Vasodilator Effect of Trace Amines, 3-Iodothyronamine, and RO5263397 in the Rat Perfused Kidney: Comparison with Tryptamine.

INTRODUCTION: Trace amine-associated receptors (TAARs) are a family of G protein-coupled receptors and are widely distributed in the body. Activation of TAAR1 by specific agonists can produce a variety of physiological effects centrally and peripherally. The objective of this study was to investigate the vasodilator effect of two selective TAAR1 agonists 3-iodothyronamine (T1AM) and RO5263397 in the isolated perfused rat kidney preparation. METHODS: Kidneys were isolated and perfused with Krebs' solution, gassed with 95% oxygen and 5% carbon dioxide, through the renal artery. RESULTS: In preparations pre-constricted with methoxamine (5 × 10-6m), T1AM (10-10 - 10-6 mol), RO5263397 (10-10 - 10-6 mol), and tryptamine (10-10 - 10-6 mol) produced dose-dependent vasodilator responses. EPPTB (1 × 10-6m), a selective TAAR1 antagonist, had no effect on vasodilator responses induced by these agonists. A higher concentration of EPPTB (3 × 10-5m) produced a sustained increase in perfusion pressure but did not affect vasodilator responses to tryptamine, T1AM, and RO5263397. Agonist-induced vasodilator responses were slightly reduced by the removal of the endothelium but were not affected by L-NAME (1 × 10-4m), an inhibitor of nitric oxide synthesis. The vasodilator responses were significantly reduced by inhibiting calcium-activated (tetraethylammonium, 1 × 10-3m) and voltage-activated (4-AP, 1 × 10-3m) potassium channels. Tryptamine-, T1AM-, and RO5263397-induced vasodilator responses were significantly reduced by BMY7378, a 5-HT1A receptor antagonist. CONCLUSION: It was concluded that vasodilator responses produced by the TAAR1 agonists, T1AM, RO5263397, and tryptamine, were not mediated via TAAR1 but were probably via activation of 5-HT1A receptors.

Hydrogen sulfide donor GYY4137 attenuates vascular complications in mesenteric bed of streptozotocin-induced diabetic rats.

Hydrogen sulfide (H2S) has been reported to have beneficial effects in different pathological conditions. OBJECTIVES: the effects of chronic treatment of diabetic rats with GYY4137 (slow releasing H2S donor) or NaHS (fast releasing H2S donor) on the reactivity of the mesenteric bed to vasoactive agonists and the changes in its downstream effectors, ERK1/2 and p38 MAP Kinase have been investigated. In addition, the levels of nitric oxide (NO) and H2S in all groups were measured. METHODS: diabetes was induced by a single intraperitoneal (ip) injection of streptozotocin (STZ; 55 mg/kg). Sprague Dawley (SD; n = 10-12/group) rats were randomly divided into six groups: control, STZ-induced diabetic rats, GYY4137-treated control, NaHS-treated control, GYY4137-treated diabetic, and NaHS-treated diabetic. After 28 days of treatment, rats were sacrificed and mesenteric beds were isolated for functional or biochemical studies. The vascular reactivity of the perfused mesenteric bed to norepinephrine, carbachol and sodium nitroprusside were determined by measurement of changes in perfusion pressure. Western blotting was performed to measure the protein expression of ERK1/2, p38, eNOS, and H2S biosynthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase. NO and H2S levels were measured in all groups in isolated mesenteric tissues or plasma. RESULTS: diabetes resulted in a significant increase in vasoconstrictor responses to norepinephrine (e.g., 129.6 ± 6.77 mmHg in diabetic vs 89.3 ± 8.48 mmHg in control at 10-7 dose), and carbachol-induced vasodilation was significantly reduced in diabetic mesenteric bed (e.g., 68.9 ± 4.8 mmHg in diabetic vs 90.6 ± 2.2 mmHg in control at 10-7 dose). Chronic treatment of the diabetic rats with GYY4137 resulted in a significant improvement in the response to norepinephrine (e.g., 86.66 ± 8.04 mmHg in GYY4137-treated diabetic vs 129.6 ± 6.77 mmHg in untreated diabetic at 10-7 dose) or carbachol (e.g., 84.90 ± 2.48 mmHg in GYY4137-treated diabetic vs 68.9 ± 4.8 mmHg in untreated diabetic at 10-7 dose). The biochemical studies showed a marked reduction of the protein expression of ERK and p38 and a significant upregulation of the expression of eNOS and H2S synthesizing enzymes after chronic treatment with GYY4137. Plasma levels of NO and H2S were significantly elevated after treatment with GYY4137. However, H2S production in the mesenteric bed showed a marginal elevation in diabetic tissues compared to controls. CONCLUSION: the results indicate that GYY4137 may be a novel therapeutic tool to prevent diabetes-associated vascular dysfunction.

