Angiotensin II-induced relaxation of anococcygeus smooth muscle via desensitization of AT1 receptor, and activation of AT2 receptor associated with nitric-oxide synthase pathway

We evaluated the role of receptor desensitization, activation of AT(2) receptors, and enzymatic degradation of angiotensin II (Ang II) by amino/neutral endopeptidases in rat anococcygeus smooth muscle (ASM) relaxation. Ang II (0.3 nM to 10 microM) produced contractions (E(max) = 21.50 +/- 5.73%) followed by passive relaxations (E(max) reduced to 9.08 +/- 2.55%). Contractions were inhibited (E(max) = 13.67 +/- 2.03%) by losartan (0.1 microM; AT(1) antagonist) but not by PD123,319 [S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid] (0.1 microM; AT(2) antagonist). Conversely, the passive relaxation was inhibited (E(max) = 18.00 +/- 3.45%) by PD123,319 but not by losartan. Ang II (0.3 microM to 100 microM) produced initial contractions (E(max) = 11.49 +/- 9.39%) followed by active relaxations [I(max) (maximum inhibition elicited by the agonist) = 47.85 +/- 4.23%] on strips precontracted by bethanechol (100 microM). A second administration of Ang II on the background of bethanechol (1 h later) resulted in stronger relaxations (I(max) = 64.03 +/- 5.47%) without the initial contractions. N(G)-Nitro-l-arginine methyl ester [nitric-oxide synthase (NOS) inhibitor], ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; guanylate cyclase inhibitor), PD123,319, and tetrodotoxin (neurotoxin) inhibited the relaxations. The presence of AT(1) and AT(2) receptors was confirmed by Western blot. Experiments with amastatin (1 microM) and thiorphan (1 microM), aminopeptidase, and neutral endopeptidase inhibitors, respectively, excluded the involvement of enzymatic degradation in Ang II-induced relaxation of ASM. In conclusion, the rat ASM relaxation by Ang II is the result of active and passive relaxations. The passive relaxation depends on desensitization of excitatory AT(1) receptors, and the active relaxation is mediated by stimulation of AT(2) receptors and activation of the neuronal NOS/soluble guanylate cyclase pathway.     

Effect of angiotensin II on primary cardiac fibroblast matrix metalloproteinase activities

Left ventricular dysfunction is associated with reperfusion injury occurring during open-heart surgery. There is an increased secretion of angiotensin II (Ang II) and increased matrix metalloproteinases (MMPs) activities associated with open-heart surgery that may affect the cardiac extracellular matrix (ECM). The goal of this study was to determine the effects of Ang II and selective angiotensin II receptor (AT1-R and AT2-R) blockers on the enzymatic activities of MMPs in primary adult murine cardiac fibroblasts (CF). Our hypothesis is that Aug II, with and without a selective receptor blocker, differentially affects CF MMPs activities. The CF were treated with Ang II (10(-6) M) and doses of AT1-R and AT2-R blockers (losartan and PD123319, respectively) at doses of 10(-7) to 10(-5) M for 48 hours. The Ang II-stimulated CF reduced collagenase activities by only 24% (p = 0.004); however, the MMP-2 and MMP-9 gelatinase activities were reduced by 42% and 39%, respectively (p = 0.022). The losartan dose dependently increased MMP-2 (p = 0.02) and MMP-9 (ns). PD123319 at 10(-5) M significantly reduced MMP-2 and MMP-9 activities compared with the Ang II group (p = 0.014 and p = 0.02, respectively). The doses of PD123319 at 10(-6) and 10(-7) M increased the MMP-2 and MMP-9 enzymatic activities significantly above the Ang II only group. Thus, Ang II and AT1-R and AT2-R differentially affect the collagenase and gelatinase MMPs activities released by cardiac fibroblasts.     

Role of AT1 receptors in the resetting of the baroreflex control of heart rate by angiotensin II in the rabbit.

