Adrian J. Hobbs

Acute Blood Pressure Lowering, Vasoprotective, and Antiplatelet Properties of Dietary Nitrate via Bioconversion to Nitrite

Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (&Dgr;max −10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a “natural” low cost approach for the treatment of cardiovascular disease.

Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans. Role for Nitrite-Derived NO

Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure–lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO3 capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO3 (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.

Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide.

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-κB, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-κB activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.

Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor.

Endothelial cells in most vascular beds release a factor that hyperpolarizes the underlying smooth muscle, produces vasodilatation, and plays a fundamental role in the regulation of local blood flow and systemic blood pressure. The identity of this endothelium-derived hyperpolarizing factor (EDHF), which is neither NO nor prostacyclin, remains obscure. Herein, we demonstrate that in mesenteric resistance arteries, release of C-type natriuretic peptide (CNP) accounts for the biological activity of EDHF. Both produce identical smooth muscle hyperpolarizations that are attenuated in the presence of high [K+], the Gi G protein (Gi) inhibitor pertussis toxin, the G protein-gated inwardly rectifying K+ channel inhibitor tertiapin, and a combination of Ba2+ (inwardly rectifying K+ channel blocker) plus ouabain (Na+/K+-ATPase inhibitor). Responses to EDHF and CNP are unaffected by the natriuretic peptide receptor (NPR)-A/B antagonist HS-142-1, but mimicked by the selective NPR-C agonist, cANF4–23. EDHF-dependent relaxation is concomitant with liberation of endothelial CNP; in the presence of the myoendothelial gap-junction inhibitor 18α-glycyrrhetinic acid or after endothelial denudation, CNP release and EDHF responses are profoundly suppressed. These data demonstrate that acetylcholine-evoked release of endothelial CNP activates NPR-C on vascular smooth muscle that via a Gi coupling promotes Ba2+/ouabain-sensitive hyperpolarization. Thus, we have revealed the identity of EDHF and established a pivotal role for endothelial-derived CNP in the regulation of vascular tone and blood flow.

L-Ng-nitro arginine inhibits non-adrenergic, non-cholinergic relaxations of guinea-pig isolated tracheal smooth muscle

The effects of l‐NG‐nitro arginine (l‐NOARG) on α‐chymotrypsin‐resistant, non‐adrenergic, non‐cholinergic (NANC) relaxations of guinea‐pig tracheal smooth muscle have been examined. l‐NOARG (1–100 μm), but not d‐NOARG (100 μm), inhibited the NANC relaxations in a concentration‐related manner. The effects of l‐NOARG were partially reversed by l‐arginine but not d‐arginine.l‐NOARG was without effect on acetylcholine‐induced contractile responses of the trachea or on relaxations produced by vasoactive intestinal peptide, sodium nitroprusside or isoprenaline. These results suggest that an endogenous nitrate may contribute to NANC relaxations of tracheal smooth muscle.

l‐NG‐monomethyl arginine and l‐NG‐nitro arginine inhibit non‐adrenergic, non‐cholinergic relaxation of the mouse anococcygeus muscle

1 The effects of l‐NG‐monomethyl arginine (l‐NMMA) and l‐NG‐nitro arginine (l‐NOARG) on non‐adrenergic, non‐cholinergic (NANC) relaxations of the mouse anococcygeus were investigated. 2 l‐NMMA (10–200 μm) produced a concentration‐related inhibition of the NANC response; the inhibitory effect of 50 μm l‐NMMA was completely reversed by l‐arginine but not d‐arginine (both 100 μm). 3 l‐NOARG (1–50 μm) also produced a concentration‐related inhibition of the NANC response and was some 30–50 times more potent than l‐NMMA; again, the effects of 10 μm l‐NOARG were reversed by 100 μm l‐, but not d‐, arginine. By itself 100 μm l‐arginine did not relax the tissue, but did cause a slight potentiation of the NANC response. 4 Sodium nitroprusside (0.01–10 μm), hydroxylamine (0.1–100 μm), sodium azide (1–100 μm) and nitric oxide (3–120 μm) all relaxed carbachol‐induced tone; relaxations to submaximal concentrations of these nitrovasodilators were unaffected by either 50 μm l‐NMMA or 10 μm l‐NOARG. 5 l‐NOARG 10 μm did not inhibit, but rather potentiated, contractions of the mouse anococcygeus due to stimulation of its sympathetic nerves. 6 The inhibitory effects of 10 μm l‐NOARG on NANC relaxations were reversed by l‐arginine (by 131%), l‐citrulline (by 75%), l‐arginine methyl ester (by 46%) and l‐homoarginine (by 22%), but were unaffected by a variety of other amino acids and their derivatives (all at 100 μm). 7 The results provide strong evidence that NANC relaxations of the mouse anococcygeus are mediated by an endogenous nitrate material, probably derived from l‐arginine, and confirm that l‐NOARG provides a very useful and potent drug for the investigation of endogenous nitrate function.

