Nitric Oxide-independent Pathway Research Articles

888 Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide independent pathways

888 Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide independent pathways

GLP-1 Receptor Agonist Exenatide Increases Capillary Perfusion Independent of Nitric Oxide in Healthy Overweight Men.

The insulinotropic gut-derived hormone glucagon-like peptide-1 (GLP-1) increases capillary perfusion via a nitric oxide-dependent mechanism in rodents. This improves skeletal muscle glucose use and cardiac function. In humans, the effect of clinically used GLP-1 receptor agonists (GLP-1RAs) on capillary density is unknown. We aimed to assess the effects of the GLP-1RA exenatide on capillary density as well as the involvement of nitric oxide in humans. We included 10 healthy overweight men (age, 20-27 years; body mass index, 26-31 kg/m(2)). Measurements were performed during intravenous infusion of placebo (saline 0.9%), exenatide, and a combination of exenatide and the nonselective nitric oxide-synthase inhibitor L-N(G)-monomethyl arginine. Capillary videomicroscopy was performed, and baseline and postocclusive (peak) capillary densities were counted. Compared with placebo, exenatide increased baseline and peak capillary density by 20.1% and 8.3%, respectively (both P=0.016). Concomitant L-N(G)-monomethyl arginine infusion did not alter the effects of exenatide. Vasomotion was assessed using laser Doppler fluxmetry. Exenatide nonsignificantly reduced the neurogenic domain of vasomotion measurements (R=-5.6%; P=0.092), which was strongly and inversely associated with capillary perfusion (R=-0.928; P=0.036). Glucose levels were reduced during exenatide infusion, whereas levels of insulin were unchanged. Acute exenatide infusion increases capillary perfusion via nitric oxide-independent pathways in healthy overweight men, suggesting direct actions of this GLP-1RA on microvascular perfusion or interaction with vasoactive factors.

Nitroxyl accelerates the oxidation of oxyhemoglobin by nitrite

Angeli’s salt (Na2N2O3) decomposes into nitroxyl (HNO) and nitrite (NO2-), compounds of physiological and therapeutic interest for their impact on biological signaling both through nitric oxide and nitric oxide independent pathways. Both nitrite and HNO oxidize oxygenated hemoglobin to methemoglobin. Earlier work has shown that HNO catalyzes the reduction of nitrite by deoxygenated hemoglobin. In this work, we have shown that HNO accelerates the oxidation of oxygenated hemoglobin by NO2-. We have demonstrated this HNO mediated acceleration of the nitrite/oxygenated hemoglobin reaction with oxygenated hemoglobin being in excess to HNO and nitrite (as would be found under physiological conditions) by monitoring the formation of methemoglobin in the presence of Angeli’s salt with and without added NO2-. In addition, this acceleration has been demonstrated using the HNO donor 4-nitrosotetrahydro-2H-pyran-4-yl pivalate, a water-soluble acyloxy nitroso compound that does not release NO2- but generates HNO in the presence of esterase. This HNO donor was used both with and without NO2- and acceleration of the NO2- induced formation of methemoglobin was observed. We found that the acceleration was not substantially affected by catalase, superoxide dismutase, c-PTIO, or IHP, suggesting that it is not due to formation of extramolecular peroxide, NO2 or H2O2, or to modulation of allosteric properties. In addition, we found that the acceleration is not likely to be related to HNO binding to free reduced hemoglobin, as we found HNO binding to reduced hemoglobin to be much weaker than has previously been proposed. We suggest that the mechanism of the acceleration involves local propagation of autocatalysis in the nitrite-oxygenated Hb reaction. This acceleration of the nitrite oxyhemoglobin reaction could affect studies aimed at understanding physiological roles of HNO and perhaps nitrite and use of these agents in therapeutics such as hemolytic anemias, heart failure, and ischemia reperfusion injury.

