Fruzsina K. Johnson

Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells.

Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited l-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like l-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in l-arginine-mediated NO synthesis. d-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated l-arginine-mediated NO synthesis, thus indicating mediation via G proteins. l-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the l-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of l-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development.

Heme Oxygenase Inhibitor Restores Arteriolar Nitric Oxide Function in Dahl Rats

Abstract—Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). CO relaxes vascular smooth muscle but inhibits nitric oxide (NO) formation. Decreased NO synthesis may contribute to salt-induced hypertension in Dahl salt-sensitive (DS) rats. The current study examines the hypothesis that elevated levels of endogenous CO contribute to NO dysfunction in salt-induced hypertensive DS rats. Male DS rats were placed on high-salt (8% NaCl, HS) or low-salt (0.3% NaCl, LS) diets for 4 weeks. With respect to the LS group, the HS group’s blood pressure and carboxyhemoglobin levels were elevated, and abdominal aortas showed 6-fold higher HO-1 protein levels. Experiments used isolated pressurized first-order gracilis muscle arterioles superfused with oxygenated modified Krebs buffer. An inhibitor of NO synthase, N &ohgr;-nitro-l-arginine methyl ester (L-NAME), caused concentration-dependent vasoconstriction in both groups, with attenuated responses in HS arterioles. HS arterioles also showed attenuated vasodilatory responses to an endothelium-dependent vasodilator, acetylcholine. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, enhanced vascular responses to L-NAME and acetylcholine in both groups but abolished the differences between HS and LS arterioles. These data show that HO-1 protein levels and CO production are increased in HS rats. Arteriolar responses to L-NAME and acetylcholine are impaired in HS rats compared with LS animals, and this difference can be abolished by an inhibitor of endogenous CO production. These results suggest that elevated levels of endogenous CO contribute to arteriolar NO dysfunction in DS rats with salt-induced hypertension.

Hypochlorous acid-induced heme oxygenase-1 gene expression promotes human endothelial cell survival.

Hypochlorous acid (HOCl) is a unique oxidant generated by the enzyme myeloperoxidase that contributes to endothelial cell dysfunction and death in atherosclerosis. Since myeloperoxidase localizes with heme oxygenase-1 (HO-1) in and around endothelial cells of atherosclerotic lesions, the present study investigated whether there was an interaction between these two enzymes in vascular endothelium. Treatment of human endothelial cells with the myeloperoxidase product HOCl stimulated a concentration- and time-dependent increase in HO-1 protein that resulted in a significant rise in carbon monoxide (CO) production. The induction of HO-1 protein was preceded by a prominent increase in HO-1 mRNA and total and nuclear factor-erythroid 2-related factor 2 (Nrf2). In addition, HOCl induced a significant rise in HO-1 promoter activity that was blocked by mutating the antioxidant response element (ARE) in the promoter or by overexpressing a dominant-negative mutant of Nrf2. The HOCl-mediated induction of Nrf2 or HO-1 was blocked by the glutathione donor N-acetyl-l-cysteine but was unaffected by ascorbic or uric acid. Finally, treatment of endothelial cells with HOCl stimulated mitochondrial dysfunction, caspase-3 activation, and cell death that was potentiated by the HO inhibitor, tin protoporphyrin-IX, or by the knockdown of HO-1, and reversed by the exogenous administration of biliverdin, bilirubin, or CO. These results demonstrate that HOCl induces HO-1 gene transcription via the activation of the Nrf2/ARE pathway to counteract HOCl-mediated mitochondrial dysfunction and cell death. The ability of HOCl to activate HO-1 gene expression may represent a critical adaptive response to maintain endothelial cell viability at sites of vascular inflammation and atherosclerosis.

Vascular effects of a heme oxygenase inhibitor are enhanced in the absence of nitric oxide

BACKGROUND Vascular endothelium and smooth muscle express heme oxygenase (HO) that metabolizes heme to biliverdin, iron and carbon monoxide (CO). Carbon monoxide promotes endothelium-independent vasodilation, but also inhibits nitric oxide formation. This study examines the hypothesis that an inhibitor of HO promotes endothelium-independent vasoconstriction, which is attenuated in the presence of unabated nitric oxide formation. METHODS In vivo studies were conducted in anesthetized male Sprague-Dawley (SD) rats instrumented with flow probes and arterial catheters. In vitro experiments were performed on pressurized first-order gracilis muscle arterioles isolated from male SD rats superfused with oxygenated modified Krebs buffer. RESULTS Vascular smooth muscle and endothelium showed positive HO-1 and HO-2 immunostaining. In anesthetized rats the HO inhibitor chromium mesoporphyrin (CrMP; 45 micromol/kg intraperitoneally) had minimal effect on hindlimb resistance. However, in animals pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 mg/kg intraperitoneally), CrMP substantially increased hindlimb resistance. In contrast, in rats infused with phenylephrine to increase blood pressure and vascular tone, CrMP had no effect on hindlimb resistance. In isolated arterioles denuded of endothelium, CrMP (15 micromol/L) caused a powerful vasoconstriction, which was abolished in the presence of a functional endothelium. In arterioles with intact endothelium pretreated with L-NAME (1 mmol/L), or with L-NAME and sodium nitroprusside (10 to 30 nmol/L), CrMP promoted a similarly powerful vasoconstriction as in vessels denuded of endothelium. CONCLUSIONS These results suggest that smooth muscle-derived CO may contribute to endothelium-independent regulation of vascular tone by providing a vasodilatory influence. Furthermore, the dilatory effects of endogenous CO are offset by a unique interaction between the CO and nitric oxide systems.

