Anti-inflammatory effect of spironolactone on human peripheral blood mononuclear cells

We evaluated the effect of alacepril, CV-11974, and spironolactone on the production of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) in cultured human peripheral blood mononuclear cells stimulated with angiotensin (Ang) II. Alacepril, CV-11974, and spironolactone significantly reduced the enhanced production of MCP-1 and TNF-alpha induced by exogenous Ang II. Specifically, 10 muM of spironolactone significantly reduced cytokine production, compared to the same dose of alacepril or CV-11974. These findings indicate that spironolactone may contribute to ameliorate the prognosis of patients with cardiovascular diseases by reducing Ang II-induced inflammation, although further exploration including determining the mechanisms would be required.     

Evidence for the AT1 subtype of the angiotensin II receptor in the rat submandibular gland

We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [3H]Ang II binding to the membrane fractions exhibited both high (Kd =0.08 nm, Bmax =2.19 fmol/mg protein) and low (Kd =4.19 nm, Bmax = 13.7 fmol/mg protein) affinity. Ang 11, Ang III and saralasin completely displaced the [3H]Ang II binding, whereas CV-11974, an AT1 receptor antagonist and PD123319, an AT2 receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [3H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a Kd of 4.22 nM and Bmax of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [3H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT1 receptor subtype.     

Angiotensin II type 2 receptor-mediated inhibition of norepinephrine release in isolated rat hearts

We investigated whether endogenous and exogenous angiotensin II (Ang II) regulates norepinephrine (NE) release from cardiac sympathetic nerves via both Ang II type 2 receptors (AT2Rs) and Ang II type 1 receptors (AT1Rs). Using isolated rat hearts, sympathetic nerves were electrically stimulated. Ang II with PD-123319 (AT2R antagonist) but not Ang II alone produced a significant increase in nerve stimulation-induced NE overflow, which was abolished by the addition of AT1R antagonist losartan. In contrast, NE overflow was markedly decreased by losartan with or without Ang II. This decrease was abolished by the combination with PD-123319, nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (NOARG), icatibant (bradykinin B2 receptor antagonist), or PKSI-527 (kininogenase inhibitor). CGP-42112A (AT2R agonist) suppressed nerve stimulation-induced NE overflow in the same way as the combination of Ang II and losartan, and this suppression was abolished by PD-123319, NOARG, icatibant, or PKSI-527. There were significant increases in NOx (NO2/NO3) contents in coronary effluent under conditions where NE overflow was suppressed. Ang II seems to function as an inhibitory modulator of cardiac noradrenergic neurotransmission via AT2Rs and well-known AT1R-mediated stimulatory actions. The inhibitory mechanism may involve local bradykinin production, its B2 receptor activation, and NO as a downstream effector.     

Effects of angiotensin II on the renal interstitial concentrations of NO2/NO3 and cyclic GMP in anesthetized rats

The present study was conducted to determine whether exogenous angiotensin II (Ang II) may increase the renal interstitial fluid concentrations of NO2/NO3 (NOx) and cyclic guanosine monophosphate (cGMP) concomitantly and which Ang II receptor subtypes may induce these changes in anesthetized rats, using a microdialysis method. Ang II (50 ng/kg per min, i.v.) significantly increased mean blood pressure (MBP), extraction rates of renal interstitial NOx from 23.9+/-1.0 to 31.2+/-1.9 pmol/min, and cGMP from 4.1+/-0.3 to 6.4+/-0.5 fmol/min, and decreased renal blood flow (RBF). The AT1-receptor antagonist CV11974 alone significantly increased RBF, but did not alter MBP, renal interstitial concentrations of NOx and cGMP. A superimposition of Ang II on CV11974 did not affect MBP and RBF, but significantly increased renal interstitial concentrations of NOx and cGMP. The AT2-receptor antagonist PD123319 alone did not change any of the parameters. However, superimposition of Ang II on PD123319 increased MBP and decreased RBF without any effects on renal interstitial concentrations of NOx and cGMP. These results suggest that Ang II stimulates NO production via the AT2-receptor in the kidney.     

Participation of angiotensin receptors in acute hypoxia in mice. II. Effects of angiotensin II nonpeptide receptor ligands losartan (DuP-753) and PD-123319.

