Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaln molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N'-methyl-L-arginlne (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipidsoluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These finding imply a role for argininederived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.Goormaghtigh (1) suggested that the macula densa is the sensor for a stimulus from tubular fluid that is conveyed to the glomerulus. Subsequently, Thurau and Schnermann (2) identified that the stimulus was the delivery and reabsorption of NaCl by this segment. This tubuloglomerular feedback response functions as a negative-feedback control mechanism, whereby glomerular filtration of NaCI, with delivery to and reabsorption by the macula densa, induces release of mediator(s) that cause afferent-arteriolar vasoconstriction and a reduction in glomerular capillary pressure and glomerular filtration rate (2). Although the signaling mechanisms or molecules inducing afferent-arteriolar vasoconstriction have not been clearly defined, the response is promoted by adenosine acting on adenosine type 1 receptors (3), angiotensin II (4), and thromboxane A2 (5)-Previous studies have established that L-arginine-derived nitric oxide (NO) is produced by several cells within the kidney, including isolated glomerular mesangial (6) and endothelial cells (7), and a renal epithelial cell line (8), but its integrative role in the control ofrenal function is not yet clear (9). In the vessel wall, ...

The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.

The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.).

Clinical assessment of the activity of tumor necrosis factor (TNF) against human cancer has been limited by a dose-dependent cardiovascular toxicity, most frequently hypotension. TNF is also thought to mediate the vascular collapse resulting from bacterial endotoxin. The present studies address the mechanism by which TNF causes hypotension and provide evidence for elevated production of nitric oxide, a potent vasodilator initially characterized as endotheliumderived relaxing factor. Nitric oxide is synthesized by several cell types, including endothelial cells and macrophages, from the guanidino nitrogen of L-arginine; the enzymatic pathway is competitively inhibited by -me yl-L-arginine. We found that hypotension induced in pentobarbital-anesthetized dogs by TNF (10 ,ug/kg, i.v., resulting in a fall in mean systemic arterial pressure from 124.7 ± 7 to 62.0 ± 22.9 mmHg; 1 mmHg = 133 Pa) was completely reversed within 2 min following administration ofNG-methyl-L-arginine (4.4 mg/kg, i.v.). In contrast, NG-methyl-L-arginine failed to reverse the hypotensive response to an equivalent depressor dose of nitroglycerin, a compound that acts by forming nitric oxide by a nonenzymatic, arginine-independent mechanism. The effect of NG-methyl-L-arginine on TNF-induced hypotension was antagonized, and the hypotension restored, by administration of excess L-arginine (100 mg/kg, i.v.). Our rmdings suggest that excessive nitric oxide production mediates the hypotensive effect of TNF.Tumor necrosis factor (TNF) is a cytotoxic protein produced by macrophages upon activation by bacterial endotoxin (1, 2). In addition to a spectrum of cytotoxic and immunologic actions, TNF causes marked hypotension in mammals (1, 3). The observations that bacterial endotoxin elicits TNF production (4, 5) and that pretreatment of animals with anti-TNF antibodies abolishes the hypotensive action of endotoxin (6) suggest that TNF is the key mediator of endotoxic shock in vivo. Although TNF is known to promote hemorrhagic necrosis of some animal tumors (7), its clinical promise as an antineoplastic agent is limited by severe dose-dependent side effects, predominantly hypotension (8, 9). Despite the clinical importance of TNF-induced hypotension, its mechanism is unknown.The present study addresses the possibility that increased nitric oxide production accounts for TNF-induced hypotension. Earlier studies established that endothelium-derived nitric oxide is a labile modulator of vascular tone (10, 11). Originally termed endothelium-derived relaxing factor (EDRF, ref. 12), nitric oxide is responsible for the vascular smooth muscle relaxation elicited by acetylcholine, bradykinin, and many other endogenous vasorelaxants. L-Arginine is the biosynthetic precursor of endothelium-derived nitric oxide (13)(14)(15)(16), and NG-methyl-L-arginine (L-MeArg) is a competitive inhibitor of this pathway (14, 15). The finding that administration of L-MeArg causes a moderate increase in blood pressure by an arginine-reversible mechanism in the anesthetized guine...

Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I-forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell-derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT 1 ) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT 1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell-deficient mice than in control hearts. Thus, mast cell-derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell-derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

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L-arginine-dependent synthesis of nitrite (NO2-) and nitrate (NO3-) by macrophages correlates with and is required for their execution of nonspecific cytotoxicity toward some tumor cells and microbes. However, the bioactive L-arginine metabolites responsible for cytotoxicity are unknown. Mammalian endothelial cells have recently been shown to release nitric oxide (NO.); we therefore determined if this reactive metabolite was synthesized by activated murine macrophages. Macrophage-derived NO. was detected by two independent methods: a bioassay for NO.-mediated relaxation of preconstricted rings of rabbit aorta; and a spectroscopic measurement of the reaction of NO. with clostridial ferredoxin, an Fe-S protein. After activation with IFN-gamma and LPS, macrophages continuously secreted a substance that relaxed rabbit aortic rings denuded of endothelium. Production of the vasorelaxant was enhanced by 0.5 mM L-arginine and inhibited reversibly by NG-methylated L-arginine analogs that block macrophage NO2-/NO3- synthesis. The vasorelaxant was scavenged by ferrous myoglobin, was labile, and was neither NO2- nor a cyclooxygenase metabolite. Activated M phi also secreted a substance that bleached Fd, a reaction carried out by NO. and NO2, but not NO2-. Macrophage bleaching of Fd correlated directly with time, cell number, and concomitant NO2-/NO3- production, required L-arginine, and was independent of reactive oxygen intermediates. Thus, activated murine M phi release NO. and/or a closely related, highly reactive nitrogen oxide such as NO2, during their conversion of L-arginine to NO2-/NO3-. NO. and NO2 may mediate L-arginine-dependent pathologic effects of M phi, as well as physiologic effects not previously considered for this widely distributed cell type.

Nitric oxide (NO) is a major endotheliumderived relaxing factor (EDRF) released in response to vasodilating amines, peptides, proteins, ionophores, and nucleotides. EDRF is an important regulator of smooth muscle tone and platelet aggregation and adhesion. Histamine and acetylcholine relax the intact norepinephrine-constricted guinea pig pulmonary artery by an EDRF-dependent mechanism in a medium free of amino acids. NaI-Monomethylarginine (NMeArg; 0.25 mM) inhibited this relaxation by 64-73%. Inhibition by N-MeArg developed rapidly and was immediately and completely reversed by excess L-arginine but not by D-arginine or by citrulfine. N-MeArg did not diminish relaxation induced by nitroprusside, an NO-generating agent, indicating that N-MeArg acts on endothelium rather than on smooth muscle. These observations strongly suggest that, in the intact guinea pig pulmonary artery, EDRF originates from enzymatic action on the guanido nitrogen(s) of an endogenous pool of arginine. This is strikingly similar to the origin of reactive nitrogen intermediates in activated macrophages.Histamine, acetylcholine, bradykinin, thrombin, ATP, and the ionophore A23187 dilate vascular smooth muscle by stimulating endothelial cells to release a relaxing factor, endothelium-derived relaxing factor (EDRF) (1-3). EDRF causes cGMP accumulation in smooth muscle cells (4, 5) and platelets (6), resulting in dilatation of blood vessels and inhibition of platelet aggregation and adhesion. EDRF has been characterized as a short-lived (7), acid-stabilized (8) moiety that is readily inactivated by superoxide anion (9) and that reacts with ozone to yield chemiluminescent species (10), with heme groups to yield nitrosyl-heme derivatives (11,12), and with sulfanilic acid to yield a diazotized product (12). EDRF is indistinguishable in each respect from nitric oxide (NO) (10); NO and NO-generating compounds like nitroprusside mimic the biologic actions of EDRF (4). However, a key obstacle to accepting the identification of NO as EDRF, and to isolating an EDRF synthetase, is the fact that no mammalian biosynthetic pathway has been identified that yields NO.As a possible clue to the origin of EDRF, we noted that immunologically activated murine macrophages oxidize the guanidino nitrogen(s) (NW) of arginine to yield nitrite (NO2-), nitrate (NO3-) (13-16), and unidentified species that react with morpholino compounds to generate nitrosamines (17). Synthesis of reactive nitrogen intermediates in macrophages is sensitive to inhibition by arginine analogs that are Nwmethylated (16,18). Therefore, we tested the ability of NW-monomethylarginine (N-MeArg) to inhibit histamine-and acetylcholine-induced relaxation ofthe guinea pig pulmonary artery, an EDRF-dependent phenomenon (19,20). The results indicate that production of EDRF in this system depends on metabolism ofarginine. Thus, the pathway of NO biosynthesis in agonist-triggered endothelial cells shares important similarities with the interferon-y-induced (15), tumor necrosis factor-enhanced (21)...