Effect of the Synadenium carinatum latex lectin (ScLL) on Leishmania (Leishmania) amazonensis infection in murine macrophages

Antiparasitic effect of a lectin isolated from Synadenium carinatum latex (ScLL) was evaluated against Leishmania (Leishmania) amazonensis promastigotes/amastigotes. Pretreatment of murine inflammatory peritoneal macrophages with ScLL reduced by 65.5% the association index of macrophages and L. (L) amazonensis promastigotes. Expression of cytokines (IL-12, IL-1 and TNF-α) was detected in infected macrophages pretreated with ScLL (10μg/mL). ScLL also reduced the growth of L. (L) amazonensis amastigote intracellular forms, showing no in vitro cytotoxic effects in mammalian host cells. ScLL treatment in infected murine inflammatory peritoneal macrophages did not induce nitric oxide production, suggesting that a nitric oxide independent pathway is activated to decrease the number of intracellular Leishmania.

The Biology and Therapeutic Potential of the DDAH/ADMA Pathway

Asymmetric dimethylarginine (ADMA) is an endogenously produced molecule that inhibits nitric oxide synthase and consequently may have adverse effects on physiology, in particular in the cardiovascular system. This review highlights the mechanisms involved in the synthesis and metabolism of ADMA and their role in the control of nitric oxide (NO) synthesis. We describe how the effects of both cellular and circulating ADMA can alter physiological function involving both NO dependent and independent pathways and go on to describe how the metabolism of ADMA by dimethylarginine dimethylaminohydrolase (DDAH) is the major endogenous mechanism by which ADMA levels are regulated. Furthermore, we discuss the association of ADMA concentrations with cardiovascular disease and how ADMA levels can be modulated therapeutically by altering its production and/or metabolism. Finally we discuss the effects of some of the current pharmaceutical therapies used to treat cardiovascular disease and their involvement in the modulation of the ADMA/DDAH pathway.

BENEFICIAL EFFECT OF NITRIC OXIDE ON LARGE ARTERIES IN HYPERTENSIVE RATS: 7D.06

Nitric oxide (NO) is a biological messenger that is known to exert a wide range of patho/physiological functions. In particular it is responsible for vasodilation and is well established that the inhibitors of NO synthases (such as L-NAME) induce vascular contraction and hypertension. Vasoconstriction of compliance arteries leads to a potentially harmful increase in heart load. Recently, a new cardiovascular parameter, augmentation index (AIx), has been described to be a biological marker of vascular tree efficiency. The aim of this study was to demonstrate a possible beneficial role of NO in large artery district. To this purpose, we measured AIx in the rat, using a recently developed technical tool enabling arterial pulse wave recording in small rodents (Samba Preclin, Samba Sensors AB, Goteborg, Sweden). To make analysis possible, we used the model of L-NAME-induced hypertension. Pulse wave was recorded in the carotid artery of anesthetized SD rats and drugs were given intravenously by jugular vein cannulation, except for L-NAME that was administered intraperitoneally. L-NAME (50 mg/kg) induced an increase in AIx of 33.9 ± 1.8%, compared to basal value. The NO donor, diethylenetriamine/NO (DETA-NO), 3-morpholinosydnonimine (SIN-1) and isoidide mononitrate (IIMN) (10 mg/kg), significantly decreased AIx (-15.5 ± 4.5%, -40.0 ± 4.93%, -22.7 ± 8.2%, respectively). Also the anti-hypertensive drugs, candesartan (3 mg/kg) and carvedilol (5 mg/kg), counteracted the L-NAME-induced increase in AIx (-16 ± 11% and 4 ± 2%, respectively), while atenolol (30 mg/kg) did not affect the L-NAME effect (data are expressed as delta from basal values). These findings show that NO plays beneficial role in large arteries, suggesting a contribution to heart load reduction. Both NO donors and drugs inducing vasodilation by NO independent pathways (such as candesartan and carvedilol) are able to counteract the increase in large artery vasoconstriction, induced by NO depletion.