Investigation of Vascular Responses in Endothelial Nitric Oxide Synthase/Cyclooxygenase-1 Double-Knockout Mice Key Role for Endothelium-Derived Hyperpolarizing Factor in the Regulation of Blood Pressure in Vivo

Background—Endothelium-dependent dilatation is mediated by 3 principal vasodilators: nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). To determine the relative contribution of these factors in endothelium-dependent relaxation, we have generated mice in which the enzymes required for endothelial NO and PGI2 production, endothelial NO synthase (eNOS) and cyclooxygenase-1 (COX-1), respectively, have been disrupted (eNOS−/− and COX-1−/− mice). Methods and Results—In female mice, the absence of eNOS and COX-1 had no effect on mean arterial blood pressure (BP), whereas BP was significantly elevated in eNOS−/−/COX-1−/− males compared with wild-type controls. Additionally, endothelium-dependent relaxation remained intact in the resistance vessels of female mice and was associated with vascular smooth muscle hyperpolarization; however, these responses were profoundly suppressed in arteries of male eNOS−/−/COX-1−/− animals. Similarly, the endothelium-dependent vasodilator bradykinin produced dose-dependent hypotension in female eNOS−/−/COX-1−/− animals in vivo but had no effect on BP in male mice. Conclusions—These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whereas NO and PGI2 are the predominant mediators in male mice. Moreover, the gender-specific prevalence of EDHF appears to underlie the protection of female eNOS−/−/COX-1−/− mice against hypertension.

Circulating Endothelial Progenitor Cells in Patients With Eisenmenger Syndrome and Idiopathic Pulmonary Arterial Hypertension

Background— Impaired endothelial homeostasis underlies the pathophysiology of pulmonary arterial hypertension (PAH). We speculated that PAH patients are deficient in circulating endothelial progenitor cells (EPCs), potentially contributing to endothelial dysfunction and disease progression. Methods and Results— We recruited 41 patients with Eisenmenger syndrome (13 with Down syndrome), 55 with idiopathic PAH, and 47 healthy control subjects. Flow cytometry and in vitro assays were used to quantify EPCs and to assess cell function. The number of circulating CD34+, CD34+/AC133+, CD34+/KDR+, and CD34+/AC133+/KDR+ progenitor cells was low in Eisenmenger patients compared with healthy control subjects, and those with Down syndrome displayed even fewer EPCs. Reductions in EPC numbers correlated with New York Heart Association functional class, 6-minute walk distance, and plasma brain-type natriuretic peptide levels. The capacity of cultured peripheral blood mononuclear cells to form colonies and incorporate into tube-like structures was impaired in Eisenmenger patients. Idiopathic PAH patients had reduced numbers of EPCs, and the number of circulating EPCs correlated with invasive hemodynamic parameters in this cohort. Levels of immune inflammatory markers, cGMP, stable nitric oxide oxidation products, and asymmetric dimethylarginine were abnormal in patients with PAH and related to numbers of EPCs. Within the idiopathic PAH population, treatment with the phosphodiesterase inhibitor sildenafil was associated with a dose-dependent rise in EPC numbers, resulting in levels consistently above those found with other therapies. Conclusions— Circulating EPC numbers are reduced in 2 well-characterized forms of PAH, which also exhibit raised levels of inflammatory mediators. Sildenafil treatment may represent a pharmacological means of increasing circulating EPC numbers long-term.