Chronic Treatment With Hydrogen Sulfide Donor GYY4137 Mitigates Microglial and Astrocyte Activation in the Spinal Cord of Streptozotocin-Induced Diabetic Rats.

Long-term diabetic patients suffer immensely from diabetic neuropathy. This study was designed to investigate the effects of hydrogen sulfide (H2S) on peripheral neuropathy, activation of microglia, astrocytes, and the cascade secretion of proinflammatory cytokines in the streptozotocin (STZ)-induced peripheral diabetic neuropathy rat model. STZ-induced diabetic rats were treated with the water-soluble, slow-releasing H2S donor GYY4137 (50 mg/kg; i.p.) daily for 4 weeks. Antiallodynic/antihyperalgesic activities were evaluated using different tests and histopathological changes and the expression of proinflammatory cytokines in the spinal cord were examined. GYY4137 treatment produced neuroprotective effects in the spinal cord of diabetic animals and modulated their sensory deficits. The treatment decreased allodynia (p < 0.05) and mechanical hyperalgesia (p < 0.01) and restored thermal hyperalgesia (p < 0.001) compared with diabetic rats. The treatment decreased the microglial response and increased astrocyte counts in spinal cord gray and white matter compared with untreated diabetic rats. Proinflammatory cytokines were reduced in the treated group compared with diabetic rats. These results suggest that H2S has a potentially ameliorative effect on the neuropathic pain through the control of astrocyte activation and microglia-mediated inflammation, which may be considered as a possible treatment of peripheral nerve hypersensitivity in diabetic patients.

Alleviation of impaired reactivity in the corpus cavernosum of STZ-diabetic rats by slow-release H2S donor GYY4137.

GYY4137 is a novel hydrogen sulfide (H2S) releasing molecule with vasodilator activity. The objectives of this study were to investigate: (1) the pharmacological effect of GYY4137 on the reactivity of the corpus cavernosum (CC) from normal and diabetic rats; (2) the contribution of ATP-sensitive potassium (K-ATP) channels and nitric oxide (NO) pathway; (3) the reactivity to vasoactive agonists following ex vivo incubation of the diabetic rat CC with GYY4137. Longitudinal strips of CC from control and diabetic male Sprague-Dawley (SD) rats (n = 5-6 animals per group) were suspended in organ-baths. Responses to GYY4137, carbachol, or phenylephrine (PE) were determined by measurement of changes in isometric tension. The effects of acute incubation of the CC strips with L-NAME (NO synthase inhibitor) or glibenclamide (K-ATP channel inhibitor) on the relaxant responses to GYY4137 were examined. The effect of ex vivo incubation with GYY4137 (10-5 M) on the responses of CC to carbachol or PE was evaluated. We found that GYY4137 provoked relaxation in the CC strips, which was significantly reduced in the presence of L-NAME or glibenclamide. Ex vivo incubation of diabetic CC with GYY4137 resulted in a significant improvement in the vascular responses to the added agonists. We conclude that GYY4137 is a relaxant agonist in SD rats CC, and the response is mediated, at least in part, by NO and K-ATP channels. Brief incubation of diabetic CC with GYY4137 markedly improved the impaired vascular reactivity, thus raising the question whether chronic in vivo treatment of diabetic animals with GYY4137 would have any protective effect, which is worth further investigation.

Angiotensin-(1-7)-dependent vasorelaxation of the renal artery exhibits unique angiotensin and bradykinin receptor selectivity.

