An in vitro investigation of aorta and corpus cavernosum from eNOS and nNOS gene-deficient mice (original) (raw)
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Nitric Oxide, 2003
Nitric oxide (NO), produced by NO-synthase (NOS), serves as an important vasodilator and inhibitory neurotransmitter. Inducible NOS (iNOS) is expressed in response to cytokine stimulation and is therefore not ordinarily present in healthy tissue. However, iNOS has been identified in certain organs, including the penis. The development of mice deficient in the iNOS gene (iNOS )/)) has provided a useful tool for the study of iNOS function. Therefore, an in vitro examination of vascular and nerve-mediated responses of corpus cavernosum (CC) and vascular responses of aorta from iNOS )/) mice and their wild-type controls was undertaken. Tissues were mounted in organ baths for agonist-and/or electrical field stimulation (EFS)-induced responses under isometric tension. CC from iNOS )/) mice developed increased sensitivity to phenylephrine (PE) and an increased maximum EFSinduced noradrenergic contraction of approximately 31%. Following PE precontraction, maximum relaxation to acetylcholine was reduced by approximately 39%; conversely, there was a 23% increase in relaxation to the NO-donor sodium nitroprusside. EFSinduced non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation was unaltered compared to control. Agonist-induced responses of aorta did not significantly differ between iNOS )/) and control mice. These results suggest that iNOS-derived NO may play a role in modulating erectile function and confirm that iNOS does not play a significant role in macrovascular function under normal physiological conditions.
PLOS ONE, 2015
Nitric oxide (NO) seems to contribute to vascular homeostasis regulating neurotransmission. This work aimed at assessing the influence of NO from different sources and respective intracellular pathways on sympathetic neurotransmission, in two vascular beds. Electrically-evoked [ 3 H]-noradrenaline release was assessed in rat mesenteric and tail arteries in the presence of NO donors or endothelial/neuronal nitric oxide synthase (NOS) inhibitors. The influence of NO on adenosine-mediated effects was also studied using selective antagonists for adenosine receptors subtypes. Location of neuronal NOS (nNOS) was investigated by immunohistochemistry (with specific antibodies for nNOS and for Schwann cells) and Confocal Microscopy. Results indicated that: 1) in mesenteric arteries, noradrenaline release was reduced by NO donors and it was increased by nNOS inhibitors; the effect of NO donors was only abolished by the adenosine A 1 receptors antagonist; 2) in tail arteries, noradrenaline release was increased by NO donors and it was reduced by eNOS inhibitors; adenosine receptors antagonists were devoid of effect; 3) confocal microscopy showed nNOS staining in adventitial cells, some co-localized with Schwann cells. nNOS staining and its co-localization with Schwann cells were significantly lower in tail compared to mesenteric arteries. In conclusion, in mesenteric arteries, nNOS, mainly located in Schwann cells, seems to be the main source of NO influencing perivascular sympathetic neurotransmission with an inhibitory effect, mediated by adenosine A 1 receptors activation. Instead, in tail arteries endothelial NO seems to play a more relevant role and has a facilitatory effect, independent of adenosine receptors activation.
Biochemical and Biophysical Research Communications, 1992
The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyI-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and 7-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock. ® 1992 Academic Pr .... Inc.
Nitric Oxide, 2009
While the unequivocal pattern of endothelial nitric oxide (NO) synthase (eNOS) inhibition in cardiovascular control has been recognised, the role of NO produced by neuronal NOS (nNOS) remains unclear. The purpose of the present study was to describe the cardiovascular effects of NO production interference by inhibition of nNOS with 7-nitroindazole (7-NI). Wistar rats (10 weeks old) were used: control and experimental rats were administered 7-NI 10 mg/kg b.w./day in drinking water for 6 weeks. Systolic blood pressure (BP) was measured by the tail-cuff plethysmographic method. Isolated thoracic aortas (TAs) were used to study vasomotor activity of the conduit artery in vitro. The BP response of anaesthetised animals was used to follow the cardiovascular-integrated response in vivo. Geometry of the TA was measured after perfusion fixation (120 mm Hg) by light microscopy. Expression of eNOS was measured in the TA by immunoblot analysis. Although 6 weeks of nNOS inhibition did not alter systolic BP, the heart/body weight ratio was decreased. Relaxation of the TA in response to acetylcholine (10 À9-10 À5 mol/L) was moderately inhibited. However, no difference in the BP hypotensive response after acetylcholine (0.1, 1, 10 lg) was observed. The contraction of TA in response to noradrenaline (10 À10-10 À5 mol/L), and the BP pressor response to noradrenaline (0.1, 1 lg) was attenuated. The inner diameter of the TA was increased, and the wall thickness, wall cross-sectional area, and wall thickness/inner diameter ratio were decreased. The expression of eNOS in the TA was increased. In summary, cardiac and TA wall hypotrophy, underlined by decreased contractile efficiency, were observed. The results suggested that two constitutive forms of NOS (nNOS, eNOS) likely participate in regulation of cardiovascular tone by different mechanisms.
