μM Losartan Research Articles

PP.17.01] ACTIVATION OF RENIN-ANGIOTENSIN SYSTEM STIMULATES THE ORGANIC CATION TRANSPORTER 2 AND INCREASES CISPLATIN TOXICITY

Objective: The intrarenal RAS is characterized by high levels of angiotensin II (AII) and wide distribution of angiotensin receptors (ATR). There is indirect data showing cross-talk between the RAS and the human organic cation transporter 2 (hOCT2). This transporter is highly expressed in the basolateral membrane of proximal tubules cells and plays an important role for uptake, accumulation, and toxicity of cisplatin. Therefore, we aimed to investigate whether AII is able to modulate cisplatin toxicity in Madin-Darby canine kidney (MDCK) cells. Design and method: The uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+, 20 μM) added to the basolateral compartment of MDCK cells stably expressing hOCT2 growing on filters was measured in the presence of 1 nM AII alone or with the AT1R blocker losartan. Following 1 hr incubation at 37C, cells were solubilized and the intracellular ASP+ concentration was measured with a microfluorimeter. Next, cell monolayers were incubated with 0.1 mM cisplatin alone or with 1 nM AII and blocker combinations. During and after incubation with different solutions trans-epithelial electrical resistance (TEER) and cell viability of MDCK cell monolayers was evaluated. All values are given as mean ± SEM. Results: Incubation with 1 nM AII significantly stimulated hOCT2 mediated ASP+ uptake (ASP+ concentration in lysates of control and AII stimulated cells: 1.8 ± 0.4 and 2.2 ± 0.2 μmol/g protein, n=7–13, respectively). This effect was abolished under 1 μM losartan. Following incubation with 0.1 mM cisplatin TEER decreased to values similar of no-cell control. AII had no additional effect on the loss of electrical resistance. However, cell viability decreased more drastically when cisplatin was applied together with AII. This effect was abolished in the presence of losartan (cisplatin alone - 46.6 ± 1.4% vs with AII - 39.2 ± 2% of control experiments which were set as 100%, p < 0.05). Application of cisplatin together with cimetidine and AII lessened the toxicity (49.1 ± 1.3% of control, p < 0.05). Conclusions: We propose that in MDCK cell system AII augments cisplatin toxicity and that this effect is related to stimulated hOCT2 function.

Heterologous Phosphorylation of the Cannabinoid Type 1 receptor by Angiotensin II in Astrocytes isolated from Spontaneously Hypertensive Rats

ObjectiveTo determine if a mediator of neuroprotection, the Cannabinoid Receptor Type 1 (CB1R), is phosphorylated by Angiotensin II (Ang II) via the Angiotension Type 1 receptor (AT1R) in astrocytes.BackgroundAT1R and CB1R have opposing roles in regulating the release of neuroinflammatory cytokines from astrocytes. While the former, a Gq G protein coupled receptor (GPCR), is pro‐inflammatory, the latter is anti‐inflammatory. Activation of Gq GPCRs have been demonstrated to inactivate the CB1R via a key phosphorylation event.MethodsAstrocytes were isolated from the brainstem and cerebellum of Spontaneously Hypertensive Rats (SHR), a model of hypertension and Attention Deficit Hyperactivity Disorder (ADHD). The cells were treated with 100 Nm Ang II for varying time periods and the extent of phosphorylation was measured using Western blotting and Flow cytometry. To determine the receptor involved, the cells were also pretreated with 10 μM Losartan and 10 μM PD123319, the AT1 and AT2 receptor antagonists respectively.ResultsBasal CB1R was significantly higher in SHR when compared to Wistar. Higher levels were observed in cerebellum. Ang II phosphorylated CB1R in both brain regions of both rat models. This effect was predominantly mediated through the AT1R. The phosphorylation event was initiated earlier and was sustained longer in SHR when compared to wistar.ConclusionHigher basal CB1R phosphorylation in SHR as opposed to Wistar is suggestive of a potential diminished protective effect in pathological conditions such as hypertension and ADHD. Also Ang II could be involved in disinhibiting CB1R actions via the AT1R by heterologous desensitization. Temporal and magnitude differences in phosphorylation of CB1R by Ang II, in SHR versus Wistar, are suggestive of a potential therapeutic target to combat neuroinflammation.Support or Funding InformationNSU HPD: Grant # 335585