Angiotensin-(1-7) [Ang-(1-7)] exhibits blood pressure lowering actions, inhibits cell growth, and reduces tissue inflammation and fibrosis which may functionally antagonize an activated Ang II-AT1 receptor axis. Since the vascular actions of Ang-(1-7) and the associated receptor/signaling pathways vary in different vascular beds, the current study established the vasorelaxant properties of the heptapeptide in the renal artery of male Wistar male rats. Ang-(1-7) produced an endothelium-dependent vasodilator relaxation of isolated renal artery segments pre-contracted by a sub-maximal concentration of phenylephrine (PE) (3×10-7M). Ang-(1-7) induced vasodilation of the rat renal artery with an ED50 of 3±1nM and a maximal response of 42±5% (N=10). The two antagonists (10-5M each) for the AT7/Mas receptor (MasR) [D-Pro7]-Ang-(1-7) and [D-Ala7]-Ang-(1-7) significantly reduced the maximal response to 12±1% and 18±3%, respectively. Surprisingly, the AT2R receptor antagonist PD123319, the AT1R antagonist losartan and B2R antagonist HOE140 (10-6M each) also significantly reduced Ang-(1-7)-induced relaxation to 12±2%, 22±3% and 14±7%, respectively. Removal of the endothelium or addition of the soluble guanylate cyclase (sGC) inhibitor ODQ (10-5M) essentially abolished the vasorelaxant response to Ang-(1-7) (10±4% and 10±2%, P <0.05). Finally, the NOS inhibitor LNAME (10-4M) reduced the response to 13±2% (p<0.05), but the cyclooxygenase inhibitor indomethacin failed to block the Ang-(1-7) response. We conclude that Ang-(1-7) exhibits potent vasorelaxant actions in the isolated renal artery that are dependent on an intact endothelium and the apparent stimulation of a NO-sGC pathway. Moreover, Ang-(1-7)-dependent vasorelaxation was sensitive to antagonists against the AT7/Mas, AT1, AT2 and B2 receptor subtypes.

Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor.

Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1-7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1-7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1-7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1-7). Ang-(1-7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1-7) that could be prevented by D-Pro7-Ang-(1-7) in VSMC. These data suggest that Ang-(1-7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.

Cationic Polyamidoamine Dendrimers as Modulators of EGFR Signaling In Vitro and In Vivo.

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling-an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine.

The dual targeting of EGFR and ErbB2 with the inhibitor Lapatinib corrects high glucose-induced apoptosis and vascular dysfunction by opposing multiple diabetes-induced signaling changes.

The epidermal growth factor receptors, EGFR and EGFR2 (ErbB2), appear important mediators of diabetes-induced vascular dysfunction. We investigated whether targeted dual inhibition of EGFR and ErbB2 with Lapatinib would be effective in treating diabetes-induced vascular dysfunction in a rat model of type 1 diabetes. In streptozotocin-induced diabetes, chronic 4-week oral or acute, ex vivo, administration of Lapatinib prevented the development of vascular dysfunction as indicated by the attenuation of the hyper-reactivity of the diabetic mesenteric vascular bed (MVB) to norephinephrine without correcting hyperglycemia. Chronic in vivo or acute ex vivo Lapatinib treatment also significantly attenuated diabetes-induced increases in phosphorylation of EGFR, ErbB2, ERK1/2, AKT, ROCK2 and IkB-alpha as well as normalized the reduced levels of phosphorylated FOXO3A, and eNOS (Ser1177) in the diabetic MVB. Similar results were observed in vascular smooth muscle cells (VSMCs) cultured in high glucose (25 mM) treated with Lapatinib or small interfering RNA (siRNA) targeting the ErbB2 receptor. Lapatinib also prevented high glucose-induced apoptosis in VSMC. Thus, Lapatinib corrects hyperglycemia-induced apoptosis and vascular dysfunction with concomitant reversal of diabetes or high glucose-induced signaling changes in EGFR/ErbB2 and downstream signaling pathways implying that targeted dual inhibition of EGFR/ErbB2 might be an effective vasculoprotective treatment strategy in diabetic patients.

Chronic treatment with Ang-(1-7) reverses abnormal reactivity in the corpus cavernosum and normalizes diabetes-induced changes in the protein levels of ACE, ACE2, ROCK1, ROCK2 and omega-hydroxylase in a rat model of type 1 diabetes.

Angiotensin-(1-7) [Ang-(1-7)] may have beneficial effects in diabetes mellitus-induced erectile dysfunction (DMIED) but its molecular actions in the diabetic corpus cavernosum (CC) are not known. We characterized the effects of diabetes and/or chronic in vivo administration of Ang-(1-7) on vascular reactivity in the rat corpus cavernosum (CC) and on protein expression levels of potential downstream effectors of the renin-angiotensin-aldosterone system (RAAS) such as angiotensin-converting enzyme (ACE), ACE2, Rho kinases 1 and 2 (ROCK1 and ROCK2), and omega-hydroxylase, the cytochrome-P450 enzyme that metabolizes arachidonic acid to form the vasoconstrictor, 20-hydroxyeicosatetraenoic acid. Streptozotocin-treated rats were chronicically administered Ang-(1-7) with or without A779, a Mas receptor antagonist, during weeks 4 to 6 of diabetes. Ang-(1-7) reversed diabetes-induced abnormal reactivity to vasoactive agents (endothelin-1, phenylepherine, and carbachol) in the CC without correcting hyperglycemia. Six weeks of diabetes led to elevated ACE, ROCK1, ROCK 2, and omega-hydroxylase and a concomitant decrease in ACE2 protein expression levels that were normalized by Ang-(1-7) treatment but not upon coadministration of A779. These data are supportive of the notion that the beneficial effects of Ang-(1-7) in DMIED involve counterregulation of diabetes-induced changes in ACE, ACE2, Rho kinases, and omega-hydroxylase proteins in the diabetic CC via a Mas receptor-dependent mechanism.

Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction.

Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu), a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor) family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem) or acute, ex-vivo (10(-6), 10(-5) M) administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB) to the vasoconstrictor, norephinephrine (NE) and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y) residues (Y1221/1222, Y1248 and Y877) in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs) and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC) that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications.

Role of angiotensin II and angiotensin-(1-7) in diabetes-induced oxidative DNA damage in the corpus cavernosum.

OBJECTIVE: To investigate diabetes mellitus (DM)-induced oxidative DNA damage, putative involvement of angiotensin (Ang) II, and possible modulatory effects of Ang-(1-7) in rat corpus cavernosum (CC). DESIGN: In vivo study. SETTING: Research laboratory. ANIMAL(S): Adult male Wistar rats. INTERVENTION(S): Streptozotocin-induced diabetic rats received either captopril, losartan (both 300 mg/L in drinking water), or Ang-(1-7) (576 µg/kg/d IP) for a 3-week period immediately before sacrifice at 6 weeks of DM. MAIN OUTCOME MEASURE(S): Histopathological changes in CC were examined in Masson's trichrome-stained tissue sections. Oxidative stress was evaluated by measuring total oxidant status and antioxidant status. The DNA damage was estimated by measuring 8-hydroxy-2'-deoxyguanosine by immunohistochemistry and ELISA. RESULT(S): The CC smooth muscle degeneration was observed in association with an increase in total oxidant status and a decrease in total antioxidant status in rats with DM. Oxidative DNA damage was significantly increased in both cytoplasm and nuclei of CC in DM. Treatment with captopril, losartan, or Ang-(1-7) inhibited these changes in rats with DM. CONCLUSION(S): The data indicate that Ang II signaling is involved in DM-induced structural changes and oxidative DNA damage in the CC and that modulation of the signaling by captopril, losartan, and Ang-(1-7) restores the effects of DM. Thus, Ang-(1-7)/MAS1 axis may be a novel therapeutic target for erectile dysfunction in DM.

On the nanotoxicity of PAMAM dendrimers: Superfect® stimulates the EGFR-ERK1/2 signal transduction pathway via an oxidative stress-dependent mechanism in HEK 293 cells.