Lipopolysaccharide Impairs Blood–Brain Barrier P-glycoprotein Function in Mice Through Prostaglandin- and Nitric Oxide-Independent Pathways

P-glycoprotein (P-gp) is a brain-to-blood efflux system that controls the ability of many drugs and endogenous substances to access the brain. In vitro work has shown that inflammatory states mediated through lipopolysaccharide (LPS) and tumor necrosis factor-alpha first impair and then stimulate P-gp activity. Here, we determined whether LPS can affect P-gp function in vivo. Mice treated with a single intraperitoneal injection of LPS (3 mg/kg) showed an inhibition of P-gp function. As assessed by brain perfusion, inhibition began 18 h after LPS administration and lasted until 36 h after administration. P-gp protein was increased by 44%, consistent with P-gp inhibition occurring through post-translational mechanisms. Unlike other effects of LPS on blood-brain barrier function, neither nitric oxide nor prostaglandin inhibition had an effect. We conclude that induction of proinflammatory states as exemplified by LPS treatment can inhibit P-gp function in vivo at the blood-brain barrier.

The modulatory role of androgens and progestins in the induction of vasorelaxation in human umbilical artery

Sex steroids have been described as protectors of the cardiovascular system and one of their relevant actions is inhibition of vascular tone. However, this information has been derived from animal models. The aim of this study was to investigate the vasorelaxant properties of several progestins and androgens on the vascular tone of human umbilical artery (HUA) to elucidate their potential regulatory role on fetoplacental blood flow. HUA rings, obtained from umbilical cords at vaginal deliveries and cesarean section from term uncomplicated pregnancies, were isometrically recorded and precontracted with either KCl or serotonin. Subsequently, dehydroepiandrosterone, testosterone, progesterone and some of their 5-reduced metabolites were added at different noncumulative concentrations on KCl-induced precontraction. There were significant differences in the vasorelaxing responses to these steroids; excluding 5α-pregnandione, the remaining steroids induced concentration-dependent vasorelaxations. In general, androgens were more potent than progestins, with 5β-dihydrotestosterone being the most potent one. These vasorelaxations remained unaffected by inhibitors of transcription and translation, selective steroid receptor antagonists, a nitric oxide synthase inhibitor or specific blockers of K + channels. Interestingly, the serotonin contraction was significantly less sensitive to steroid-induced vasorelaxation. Moreover, the contraction evoked by Ca 2+ in depolarized tissues (by KCl–Ca 2+ free solution) was prevented by steroids. These data, taken together, suggest that sex steroids (particularly androgens) induce an acute (nongenomically-mediated) vasorelaxing effect on the HUA which may be mediated by: (i) a nitric oxide-independent pathway; and/or (ii) a decrease in external Ca 2+ influx by inactivating Ca 2+ channels, but not by activating K + channels.

Selective Impairment of Nuclear Factor-κB-Dependent Gene Transcription in Adult Cardiomyocytes

The ability of neonatal and adult cardiomyocytes to activate the nuclear factor (NF)-kappaB pathway in response to lipopolysaccharide and interleukin-1beta challenge has been investigated and compared with that of peritoneal macrophages. The activation of the IkappaB kinase and the phosphorylation and degradation of IkappaBalpha and IkappaBbeta was much lower in adult cardiomyocytes than in the neonatal counterparts and macrophages. This restricted activation of the NF-kappaB pathway resulted in a significant reduction in the time of nuclear activation of NF-kappaB, as deduced by electrophoretic mobility shift assays and in the transcription of target genes, such as IkappaBalpha, cyclooxygenase-2 (COX-2) and nitric-oxide synthase-2 (NOS-2). Studies on chromatin immunoprecipitation showed binding of NF-kappaB proteins to the regulatory kappaB sites identified in the promoters of the IkappaBalpha, COX-2, and NOS-2 genes in macrophages and, to a lower extent, in neonatal cardiomyocytes. The binding to these kappaB sites in adult cardiomyocytes was observed only in the IkappaBalpha promoter and was minimal or absent in the COX-2 and NOS-2 promoters, respectively, suggesting a restricted activation of NF-kappaB-regulated genes in these cells. These data indicate that the function of the NF-kappaB pathway in adult cardiomyocytes is limited in time, which results in the expression of a reduced number of genes and provides a functional explanation for the absence of NOS-2 inducibility in these cells under proinflammatory conditions.