The effects of intracerebroventricularly (i.c.v.) administered angiotensin nonpeptide receptor ligands losartan (DuP-753) and PD-123319 and their interactions with Ang II were examined on acute anoxic hypoxia in male mice. Results showed that losartan and PD-123319 exerted antihypoxic effects including increased latency to convulsive seizures and survival time and pretreatment with both drugs enhanced Ang II-increased survival time. Taken together, the results suggest that the antihypoxic effect of losartan and PD-123319 is most likely due to action on brain AT1 and AT2 receptors with both drugs behaving as partial agonists.     

The inhibitory effect of KT3-671, a nonpeptide angiotensin-receptor antagonist, on rabbit and rat isolate vascular smooth muscles: a possible involvement of K(ATP) channels

The vasoinhibitory effect of KT3-671, a recently synthesized nonpeptide angiotensin II (Ang II), AT1-receptor antagonist, and the factors affecting insurmountable antagonism of Ang II were examined in rabbit and rat isolated vascular smooth muscle preparations. In rabbit and rat aortic rings, KT3-671 caused insurmountable antagonism of Ang II. In addition, KT3-671 inhibited contractile responses to angiotensin III (Ang III). In rabbit isolated smooth muscles, KT3-671 was most effective in reducing the maximal contraction induced by Ang II in the renal artery followed by the basilar artery and the aorta. In rat renal arterial rings, KT3-671 (10(-5) M) inhibited the concentration-response curves of prostaglandin F2alpha and STA2. In rabbit and rat aortic rings without endothelium, the insurmountable antagonisms of Ang II by KT3-671 and EXP 3174 were changed to surmountable antagonism by pretreatment with DuP 753 and KT3-671, respectively. In addition, KT3-671 abolished the inhibitory effect of CV- 11974 in the rat aorta but not in the rabbit aorta. Indomethacin (10(-5) M) or the removal of endothelium did not affect the inhibitory effect of Ang II by CV-11974 or EXP 3174 but enhanced the insurmountable antagonism by KT3-671. ODQ (3 x 10(-6) M), N(G)-nitro-L-arginine (3 x 10(-4) M), 4-aminopyridine (3 x 10(-3) M), tetraethylammonium (TEA; 10(-3) M), or iberiotoxin (10(-7) M) did not affect the inhibitory action of KT3-671 or CV-11974. Methylene blue (3 x 10(-6) M), KCl (10(2) M), TEA (10(-2) M), or BaC12 (10(-4) M) changed the insurmountable antagonism by KT3-671 to surmountable antagonism and abolished the inhibitory effect of CV-11974. However, glibenclamide (3 x 10(-6) M) did not affect the inhibitory action of KT3-671 but reduced the insurmountable antagonism by CV- 11974. These results indicate that KT3-671 is an insurmountable antagonist of Ang II in the rabbit and rat aorta. The results in the rat aorta also suggest that K(ATP) channels may be involved in insurmountable antagonism of Ang II by KT3-671 and CV-11974. Key Words: KT3-671-Rabbit-Rat-Vascular smooth muscle-Angiotensin II-Insurmountable antagonist-K(TP)channels.     

Role of angiotensin in cardioprotective effect of ischemic preconditioning

This study was designed to investigate the role of angiotensin (Ang II) in the cardioprotective effect of ischemic preconditioning. Isolated perfused rat heart was subjected to global ischemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK) release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically by using triphenyl tetrazolium chloride (TTC) staining. Four episodes of ischemic/Ang II preconditioning markedly reduced LDH and CK release in the coronary effluent and decreased myocardial infarct size. The cardioprotective effect of Ang II preconditioning was abolished by CV 11974, AT1-receptor antagonist, whereas no such effect was noted with CV 11974 in ischemic preconditioning. PD 123319, AT2-receptor antagonist, produced no marked effect on Ang II preconditioning and ischemic preconditioning induced reduction in myocardial injury. On the basis of these results, it may be concluded that activation of AT1 receptors may be involved in angiotensin-induced pharmacologic preconditioning. But it may not be involved in the cardioprotective effect of ischemic preconditioning in isolated rat heart.     

Changes of gene expression profiles in macrophages stimulated by angiotensin II--angiotensin II induces MCP-2 through AT1-receptor.

INTRODUCTION: Macrophages play critical roles in the development of atherosclerosis and diabetic nephropathy as well as many inflammatory diseases. Angiotensin II type 1 receptor antagonists (AIIA) are beneficial for the prevention of atherosclerosis and diabetic nephropathy suggesting that angiotensin II (Ang II) promotes the development of these diseases. It has recently been reported that Ang II exerts proinflammatory actions in vivo and in vitro. This study was aimed to clarify the direct effects of Ang II on monocytes/macrophages. MATERIALS AND METHODS: PMA-treated THP-1 cells, a human monocytic leukaemia cell line, were treated with Ang II (10-6 mol/L) for 24 hours with or without AIIA (CV11974). We evaluated gene expression profiles of these cells using DNA microarray system and quantified them by real-time RT-PCR. RESULTS: DNA microarray revealed that in total 19 genes, including monocyte chemoattractant protein (MCP)-2, were up-regulated by Ang II and down-regulated by AIIA. Real-time RT-PCR showed that up-regulation of MCP-2 with Ang II is blocked by the AIIA (CV11974) but not by an AT2-receptor antagonist. CONCLUSIONS: These results suggest that Ang II directly stimulates MCP-2 expression through AT1-receptors in activated macrophages. Ang II may contribute to the persistence or amplification of microinflammation in vessel walls, heart and kidney. Vasculoprotective or renoprotective effects of AIIA might partly depend on direct anti-inflammatory effects on macrophages.     

A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells.

In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [Sar1,Ile8]Ang II but not by the AT1 antagonist DuP 753 or the AT2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang II receptor subtype in differentiated Neuro-2A cells.     

Differential regulation by AT(1) and AT(2) receptors of angiotensin II-stimulated cyclic GMP production in rat uterine artery and aorta

1. In the present study we determined whether angiotensin II (Ang II) could increase cyclic GMP levels in two blood vessels that exhibit markedly different angiotensin II receptor subtype expression: rat uterine artery (UA; AT(2) receptor-predominant) and aorta (AT(1) receptor-predominant), and investigated the receptor subtype(s) and intracellular pathways involved. 2. UA and aorta were treated with Ang II in the absence and presence of losartan (AT(1) antagonist; 0.1 microm), PD 123319 (AT(2) antagonist; 1 microm), NOLA (NOS inhibitor; 30 microm), and HOE 140 (B(2) antagonist; 0.1 microm), or in combination. 3. Ang II (10 nm) induced a 60% increase in UA cyclic GMP content; an effect that was augmented with PD 123319 and HOE 140 pretreatment, and abolished by cotreatment with losartan, as well as by NOLA. 4. In aorta, Ang II produced concentration-dependent increases in cyclic GMP levels. Unlike effects in UA, these responses were abolished by PD 123319 and by NOLA, whereas losartan and HOE 140 caused partial inhibition. 5. Thus, in rat UA, Ang II stimulates cyclic GMP production through AT(1) and, to a less extent, AT(2) receptors. In rat aorta, the Ang II-mediated increase in cyclic GMP production is predominantly AT(2) receptor-mediated. In both preparations, NO plays a critical role in mediating the effect of Ang II, whereas bradykinin has differential roles in the two vessels. In UA, B(2) receptor blockade may result in a compensatory increase in cyclic GMP production, whilst in aorta, bradykinin accounts for approximately half of the cyclic GMP produced in response to Ang II.     

Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta

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.     

Prejunctional and postjunctional inhibitory actions of eprosartan and candesartan in the isolated rabbit mesenteric artery

Effects of angiotensin II type 1 (AT1) receptor antagonists eprosartan and candesartan and AT2 receptor antagonist PD123319 on Ang II-induced facilitation of noradrenergic neurotransmission were investigated in isolated rabbit mesenteric artery under isometric conditions. Sympathoinhibitory potency of AT1 blockers was compared with their potency concerning inhibition of direct vasoconstrictor effect of Ang II. To investigate blockade of presynaptic AT1 and AT2 receptors, effects of Ang II on electrical field stimulation (EFS)-induced contractions in presence or absence of eprosartan, candesartan, or PD123319 were studied. To investigate blockade of postsynaptic AT1 receptors, effects of either eprosartan or candesartan on concentration-response curves of Ang II were studied. In addition, effect of Ang II on postsynaptic alpha-adrenoceptor-mediated responses was studied using noradrenaline. EFS (1, 2, and 4 Hz) caused an increase of contractile force. At stimulation frequencies of 1, 2, and 4 Hz, a subpressor concentration of Ang II (0.5 nM) increased stimulation-induced vasoconstrictor responses by 2.8 +/- 0.5, 2.4 +/- 0.4, and 1.6 +/- 0.1 of control values, respectively (p < 0.05 compared with control for all frequencies). The enhancement could be antagonized by eprosartan (1 nM-0.1 microM) and candesartan (1 nM-0.1 microM). The AT2 antagonist PD123319 (10 nM) did not influence Ang II-induced facilitation of stimulation-induced contractions. Contractile responses to exogenous noradrenaline were unaltered in presence of Ang II 0.5 nM. Ang II (1 nM-0.3 microM) caused a concentration-dependent increase in contractile force, which could be antagonized by eprosartan (pD2; 8.8 +/- 0.19) and candesartan (pD2; 11.3 +/- 0.23). Thus, the facilitating effect of Ang II on noradrenergic neurotransmission is mediated by presynaptically located AT1 receptors and not by AT2 receptors. For eprosartan, sympathoinhibition was achieved at concentrations that also block AT1 receptors on vascular smooth muscle. In contrast, for candesartan, presynaptic inhibitory concentrations were considerably higher than those required for postsynaptic inhibition.     

Prejunctional AT(1) receptor subtype-dependent modification of neurotransmitter releases in canine isolated splenic arteries

1. The regulation by angiotensin II (Ang II) formed locally on nerve-stimulated purinergic and adrenergic components of double-peaked vasoconstrictions in the canine splenic artery and Ang II receptor subtypes involved were investigated. 2. The perfusion of the precursor angiotensin I (Ang I, 0.1-1 nm) did not affect the vasoconstrictor responses to noradrenaline (NA, 0.03-1 nmol) and adenosine 5'-triphosphate (ATP, 0.03-1 micromol). The second component vasoconstrictor response to nerve stimulation was dose dependently potentiated by Ang I (0.1-1 nm). The first peaked constriction was slightly, but insignificantly increased. The potentiating effects of Ang I were abolished by KRH-594 (10 nm), a selective AT(1) receptor antagonist, but not by PD 123319 (1-10 nm), an AT(2) receptor antagonist. KRH-594 (10 nm) or PD 123319 (10 nm) never affected the vasoconstrictions to either NA or ATP. 3. The treatment with KRH-594 (1-10 nm) produced a greater inhibition on the second peaked response than the first one, although both of them were dose dependently inhibited. PD 123319 (1-10 nm) did not affect the vasoconstrictor responses induced by nerve stimulation. 4. Inhibition of angiotensin-converting enzyme with 10 nm enalaprilat reduced the second peaked response, having no significant inhibition on the first peaked response. A higher dose of enalaprilat (100 nm) produced a greater inhibition of the second peak than the first one. It reduced the second peak by approximately 65%, while the first peak was decreased approximately 35%. After treatment with enalaprilat, Ang I (1 nm) failed to enhance the neuronal vascular response. Enalaprilat at doses used did not affect the vasoconstrictions to either NA or ATP. 5. The present results indicate that endogenously generated Ang II may produce a more marked potentiation of adrenergic transmission than purinergic transmission via activation of prejunctional AT(1) receptors.     

Superoxide anion mediates angiotensin II-induced potentiation of contractile response to sympathetic stimulation

Angiotensin II is known to potentiate vasoconstriction induced by electrical field stimulation (EFS), but the underlying mechanisms for this potentiation are not fully understood. This study was designed to investigate the role of superoxide anion in the potentiation effects of angiotensin II. Contraction of rat mesenteric arterial segments was induced by perivascular nerve stimulation with EFS, and superoxide production was measured with lucigenin-enhanced chemiluminescence. Extracellular signal-regulated kinase (ERK) phosphorylation was determined in cultured smooth muscle cells with Western blot. Angiotensin II concentration dependently potentiated the contraction of rat mesenteric arteries to EFS, which is frequency-dependent. This potentiation was blunted by an angiotensin AT(1) receptor antagonist (2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid, CV-11974), NAD(P)H oxidase inhibitor (apocynin), superoxide dismutase (SOD) and its mimetic tiron, but not affected by angiotensin AT(2) receptor antagonist and inhibitors of xanthine oxidase, cytochrome P450, and cyclooxygenase. Angiotensin II increased superoxide production by mesenteric arteries, which was blunted by angiotensin AT(1) receptor antagonist CV-11974, and NAD(P)H oxidase inhibitor apocynin. Superoxide generating compound pyrogallol mimicked the effects of angiotensin II. Tyrosine kinase inhibitor (tyrphostin A25) and mitogen-activated protein kinase (MAPK)/ERK inhibitors (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene (U 0126)) inhibited angiotensin II- and pyrogallol-induced potentiation of EFS-induced contraction, while inactive forms of these inhibitors did not show any inhibitory effects. In cultured smooth muscle cells from mesenteric arteries, angiotensin II and superoxide similarly induced ERK phosphorylation. These results showed that superoxide mediated angiotensin II-induced potentiation of contractile response to EFS and tyrosine kinase-MAPK/ERK activation was involved.     

Angiotensin II stimulates arachidonic acid release from bone marrow stromal cells.

INTRODUCTION: Angiotensin II (Ang II) is recognised as a regulator of haematopoiesis, but its actions within the bone marrow are not fully understood. Support of haematopoiesis by bone marrow stromal cells (MSC) is dependent on factors that include arachidonic acid and macrophage colony stimulating factor (MCSF), both of which are increased by Ang II stimulation in other tissues. To further elucidate the mechanisms of Ang II-regulated haematopoiesis, we determined whether Ang II-stimulation alters arachidonic acid release and MCSF secretion from MSC. METHODS: Cynomolgus monkey MSC isolated from bone marrow aspirates and the human HS-5 stromal cell line were studied for Ang II-mediated arachidonic acid (AA) release, while secretion of MCSF in response to Ang II was studied in HS-5 cells. Cells were labelled overnight with 3H-AA and the release of 3H-AA was measured in culture medium following 20 minutes stimulation with Ang II, alone or in combination with the AT1- or AT2-receptor antagonists, losartan and PD 123319, respectively. MCSF secretion into culture medium was measured using an enzyme immunoassay following 24 hours of treatment with Ang II alone or in combination with losartan or PD 123319. Phorbol-myristate-acetate, known to stimulate release of AA and MCSF, was used as a positive control in both experiments. RESULTS: In response to Ang II, release of 3H-AA from monkey and human MSC was increased (p<0.05) to 147+/-4% and 124+/-3% of control, respectively. The AT1- and AT2-receptor antagonists, losartan and PD 123319, individually reduced Ang II-stimulated 3H-AA release. In contrast, Ang II had no effect on secretion of MCSF from HS-5 cells. CONCLUSIONS: These results provide mechanistic evidence for Ang II-mediated haematopoiesis through AA release that may, in part, explain Ang II-facilitated recovery of haematopoiesis in experimental myelosuppression and the anaemias associated with Ang II receptor blockade.     

Comparative effects of angiotensin II AT-1-type receptor antagonists in vitro on human platelet activation

A recent study has shown that losartan, an AT-1-receptor antagonist, interacts with thromboxane A2 (TxA2)/prostaglandin H2 (PGH2) receptors in human platelets. The aim of this study was to analyze the ability of different angiotensin II (Ang II) AT-1-receptor antagonists to inhibit TxA2-dependent human platelet activation. Platelets were obtained from healthy volunteers. Platelets were stimulated with the TxA2 analogue, U46619 (10(-6) M). U46619-stimulated platelet activation was significantly reduced by both losartan and irbesartan in a dose-dependent manner. Only maximal doses of valsartan (5 x 10(-6) M) and the main metabolite of losartan, EXP3174 (5 x 10(-6) M), reduced U46619-induced platelet activation. Whereas the active form of candesartan cilexetil (candesartan, CV-11974) failed to modify platelet activation involved by TxA2, telmisartan showed a higher effect than valsartan and EXP3174 but lower than either losartan and irbesartan. Losartan or irbesartan reduced the binding of [3H]-U46619 to platelets, an effect that was observed with lower ability with the other AT-1 antagonists. Although platelets expressed AT-1-type receptors, exogenous Ang II did not modify platelet activation. This effect was not modified by blocking the AT-2 receptor with PD123319. These results suggest that some AT-1-receptor antagonists reduce TxA2-dependent activation independent of Ang II involvement.     

Effect of angiotensin Ⅱ type 1 receptor on delayed rectifier potassium current in catecholaminergic CATH.a cells

AIM: To study the modulatory effects of angiotensin Ⅱ (Ang Ⅱ) on the delayed rectifier potassium (Kv) current (IKv) and its underlying intracellular mechanism in the catecholaminergic system of rats. METHODS: AT1 and AT2 receptors of the differentiated and undifferentiated CATH.a cells were determined by radioligands binding assay. The IKv was recorded with the whole cell patch-clamp configuration in voltage clamp mode on CATH.a cells. RESULTS: The Ang Ⅱ receptor proteins including AT1 and AT2 receptors were expressed in CATH.a cells, and the number of the former was significantly more than the latter (P<0.01). The IKv of CATH.a cells was reduced by superfusion with the Ang Ⅱ (100 nmol/L) (P<0.05) in the presence of the AT2 receptor antagonist PD123319, but was not affected by only superfusion with PD123319. The effect of Ang Ⅱ on IKv in CATH.a cells was completely inhibited by addition of AT1 receptor antagonist losartan. Superfusion with Ang Ⅱ (100 nmol/L) plus U73122, an inhibitor of     

Stimulation of angiotensin subtype 2 receptor reduces basal cGMP levels in the neointima of rat aorta after balloon injury

• 1.1. The association between the stimulation of the angiotensin subtype 2 receptor (AT2-R) and the change in tissue levels of cyclic nucleotide was assessed on neointima formation in rat aorta following aortic balloon injury.
• 2.2. Tissue levels of guanosine 3′,5′-cyclic monophosphate (cGMP) and adenosine 3′,5′-cyclic monophosphate levels (cAMP) in the injured and uninjured aorta was determined by enzyme immunoassay at baseline and again 30 s after administration of 10⁻⁷ M angiotension II.
• 3.3. Injured and uninjured aorta showed no difference in basal levels of cGMP. Angiotension II reduced the basal level of cGMP in the injured aorta only.
• 4.4. This decrease was blocked by a selective AT2-R antagonist (PD123319) and by a nonselective angiotensin II antagonist (angiotensin II antipeptide), but not by a selective angiotensin subtype 1 antagonist (CV-11974).
• 5.5. Stimulation with a selective AT2-R caused no change in the level of cAMP in the injured or uninjured aorta.
• 6.6. Results suggest that stimulation of AT2-R in proliferative neointima leads to a decreased tissue level of cGMP.

     

Genomic and nongenomic effects of aldosterone in the rat heart: why is spironolactone cardioprotective?

1. Mineralocorticoid receptor (MR) antagonism with spironolactone reduces mortality in heart failure on top of ACE inhibition. To investigate the underlying mechanism, we compared the actions of both aldosterone and spironolactone to those of angiotensin (Ang) II in the rat heart. 2. Hearts of male Wistar rats were perfused according to Langendorff. Ang II and aldosterone increased left ventricular pressure (LVP) by maximally 11+/-4 and 9+/-2%, and decreased coronary flow (CF) by maximally 36+/-7 and 20+/-4%, respectively. Spironolactone did not significantly affect LVP or CF. 3. In hearts that were exposed to a 45-min coronary artery occlusion and 3 h of reperfusion, a 15-min exposure to spironolactone prior to occlusion reduced infarct size (% of risk area) from 68+/-2 to 45+/-3%, similar to the reduction (34+/-2%) observed following 'preconditioning' (15 min occlusion followed by 10 min reperfusion) prior to the 45-min occlusion. Aldosterone exposure did not affect infarct size (71+/-5%). 4. In cardiomyocytes, aldosterone decreased [(3)H]thymidine incorporation maximally by 73+/-3%, whereas in cardiac fibroblasts it decreased [(3)H]proline incorporation by 33+/-7%. Spironolactone inhibited both effects. Ang II increased DNA and collagen synthesis, and these effects were reversed by aldosterone. 5. In conclusion, aldosterone induces positive inotropic and vasoconstrictor effects in a nongenomic manner, and these effects are comparable to those of Ang II. Aldosterone reduces DNA and collagen synthesis via MR activation, and counteracts the Ang II-induced increases in these parameters. MR blockade reduces infarct size and increases LVP recovery following coronary artery occlusion. The MR-related phenomena may underlie, at least in part, the beneficial actions of spironolactone in heart failure.