Abstract P127: Angiotensin II Priming Enhances Prostaglandin E2 Vasoconstrictor Effects

Prostaglandins are key modulators of blood pressure and arterial tone. Prostaglandin E 2 (PGE 2 ), is a prostanoid that has vasodepressor effects; however, under certain circumstances PGE 2 can induce vasopressor responses. Recent reports demonstrated that sub-threshold concentrations of vasoconstrictors augment PGE 2 -mediated constriction in rat femoral arteries. However, whether angiotensin II (Ang II) could affect PGE 2 -mediated contraction is not known. Using a wire myograph, we demonstrated that PGE 2 had no significant effect on mouse femoral arterial rings at doses up to 1 μM. However, priming of arterial rings with 1 nM Ang II potentiated PGE 2 -evoked constriction in a concentration dependent manner (Area Under the Curve, AUC untreated 1.784 ± 0.353, AUC Ang II 23.27± 9.820, P&lt;0.05). We tested femoral arteries from EP1, EP2, and EP3 receptor knockout mice. Only the EP3-/- arteries were unable to respond to PGE 2 after Ang II priming (figure below). Pretreatment of arterial rings with 1 μM losartan, an angiotensin receptor antagonist, blocked PGE 2 -induced constrictor effects primed with Ang II (% of KCl, Ang II 21.72 ± 5.296, Ang II + losartan 3.025 ± 1.046, n=3). We have determined that re-addition of extracellular Ca 2+ to a Ca 2+ -free artery restores PGE 2 -induced contractions (n=5) and that the Rho-kinase inhibitor Y-27632 blocks contraction (n=3). Taken together these data are consistent with angiotensin AT1 and prostaglandin EP3 receptors mediating a synergistic Rho-kinase-dependent contractile response. We are continuing to investigate the relationship between Ang II and PGE 2 to determine the physiological relevance this may have in modulating blood pressure.

Mechanism of Angiotensin II ‐ Mediated Changes in Glomeruli Permeability and Calcium Influx in Podocytes

Podocytes maintain glomerular structure and permeability and play a key role in the pathogenesis of various renal diseases. It was previously shown that angiotensin II (Ang II), the key peptide in the RAAS, produces an increase in intracellular Ca2+ in cultured podocytes. Members of the TRPC ion channels family were proposed to be responsible for this calcium flux. We examined here the effects of Ang II on the function of the podocytes of freshly isolated rat glomeruli using ratiometric epifluorescence calcium imaging with Fura2-AM, patch clamp electrophysiology and a novel protocol designed to measure dynamic changes in glomerular permeability to albumin. To assess the effects of Ang II on glomerular function we measured real-time albumin permeability changes using two fluorescently labeled dextrans and confocal microscopy. We found that Ang II rapidly reduced glomerular volume resulting in increased glomerular permeability to albumin. This response was attenuated by treatment with 10 µM losartan and 1 µM PD 123319, specific inhibitors of AT1 and AT2 receptors, respectively. Single channel patch-clamp analysis demonstrated that Ang II acutely activates native TRPC6 channels in the podocytes; the effect is mediated by changes in the channel Po. Depletion of internal Ca2+ stores with thapsigargin did not affect calcium influx activated by Ang II, whereas TRPC channel blocker SKF 96365 decreased this response. Overall, our data indicate that Ang II induces glomeruli albumin permeability changes likely via both AT1 and AT2 receptors, and Ang II-dependent activation of calcium influx in podocytes might play a significant role in the pathogenesis of kidney diseases via effects on glomeruli permeability.

Drug-drug interaction of losartan and glimepiride metabolism by recombinant microsome CYP2C9*1, 2C9*3, 2C9*13, and 2C9*16 in vitro.

