Georgette M. Buga

Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical

The objective of this study was to elucidate the close similarity in properties between endothelium-derived relaxing factor (EDRF) and nitric oxide radical (NO). Whenever possible, a comparison was also made between arterial and venous EDRF. In vascular relaxation experiments, acetylcholine and bradykinin were used as endothelium-dependent relaxants of isolated rings of bovine intrapulmonary artery and vein, respectively, and NO was used to relax endothelium-denuded rings. Oxyhemoglobin produced virtually identical concentration-dependent inhibitory effects on both endothelium-dependent and NO-elicited relaxation. Oxyhemoglobin and oxymyoglobin lowered cyclic guanosine monophosphate (cGMP) levels, increased tone in unrubbed artery and vein, and abolished the marked accumulation of vascular cGMP caused both by endothelium-dependent relaxants and by NO. The marked inhibitory effects of Oxyhemoglobin on arterial and venous relaxant responses and cGMP accumulation as well as its contractile effects were abolished or reversed by carbon monoxide. These observations indicate that EDRF and NO possess identical properties in their interactions with oxyhemoproteins. Both EDRF from artery and vein and NO activated purified soluble guanylate cyclase by heme-dependent mechanisms, thereby revealing an additional similarity in heme interactions. Spectrophotometric analysis disclosed that the characteristic shift in the Soret peak for hemoglobin produced by NO was also produced by an endothelium-derived factor released from washed aortic endothelial cells by acetylcholine or A23187. Pyrogallol, via the action of superoxide anion, markedly inhibited the spectral shifts, relaxant effects, and cGMP accumulating actions produced by both EDRF and NO. Superoxide dismutase enhanced the relaxant and cGMP accumulating effects of both EDRF and NO. Thus, EDRF and NO are inactivated by superoxide in a closely similar manner. We conclude, therefore, that EDRF from artery and vein is either NO or a chemically related radical species.

Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle.

In the presence of functional adrenergic and cholinergic blockade, electrical field stimulation relaxes corpus cavernosum smooth muscle by unknown mechanisms. We report here that electrical field stimulation of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of nitric oxide (NO), nitrite, and cyclic GMP. Corporal smooth muscle relaxation in response to electrical field stimulation, in the presence of guanethidine and atropine, was abolished by tetrodotoxin and potassium-induced depolarization, and was markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but was unaffected by indomethacin. The inhibitory effects of NG-substituted analogs of L-arginine were nearly completely reversed by addition of excess L-arginine but not D-arginine. Corporal smooth muscle relaxation elicited by electrical field stimulation was accompanied by rapid and marked increases in tissue levels of nitrite and cyclic GMP, and all responses were nearly abolished by NG-nitro-L-arginine. These observations indicate that penile erection may be mediated by NO generated in response to nonadrenergic-noncholinergic neurotransmission.

Shear stress-induced release of nitric oxide from endothelial cells grown on beads.

An in vitro bioassay system was developed to study endothelium-mediated, shear stressinduced, or flow-dependent generation of endothelium-derived relaxing factor (EDRF). Monolayers of aortic endothelial cells were grown on a rigid and large surface area of microcarrier beads and were packed in a small column perfused with Krebs bicarbonate solution. The perfusate was allowed to superfuse three endothelium-denuded target pulmonary arterial strips arranged in a cascade. Fluid shear stress caused a flow-dependent release of EDRF from the endothelial cells. The action of EDRF was abolished by oxyhemoglobin and methylene blue, and the generation of EDRF in response to shear stress was markedly inhibited or abolished by NG-nitro-L-arginine, by NG-amino-L-arginine, by calcium-free extracellular medium, and by depleting endothelial cells of endogenous L-arginine. Addition of L-arginine to arginine-deficient but not arginine-containing endothelial cells rapidly restored the capacity of shear stress and bradykinin to generate EDRF. These observations indicate that fluid shear stress causes the generation of EDRF with properties of nitric oxide from aortic endothelial cells and that the bioassay system described may be useful for studying the mechanism of mechanochemical coupling that leads to nitric oxide generation.