Angiotensin II (Ang II) resets the baroreflex control of heart rate to a higher blood pressure. This action is apparently mediated via Ang II receptors in the area postrema, but it is not known if these are of the AT1 or AT2 subtype. In the present study the effects of losartan, a selective AT1 receptor antagonist, and PD 123319, a selective AT2 antagonist, on the cardiac baroreflex response to Ang II were investigated in conscious rabbits with chronically implanted arterial and venous catheters. Baroreflex curves were generated with intravenous infusions of phenylephrine and nitroprusside (2.6-25 micrograms/kg per min) and analyzed using a four-parameter logistic model to yield their upper and lower plateaus, arterial pressure at the midpoint of the heart rate range (BP50), and slope coefficient. From these four parameters, the gain and range of the baroreflex were calculated. Background intravenous infusion of Ang II at 10 ng/kg per min increased mean arterial pressure by 17 mmHg but did not change heart rate. Ang II shifted the baroreflex curve to the right as indicated by an increase in BP50 from 70.9 +/- 2.0 to 89.3 +/- 2.7 mmHg (P < 0.05), but did not change baroreflex gain significantly. Ang II did not alter the upper plateau of the baroreflex, but decreased the lower plateau from 119.4 +/- 10.3 to 73.6 +/- 11.5 beats per minute (bpm) (P < 0.05), extending the heart rate range by 52.5 bpm. Pretreatment with losartan completely abolished the pressor and cardiac baroreflex responses to Ang II. In contrast, PD 123319 had no effect on these responses. Administration of losartan alone to block endogenous Ang II shifted the baroreflex curve to the left as indicated by a decrease in BP50 from 71.2 +/- 2.7 to 64.7 +/- 2.5 mmHg (P < 0.05). These results demonstrate that the resetting of the baroreflex control of heart rate by Ang II is mediated by AT1 receptors, and that basal levels of endogenous Ang II exert a tonic action on the cardiac baroreflex to increase the setpoint around which the baroreflex regulates heart rate.     

Chronic blockade of AT2-subtype receptors prevents the effect of angiotensin II on the rat vascular structure.

Angiotensin II (Ang II) is both a vasoactive and a potent growth-promoting factor for vascular smooth muscle cells. Little is known about the in vivo contribution of AT1 and AT2 receptor activation to the biological action of Ang II. Therefore, we investigated the effect of AT1 or AT2 subtype receptor chronic blockade by losartan or PD123319 on the vascular hypertrophy in rats with Ang II-induced hypertension. Normotensive rats received for 3 wk subcutaneous infusions of Ang II (120 ng/kg per min), or Ang II + PD 123319 (30 mg/kg per d), or Ang II + losartan (10 mg/kg per d) or PD 123319 alone, and were compared with control animals. In normotensive animals, chronic blockade of AT2 receptors did not affect the plasma level of angiotensin II and the vascular reactivity to angiotensin II mediated by the AT1 receptor. Chronic blockade of AT1I in rats receiving Ang II resulted in normal arterial pressure, but it induced significant aortic hypertrophy and fibrosis. Chronic blockade of AT2 receptors in Ang II-induced hypertensive rats had no effect on arterial pressure, but antagonized the effect of Ang II on arterial hypertrophy and fibrosis, suggesting that in vivo vasotrophic effects of Ang II are at least partially mediated via AT2 subtype receptors.     

Angiotensin receptor subtype 1 mediates angiotensin II enhancement of isoproterenol-induced cyclic AMP production in preglomerular microvascular smooth muscle cells