‘Johnston and Hagberg reply’

Developmental Medicine & Child NeurologyVolume 49, Issue 6 p. 477-478 Free Access ‘Johnston and Hagberg reply’ MV Johnston MD, Corresponding Author MV Johnston MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine, Baltimore, MD, USA *Correspondence to: [email protected]Search for more papers by this authorH Hagberg MD PhD, H Hagberg MD PhD Perinatal Center and Department of Obstetrics and Gynaecology, Sahlgrenska Academy, Göteborg University, Göteborg, SwedenSearch for more papers by this author MV Johnston MD, Corresponding Author MV Johnston MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine, Baltimore, MD, USA *Correspondence to: [email protected]Search for more papers by this authorH Hagberg MD PhD, H Hagberg MD PhD Perinatal Center and Department of Obstetrics and Gynaecology, Sahlgrenska Academy, Göteborg University, Göteborg, SwedenSearch for more papers by this author First published: 21 May 2007 https://doi.org/10.1111/j.1469-8749.2007.00477a.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 Renolleau S, Fau S, Goyenvalle C, Joly LM, Chauvier D, Jacotot E, Mariani J, Charriaut-Marlangue C. (2007) Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rats: a role for gender. J Neurochem. 100: 1062– 1071. 2 Nijboer CH, Groenendaal F, Kavelaars A, Hagberg HH, Van Bel F, Heijnen CJ. (2007) Gender-specific neuroprotection by 2-iminobiotin after hypoxia-ischemia in the neonatal rat via a nitric oxide independent pathway. J Cereb Blood Flow Metab. 27: 282– 292. Citing Literature Volume49, Issue6June 2007Pages 477-478 ReferencesRelatedInformation

Gender-Specific Neuroprotection by 2-Iminobiotin after Hypoxia—Ischemia in the Neonatal Rat via a Nitric Oxide Independent Pathway

We have shown earlier that 2-iminobiotin (2-IB) reduces hypoxia-ischemia (HI)-induced brain damage in neonatal rats, and presumed that inhibition of nitric oxide synthases (NOS) was the underlying mechanism. We now investigated the effect of 2-IB treatment in P7 rat pups to determine the role of gender and the neuroprotective mechanism. Pups were subjected to HI (occlusion of right carotid artery and 120 mins FiO(2) 0.08) and received subcutaneous (s.c.) 10 mg/kg 2-IB at 0, 12 and 24 h after hypoxia. After 6 weeks, neuronal damage was assessed histologically. We determined cerebral nitrite and nitrate (NO(x)) and nitrotyrosine, heat-shock protein 70, cytosolic cytochrome c, cleaved caspase 3, nuclear translocation of apoptosis-inducing factor (AIF) and the effect of 2-IB on NOS activity in cultured cells. 2-Iminobiotin treatment reduced long-term brain damage in female but not male rats. Unexpectedly, 2-IB treatment did not reduce cerebral NO(x) or nitrotyrosine levels, and did not inhibit NOS activity in vitro. The gender-dependent neuroprotective effect of 2-IB was reflected in inhibition of the HI-induced increase in cytosolic cytochrome c and cleaved caspase 3 in females only. Hypoxia-ischemia-induced activation of AIF was observed in males only and was not affected by 2-IB. Post-HI treatment with 2-IB provides gender-specific long- and short-term neuroprotection in female P7 rats via inhibition of the cytochrome c-caspase 3 neuronal death pathway. 2-Iminobiotin did not alter cerebral NO(x) nor inhibited NOS in intact cells. Therefore, we conclude that it is highly unlikely that the neuroprotective effect of 2-IB involves NOS inhibition.