Polyamidoamine (PAMAM) dendrimers are cationic branch-like macromolecules that may serve as drug delivery systems for gene-based therapies such as RNA interference. For their safe use in the clinic, they should ideally only enhance drug delivery to target tissues and exhibit no adverse effects. However, little is known about their toxicological profiles in terms of their interactions with cellular signal transduction pathways such as the epidermal growth factor receptor (EGFR). The EGFR is an important signaling cascade that regulates cell growth, differentiation, migration, survival and apoptosis. Here, we investigated the impact of naked, unmodified Superfect (SF), a commercially available generation 6 PAMAM dendrimer, on the epidermal growth factor receptor (EGFR) tyrosine kinase-extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway in human embryonic kidney (HEK 293) cells. At concentrations routinely used for transfection, SF exhibited time and dose-dependent stimulation of EGFR and ERK1/2 phosphorylation whereas AG1478, a selective EGFR tyrosine kinase antagonist, inhibited EGFR-ERK1/2 signaling. SF-induced phosphorylation of EGFR for 1h was partly reversible upon removal of the dendrimer and examination of cells 24 later. Co-treatment of SF with epidermal growth factor (EGF) ligand resulted in greater EGFR stimulation than either agent alone implying that the stimulatory effects of SF and the ligand are synergistic. Dendrimer-induced stimulation of EGFR-ERK1/2 signaling could be attenuated by the antioxidants apocynin, catalase and tempol implying that an oxidative stress dependent mechanism was involved. These results show for the first time that PAMAM dendrimers, aside from their ability to improve drug delivery, can modulate the important EGFR-ERK1/2 cellular signal transduction pathway - a novel finding that may have a bearing on their safe application as drug delivery systems.

Chronic treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase, attenuates ischemia/reperfusion-induced cardiac dysfunction in normotensive and hypertensive diabetic animals.

Diabetes is associated with increased incidence of cardiovascular disease. Mechanisms that contribute to development of diabetic cardiopathy are not well understood. Phosphatidylinositol 3-kinase (PI3K) is a family of protein kinases that play an important role in regulation of cardiac function. It has been shown that inhibition of certain PI3K enzymes may produce cardiovascular protection. The aim of the present study was to determine whether chronic treatment with LY294002, an inhibitor of PI3K, can attenuate diabetes-induced cardiac dysfunction in isolated hearts obtained from normotensive and hypertensive rats. Recovery of cardiac function after 40 min of global ischemia and 30 min of reperfusion, measured as left ventricular developed pressure, left ventricular end-diastolic pressure, coronary flow and coronary vascular resistance, was worse in hearts obtained from diabetic and/or hypertensive animals compared to their respective controls. Treatment with LY294002 (1.2 mg/kg/day) for 4 weeks significantly prevented diabetes-induced cardiac dysfunction in both normotensive and hypertensive rats. Treatment with LY294002 did not significantly alter blood pressure or blood glucose levels. These results suggest that inhibition of PI3K signaling pathways can prevent ischemia/reperfusion-induced cardiac dysfunction in normotensive and hypertensive rats without correcting hyperglycemia or high blood pressure.

Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury.

This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.

Characterization of Angiotensin-(1-7) effects on the cardiovascular system in an experimental model of type-1 diabetes.

Although exogenous administration of Angiotensin-(1-7) [Ang-(1-7)] can prevent development of diabetes induced end-organ damage, little is known about the role of endogenous Ang-(1-7) in diabetes and requires further characterization. Here, we studied the effects of chronically inhibiting endogenous Ang-(1-7) formation with DX600, a selective angiotensin converting enzyme-2 (ACE2) inhibitor, on renal and cardiac NADPH oxidase (NOX) activity, vascular reactivity and cardiac function in a model of Type-1 diabetes. The contribution of endogenous Ang-(1-7) to the protective effects of Losartan and Captopril and that of prostaglandins to the cardiovascular effects of exogenous Ang-(1-7) were also examined. Cardiac and renal NOX activity, vascular reactivity to endothelin-1 (ET-1) and cardiac recovery from ischemia/reperfusion (I/R) injury were evaluated in streptozotocin-treated rats. Chronic treatment with DX600 exacerbated diabetes-induced increase in cardiac and renal NOX activity. Diabetes-induced abnormal vascular reactivity to ET-1 and cardiac dysfunction were improved by treatment with Ang-(1-7) and worsened by treatment with DX600 or A779, a Mas receptor antagonist. Ang-(1-7)-mediated improvement in cardiac recovery or vascular reactivity was attenuated by Indomethacin. Captopril and Losartan-induced improvement in cardiovascular function was attenuated when these drugs were co-administered with A779. Ang-(1-7)-mediated decrease in renal NOX activity was prevented by indomethacin. Losartan also decreased renal NOX activity that could be attenuated with A779 co-treatment. In conclusion, endogenous Ang-(1-7) inhibits diabetes-induced cardiac/renal NOX activity and end-organ damage, and mediates the actions of Captopril and Losartan. Further, prostaglandins are important intermediaries in the beneficial effects of Ang-(1-7) in diabetes. Combining either Losartan or Captopril with Ang-(1-7) had additional beneficial effects in preventing diabetes-induced cardiac dysfunction and this may represent a novel therapeutic strategy. Collectively, these data shed new insights into the likely mechanism of action through which the ACE2/Ang-(1-7)/Mas receptor axis prevents Type 1 diabetes-induced cardiovascular dysfunction.

