Are polyamines involved in the contractile effects of angiotensin II in the rat aorta? (Polyamines and angiotensin II in rat aorta).

Angiotensin II (AII) is a central factor involved in the pathophysiology of arterial hypertension and atherosclerosis. On the other hand, polyamines represent a family of organic cations with low molecular weight, playing intracellular regulatory roles essential for the cellular growth and differentiation. The cellular contents, the synthesis and the transport of polyamines are increased following the actions of AII, as well as of other cellular growth factors. Our results show that the administration of polyamines as pre-treatment modulates the contractile effects of extracellular AII (80 nM). This modulation is concentration-dependent and dual: the lower concentrations amplify and the higher concentrations reduce the effects of AII in the isolated rat aorta rings without endothelium. Moreover, DL-alpha-Difluoromethylomithine (DFMO), a specific inhibitor of ornithine decarboxylase, does not significantly modify the contractile effects of AII. Thus, these data suggest that polyamines generated through this metabolic pathway are not involved in the contractile effects of AII in rat aortic vascular smooth muscle.     

Endothelium modulates contractile response to simvastatin in rat aorta

Simvastatin is an inhibitor of HMG-CoA reductase used in the treatment of hypercholesterolemia. In the present study simvastatin-induced contraction was observed in rat aortic thoracic rings, this effect increased when the endothelium was removed and when NO synthase was blocked by L-NOARG (3 x 10(-5) M). The contractile effect of simvastatin on intact aortic rings diminished when cyclo-oxygenase was inhibited with indomethacin (10(-5) M). Also in the presence of endothelium, pretreatment with mevalonate (1 mM), the product of HMG-CoA reductase activity, significantly inhibited the contraction. In other experiments carried out on endothelium-removed preparations and in medium containing the calcium antagonist, diltiazem (10(-5) and 10(-6) M), the contraction dose-response curves were significantly reduced and the same happened in the presence of the inhibitor of sarcoplasmic reticulum Ca-2+-ATPase, cyclopiazonic acid (CPA) (3 x 10(-6) M). The results suggest that simvastatin might increase intracellular calcium concentration. This effect could lead to an activation of NO synthase and cyclooxygenase pathways in endothelial cells and to contraction in vascular smooth muscle cells. This rise in Ca2+ concentration could be due to an inhibition of isoprenoid synthesis prevented by mevalonate.     

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.     

17-Octadecynoic acid improves contractile response to angiotensin II by releasing vasocontrictor prostaglandins

The present study investigated the role of CYP-enzymes in the modulation of vasoconstrictor responses to angiotensin II in rabbit aortae. In arteries with the endothelium-intact (E+) the CYP-inhibitor, 17-octadecynoic acid (17 ODYA), increased the efficacy to angiotensin II (17-ODYA-effect) as well as simultaneous incubation with miconazole (epoxygenase-inhibitor) and CAY 10434 (ω-hydroxylase-inhibitor). The removal of endothelium (E-) caused potentiation of the 17 ODYA-effect. Therefore, endothelium-dependent and -independent mechanisms would be involved. 17-ODYA and miconazole reduced Ach-relaxation. Indomethacin blocked the 17-ODYA-effect in E+ and E- arteries but blunted the response to angiotensin II only in E+ arteries. NS 398 (cyclooxygenase-2-inhibitor) blocked the 17-ODYA-effect and reduced angiotensin II affinity as well as SQ 29548 (thromboxane-prostanoid (TP) receptor-inhibitor). In E- arteries, CAY 10434 enhanced angiotensin II response as well as 17-ODYA. SC 560 (cyclooxygenase-1-inhibitor) and NS 398 partially blocked the 17-ODYA-effect. In conclusion, 17-ODYA induced endothelial dysfunction by inhibiting CYP-epoxygenase and thus improves vasoconstrictor cyclooxygenase-2 metabolites release acting through TP receptors. The endothelium-independent mechanism of 17-ODYA-effect may involve increase of vasoconstrictor cyclooxygenase-metabolites induced by prostaglandin-ω-hydroxylase-inhibition.     

Effect of phorbol ester on contractile response of aorta from endotoxic rats

The influence of phorbol ester on the isometric contractile response of aorta from endotoxic rats was examined. In endotoxic rat aorta, the contractile responses to KCl and phorbol 12,13-dibutyrate (PDBu) were both remarkably diminished, compared to those in control rat aorta. Preincubation with PDBu augmented the aortic contractile response to KCl in both control and endotoxic rats. This augmentative effect of PDBu was significantly more pronounced in endotoxic rats than in controls. When the contractile response to 80 mM KCl reached a plateau after PDBu pretreatment, addition of 5 mM CaCl2 (final concentration) to the organ bath completely reversed the diminished contractile response of endotoxic rat aorta to the control level. These results suggest that the hyporesponsiveness of endotoxic rat aorta to KCl may be caused by decreases in both protein kinase C mediated response and calcium sensitivity of vascular smooth muscle cells.     

ERK activation contributes to regulation of spontaneous contractile tone via superoxide anion in isolated rat aorta of angiotensin II-induced hypertension

Arteries from hypertensive animals and humans have increased spontaneous tone. Increased superoxide anion (superoxide) contributes to elevated blood pressure (BP) and spontaneous tone in hypertension. The association between the extracellular signaling-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK) signaling pathway and generation of superoxide and spontaneous tone in isolated aorta was studied in angiotensin II (ANG II)-infused hypertensive (HT) rats. Systolic BP, phosphorylation of ERK, aortic superoxide formation, and aortic spontaneous tone were compared in sham normotensive and HT rats. Infusion of ANG II (0.5 mg x kg(-1) x day(-1) for 6 days) significantly elevated the systolic BP (P<0.01). The phosphorylation of ERK1/2 vs. total ERK1/2 in thoracic aorta was enhanced, and superoxide was increased in the HT vs. the sham group (P<0.01). Spontaneous tone developed in the HT group, but not in the normotensive group. MAPK/ERK1/2 (MEK1/2)-ERK1/2 signaling pathway inhibitors, PD-98059 (10 micromol/l), and U-0126 (10 micromol/l), significantly reduced the phosphorylation of ERK1/2, superoxide generation (P<0.01), and spontaneous tone (P<0.01) in HT. These findings suggest that ANG II infusion induces the production of superoxide and spontaneous tone and that both are dependent on ERK-MAPK activation. In endothelium-denuded aorta, however, MEK1/2 inhibitors did not inhibit the spontaneous tone, even though they significantly reduced superoxide generation similar to endothelium-intact aorta. These data suggest that inhibition of ERK1/2 signaling pathway, via PD-98059 or U-0126, may regulate spontaneous tone in an endothelium-dependent manner. In conclusion, these findings support the importance of the ERK1/2 signaling pathway in modulating vascular oxidative stress and subsequently mediating spontaneous tone in HT.     

Effect of rat cardionatrin I (rat ANF 99-126) on the response of toad skin to angiotensin II

The atrial natriuretic peptide cardionatrin I (cardionatrin I is ANF 99-126) was used in studies directed to assess its effects on osmotic water permeability (Posm) and short-circuit current (SCC) in isolated toad skin. Results showed that ANF 99-126 (10(-7) M) added to the dermal side of the skin had no effect on basal Posm or SCC. However, ANF 99-126 (3.3 x 10(-8) M) was able to produce a 50% reversible inhibition of the maximal Posm response to angiotensin II (AII) (3.2 x 10(-8) M). These effects were seen when the skins were preincubated with ANF 99-126 for 10 min or less before the addition of AII. Longer preincubation appeared to inactivate ANF 99-126 through proteolysis. ANF 99-126(10(-7) M) failed to inhibit the SCC response to AII (10(-5) M) in toad skin. These results are compatible with a modulatory function for ANF on several systems including those involved in the regulation of extracellular fluid volume.     

Effect of aldosterone on vascular angiotensin II receptors in the rat

The effect of aldosterone on the density and affinity of binding sites for 125I-labelled angiotensin II was investigated in a particulate fraction prepared from the rat mesenteric arteriolar arcades. The infusion of aldosterone 6.6 micrograms/h intraperitoneally via Alzet osmotic minipumps for 6 d produced an increase in the density of binding sites for 125I-labelled angiotensin II without change in affinity. After sodium depletion, mesenteric artery angiotensin II receptors were down-regulated as expected. An increase in the number of binding sites could be found when aldosterone was infused into sodium-depleted rats with no change in the elevated plasma renin activity. The intraperitoneal infusion of angiotensin II (200 ng X kg-1 X min-1 for 6 d) simultaneously with aldosterone resulted in down-regulation of vascular angiotensin II receptors, whereas after intravenous angiotensin II infusion (at 60 ng X kg-1 X min-1) the density of angiotensin II binding sites rose with aldosterone infusion. Plasma renin activity (PRA) was reduced and plasma angiotensin II increased in a dose-dependent fashion after angiotensin II infusion. An aldosterone concentration of 3 ng/mL for 18 h produced an increase in the number of angiotensin II binding sites in rat mesenteric artery smooth muscle cells in culture. We conclude that increased plasma aldosterone may result in up-regulation of vascular angiotensin II receptors independently of changes in plasma renin activity, and may in certain physiological states effectively antagonize the down-regulating action of angiotensin II.     

Increased local angiotensin II formation in aneurysmal aorta

We investigated the levels and locations of angiotensin II-forming enzymes, angiotensin converting enzyme (ACE) and chymase, in aneurysmal and normal aortas. Aneurysmal aortic specimens (n = 14) were obtained at the time of operative aneurysm repair from 14 patients ranging in age from 57 to 84 y. Normal aortic specimens (n = 16) were obtained from 16 patients (48 to 72 y) who underwent coronary artery bypass surgery. The ACE and chymase activities were determined using each specimen. Sections of each specimen were immunostained with antibodies for ACE and chymase. The ACE activities in the aneurysmal and normal aortas were 0.82 +/- 0.10 and 0.14 +/- 0.05 mU/mg protein, respectively, and this difference was significant. The chymase activities in the aneurysmal and normal aortas were 17.9 +/- 2.40 and 1.02 +/- 0.18 mU/mg protein, respectively, and this difference was also significant. In the aneurysmal aorta, ACE-positive cells were detected with macrophages in the intima and media and chymase-positive cells were detected with mast cells in the media and adventitia, whereas positive ACE and chymase cells in the normal aorta were located only in the endothelium and adventitia, respectively. Angiotensin II-forming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased angiotensin II may be associated with the development of aneurysmal formations.     

Aldosterone response to angiotensin II during hypoxemia

Exercise in humans causes increases in plasma renin activity (PRA) and plasma aldosterone concentrations (PAC) except when performed at high altitude or while the subjects breathe hypoxic gas. Under those conditions, PRA increases with exercise but PAC does not. We speculated that the PAC suppression during hypoxemic exercise was due to hypoxemia-induced release of a circulating inhibitor of angiotensin II-mediated aldosterone secretion. To test this hypothesis, we measured the PAC response to graded infusions of angiotensin II during hypoxemia and normoxemia. Eight normal volunteers were given increasing doses of angiotensin II (first 2 ng X kg-1 X min-1 and then 4, 8, and finally 12 ng X kg-1 X min-1, each for 20-min periods) on 2 separate days, once while breathing room air and the other day while breathing hypoxic gas adjusted to maintain the subjects' hemoglobin saturation at 90%. The PAC response to different doses of angiotensin II did not significantly differ during hypoxemia from normoxemia. We conclude that our model of hypoxemia does not cause release of an inhibitor of angiotensin II-mediated aldosterone release.     

Potentiation of pressor response to angiotensin II at the preoptic area in spontaneously hypertensive rat

Cardiovascular responses to angiotensin II(AII) at the preoptic area (POA) were compared between normotensive Wistar Kyoto rat (WKY) and spontaneously hypertensive rat(SHR) by measuring blood pressure and heart rate under unrestrained, conscious state via a catheter implanted chronically into the abdominal aorta and by injection of drugs into POA through a chronic guide cannula. AII injected into POA at doses of 0.3 ng and 1 ng produced a dose-dependent pressor response, accompanied with a slight decrease of heart rate, in both WKY and SHR. However, in SHR, the pressor response to AII was more than 2 times greater than that in WKY and was quick in onset and lasted about 30 min. When AII in combination with [Sar1, Ile8]-angiotensin II (0.5 microgram), an AII receptor antagonist, were simultaneously administered to POA, the pressor response to AII was strongly inhibited in both WKY and SHR. The results suggest that the pressor response to AII due to its receptor stimulation at POA is markedly potentiated in SHR.     

Differential response of human cardiac fibroblasts to angiotensin I and angiotensin II

The vasoactive peptide angiotensin II (Ang II) has been implicated as a mediator of myocardial fibrosis. We carried out a comparative investigation of the effects of Ang II and its precursor Ang I on collagen metabolism and proliferation in cultured human cardiac fibroblasts. Cardiac fibroblasts responded to both Ang I and Ang II with concentration-dependent increases in collagen synthesis but no proliferation. The stimulatory effect of Ang II was abolished by the AT(1) receptor antagonist losartan but not the AT(2) receptor antagonist PD123319. The response to Ang I was not affected by either antagonist, nor by the angiotensin-converting enzyme (ACE) inhibitor captopril. In conclusion, Both Ang I and Ang II stimulate collagen synthesis of human cardiac fibroblasts, the effect of Ang II occurring via the AT(1) receptor whilst Ang I appears to exert a direct effect through non-Ang II-dependent mechanisms. These results suggest distinct roles for angiotensin peptides in the development of cardiac fibrosis.