In a previous study, we found that angiotensin (Ang) II enhances beta-adrenoceptor-induced cAMP production in cultured preglomerular microvascular smooth muscle cells (PMVSMCs) obtained from spontaneously hypertensive rats. The purpose of the present investigation was to identify the Ang receptor subtypes that mediate this effect. In our first study, we compared the ability of Ang II, Ang III, Ang (3-8), and Ang (1-7) to increase cAMP production in isoproterenol (1 microM)-treated PMVSMCs. Each peptide was tested at 0.1, 1, 10, 100, and 1000 nM. Both Ang II and Ang III increased intracellular (EC50s, 1 and 11 nM, respectively) and extracellular (EC50s, 2 and 14 nM, respectively) cAMP levels in a concentration-dependent fashion. In contrast, Ang (3-8) and Ang (1-7) did not enhance either intracellular or extracellular cAMP levels at any concentration tested. In our second study, we examined the ability of L 158809 [a selective Ang receptor subtype 1 (AT1) receptor antagonist] to inhibit Ang II (100 nM) and Ang III (100 nM) enhancement of isoproterenol (1 microM)-induced cAMP production in PMVSMCs. L 158809 (10 nM) abolished or nearly abolished (p <.001) Ang II and Ang III enhancement of isoproterenol-induced intracellular and extracellular cAMP levels. In contrast, PD 123319 (300 nM; a selective AT2 receptor antagonist) did not significantly alter Ang II enhancement of isoproterenol-induced intracellular or extracellular cAMP levels. We conclude that AT1 receptors, but not AT2, Ang (3-8), nor Ang (1-7) receptors mediate Ang II and Ang III enhancement of beta-adrenoceptor-induced cAMP production in cultured PMVSMCs.     

Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV.

Recent studies from this laboratory have demonstrated that angiotensin II (Ang II) stimulates the expression of plasminogen activator inhibitor 1 (PAI-1) in cultured endothelial cells. This response does not appear to be mediated via an interaction with either the AT1 or the AT2 receptor subtype. Since a novel angiotensin receptor has been identified in a variety of tissues that specifically binds the hexapeptide Ang IV (Ang II, [3-8]), we therefore examined the effects of Ang IV on the expression of PAI-1 mRNA in bovine aortic endothelial cells. Ang IV stimulated dose- and time-dependent increases in the expression of PAI-1 mRNA. The effect of Ang IV (10 nM) was not inhibited by Dup 753 (1.0 microM), a highly specific antagonist of the AT1 receptor, or by PD123177 (1.0 microM), a highly specific antagonist of the AT2 receptor. In contrast, the AT4 receptor antagonist, WSU1291 (1.0 microM), effectively prevented PAI-1 expression. Although larger forms of angiotensin (i.e., Ang I, Ang II, and Ang III) are capable of inducing PAI-1 expression, this property is lost in the presence of converting enzyme or aminopeptidase inhibitors. These results indicate that the hexapeptide Ang IV is the form of angiotensin that stimulates endothelial expression of PAI-1. This effect appears to be mediated via the stimulation of an endothelial receptor that is specific for Ang IV.     

AT1 receptor antagonist telmisartan administered peripherally inhibits central responses to angiotensin II in conscious rats.

The effects of systemic treatment with the AT1 receptor antagonist telmisartan on central effects of angiotensin II (Ang II), namely, increase in blood pressure, vasopressin release into the circulation, and drinking response, were investigated in conscious, normotensive rats. The central responses to i.c.v. Ang II (30 ng/kg) were measured at 0.5, 2, 4, and 24 h following acute i.v. or acute and chronic oral telmisartan application. At a dose of 10 mg/kg i.v., the drinking response to i.c.v. Ang II was completely blocked over 4 h, while the pressor response and the release of vasopressin in response to i.c.v. Ang II were blocked by 60 to 80%. The inhibition of the centrally mediated pressor and drinking response to Ang II was sustained over 24 h. The lower doses of telmisartan (0.3 and 1 mg/kg) significantly inhibited the Ang II-induced actions over 4 h. A consistent 24-h inhibition of the central responses to i.c.v. Ang II was obtained after acute and chronic oral treatment with 30 mg/kg telmisartan. Oral treatment with 1 and 3 mg/kg telmisartan produced a slight but inconsistent inhibition of the central actions of Ang II. Telmisartan concentrations measured in the cerebrospinal fluid following 8 days of consecutive daily oral treatment (1-30 mg/kg) ranged from 0.87 +/- 0.27 ng/ml (1 mg/kg/day) to 46.5 +/- 11.6 ng/ml (30 mg/kg/day). Our results demonstrate that, following peripheral administration, the AT1 receptor antagonist telmisartan can penetrate the blood-brain barrier in a dose- and time-dependent manner to inhibit centrally mediated effects of Ang II.     