Y-27632, A Rho-Kinase Inhibitor, Inhibits Proliferation and Adrenergic Contraction of Prostatic Smooth Muscle Cells

Benign prostatic hyperplasia (BPH) causes mechanical urinary flow obstruction by 2 components, namely an enlarged prostate (static component) and elevated smooth muscle tone (dynamic component). Currently available treatments for BPH aim to inhibit the proliferation of prostatic cells or decrease the elevated tone. To our knowledge no single agent that can achieve these 2 ends has yet been identified. A specific inhibitor of Rho-kinase, Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), has been demonstrated to cause smooth muscle relaxation and inhibit smooth muscle cell proliferation. Therefore, we investigated the effect of Y-27632 on prostatic smooth muscle proliferation and tone. Rho-kinase expression was investigated by immunocytochemistry and immunoblotting in smooth muscle cells obtained from rat and human prostates. The effect of Y-27632 was examined on the proliferation of these cells and on the contractions elicited by electrical field stimulation and exogenous phenylephrine in rat prostatic strips. Immunoblot and immunofluorescence analysis showed that Rho-kinase is present in the cytosol and located in the perinuclear region in human and rat prostatic smooth muscle cells. Y-27632 decreased the proliferation of human and rat prostatic smooth muscle cells, and inhibited noradrenergic contractions elicited by electrical field stimulation and exogenous phenylephrine in rat prostatic strips (EC50 17.8 +/- 4.8 and 7.8 +/- 2.1 microM, respectively). To our knowledge we report the first demonstration of the presence of Rho-kinase in prostatic smooth muscle cells, and of the relaxant and antiproliferative effect of a Rho-kinase inhibitor. We suggest a novel use for Rho-kinase inhibitors in the treatment of BPH as a single agent with dual action.

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway.

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.

Differences in cellular responses to mitogens in arterial smooth muscle cells derived from patients with moyamoya disease.

Moyamoya disease is a progressive cerebrovascular occlusive disease affecting primarily children. The etiology remains unknown. We examined the chemotactic and proliferative activities of inflammatory cell products from arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those from control subjects. We used 12 SMC strains from moyamoya patients and eight from control subjects. SMC migration was examined in a micro chemotaxis chamber. DNA synthesis was measured by an immunoperoxidase technique. Platelet-derived growth factor (PDGF)-BB markedly stimulated cell migration and DNA synthesis in control SMCs. PDGF-AA stimulated only DNA synthesis in control SMCs. In moyamoya SMCs, PDGF-AA and PDGF-BB stimulated cell migration but not DNA synthesis. Basic fibroblast growth factor had little migratory activity but stimulated DNA synthesis in moyamoya SMCs and control SMCs. Conversely, hepatocyte growth factor stimulated cell migration but not DNA synthesis in moyamoya SMCs and control SMCs. In contrast, interleukin-1 beta (IL-1 beta) significantly stimulated the migration and DNA synthesis of control SMCs, while it inhibited moyamoya SMC migration. The levels of IL-1 beta-induced nitric oxide production did not differ between moyamoya SMCs and control SMCs, suggesting that IL-1 beta inhibits the migration of moyamoya SMCs through a nitric oxide-independent pathway. The differences in responses to PDGF and IL-1 in moyamoya SMCs are involved in the mechanism by which intimal thickening develops in moyamoya disease.

Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.

Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.

Androgenic maintenance of the rat erectile response via a non-nitric-oxide-dependent pathway.

Prior studies have demonstrated that the erectile response in the rat penis is androgen dependent and is mediated by nitric oxide (NO), the neurotransmitter synthesized by the enzyme nitric oxide synthase (NOS). The present studies used L-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, to determine if androgens also regulate alternative pathways leading to the erectile response but not mediated by NO. Castrated rats that were treated with L-NAME (L-NAME CASTRATE) exhibited little or no increase in intracavernosal pressure in response to stimulation of the major pelvic ganglion. This ganglion controls blood flow into the penis and, when stimulated, normally leads to erection. However, when castrated animals were treated with testosterone along with L-NAME (L-NAME TESTO), the animals responded to the ganglionic stimulation with increased intracavernosal pressure. This finding suggests that there are other androgen-dependent pathways that lead to penile erection but are not mediated by NO. Erection occurred in both L-NAME CASTRATE and L-NAME TESTO rats in response to intracavernosal injection of sodium nitroprusside (an NO donor drug), proving that the NO responsive mechanisms were unaffected by the inhibition of NOS activity. To investigate further the nature of this NO independent pathway, L-NAME CASTRATE and L-NAME TESTO rats were treated with either zaprinast (a specific phosphodiesterase 5 inhibitor), which would block the breakdown of cGMP to 5'GMP, or methylene blue (an inhibitor of guanylate cyclase) to prevent the synthesis of cGMP. Zaprinast treatment led to increased erectile response in L-NAME TESTO rats but not in L-NAME CASTRATE rats, demonstrating that androgen-sensitive alternative pathways increased guanylate cyclase activity. Methylene blue inhibited the erectile response in all treatment groups, showing that cyclic GMP is critical to the NO-independent pathway as well as the NO-dependent pathway. Taken together, these results support the hypothesis that androgens maintain the erectile response by alternate pathways, including one that is independent of NO but involves the synthesis of cyclic GMP.