Nitric oxide-cyclic GMP signal transduction system.

Publisher Summary This chapter provides an overview of nitric oxide (NO)–guanylate cyclase–cyclic guanosine monophosphate (cGMP) signal transduction and discusses its physiological significance. The methods used extensively to study and elucidate the mechanisms involved in cellular communication mediated by this pathway is discussed; these include assays for detecting the enzymatic formation of NO and its coproduct L -citrulline, and purification and assay of soluble guanylate cyclase. The NO–guanylate cyclase–cGMP transduction system represents a remarkable example of how a multitude of cellular responses to a multiple array of biological messengers can be mediated by a single signaling system. The role of NO as an inter- and intracellular messenger molecule, coupled with the identification of the atrial natriuretic peptides (ANPs) as activators of a second, membrane-bound isoform of guanylate cyclase, have further elevated the significance of cGMP as a second messenger. The spectrum of biological effects mediated by NO and cGMP include diverse actions such as anticoagulation, nonadrenergic-noncholinergic (NANC) neurotransmission, phototransduction, and vascular and nonvascular smooth muscle relaxation.

Negative Modulation of Nitric Oxide Synthase by Nitric Oxide and Nitroso Compounds

These observations clearly indicate that NO inhibits NOS activity and that nNOS and eNOS are more sensitive than iNOS to the inhibitory action of NO. Not only exogenously added NO but also enzymatically generated NO inhibits the activity of nNOS and eNOS. The mechanism by which NO inhibits NOS appears to involve the heme iron prosthetic group of NOS. Moreover, the oxidation state of the heme iron is critical in determining the magnitude of inhibition of NOS by NO. Conditions that favor the higher oxidation state of FeIII markedly increase the inhibitory action of NO, whereas conditions that favor the lower oxidation state of FeII markedly decrease the inhibitory action of NO. One of the cofactor roles of tetrahydrobiopterin may be to reduce the negative-feedback effect of NO on NOS by favoring the formation of the ferrous heme state in NOS. The inhibitory influence of NO on eNOS, albeit indirectly, was also observed in vascular endothelial cells, arterial rings, and in vivo in the perfused rabbit hindquarters vascular bed. Excess NO in the form of NO donor compounds inhibited the endothelium-dependent formation of EDRF/NO in response to endothelium-dependent vasorelaxants such as acetylcholine and bradykinin without influencing the relaxant effect of NO itself. These studies are consistent with the view that enzymatically generated NO may play an important negative-feedback regulatory role on eNOS, and therefore on vascular endothelial cell function. Several biological implications of a negative-feedback modulatory effect by NO on constitutive isoforms of NOS are evident. In nonadrenergic-noncholinergic transmission, in which NO is believed to be the principal inhibitory neurotransmitter (Sanders and Ward, 1992; Rand, 1992; Rajfer et al., 1992), NO may regulate its own synthesis, and therefore the neurotransmission process. Excess NO production may be undesirable because of the potential of NO or a reaction product of NO to elicit cytotoxic effects. Many extraneuronal factors could also contribute to decreasing the potentially cytotoxic actions of NO. For example, reduced hemoproteins such as hemoglobin, myoglobin, and/or their oxygen adducts could inactivate NO, as could superoxide anion generated in the vicinity of NO. In vascular endothelial cells either enzymatically generated NO or the presence of exogenously added NO in the form of nitrovasodilator drugs could diminish the vasodilator responses to endothelium-dependent relaxants and flow or shear stress. Although iNOS is less sensitive than either eNOS or nNOS to inhibition by NO, the generation of relatively large quantities of NO by iNOS within the confines of a cell may lead to a negative-feedback effect. The concomitant generation of superoxide anion by the same or adjacent cells could result in a diminished negative-feedback effect because of the rapid reaction between NO and superoxide anion to form peroxynitrite. Thus, NO production would increase and there would be increased peroxynitrite formation as well, which would result in enhanced cytotoxicity, provided that peroxynitrite is a cytotoxic species. Alternatively, iNOS may be conveniently insensitive to NO in order to allow for the generation of large quantities of NO for the purpose of producing cytotoxic effects.