Angiotensin II receptor-mediated function unmasked by gene-engineered animals]

The receptors for angiotensin (Ang) II are classified into two subtypes (AT1-R and AT2-R) by the discovery of non-peptidic ligands and AT1-R mediates most of the cardiovascular actions of Ang II. AT2-R is expressed at very high levels in the developing fetus, whereas in the adult its expression in the cardiovascular system is very low. Cardiac myocyte- or vascular smooth muscle-specific overexpression mice of AT2-R display an inhibitory effect on Ang II-induced chronotropic or pressor actions, suggesting the role of AT2-R on the activity of cardiac pacemaker cells or maintenance of vascular resistance. AT2-R also activates the kinin/nitric oxide/cGMP system in the cardiovascular and renal system, resulting in the AT2-R-mediated cardioprotection, vasodilation and pressure natriuresis. These effects transmitted by AT2-R are mainly exerted by stimulation of protein tyrosine or serine/threonine phosphatases in Gi-protein dependent manner. The expression level of AT2-R is much higher in human hearts than in those of rodents, and the AT2-R-mediated actions are likely enhanced, especially by clinical application of AT1-R antagonists.     

Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects.

Angiotensin (Ang) II has two major receptor isoforms, AT1 and AT2. Currently, AT1 antagonists are undergoing clinical trials in patients with cardiovascular diseases. Treatment with AT1 antagonists causes elevation of plasma Ang II which selectively binds to AT2 and exerts as yet undefined effects. Cardiac AT2 level is low in adult hearts, whereas its distribution ratio is increased during cardiac remodeling and its action is enhanced by application of AT1 antagonists. Although in AT2 knock-out mice sensitivity to the pressor action of Ang II was increased, underlying mechanisms remain undefined. Here, we report the unexpected finding that cardiac-specific overexpression of the AT2 gene using alpha-myosin heavy chain promoter resulted in decreased sensitivity to AT1-mediated pressor and chronotropic actions. AT2 protein undetectable in the hearts of wild-type mice was overexpressed in atria and ventricles of the AT2 transgenic (TG) mice and the proportions of AT2 relative to AT1 were 41% in atria and 45% in ventricles. No obvious morphological change was observed in the myocardium and there was no significant difference in cardiac development or heart to body weight ratio between wild-type and TG mice. Infusion of Ang II to AT2 TG mice caused a significantly attenuated increase in blood pressure response and the change was completely blocked by pretreatment with AT2 antagonist. This decreased sensitivity to Ang II-induced pressor action was mainly due to the AT2-mediated strong negative chronotropic effect and exerted by circulating Ang II in a physiological range that did not stimulate catecholamine release. Isolated hearts of AT2 transgenic mice perfused using a Langendorff apparatus also showed decreased chronotropic responses to Ang II with no effects on left ventricular dp/dt max values, and Ang II-induced activity of mitogen-activated protein kinase was inhibited in left ventricles in the transgenic mice. Although transient outward K+ current recorded in cardiomyocytes from AT2 TG mice was not influenced by AT2 activation, this study suggested that overexpression of AT2 decreases the sensitivity of pacemaker cells to Ang II. Our results demonstrate that stimulation of cardia AT2 exerts a novel antipressor action by inhibiting AT1-mediated chronotropic effects, and that application of AT1 antagonists to patients with cardiovascular diseases has beneficial pharmacotherapeutic effects of stimulating cardiac AT2.     