Multiple pathways underlying endothelium‐dependent relaxation in the rabbit isolated femoral artery

1. In isolated segments of the rabbit femoral artery stimulated with noradrenaline, both acetylcholine (1 nM-10 microM) and the calcium ionophore A23187 (1 nM-100 microM) evoked endothelium-dependent smooth muscle relaxation and hyperpolarization while bradykinin (0.01-100 nM) had no effect. 2. The nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NOARG; 100 microM; 20 min) or NG-nitro-L-arginine methyl ester (L-NAME; 100 microM; 20 min) each abolished the hyperpolarization and the majority of the relaxation to acetylcholine (maximal response reduced from 96.8 +/- 2.3% to 2.0 +/- 1.4%). 3. The potassium channel blocker, glibenclamide (10 microM; 10 min) also abolished the change in membrane potential to acetylcholine but did not modify the smooth muscle relaxation. 4. In contrast, neither L-NAME nor glibenclamide modified the comparable responses of the femoral artery to A23187, which were also unaffected by the cyclo-oxygenase inhibitor, indomethacin (10 microM). 5. In artery segments stimulated with potassium chloride (25 mM), the maximal change in tension and membrane potential evoked by A23187 (100 microM) was significantly reduced from 95.0 +/- 4.5% and 23.0 +/- 2.0 mV to 69.0 +/- 10.1% and 12.0 +/- 1.5 mV, respectively. Under these conditions L-NAME further reduced the relaxation but not the accompanying hyperpolarization to A23187. 6. Endothelium-denuded arterial segments sandwiched with endothelium-intact 'donor' segments gave qualitatively similar relaxant responses to those described above for acetylcholine and A23187. 7. Exogenous nitric oxide (0.5-10 microM) stimulated a transient relaxation in pre-contracted artery segments, which at concentrations above 5 microM was accompanied by smooth muscle hyperpolarization(maximum 8.5 +/- 3.2 mV; n = 4). The hyperpolarization but not the relaxation to nitric oxide was abolished by either glibenclamide or 25 mM potassium.8. These data indicate that in the femoral artery, acetylcholine-induced relaxation can be attributed solely to the release of nitric oxide from the endothelium, which then stimulates relaxation independently of a change in smooth muscle membrane potential. In contrast, both the relaxation and hyperpolarization evoked by A23187 appear to be mediated predominantly by nitric oxide-independent pathways which appear to involve a diffusible factor released from the endothelium. The results suggest that this diffusible hyperpolarizing factor can be released from endothelial cells in the femoral artery by A23187 but not by acetylcholine.

Bacterial Lipopeptides Induce Nitric Oxide Synthase and Promote Apoptosis through Nitric Oxide-independent Pathways in Rat Macrophages

Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-C ysSerLys4 or S(-)[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-++ +CysAlaLys4, two synthetic bacterial lipopeptides, promoted the expression of the inducible form of nitric oxide synthase, exhibiting a temporal pattern of nitric oxide release that was delayed with respect to the induction elicited by bacterial lipopolysaccharide. Treatment of macrophages with genistein blocked the nitric oxide synthesis triggered by the lipopeptides or lipopolysaccharide. Simultaneous incubation with lipopolysaccharide and lipopeptide resulted in an antagonistic effect on nitric oxide synthase mRNA levels and on nitrite plus nitrate release to the medium. Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells.