not by normal human lung fibroblasts. Specific ELISA detected angiotensin II (ANG II) at concentrations sixfold higher in HIPF-conditioned medium than in normal fibroblast-conditioned medium. Pretreatment of the concentrated medium with purified renin plus purified angiotensin-converting enzyme (ACE) further increased the ELISA-detectable ANG II eightfold. Apoptosis of AECs in response to HIPF-conditioned medium was completely abrogated by the ANG II receptor antagonist saralasin (50 microg/ml) or anti-ANG II antibodies. These results identify the protein inducers of AEC apoptosis produced by HIPF fibroblasts as ANGEN and its derivative ANG II. They also suggest a mechanism for AEC death adjacent to HIPF myofibroblasts [B. D. Uhal, I. Joshi, C. Ramos, A. Pardo, and M. Selman. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1192-L1199, 1998]. (+info)
Interaction between Mas and the angiotensin AT1 receptor in the amygdala.
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The Mas-protooncogene is a maternally imprinted gene encoding an orphan G protein-coupled receptor expressed mainly in limbic structures of the rodent CNS. Because Mas and the product of the Mas-related gene enhance the effects of angiotensins on cells expressing angiotensin receptors of the AT1 subtype, we first compared the distribution of cells expressing AT1 receptors in different limbic and thalamic brain structures in Mas-knockout mice and in wildtype mice by an immunohistochemical approach. No significant differences could be found between the two strains. The Mas-protooncogene seems to be implicated in the signal transduction of angiotensin receptors and is expressed in the amygdala. Therefore we then analyzed whether field potentials are altered by angiotensin II in brain slices of the basolateral amygdala. An opposite action of angiotensin II was obtained in mice lacking the Mas-protooncogene in comparison to wildtype mice. The use of different angiotensin receptor antagonists provides the first in vitro evidence for a functional interaction between the Mas-protooncogene and the AT1 receptor. (+info)
Role of adrenal renin-angiotensin system in the control of aldosterone secretion in sodium-restricted rats.
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This study examined the effect of the pharmacological manipulation of adrenal renin-angiotensin system (RAS) on aldosterone secretion from in situ perfused adrenals of rats kept on a normal diet and sodium restricted for 14 days. Neither the angiotensin-converting enzyme inhibitor captopril nor the nonselective angiotensin II receptor antagonist saralasin and the AT(1) receptor-selective antagonist losartan affected basal aldosterone output in normally fed rats. In contrast, they concentration dependently decreased aldosterone secretion in sodium-restricted animals, with maximal effective concentration ranging from 10(-7) to 10(-6) M. Captopril (10(-6) M), saralasin (10(-6) M), and losartan (10(-7) M) counteracted aldosterone response to 10 mM K(+) in sodium-restricted rats but not in normally fed animals. Collectively, these findings provide evidence that adrenal RAS plays a role in the regulation of aldosterone secretion, but only under conditions of prolonged stimulation of zona glomerulosa probably leading to overexpression of adrenal RAS. (+info)