Co-administration of anti-hypertension and anti-diabetic drugs is common in clinical settings. In this study, we characterized the drug-drug interactions of losartan (LOS) and glimepiride (GLP) using recombinant cytochrome P450 (CYP) 2C9 enzymes (CYP2C9*1, CYP2C9*3, CYP2C9*13, and CYP2C9*16). Metabolism of losartan by recombinant CYP2C9* 1, CYP2C9*3, CYP2C9*13, and CYP2C9* 16 was inhibited by glimepiride competitively with IC50 values of 0.669 ± 0.055 µM, 0.424 ± 0.032 µM, 2.557 ± 0.058 µM, and 0.667 ± 0.039 µM, respectively. The inhibitory effect of glimepiride on losartan metabolism by CYP2C9*13 was marginal. The apparent Ki value of glimepiride with CYP2C9*3 (0.0416 ± 0.0059 µM) was significantly lower than with CYP2C9*1 (0.1476 ± 0.0219 µM) and CYP2C9*16 (0.2671 ± 0.0456 µM). On the other hand, losartan weakly inhibited the hydroxylation of glimepiride by P450 2C9 enzymes competitively. The potencies for inhibition of glimepiride hydroxylation were determined to be CYP2C9*1~CYP2C9*3~CYP2C9*16 > CYP2C9*13 by 4 µM losartan. No significant inhibition was observed when 0.5 µM losartan was used. Given these results, the potential inhibition of losartan metabolism by CYP2C9*3, CYP2C9*13, and CYP2C9*16 in vivo by glimepiride deserves further investigation. These results may provide valuable information for optimizing the anti-hypertension efficacy of losartan when glimepiride is co-administered to patients.

Abstract 202: Involvement of neuronal high mobility group box 1 (HMGB1) in Angiotensin II-mediated neuronal-microglial interaction

We have demonstrated that activation of microglia in the autonomic brain regions, primarily in the paraventricular nucleus (PVN), is critical in the development and establishment of hypertension. Neuroimmune synaptic communication is implicated in activating microglia and inducing neuroinflammation in many neuropathophysiological diseases. Specifically, HMGB1 secreted by neurons is a critical mediator in neuroimmune crosstalk. Thus we hypothesized that HMGB1 is a key regulator involved in microglia activation in Ang II-induced hypertension. Neuronal and microglial cells in primary culture were established for this study. Effect of Ang II on microglial movement was recorded and quantified by time-lapse microscopy and video analysis. In addition, the expression, translocation and secretion of HMGB1 in neurons were measured by qPCR, Western blot and immunocytochemistry. Ang II treatment of neuronal-microglial co-culture resulted in a 5-fold increase of microglial migration towards neurons, which was completely abolished by treatment with 1 μM Losartan, an Ang II receptor type 1 antagonist. The presence of neurons was found to be essential for microglial movement since Ang II failed to induce these effects in the absence of neurons. Neurons express high levels of HMGB1, which was predominantly localized in the nucleus. Ang II treatment resulted in a time-dependent translocation of nuclear hMGB1 into the cytoplasm and ultimately into the medium. Both translocation and release of Ang II-induced HMGB1 was blocked by Losartan. Furthermore treatment of microglia with 500ng/ml recombinant HMGB1 caused ~2 fold increase of proinflammatory cytokines (TNF-α and IL-1β). The importance of HMGB1 is further underscored by our observation that its protein level was increased ~2 fold in the PVN but not in the nucleus of the solitary tract (NTS) or rostral ventrolateral medulla (RVLM) of the spontaneously hypertensive rats (SHR) compared with the normotensive WKY rats. Taken together, these observations suggest that Ang II primarily activates the neuronal AT1R to increase the expression, cytosolic translocation and release of HMGB1. The released HMGB1 primes microglial activation and release of cytokines, two key events in neuroinflammation in hypertension.

Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells.

Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 μM) and by a Src kinase inhibitor PP2 (10 μM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 μM losartan and 10 μM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway.

Abstract 423: Angiotensin II Type 1 Receptor (AT1R) Activating Autoantibodies Contribute to the Pathophysiology of Primary Hyperaldosteronism

Objectives: Activating autoantibodies (AA) to the AT1R second extracellular loop (ECL2) are present in patients with primary aldosteronism (PA). We investigated whether AA from PA and from AT1R-ECL2 immunized rabbits activate AT1R and thereby contribute to hypertension by a direct contractile effect on the vasculature and stimulate adrenal aldosterone production. Methods: We studied 5 normal controls (NC) and 15 subjects with biochemically confirmed PA [4 APA, 4 IAH non- and 7 indeterminate (IPA)]. AT1R-AA activity in serum or IgG was analyzed in AT1R transfected CHO cells. Contractile effects were assayed in isolated perfused rat cremaster arteries. We determined the effects of an ARB and the L-aa AT1R-ECL2 target epitope on in vitro AT1R cremaster artery responsiveness to patient sera and to MAP-L-aa (AFHYESQ) peptide immunized rabbit serum. We examined the effect of IgG from 7 PA subjects on aldosterone production in HAC15 cells in vitro w/wo 1.0 and 10 nM Ang II; and w/wo ARB. Results: Serum from PA significantly (p&lt;0.05) increased Ang II-equivalent activity in AT1R-CHO cells in IAH 199± x pM, n=4; APA 177±y pM, n=3; IPA 187±z pM, n=7; vs NC 83±a pM, n=5 and was blocked by 10 μM losartan. IgG from PA decreased perfused rat arteriole diameter in IAH by 22±8(SD)% of a 100% (normalized) baseline, n=4; APA 14±0.8%, n=3; IPA 21±6%, n=7; and NC 7±3.5%, n=5 and was blocked by 10 μM losartan. IgG from 7 PA subjects increased HAC15 aldosterone (aldo) 1.8 fold over baseline (p&lt;0.01) and markedly increased 1nM and 10 nM Ang II-induced aldo by 3.5 fold (p&lt;0.01); and was blocked by ARB. BP in hi Na rabbits rose from 80/60 to 140/90 6 weeks after immunization with AT1R MAP-L-aa AFHYESQ peptide. Arteriole contraction rose from 5% (preimmune) to 30% (post-immunization). Preincubation with the L-aa 2 nd ECL target reduced contractility to 8%; not different from baseline. Conclusions: AA from PA and AT1R-immunized rabbits activates AT1R transfected CHO cells, increases in vitro arterial contractility and stimulates basal and Ang II induced HAC15 aldosterone secretion. ARBs and decoy peptides were effective in blocking AA effects in vitro . These AA have important etiological and therapeutic implications.

Effect of 36 CYP2C9 variants found in the Chinese population on losartan metabolism in vitro

1. CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Among these variants, we recently identified 21 novel alleles (*36–*56) in the Han Chinese population. The aim of this study was to assess the catalytic activities of 36 CYP2C9 variants found in the Chinese population toward losartan in vitro.2. Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.5–25 μM losartan for 30 min at 37 °C. Next, the products were extracted, and signal detection was performed using high-performance liquid chromatography.3. Compared with wild-type CYP2C9.1, the intrinsic clearance (Vmax/Km) values of all variants except for CYP2C9.56 were significantly altered. One variant exhibited markedly increased values (>250%), whereas 33 variants exhibited significantly decreased values (from 20 to 96%) due to increased Km and/or decreased Vmax values.4. These findings suggest that more attention should be paid to subjects carrying these infrequent CYP2C9 alleles when administering losartan in the clinic.

