Gamal Abou-Mohamed

Experimental Diabetes Causes Breakdown of the Blood-Retina Barrier by a Mechanism Involving Tyrosine Nitration and Increases in Expression of Vascular Endothelial Growth Factor and Urokinase Plasminogen Activator Receptor

The purpose of these experiments was to determine the specific role of reactive oxygen species (ROS) in the blood-retinal barrier (BRB) breakdown that characterizes the early stages of vascular dysfunction in diabetes. Based on our data showing that high glucose increases nitric oxide, superoxide, and nitrotyrosine formation in retinal endothelial cells, we hypothesized that excess formation of ROS causes BRB breakdown in diabetes. Because ROS are known to induce increases in expression of the well-known endothelial mitogen and permeability factor vascular endothelial growth factor (VEGF) we also examined their influence on the expression of VEGF and its downstream target urokinase plasminogen activator receptor (uPAR). After 2 weeks of streptozotocin-induced diabetes, analysis of albumin leakage confirmed a prominent breakdown of the BRB. This permeability defect was correlated with significant increases in the formation of nitric oxide, lipid peroxides, and the peroxynitrite biomarker nitrotyrosine as well as with increases in the expression of VEGF and uPAR. Treatment with a nitric oxide synthase inhibitor (N-omega-nitro-L-arginine methyl ester, 50 mg/kg/day) or peroxynitrite scavenger (uric acid, 160 mg/kg/day) blocked the breakdown in the BRB and prevented the increases in formation of lipid peroxides and tyrosine nitration as well as the increases in expression of VEGF and uPAR. Taken together, these data indicate that early diabetes causes breakdown of the BRB by a mechanism involving the action of reactive nitrogen species in promoting expression of VEGF and uPAR.

Neuroprotective Effect of(−)Δ9-Tetrahydrocannabinol and Cannabidiol in N-Methyl-d-Aspartate-Induced Retinal Neurotoxicity : Involvement of Peroxynitrite

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.

Downregulation of nitrovasodilator‐induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin‐1β

1 Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO‐induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin‐1β (IL‐1β). 2 Exposure of rat aortic smooth muscle cells to SNP (10 μm) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30–70% in IL‐1β‐ or LPS‐pretreated cells, in a pretreatment time‐and IL‐1β‐ or LPS‐concentration‐dependent manner. When, however, cells were exposed to IL‐1β or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3 Pretreatment of rats with LPS (5 mg kg−1, i.v.) for 6 h led to a decrease in aortic ring SNP‐induced cyclic GMP accumulation. 4 The IL‐1β‐induced reduction in SNP‐stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5 Reverse‐transcriptase‐polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the α1 subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS‐ or IL‐1β‐treated cells, as compared to vehicle‐treated cells. 6 Protein levels of the α1 sGC subunit remained unaltered upon exposure to LPS or IL‐1β, suggesting that the early decreased cyclic GMP accumulation in IL‐1β‐ or LPS‐pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.

Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin

The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate L‐arginine in development of tolerance to nitroglycerins (GTN) vasodilator actions. GTNs effects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by ∼40%, comparable with acetylcholine (ACh)‐treated controls. Both effects were blocked by L‐NMMA. Photometric assays showed that both GTN and ACh‐stimulated NO formation. Both effects were potentiated by L‐arginine and inhibited by L‐NAME. L‐NAME inhibited ACh responses ∼80% compared with ∼40% for GTN responses. The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTNs vasodilating effects as evidenced by a 38 fold rightward shift of the concentration‐relaxation curve. In contrast to D‐arginine, addition of L‐arginine substantially inhibited this effect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L‐arginine tissue levels. GTN had a biphasic effect on BAEC uptake of L‐arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L‐arginine. Prolonged GTN exposure reduces GTNs vasodilator actions, decreases L‐arginine tissue levels and depresses BAECs uptake of L‐arginine. Supplementation of L‐arginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

Zn2+ inhibits nitric oxide formation in response to lipopolysaccharides: implication in its anti-inflammatory activity.

There is compelling evidence to indicate an anti-inflammatory action of Zn2+. Most inflammatory diseases are associated with an increase of the inducible form of nitric oxide (NO) synthase. Additionally, inflammatory mediators such as histamine or bradykinin stimulate the constitutive NO synthase. Thus, the present study was undertaken to investigate whether Zn2+ inhibits production of inducible NO synthase and/or constitutive NO synthase activity to produce NO. Lipopolysaccharide, 5 mg/kg i.v., administered to Zn2+-deficient (ZD) rats, rats supplemented with Zn2+ sulfate (ZG), 10 mg/kg s.c., or controls resulted in a significant reduction of their serum Zn2+. The levels of N(G)-nitro-L-arginine methylester (L-NAME)-sensitive cyclic GMP (cGMP) in aortas isolated from ZD or ZG were significantly lower than those obtained from control animals. Zinc (100-150 microM) produced a dose-dependent inhibition of lipopolysaccharide or interleukin-1beta-induced NO formation in isolated rat aortic smooth muscle cells. Compared to cyclohexamide or actinomycin-D, the time course of inhibition of NO formation by 150 microM Zn2+ did not suggest an effect of Zn2+ on inducible NO synthase protein synthesis. Moreover, Zn2+ (150 microM) significantly reduced the rate of conversion of [3H]arginine to [3H]citrulline in lung homogenates from lipopolysaccharide-treated rats. Incubation of rat aortic smooth muscle cells and bovine pulmonary artery endothelial cell co-cultures with Zn2+ (150 microM) caused a significant reduction in basal and bradykinin- or A-23187-induced formation of cGMP. Thus, our results indicate that Zn2+ is capable of inhibiting lipopolysaccharide- or interleukin-1beta-induced NO formation as well as NO formation by constitutive NO synthase basally or in response to bradykinin or A-23187, and may explain the reported anti-inflammatory activity of Zn2+.

Effect of Zinc on the Anti-Inflammatory and Ulcerogenic Activities of Indometacin and Diclofenac

In the present study, the potential anti-inflammatory activity of zinc sulfate (zinc) has been examined in rats with acute and chronic inflammation. Additionally, we studied the effect of the concurrent administration of zinc on the anti-inflammatory activity of indometacin and diclofenac and their gastric side effects. Oral or subcutaneous administration of zinc (25 and 15 mg/kg, respectively) significantly reduced carrageenan-induced paw edema. Subcutaneous co-administration of zinc (15 mg/kg) and indometacin (5 mg/kg) or diclofenac (10 mg/kg) resulted in a further reduction in paw edema which was more than either that produced by either agent alone. However, after oral co-administration of zinc and diclofenac the reduction in paw edema was not significantly different from that produced by either zinc or diclofenac alone. In rats with chronic inflammation, the administration of zinc (5 mg/kg s.c. for 7 days) proved as effective as either indometacin (3 mg/kg) or diclofenac (5 mg/kg). Co-administration of zinc with indometacin or diclofenac did not affect the level of activity of either drug. Co-administration of zinc did not affect the ulcerogenic effect of indometacin expressed as the ulcer index. In contrast to indometacin, administration of zinc markedly reduced the ulcerative action of diclofenac. In conclusion, zinc supplementation may contribute significantly to the treatment of inflammation. The combination of zinc with other anti-inflammatory drugs may provide beneficial additive effects and reduce their gastric hazards, particularly with diclofenac.

Estradiol Relaxes Rat Aorta via Endothelium-Dependent and -Independent Mechanisms

The effects of estrogen on arterial function are heterogeneous with respect to vessel and/or species. We have investigated 17β-estradiol-induced relaxation in isolated rat aorta with regard to the role of the vascular endothelium and ionic mechanisms. Estrogen induced a concentration-dependent relaxation of 46.5 ± 7.9% and 70.1 ± 12.2% (10–8 and 10–7M), which was reduced by endothelial denudation. Furthermore, L-nitroarginine methyl ester completely abrogated this effect; however, estradiol did not relax KCl-contracted rings. Tetraethyl ammonium (1 mmol/l) completely blocked estradiol-induced relaxation. Estradiol increased [cGMP] in isolated aortic rings via NO, but did not significantly affect NOS activity in endothelial cells. Thus, estrogen can relax rat aorta in vitro via both endothelium-dependent and -independent mechanisms involving the NO/cGMP and potassium channel signaling system.

Characterization of the adrenergic activity of arbutamine, a novel agent for pharmacological stress testing.

SummaryIn this study, we characterized the interactions of arbutamine, a novel catecholamine developed for use as a cardiac stress testing agent, with different adrenergic receptor subtypes in vitro. These effects were compared with those of isoproterenol. In the electrically stimulated left atria of rats, arbutamine increased contractile force. The pD2 values (-log of the dose that produces 50% of the maximal responses) for arbutamine and isoproterenol were 8.45±0.15 and 8.55±0.02, respectively. Metoprolol shifted the concentration-effect curves for both isoproterenol and arbutamine to the right with a pA2 value (-log of the dose of the antagonist that reduces the maximal responses of an agonist to 50%) of 7.22–7.5. Both arbutamine and isoproterenol increased the rate of spontaneously beating rat right atria with pD2 values of 9.0±0.19 and 8.82±0.18, respectively. The affinity constants (KA) of arbutamine and isoproterenol for cardiac beta1-adrenergic receptors, as determined by competition binding assays, were found to be 7.32 and 6.04, respectively. In guinea pig trachea, arbutamine and isoproterenol produced a concentration-dependent relaxation that was blocked by propranolol. Their pD2 values were 7.9±0.1 and 8.2±0.1, respectively. Arbutamine contracted isolated rat aortic rings with a maximal increase of 38.1±6.7% that of 10 μM of norepinephrine. In rat white adipocytes, arbutamine, isoproterenol, and BRL-37344 stimulated glycerol release, with the order of potency being BRL-37344 > arbutamine > isoproterenol. In hamster brown adipocytes, the order was arbutamine > isoproterenol > BRL-37344. Moreover, arbutamine stimulated beta3-adrenergic receptors in guinea pig ileum. In conclusion, arbutamine is a novel catecholamine with similar potency and efficacy to that of isoproterenol. It stimulates cardiac beta1-, tracheal beta2-, and adiopocyte beta3-adrenergic receptors. Arbutamine does not stimulate alpha-adrenergic receptors at concentrations that wer high enough to maximally activate the beta-adrenergic receptors.

A novel catecholamine, arbutamine, for a pharmacological cardiac stress agent

SummaryArbutamine, developed for use as a cardiac stress agent, was compared with isoproterenol and dobutamine in anesthetized dogs for cardiovascular actions prior to and after beta-adrenergic blockade with propranolol. The efficacy and safety of arbutamine were also evaluated in a canine model of myocardial ischemia obtained by partially occluding the left anterior descending coronary artery. Comparison of hemodynamic variables in normal dogs showed that arbutamine was approximately equipotent to isoproterenol in increasing heart rate and cardiac contractility, and in decreasing total peripheral vascular resistance and mean arterial blood pressure. Arbutamine was 210 times more potent than dobutamine in increasing cardiac contractility by 70%; however, at this dose dobutamine exhibited a negative chronotropic response. Beta-adrenergic blockade with propranolol shifted the agonists dose-response curves for heart rate and contractility to the right; however, low doses of dobutamine exhibited a negative chronotropic effect and increased the total peripheral vascular resistance. In dogs subjected to partial left anterior descending coronary artery occlusion, arbutamine produced significant ST-segment deflections, beginning at a dose of 0.1 nmol/kg/min. Impairment of segment shortening, reflecting cardiac wall motion abnormality, was evident at a dose of 0.3 nmol/kg/min. Isoproterenol did not cause significant changes in these parameters. These results show that arbutamine is capable of producing graded increments in cardiac contractility and rate before and after betaadrenergic blockade in normal dogs. In dogs subjected to coronary artery occlusion, it is capable of provoking myocardial ischemia at dose levels devoid of toxicity.

A Novel Peptide Inhibits Induction of Nitric Oxide Synthase

Alpha-melanocyte stimulating hormone has been shown to prevent endotoxin shock. A heptapeptide analog (HPA) has recently been synthesized and shown to be an even more potent protective agent. Since the hypotensive and toxic actions of endotoxin lipopolysaccharide (LPS) appear to involve the induction of nitric oxide synthase, we have examined the actions of HPA on induction of iNOS using an endotoxemia model in conscious rats. In vascular smooth cells, we also determined whether the HPA could directly alter the actions of LPS, TNFα or I1-1β on NOS induction. Rats were instrumented for measurement of blood pressure and i.v. administration of agents prior to the day of experiment. In the conscious state, rats were given E. coli LPS (5 mg/kg, i.v.) and monitored for 6 hrs. One group of rats received HPA (30 μg/kg) 0.5 hr before LPS. Using L-NAME-sensitive cGMP production as an estimate of NOS activity, we determined that LPS increases iNOS and that HPA pretreatment markedly reduces this response. In isolated aortic cells, LPS and I1-1β, but not that of LPS. This former action was reversed by L-arginine. In conclusion, HPA can prevent the in vivo induction of NOS by LPS. It does not appear to directly prevent this action of LPS. Possibly, a metabolite of HPA or a secondary mediator is involved.