Negative feedback regulation of endothelial cell function by nitric oxide.

The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelial cell function by inhibiting NO synthase in vascular endothelial cells. The rationale for this approach was a previous study from this laboratory, which revealed that NO inhibits neuronal NO synthase from rat cerebellum. In the present study, NO and NO-donor agents noncompetitively inhibited NO synthase derived from bovine aortic endothelial cells. Oxyhemoglobin blocked the inhibitory action of NO and by itself increased NO synthase activity. This finding suggests that NO acts as a negative feedback modulator of NO synthase. In intact aortic endothelial cells grown on microcarrier beads and perfused in a bioassay cascade system, pretreatment of cells with NO-donor agents caused a marked inhibition of endothelial NO biosynthesis in response to bradykinin and increased fluid shear or flow. When isolated bovine pulmonary arterial rings precontracted by phenylephrine were used, pretreatment of arterial rings with NO-donor agents diminished endothelium-dependent arterial relaxation involving the L-arginine-NO pathway without altering endothelium-independent relaxation to NO itself. On the basis of these studies, NO is suggested to play an important negative feedback regulatory role on endothelial NO synthase and, therefore, vascular endothelial cell function.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. NG-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1–3 μM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as α-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

Nitric Oxide is a Potent Relaxant of Human and Rabbit Corpus Cavernosum

Nitric oxide (NO) caused a potent, marked, and transient relaxation of precontracted strips of corpus cavernosum isolated from humans and rabbits. The relaxation response elicited by NO was very similar to the relaxation evoked by electrical field stimulation via the nonadrenergic-noncholinergic pathway. Sodium nitroprusside, nitroglycerin, and S-nitroso-N-acetylpenicillamine, which are nitrovasodilators known to generate NO, also caused marked concentration-dependent relaxation of corpus cavernosum. Relaxant responses to NO were enhanced by the cyclic GMP phosphodiesterase inhibitor M&B 22,948 and inhibited by oxyhemoglobin. Similarly, relaxation of corpus cavernosum in response to electrical field stimulation or acetylcholine was enhanced by M&B 22,948 and inhibited by oxyhemoglobin. NO stimulated cyclic GMP formation in corpus cavernosum and a close positive correlation was found between the magnitudes of relaxation and cyclic GMP formation. The data suggest that NO-elicited activation of guanylate cyclase and cyclic GMP formation represents the signal transduction mechanism responsible for relaxation and nonadrenergic-noncholinergic-mediated penile erection. These observations indicate that NO is a potent relaxant of human and rabbit corpus cavernosum and support our hypothesis that endogenous NO is the principal mediator of penile erection caused by nonadrenergic-noncholinergic stimulation.

Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide.

The principal finding in this study is that vascular smooth muscle generates a labile relaxing factor that possesses pharmacological and chemical properties that are similar to those of authentic nitric oxide. MDRF was generated by perfusion of endothelium-denuded bovine pulmonary artery as assessed by bioassay. In addition, endothelium-denuded arterial rings that were incubated at 37 degrees C for 24 hr to lower endogenous L-arginine levels relaxed in response to L-arginine but not D-arginine. Freshly mounted, endothelium-denuded arterial rings were not relaxed by L-arginine but did relax in response to the dipeptide L-arginyl-L-alanine. Relaxant responses were accompanied by increases in smooth muscle levels of cyclic GMP and nitrite, and were inhibited by oxyhemoglobin, methylene blue, and NG-nitro-L-arginine. NG-Nitro-L-arginine also caused endothelium-independent contractile responses. Thus, a relaxing factor with the properties of nitric oxide can be generated from vascular smooth muscle.

Endothelium-derived nitric oxide relaxes nonvascular smooth muscle.

A bioassay cascade superfusion procedure was used to compare and contrast the actions of arterial and venous endothelium-derived relaxing factor (EDRF) with authentic nitric oxide (NO) on several nonvascular smooth muscle preparations. EDRF was released from human umbilical vein or bovine pulmonary artery by A23187 and allowed to superfuse two nonvascular and one vascular precontracted smooth muscle strips arranged in a cascade. NO or S-nitroso-N-acetylpenicillamine was delivered by superfusion. Both EDRF and NO relaxed bovine trachea, although artery was 10 times more sensitive than trachea to either relaxant. Similarly, rabbit taenia coli and rat fundus relaxed in response to high concentrations of NO or large amounts of EDRF released from umbilical vein. Vascular and nonvascular relaxant responses to both EDRF and NO were inhibited by oxyhemoglobin, methylene blue or superoxide, and were enhanced by superoxide dismutase. Perfusion of pulmonary artery or umbilical vein with A23187 resulted in contraction of guinea pig ileum and relaxation of pulmonary artery, whereas NO relaxed both preparations. Oxyhemoglobin enhanced the contractile and abolished the relaxant responses. Thus, ileum is more sensitive to endothelium-derived contracting factor(s) than to EDRF. NO raised cyclic GMP levels in all smooth muscle preparations, but a greater fold increase was observed in artery than in nonvascular smooth muscle. EDRF released from human umbilical vein was identified chemically as NO or a nitroso compound, as was done previously for EDRF from bovine pulmonary artery and vein. These observations support the view that one EDRF from artery and vein is NO or a labile nitroso compound.

NG-hydroxy-l-arginine and nitric oxide inhibit Caco-2 tumor cell proliferation by distinct mechanisms

The objective of this study was to elucidate the role and mechanism of nitric oxide (NO) synthase (NOS) in modulating the growth of the Caco-2 human colon carcinoma cell line. The two novel observations reported here are, first, that N G-hydroxy-l-arginine (NOHA) inhibits Caco-2 tumor cell proliferation, likely by inhibiting arginase activity, and, second, that NO causes cytostasis by mechanisms that might involve inhibition of ornithine decarboxylase (ODC) activity. Both arginase and ODC are enzymes involved in the conversion of arginine to polyamines required for cell proliferation. Cell growth was monitored by cell count, cell protein analysis, and DNA synthesis. NOHA (1-30 μM) and NO in the form of DETA/NO (1-30 μM) inhibited cell proliferation by 30-85%. The cytostatic effect of NOHA was prevented by addition of excess ornithine, putrescine, spermidine, or spermine to cell cultures, whereas the cytostatic effect of NO (DETA/NO) and α-difluoromethylornithine (ODC inhibitor) was unaffected by ornithine but was prevented by putrescine, spermidine, or spermine. The cytostatic effect of NOHA appeared to be independent of its conversion to NO, and the effect of NO appeared to be independent of cGMP. NOHA inhibited urea production by Caco-2 cells and inhibited arginase catalytic activity (85% at 3 μM), whereas NO (DEA/NO and SNAP) inhibited ODC activity (≥60% at 30 μM) without affecting arginase activity. Coculture of Caco-2 cells with lipopolysaccharide/cytokine-activated rat aortic endothelial cells markedly slowed Caco-2 cell proliferation, and this was blocked by NOS inhibitors. These observations that NOHA and NO may inhibit sequential steps in the arginine-polyamine pathway suggest a novel biological role for NOS in the inhibition of cell proliferation of certain tumor cells and possibly other cell types.

Characterization and actions of human umbilical endothelium derived relaxing factor

1 A bioassay cascade superfusion technique was utilized to study the properties of endothelium derived relaxing factor (EDRF) from human umbilical vein (HUV) and compare its actions on umbilical, chorionic plate and bovine pulmonary arterial strips. 2 Histamine (1 μm), bradykinin (1 μm) and A‐23187 (0.3 μm, 1 μm) but not acetylcholine (1 μm) released EDRF. 3 The non‐innervated human foetoplacental vessels, i.e., umbilical and chorionic plate arteries, do relax to EDRF by a guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP)‐mediated mechanism. 4 The sensitivity of the human umbilical arterial strips to EDRF was less than that of the chorionic plate arterial strips. Bovine pulmonary arterial strips were the most sensitive to the relaxant actions of human umbilical EDRF.