Angiotensin II Type 1 receptor Regulates BK Channels Independent of G-Protein Activation in Rat Renal Arterial Smooth Muscle Cells

In vascular smooth muscles cells (SMCs),Angiotensin II (Ang II) is known to be involved in numerous biological events, including cell contraction, proliferation, migration, and senescence, through the activation of canonical G-protein-dependent signaling pathways. We previously identified a physical interaction between expressed angiotensin II type 1 receptor (AT1R) and large-conductance calcium-activated potassium (BK) channels. In the present study, we assessed the contribution of AT1R and G-protein activation in Ang II-mediated inhibition of BK currents using losartan, an antagonist of AT1R and GDPβS, an inhibitor of G-protein activation. First, we tested the effectiveness of losartan in preventing AT1R activation in HEK293T cells by measuring ERK1/2 phosphorylation and confirmed that 10 μM losartan could fully inhibit the activation induced by 1 μM Ang II. In freshly isolated rat renal arterial smooth muscle cells (SMCs), extracellular application of 1 μM Ang II reduced whole-cell BK currents by 42.9±6.5% (n=5). Supporting the role of AT1R, BK currents were unaffected after Ang II stimulation in losartan-pretreated SMCs and remained at 99.5±2.8% (n=9) of their original amplitude. The full antagonistic action of losartan was also observed in Ang II pretreated HEK293T cells concurrently expressing AT1R and BK channels (n=6). On the other hand, intracellular application of 500 μM GDPβS via the pipette failed to prevent Ang II-induced inhibition of BK currents supporting a process that is independent of G-protein activation. In summary, the results demonstrate that AT1R mediates the Ang II-induced inhibition of BK currents in renal arterial SMCs, and support the hypothesis that the Ang II inhibitory effect on BK currents is mediated by an intimate interaction between AT1R and BK channels. Supported by NIH.

A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport

Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.

MTOR/S6K Signaling: A Novel Effector of Neuronal Action of Angiotensin II

The renin‐angiotensin system (RAS) is critically involved in arterial pressure regulation. Angiotensin II (Ang II), the main effector of the RAS, acts in several brain nuclei including the subfornical organ (SFO) to increase arterial pressure by stimulating Ang II type 1 receptors (AT1R). However, the signaling mechanisms of neuronal AT1R remain poorly defined. Neuronal mammalian target of rapamycin (mTOR) and its downstream effector, p70S6 kinase (S6K), have emerged as important regulators of arterial pressure. Thus, we hypothesized that mTOR/S6K signaling mediates brain Ang II actions. In hypothalamic neuronal cell line (GT1‐7), transfected with AT1aR, Ang II (100 nM) caused a time‐dependent increase in S6K activity with a maximum effect at 15 min (3.4‐fold; P&lt;0.05 vs. vehicle). Pre‐treatment with AT1R blocker (1 μM losartan) completely abolished Ang II‐induced activation of S6K. We also examined, by immunohistochemistry, the level of phospho‐S6 (a readout of S6K activity) in the brain of transgenic sRA mice which have overactive brain RAS. The SFO of female sRA mice exhibited significantly (P=0.02) elevated phospho‐S6 positive cells (64±6) relative to littermate controls (36±8). This increase was specific to the SFO as there was no difference in phospho‐S6 levels in other forebrain nuclei in sRA vs. control mice. This study has identified mTOR/S6K as a novel mechanism of AT1R signaling in neurons.

Angiotensin III stimulates ERK1/2 mitogen-activated protein kinases and astrocyte growth in cultured rat astrocytes

Angiotensin (Ang) III is a biologically active metabolite of Ang II with similar effects and receptor binding properties as Ang II. Most Ang III studies delineate physiological effects of the peptide but, the intracellular pathways leading to the actions are unknown and are a focus of these studies. We investigated in cultured brainstem and cerebellum rat astrocytes whether Ang III stimulates ERK1/2 mitogen activated protein (MAP) kinases and astrocyte growth. Ang III significantly stimulated ERK1/2 MAP kinases in a dose- and time-dependent manner. The maximal stimulation occurred with 100 nM Ang III (2.8±0.3 and 2.3±0.1-fold over basal, in brainstem and cerebellum astrocytes, respectively). This stimulation occurred as early as 1 min, and was sustained for at least 15 min. Moreover, inhibition of the ERK1/2 MAP kinase pathway by 10 μM PD98059 attenuated Ang III-induced ERK1/2 phosphorylation. Ang III induction of ERK1/2 occurred via stimulation of the Ang AT(1) receptor since pretreatment with 10 μM Losartan, a selective AT(1) receptor blocker, prevented Ang III-induced ERK1/2 phosphorylation. The selective AT(2) Ang receptor blocker PD123319 was ineffective. Comparable to Ang II, Ang III also stimulated astrocyte growth in a concentration-dependent manner, an effect that occurred via activation of the AT(1) receptor as well. These findings suggest that Ang III has similar effects as Ang II in astrocytes since it rapidly stimulates the phosphorylation of the ERK1/2 MAP kinases and induces astrocyte proliferation through activation of the AT(1) receptor. These studies are important in establishing signaling pathways for Ang III and provide validation of the central role of Ang III.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Endothelium-dependent vasomotor effects of telmisartan in isolated rat femoral arteries

AT 1 receptor antagonists (ARBs) are drugs widely used for preventing and/or treating major cardiovascular diseases. Some of these drugs also show AT 1 receptor-independent effects that may have patho-physiological significance, such as Peroxisome Proliferator-Activated Receptors gamma (PPARγ) stimulation. Here we investigated the effect of telmisartan (that also stimulates PPARγ) on vasomotor responses of femoral arteries isolated from rat, in comparison to losartan. Femoral artery segments were mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE) and acetylcholine (ACh) after 30-min incubation in the absence or presence of 30 μM telmisartan or 30 μM losartan. Vasomotor responses were not significantly changed by losartan, whereas telmisartan reduced vasoconstriction to PE and increased vasodilatation to ACh. Incubation with 0.1 mM N G-nitro- l-arginine abolished relaxation to ACh in untreated controls as well as in losartan-treated preparations, but did not in telmisartan-treated preparations (were 20% relaxation subsisted); this residual relaxing effect was abolished by indomethacin and by endothelium removal. Incubation with 30 μM GW9662 (PPARγ antagonist), 10 μM PD123319 (AT 2 antagonist) or 30 μM A779 (angiotensin(1-7)/Mas antagonist) did not change the effect of telmisartan on vasomotor responses in preparations with intact endothelium. We conclude that telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. The present effect of telmisartan, however, does not seem to involve PPARγ, AT 2 or angiotensin(1-7)/Mas.

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT1 and AT4 receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of 125I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ∼1 nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT1 and AT4 receptor antagonists, respectively). The insulin release was accompanied by a 45Ca2+ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT4 receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT1 and AT4 receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca2+. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.

Distribution of a novel binding site for angiotensins II and III in mouse tissues

A novel binding site for angiotensins II and III that is unmasked by parachloromercuribenzoate has been reported in rat, mouse and human brains. Initial studies of this binding site indicate that it is not expressed in the adrenal, liver or kidney of the rat and mouse. To determine if this binding site occurs in other mouse tissues, 8 tissues were assayed for expression of this binding site by radioligand binding assay and compared with the expression of this binding site in the forebrain. Particulate fractions of homogenates of testis, epididymis, seminal vesicles, heart, spleen, pancreas, lung, skeletal muscle, and forebrain were incubated with 125I-sarcosine 1, isoleucine 8 angiotensin II in the presence or absence of 0.3 mM parachloromercuribenzoate plus 10 µM losartan and 10 µM PD123319 (to saturate AT 1 and AT 2 receptors). Specific (3 µM angiotensin II displaceable) high affinity binding occurred in the testis > forebrain > epididymis > spleen > pancreas > lung when parachloromercuribenzoate was present. Binding could not be reliably observed in heart, skeletal muscle and seminal vesicles. High affinity binding of 125I-sarcosine 1, isoleucine 8 angiotensin II was observed in the absence of parachloromercuribenzoate in the pancreas on occasion. This suggests that this novel angiotensin binding site may have a functional role in these tissues.

Angiotensin II enhances hyperpolarization-activated currents in rat aortic baroreceptor neurons: involvement of superoxide

As an endogenous physiologically active peptide, angiotensin (ANG) II plays an important role in the maintenance of blood pressure. In the arterial baroreceptor reflex (a pivotal regulator of blood pressure), aortic baroreceptor (AB) neurons located in the nodose ganglia (NG) are a primary afferent limb of the baroreflex. Hyperpolarization-activated currents (I(h)) in the AB neurons contribute to the excitability of the AB neurons. Therefore, the present study was to measure the modulating effect of ANG II on the I(h) in the primary AB neurons isolated from rats. Data from immunofluorescent and Western blot analyses showed that protein of AT(1) and AT(2) receptors was expressed in the nodose neurons. In the whole cell patch-clamp recording, ANG II concentration dependently enhanced the I(h) density in the AB neurons (100 nM ANG II-induced 53.8 +/- 3.8% increase for A-type AB neurons and 30.4 +/- 7.7% increase for C-type AB neurons at test pulse -140 mV, P < 0.05). ANG II also decreased membrane excitability in the AB neurons. AT(1) receptor antagonist (1 muM losartan) but not AT(2) receptor antagonist (1 muM PD-123,319) totally abolished the effect of ANG II on the I(h) and neuronal excitability. In addition, NADPH oxidase inhibitor (100 muM apocynin) and superoxide scavenger (1 mM tempol) also significantly blunted the ANG II-induced increase of the I(h) and decrease of the membrane excitability in the AB neurons. Furthermore, losartan, apocynin, or tempol significantly attenuated the superoxide overproduction in the NG tissues induced by ANG II. These results suggest that ANG II-NADPH oxidase-superoxide signaling can activate the I(h) and subsequently decrease the membrane excitability of rat AB neurons.

Tissue distribution of a novel non‐AT1, non‐AT2 binding site for angiotensins in the mouse

We recently reported the existence of a novel, non‐AT1, non‐AT2 binding site for angiotensins II (Ang II) and III (Ang III) in the rat, mouse and human brain. While the absence of this binding site in the liver, adrenal, and kidney suggested that this binding site was brain‐specific, we now report its presence in other mouse tissues. 125I‐Sar1,Ile8 Ang II binding was determined as described previously (Karamyan et al., Eur J. Pharmacol., 2008, 590: 87) in tissue homogenates with or without 0.3 mM parachloromercuribenzoate (PCMB), in the presence of 10 μM losartan and 10 μM PD123319 to saturate AT1 and AT2 receptors, respectively. In the absence of 0.3 mM PCMB specific (3 μM Ang II displaceable) binding of 125I‐ Sar1,Ile8 Ang II could not be measured reliably. In the presence of 0.3 mM PCMB, saturable, high affinity binding was observed in the testis (4.1±2.2 pmol/g, KD = 5.0±2.7 nM) with comparable density to the brain (4.2±2.0 pmol/g), but slightly lower affinity (KD = 2.7±1.2 nM, p&lt;0.05). Measurable binding was present in 5 of 6 pancreas and 4 of 7 spleen samples with KD values similar to the brain, but at lower (23 and 30%, respectively) binding density. Binding was generally undetectable in the lung and heart. The presence of this binding site in testis, pancreas and spleen may indicate additional functions for angiotensins in these tissues. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides

Aims To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. Main methods Radioligand binding assays of 125I-sarcosine 1, isoleucine 8 angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 µM losartan to saturate AT1 receptors, 10 µM PD123319 to saturate AT2 receptors, with or without 10 µM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. Key findings The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77 ± 0.16 and 1.52 ± 0.17 fmol/mg initial wet weight in female and male brains, respectively. The K D values, 1.79 ± 0.09 nM for females, and 1.53 ± 0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine 1, isoleucine 8 angiotensin II > angiotensin III > angiotensin II > angiotensin I ’ angiotensin IV > angiotensin 1–7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. Significance The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.