Cross-talk between AT(1) and AT(2) angiotensin receptors in rat anococcygeus smooth muscle

Schild regressions for the selective AT(1) and AT(2) receptor antagonists, losartan and PD123319 (S-[+]-1-[(4-dimethylamino]-3-methylphenyl)methyl]-5-[diphenylacetyl]-4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxilic acid), respectively, were calculated to analyze the heterogeneity of receptor populations in the rat anococcygeus muscle. For a one-receptor system, the Schild regression has a slope of unity and an intercept of K(B) for competitive antagonists. However, in a two-receptor system, a deviation from the single-receptor plot will occur. This is predicated on the assumption that the secondary receptor is less sensitive to the antagonist than the primary receptor. Results showed that the Schild regression for losartan did not produce a slope of unity, and PD123319 did not produce any effect. However, tissue incubation with losartan plus PD123319 resulted in a Schild regression that has a slope of unity and a pK(B) of 9.32. In the presence of prazosin, an alpha(1)-adrenoceptor antagonist, losartan did not produce any effect. Conversely, PD123319 enhanced the angiotensin II (Ang II)-induced contraction in a concentration-dependent fashion, suggesting an inhibitory AT(2)-mediated effect. This effect was confirmed with assays that showed a relaxant response induced by Ang II on precontracted tissues incubated with prazosin. PD123319 and N(G)-nitro-L-arginine methyl ester [nitric-oxide (NO) synthase inhibitor)] markedly inhibited the relaxant response of Ang II. In contrast, losartan did not produce any significant effect. Consequently, results show that the mechanism underlying the AT(2)-mediated effect is highly dependent on NO generation. Results indicate the presence of a heterogeneous angiotensin receptor population in the rat anococcygeus muscle following a negative cross-talk relationship between the AT(1) and AT(2) subtypes.     

Inhibition of angiotensin II- and endothelin-1-stimulated proliferation by selective MEK inhibitor in cultured rabbit gingival fibroblastsdagger

We investigated the implication of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the proliferation stimulated by angiotensin II (Ang II) and endothelin-1 (ET-1) in cultured rabbit gingival fibroblasts (CRGF). Ang II stimulated activation of ERK1/2 and the activation was inhibited by CV-11974, an AT1 antagonist, and saralasin, an AT1/AT2 antagonist, but not by PD123,319, an AT2 antagonist in the CRGF. Ang II-stimulated proliferation was inhibited by PD98059 or U0126, selective MEK inhibitors. Furthermore, ET-1 stimulated proliferation via G-protein-coupled ETA receptors, which were identified by Western blot analysis of membrane protein from the CRGF. ET-1 also stimulated activation of ERK1/2 and the activation was inhibited by BQ-123, an ETA inhibitor, and TAK044, an ETA/ETB inhibitor, but not by BQ-788, an ETB inhibitor. ET-1-stimulated proliferation was inhibited by PD98059 or U0126. These findings suggest that ERK1/2 play a role in the signaling process leading to proliferation stimulated by Ang II and ET-1 via G-protein-coupled receptors, AT1 and ETA in CRGF.     

Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice

Angiotensin II (Ang II) is a bioactive peptide that plays important roles in blood pressure regulation and salt–water homeostasis. Recently, Ang II was reported to function in the promotion of angiogenesis. Since the wound healing process is highly dependent upon angiogenesis, we employed Ang II receptor knockout mice (AT1a^−/−) to investigate whether or not Ang II facilitates angiogenesis and wound healing via AT1a receptor signaling. In comparison to wild-type (WT) mice, wound healing and wound-induced angiogenesis were significantly suppressed in AT1a^−/− mice, and these mice exhibited reduced expression of CD31 in wound granulation tissues. In comparison to vehicle-treated mice, wound healing was delayed significantly in mice treated with an AT1-R antagonist and this delay was accompanied by the reduced expression of vascular endothelial growth factor in wound granulation tissues. These findings suggest that Ang II–AT1a signaling plays a crucial role in wound healing and wound-induced angiogenesis.     

The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats.