Angiotensin-(1-7) inhibits epidermal growth factor receptor transactivation via a Mas receptor-dependent pathway.

BACKGROUND AND PURPOSE: The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(1-7) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro. EXPERIMENTAL APPROACH: Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG1478, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs). KEY RESULTS: Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y1068, Y1086, Y1148, as well as ERK1/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(1-7) or AG1478 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(1-7) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(1-7) could be prevented by a selective Mas receptor antagonist, D-Pro7-Ang-(1-7). CONCLUSIONS AND IMPLICATIONS: These results show for the first time that Ang-(1-7) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction.

Anti-tussive and bronchodilator mechanisms of action for the enaminone E121.

AIMS: In this study, we investigated whether the enaminone, E121, has anti-tussive effects in a guinea pig model of cough, and if so, whether this effect is mediated via a central or peripheral site of action. We also assessed whether E121 has bronchodilator effects and the molecular mechanisms underlying any anti-tussive and/or bronchodilator effects. MAIN METHODS: Whole body plethysmography was used to assess both cough and airway obstruction. A stereotaxic apparatus was used to administer drugs intracerebroventricularly (i.c.v.). Effects of E121 were examined in vitro on contractile effects in guinea pig bronchioles. KEY FINDINGS: Pre-treatment of animals with E121 resulted in a significant inhibition in the citric acid-induced cough and airway obstruction compared to vehicle-pretreated animals. The K(ATP) antagonist, glibenclamide, significantly inhibited the anti-tussive and bronchoprotective effects of E121. Also, intra-tracheal administration of E121 resulted in a significant inhibition of both the citric acid-induced cough response and airway obstruction compared to vehicle-pretreated animals. By contrast, i.c.v. administration had no effect. Finally, E121 significantly inhibited carbachol-induced airway smooth muscle contractions, an effect that was reduced by both glibenclamide and propranolol. Interestingly, E121 enhanced histamine-induced cAMP release in human eosinophils although it did not directly elevate cAMP levels. SIGNIFICANCE: The enaminone, E121, has anti-tussive and bronchodilatory effects and is topically, but not centrally, active. The anti-tussive mechanism of action of E121 seems to be K(ATP) channel dependent, whereas its bronchodilatory effects appear to be mediated via activation of both K(ATP) channels and ß(2) receptors. Therefore, E121 may potentially represent a novel therapy for cough, particularly cough associated with airway obstruction.

Angiotensin-(1-7) blockade attenuates captopril- or hydralazine-induced cardiovascular protection in spontaneously hypertensive rats treated with NG-nitro-L-arginine methyl ester.

We assessed the contribution of angiotensin-(1-7) [Ang-(1-7)] to captopril-induced cardiovascular protection in spontaneously hypertensive rats (SHRs) chronically treated with the nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (SHR-l). NG-nitro-L-arginine methyl ester (80 mg/L) administration for 3 weeks increased mean arterial pressure (MAP) from 196 ± 6 to 229 ± 3 mm Hg (P < 0.05). Treatment of SHR-l with Ang-(1-7) antagonist [d-Ala7]-Ang-(1-7) (A779; 744 µg·kg(-1)·d(-1) ip) further elevated MAP to 253 ± 6 mm Hg (P < 0.05 vs SHR-l or SHR). Moreover, A779 treatment attenuated the reduction in MAP and proteinuria by either captopril (300 mg/L in drinking water) or hydralazine (1.5 mg·kg(-1)·d(-1) ip). In isolated perfused hearts, the recovery of left ventricular function from global ischemia was enhanced by captopril or hydralazine treatment and was exacerbated with A779. The Ang-(1-7) antagonist attenuated the beneficial effects of captopril and hydralazine on cardiac function. Recovery from global ischemia was also improved in isolated SHR-l hearts acutely perfused with captopril during both the perfusion and reperfusion periods. The acute administration of A779 reduced the beneficial actions of captopril to improve recovery after ischemia. We conclude that during periods of reduced nitric oxide availability, endogenous Ang-(1-7) plays a protective role in effectively buffering the increase in blood pressure and renal injury and the recovery from cardiac ischemia. Moreover, Ang-(1-7) contributes to the blood pressure lowering and tissue protective actions of captopril and hydralazine in a model of severe hypertension and end-organ damage.

Role of 20-hydroxyeicosatetraenoic and epoxyeicosatrienoic acids in the regulation of vascular function in a model of hypertension and endothelial dysfunction.

The objective of this study was to determine if acute inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis or reduced inactivation of epoxyeicosatrienoic acids (EETs) can correct L-N(G)-nitro-arginine-methyl-ester (L-NAME)-induced abnormal vascular reactivity in the perfused mesenteric bed and the carotid artery of spontaneously hypertensive rats (SHR). Administration of L-NAME in drinking water (80 mg/l) to SHR for 3 weeks resulted in abnormal vascular reactivity to norepinephrine and carbachol in the perfused mesenteric vascular bed and carotid artery, and significantly elevated mean arterial blood pressure (244 +/- 9 mm Hg) as compared to SHR controls drinking regular water (176 +/- 3 mm Hg). In the perfused mesenteric vascular bed, the impaired vascular responsiveness to norepinephrine was corrected by acute treatment with N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016), an inhibitor of 20-HETE formation, but not by 1-cyclohexyl-3-dodecyl urea (CDU), an inhibitor of soluble epoxide hydrolase. Treatment with either HET0016 or CDU did not improve impaired carbachol-induced vasodilation in the perfused mesenteric vascular bed. In the isolated carotid artery, treatment with HET0016 corrected the L-NAME-induced increase in norepinephrine-induced vasoconstriction, whereas only CDU treatment could improve impaired carbachol-induced vasodilation. Results of this study indicate that vascular function in a state of compromised nitric oxide formation is differentially modulated by 20-HETE and EETs, and that treatment with HET0016 or CDU may improve vascular function in a state of high blood pressure and endothelial dysfunction.

Angiotensin-(1-7) prevents diabetes-induced attenuation in PPAR-gamma and catalase activities.

The mechanisms by which angiotensin-(1-7) [Ang-(1-7)] exerts its beneficial effects on end-organ damage associated with diabetes and hypertension are not well understood. The purpose of this study was A) to compare the effects of apocynin with Ang-(1-7) on renal vascular dysfunction and NADPH oxidase activity in a combined model of diabetes and hypertension and B) to further determine whether chronic treatment with Ang-(1-7) can modulate renal catalase, and peroxisome proliferator activated receptor- gamma (PPAR-gamma) levels in streptozotocin-induced diabetes in both normotensive Wistar Kyoto rats (WKY) and in spontaneously hypertensive rats (SHR). Apocynin or Ang-(1-7) treatment for one month starting at the onset of diabetes similarly attenuated elevation of renal NADPH oxidase activity in the diabetic SHR kidney and reduced the degree of proteinuria and hyperglycemia, but had little or modest effect on reducing mean arterial pressure. Both drugs also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-1. Induction of diabetes in WKY and SHR animals resulted in significantly reduced renal catalase activity and in PPAR-gamma mRNA and protein levels. Treatment with Ang-(1-7) significantly prevented diabetes-induced reduction in catalase activity and the reduction in PPAR-gamma mRNA and protein levels in both animal models. Taken together, these data suggest that activation of Ang-(1-7)-mediated signaling could be an effective way to prevent the elevation of NADPH oxidase activity and inhibition of PPAR-gamma and catalase activities in diabetes and/or hypertension.