The effects of carbon monoxide as a neurotransmitter.

Neural tissues generate carbon monoxide. Although neuronal carbon monoxide does not appear to be released in a directed manner, heme-derived carbon monoxide affects neuronally mediated activities. This rather suggests that endogenously formed carbon monoxide is an important neuromodulator. In addition, it appears that carbon monoxide may contribute to various neuropathological conditions.

Influence of the heme-oxygenase pathway on cerebrocortical blood flow.

Heme-oxygenase (HO)-derived carbon monoxide (CO) is generated in the cardiovascular and in the central nervous systems. Endogenous CO exerts direct vascular effects and has also been shown to inhibit nitric oxide synthase (NOS). In the current study, the heme-oxygenase blockade [zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG), 45 μmol/kg intraperitoneally] decreased cerebral CO production and increased cerebrocortical blood flow (CBF) in anesthetized rats. This latter effect was abrogated by the NOS blockade (50 mg/kg L-NAME intravenously). Furthermore, inhibition of CO production had no effect on stepwise hypoxia/hypercapnia-stimulated increases in CBF. Our results indicate that endogenous CO reduces the resting CBF via inhibition of NOS but fails to influence the CBF response to hypoxia and hypercapnia in adult rats.

62 HEME-DERIVED CARBON MONOXIDE PROMOTES ENDOTHELIAL DYSFUNCTION AND HYPERTENSION IN OBESE ZUCKER RATS

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelial dysfunction (ED) in salt-induced hypertension. The obese Zucker rat (ZR) is a model of type 2 diabetes showing hypertension and vascular ED. This study tests the hypothesis that HO-derived CO contributes to arteriolar ED and hypertension in obese ZR. Male obese (548 ± 12 g, n = 22) and lean (309 ± 14 g, n = 12) ZR were used for the study. Obese ZR had elevated mean arterial blood pressure (154 ± 3 mm Hg vs. 121 ± 4 mm Hg), blood glucose (186 ± 7 mg/dL vs. 140 ± 3 mg/dL) and blood carboxyhemoglobin (HbCO) levels (3.9 ± 0.1% vs. 3.0 ± 0.1%). Experiments used isolated skeletal muscle arterioles with constant (80 mm Hg) pressure and no flow, or constant midpoint, but altered endpoint pressures to establish graded levels of luminal flow (0-50 μL/min). In obese ZR arterioles, responses to an endothelium-dependent vasodilator, acetylcholine (ACh, 1 nmol/L - 3 μmol/L) (Δmax 32 ± 4 μm, n = 8 vs. 62 ± 7 μm, n = 5) and flow (Δmax-1 ± 1 μm, n = 4 vs. 21 ± 2 μm, n = 4) were attenuated. Acute in vitro pretreatment with a HO inhibitor, chromium mesoporphyrin (CrMP, 15 μmol/L), enhanced ACh (Δmax 59 ± 8 μm, n = 7 vs. 58 ± 13 μm, n = 5) and flow-induced dilation (Δmax 19 ± 2 μm, n = 4 vs. 20 ± 3 μm, n = 3) and abolished the differences between lean and obese ZR arterioles. Furthermore, exogenous CO (100 μmol/L) prevented the restoration of flow-induced dilation by the HO inhibitor (Δmax-1 ± 3 μm, n = 4) in obese ZR arterioles. In awake obese ZR instrumented with chronic femoral arterial catheters, administration of a HO inhibitor (25 μmol/kg/day zinc deuteroporphyrin 2,4-bisglycol IP for 3 days) lowered the blood pressure (151 ± 1 to 109 ± 1 mm Hg, n = 3). These data show that HbCO levels are increased, and arteriolar endothelium-dependent vasodilation is decreased in obese ZR. Acute in vitro treatment with a HO inhibitor restores endothelium-dependent responses to lean ZR levels, but exogenous CO prevents this effect. Furthermore, HO inhibition lowers blood pressure in obese ZR. These results suggest that heme-derived CO production is increased and contributes to arteriolar ED and hypertension in obese ZR, and hence identify vascular HO as a novel therapeutic target to prevent ED and hypertension in obesity and type 2 diabetes.

65 L-ARGININE RESTORES CORONARY ENDOTHELIAL FUNCTION IN OBESE ZUCKER RATS

The release of nitric oxide (NO) through the oxidation of L-arginine by endothelial NO synthase (eNOS) promotes relaxation of vascular smooth muscle and plays a pivotal role in the maintenance of vascular homeostasis. Since L-arginine is an exclusive substrate for eNOS, its availability can regulate NO formation. Endothelial dysfunction, due to decreased NO function, is a prominent feature of obesity and type 2 diabetes and contributes to cardiovascular pathology. While eNOS expression is unchanged, vascular NO levels are decreased and L-arginine administration lowers blood pressure in patients with type 2 diabetes. This study tests the hypothesis that L-arginine restores coronary arterial endothelial function in obese Zucker rats (ZR), experimental models of type 2 diabetes. Male obese ZR (548 ± 12 g) and lean Sprague-Dawley (SD) rats (300 g) were used for the study. Mean arterial blood pressure (154 ± 3 mm Hg) and fasting blood glucose levels (186 ± 7 mg/dL) were elevated in obese ZR. Experiments were performed on isolated pressurized (80 mm Hg no flow conditions) small septal coronary arteries (200-300 μm) superfused with oxygenated (14% O2, 5% CO2 balanced with N2) Krebs buffer. Responses to an endothelium- and NO-dependent vasodilator, acetylcholine (1 nmol/L-3 μmol/L) were greatly attenuated in obese ZR coronary vessels (Δmax 5 ± 2 μm vs. 54 ± 10 μm). Acute in vitro pretreatment with the NOS substrate, 1 mmol/L L-arginine, enhanced acetylcholine-induced dilation and abolished the differences between obese ZR and SD arteries (Δmax 48 ± 18 μm vs. 61 ± 18 μm). These data show that blood pressure and blood glucose levels are elevated in obese ZR. Acetylcholine-induced endothelium-dependent vasodilation is greatly attenuated in small coronary arteries isolated from obese ZR. In addition, acute in vitro administration of L-arginine enhanced coronary endothelium-dependent responses and abolished the differences between obese ZR and SD coronary vessels. These results suggest that decreased L-arginine availability may contribute to coronary arterial endothelial dysfunction in obese ZR. Thus, L-arginine supplementation may present a novel therapeutic method to deter coronary vascular endothelial dysfunction and cardiovascular complications in patients with obesity and type 2 diabetes.

76 NEURAL ENDOGENOUS CARBON MONOXIDE LOWERS BLOOD PRESSURE VIA SUPPRESSION OF PERIPHERAL SYMPATHETIC NEURONS

Carbon monoxide (CO) is endogenously produced from the degradation of heme, in a reaction that is catalyzed by heme oxygenase (HO). Systemic blockade of HO acutely decreases neural HO activity, and this increases arterial pressure in a manner that is reversed by CO microinjections into the nucleus tractus solitarii (NTS) of the brain stem; such findings are the original evidence that endogenous CO in the NTS serves as a tonic vasodepressor. Whilst the NTS coordinates the balance and distribution of sympathetic and parasympathetic influences on the cardiovascular system, the actions of the endogenous CO on these autonomic components have not been explored. The purpose of the current study was to identify the sympathetic and parasympathetic influences that contribute to the vasoregulatory actions of the endogenous CO system. Towards this end, male Sprague-Dawley rats were fitted with chronic femoral catheters and permitted four days recovery before conducting awake experiments. The acute blood pressure effects of an HO inhibitor, zinc deuteroporphyrin 2,4-bisglycol (ZnDPBG), were examined in animals pretreated with atropine, prazosine or propranolol to block the muscarinic, α1- or β-adrenergic receptors, respectively. Administration of ZnDPBG (45 μmol/kg, IP) increased arterial pressure from 123 ± 2 to 133 ± 3 mm Hg (p < .05) but did not affect heart rate. The actions of ZnDPBG were not affected by pretreatment with atropine (50 mg/kg, IP) but were abolished by pretreatment with prazosine (5 mg/kg, IP). In addition, pretreatment with a beta-blocker, propranolol (15 mg/kg, IP), resulted in an abbreviated pressor response to ZnDPBG. Immunohistochemical staining and Western blot analysis confirmed that HO is present in both the NTS and adrenal medulla, but complementary experiments revealed that ZnDPBG treatment did not affect circulating levels of epinephrine. This study shows that HO is present in NTS and that the actions of ZnDPBG are independent of parasympathetic changes. While HO is present in the adrenal medulla and the pressor effect of ZnDPBG apparently arises from enhanced sympathetic tone, the HO inhibitor does not increase plasma levels of epinephrine. These findings suggest that inhibition of heme-derived CO in the NTS leads to activation of peripheral sympathetic neurons and that the consequent pressor effect arises from α1-adrenergic-mediated vasoconstriction and β1-adrenergic-mediated increases in cardiac performance.