The angiotensin AT2 receptor modulates renal production of cyclic guanosine 3',5'-monophosphate (cGMP; J. Clin. Invest. 1996. 97:1978-1982). In the present study, we hypothesized that angiotensin II (Ang II) acts at the AT2 receptor to stimulate renal production of nitric oxide leading to the previously observed increase in cGMP. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) cGMP in response to intravenous infusion of the AT2 receptor antagonist PD 123319 (PD), the AT1 receptor antagonist Losartan, the nitric oxide synthase (NOS) inhibitor nitro--arginine-methyl-ester (-NAME), the specific neural NOS inhibitor 7-nitroindazole (7-NI), or Ang II individually or combined in conscious rats during low or normal sodium balance. Sodium depletion significantly increased RIF cGMP. During sodium depletion, both PD and -NAME caused a similar decrease in RIF cGMP. Combined administration of PD and -NAME decreased RIF cGMP to levels observed with PD or -NAME alone or during normal sodium intake. During normal sodium intake, Ang II caused a twofold increase in RIF cGMP. Neither PD nor -NAME, individually or combined, changed RIF cGMP. Combined administration of Ang II and either PD or -NAME produced a significant decrease in RIF cGMP compared with that induced by Ang II alone. Combined administration of Ang II, PD, and -NAME blocked the increase in RIF cGMP produced by Ang II alone. During sodium depletion, 7-NI decreased RIF cGMP, but the reduction of cGMP in response to PD alone or PD combined with 7-NI was greater than with 7-NI alone. During normal sodium intake, 7-NI blocked the Ang II-induced increase in RIF cGMP. PD alone or combined with 7-NI produced a greater inhibition of cGMP than did 7-NI alone. During sodium depletion, 7-NI (partially) and -NAME (completely) inhibited RIF cGMP responses to -arginine. These data demonstrate that activation of the renin- angiotensin system during sodium depletion increases renal nitric oxide production through stimulation by Ang II at the angiotensin AT2 receptor. This response is partially mediated by neural NOS, but other NOS isoforms also contribute to nitric oxide production by this pathway.     

Analysis of responses to angiotensin I (3-10) and Leu3 angiotensin (3-8) in the pulmonary vascular bed of the cat

Pulmonary vascular responses to angiotensin (3-8) (Ang IV), leu3 angiotensin (3-8) (LeuAng IV), an Ang IV analog and angiotensin I (3-10) [Ang I (3-10)], the precursor for Ang IV, were investigated in the intact-chest cat under conditions of controlled blood flow. Intralobar injections of Ang IV, LeuAng IV, and Ang I (3-10) caused dosage-related increases in lobar arterial pressure. When responses were compared, Ang IV, LeuAng IV, and Ang I (3-10) were equipotent and were approximately 100- to 300-fold less potent than Ang II when dosages are expressed on a nanomolar basis. DuP 753, an angiotensin II type 1 (AT1 ) receptor antagonist, attenuated pulmonary vasoconstrictor responses to LeuAng IV, Ang IV, and its precursor, Ang I (3-10), but did not significantly change pressor responses to serotonin, norepinephrine, or U46619. PD 123319, an angiotensin II type 2 (AT2 ) receptor antagonist, and WSU 3033, a putative angiotensin II type 4 (AT4 ) receptor antagonist, did not significantly change pressor responses to LeuAng IV, Ang IV, and its precursor, Ang I (3-10). Captopril, an angiotensin-converting enzyme (ACE) inhibitor, decreased pulmonary vasoconstrictor responses to Ang I (3-10) but did not significantly change responses to serotonin, norepinephrine, U46619, LeuAng IV, or Ang IV. These data show that LeuAng IV, Ang IV, and its precursor, Ang I (3-10), increase pulmonary vascular resistance by activating AT1 receptors, and that Ang I (3-10) is rapidly and efficiently converted by an ACE-dependent pathway into an active peptide. The present data suggest that Ang IV and LeuAng IV increase pulmonary vascular resistance by activating AT1 receptors and that activation of AT2 or AT4 are not involved in mediating or modulating responses to these peptides. These data provide support for the hypothesis that Ang I (3-10) is converted into an active peptide by ACE at or near the site of action within the pulmonary vascular bed.     

Effects of AT1 and AT2 receptor blockade on angiotensin II induced apoptosis of human renal proximal tubular epithelial cells

BACKGROUND: Tubular atrophy is a common histological feature of chronic renal failure, and epithelial cell death by apoptosis might play an important role in its pathogenesis. Angiotensin II contributes to the progressive nature of many kidney diseases and treatment with angiotensin converting enzyme inhibitors preserves the structure of the tubulointerstitial compartment in human and experimental renal diseases. METHODS: Primary cultures of human renal proximal tubular epithelial cells were co-incubated with angiotensin II alone or in combination with the angiotensin II AT1 receptor antagonist losartan or/and the AT2 antagonist PD123319. Apoptosis was determined after 20 hours by TUNEL staining and flow cytometry. RESULTS: Angiotensin II at concentrations of 10(-9) M induced apoptosis (control vs. angiotensin II 4 +/- 3% vs. 73 +/- 11%; p < 0.05). This effect was completely offset by co-incubation with the angiotensin II AT2 receptor blocker at concentrations 10(-7) M (control vs. PD123319 4 +/- 3% vs. 8 +/- 3%; p < 0.05); AT1 blockade was ineffective in apoptosis inhibition. When both angiotensin receptors were blocked, no additional effect on apoptosis inhibition could be detected. CONCLUSION: We provided evidence, that physiological concentrations of angiotensin II can induce apoptosis of human renal proximal tubule epithelial cells. This effect is mediated via AT2 receptors.     

Isoflurane is a potent modulator of extrasynaptic GABA(A) receptors in the thalamus

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250-500 microM), enhances fast synaptic inhibition in the brain mediated by GABA(A) receptors (GABA(A)-Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25-85 microM) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl(-) equilibrium potential and were totally blocked by the GABA(A)-R antagonist gabazine. Isoflurane (25-250 microM) produced no sustained current in VB neurons from GABA(A)-R alpha(4)-subunit knockout (Gabra4(-/-)) mice, although 250 microM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4(-/-) mice. These data indicate an obligatory requirement for alpha(4)-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated alpha(4)beta(2)delta GABA(A)-Rs expressed in human embryonic kidney 293 cells, and it was more potent at alpha(4)beta(2)delta than at alpha(1)beta(2)gamma(2) receptors (the presumptive extrasynaptic and synaptic GABA(A)-R subtypes in VB neurons). We conclude that the extrasynaptic GABA(A)-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABA(A)-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.     

Effect of chronic AT(1) receptor antagonism on postischemic functional recovery and AT(1)/AT(2) receptor proteins in isolated working rat hearts

To determine whether chronic angiotensin II (Ang II) type I receptor (AT(1)R) antagonism improves recovery of left ventricular (LV) function after ischemia-reperfusion (IR) and increases AT(1)R and Ang II type 2 receptor (AT(2)R) protein expression in isolated working rat hearts, rats were randomized to pretreatment with either losartan (30 mg/kg/day) or UP269-6 (3 mg/kg/day), or no drug (control), for 1 week or 3 weeks before IR (50 min perfusion, 25 min ischemia, 40 min reperfusion). In vitro LV work and power and ex vivo AT(1)R and AT(2)R proteins (immunoblots) were measured. Compared to baseline perfusion, LV work and power showed variable recovery in control, losartan, and UP269-6 groups. Compared to control, losartan preserved recovery of LV work and power while UP269-6 showed less recovery after IR at both 1 week and 3 weeks. Both antagonists increased AT(2)R but not AT(1)R protein. The duration of pretreatment did not affect the expression of AT(1)R or AT(2)R proteins. The results indicate that chronic AT(1)R blockade over 1 or 3 weeks increases AT(2)R (not AT(1)R) protein expression and may preserve but not improve postischemic functional recovery compared to controls in isolated working rat hearts.