European Journal of Pharmacology, 1991
The effects of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine (NOLA), have been examined in perfused segments of rat tail artery. NOLA (1 and 10/.tM) significantly enhanced the vasoconstrictor responses to perivascular nerve stimulation (5 Hz, I0 s) and noradrenaline (10 ng). The enhancing effects of NOLA were prevented by L-arginine, but not by o-arginine, and were absent in endothelium-denuded artery segments. The results suggest that nitric oxide derived from endothelial cells attenuates vasoconstrictor responses to both nerve stimulation and noradrenaline.
British Journal of Pharmacology, 1999
Acetycholine-mediated relaxations in phenylephrine-contracted aortas, femoral and mesenteric resistance arteries were studied in vessels from endothelial nitric oxide synthase knockout (eNOS 7/7) and the corresponding wild-type strain (eNOS +/+) C57BL6/SV19 mice. 2 Aortas from eNOS (+/+) mice relaxed to acetylcholine in an endothelium-dependent N G-nitro-L-arginine (L-NOARG) sensitive manner. Aortas from eNOS (7/7) mice did not relax to acetylcholine but demonstrated enhanced sensitivity to both authentic NO and sodium nitroprusside. 3 Relaxation to acetylcholine in femoral arteries was partially inhibited by L-NOARG in vessels from eNOS (+/+) mice, but relaxation in eNOS (7/7) mice was insensitive to a combination of L-NOARG and indomethacin and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The L-NOARG/ODQ/indomethacin-insensitive relaxation to acetylcholine in femoral arteries was inhibited in the presence of elevated (30 mM) extracellular KCl. 4 In mesenteric resistance vessels from eNOS (+/+) mice, the acetylcholine-mediated relaxation response was completely inhibited by a combination of indomethacin and L-NOARG or by 30 mM KCl alone. In contrast, in mesenteric arteries from eNOS (7/7) mice, the acetylcholine-relaxation response was insensitive to a combination of L-NOARG and indomethacin, but was inhibited in the presence of 30 mM KCl. 5 These data indicate arteries from eNOS (7/7) mice demonstrate a supersensitivity to exogenous NO, and that acetylcholine-induced vasorelaxation of femoral and mesenteric vessels from eNOS (7/7) mice is mediated by an endothelium-derived factor that has properties of an EDHF but is neither NO nor prostacyclin. Furthermore, in mesenteric vessels, there is an upregulation of the role of EDHF in the absence of NO.
British Journal of Pharmacology, 2005
Prolonged exposure (6-12 h) of rat aorta to alpha1-adrenergic receptor (a 1 AR) agonist phenylephrine (Phe) leads to a decrease in a 1 AR-mediated vasoconstriction. This reduced responsiveness to a 1 AR stimulation was strongly dependent on the intactness of the endothelium. 2 We examined the effect of Phe on nitric oxide synthase (NOS) activity by measuring the conversion of [ 3 H]L-arginine to [ 3 H]L-citrulline in rat aorta or in endothelial cells isolated from rat aorta. Phe stimulation increased NOS activity in control aortas. This response was antagonized by prazosin. However, Phe increased neither the activity of NOS nor intracellular Ca 2 þ in the isolated endothelial cells from the control aortas, whereas acetylcholine (Ach) was able to stimulate both responses in these cells. This result suggests that Phe stimulates a 1 AR on vascular smooth muscle cells and has an indirect influence on endothelial cells to increase NOS activity. 3 In Phe-exposed aortic rings, basal NOS activity was found to have increased compared to vehicleexposed control rings. Stimulation with Phe or Ach caused a small increase over basal NOS activity in these preparations. Prolonged exposure to Phe also caused an enhancement of Ach-mediated vasorelaxation in rat aorta. 4 Immunoblot and reverse transcription-polymerase chain reaction experiments showed that prolonged exposure of rat aorta to Phe resulted in an increased expression of eNOS, but not iNOS. This increase was antagonized by nonselective antagonist prazosin. Immunohistochemical staining experiments also showed that expression of eNOS increased in endothelial cells after Phe exposure of the aortas. 5 These results, all together, showed that prolonged exposure of rat aorta to a l AR agonist Phe enhanced the expression of eNOS and basal NOS activity, which probably causes a decreased vasocontractile response to Phe or to other agonists such as 5HT (5-hydroxytryptamine) in rat aorta. 6 This phenomenon can be considered more as a functional antagonism of vasocontractile response to agonists mediated by endothelium than a specific desensitization of a 1 AR-mediated signalling in vascular smooth muscle cells.
The Vasoactive Role of Nitric Oxide: Physiological and Morphological Aspects
Current Pharmaceutical Biotechnology, 2011
Nitric oxide (NO) participates in the control of the cardiovascular system where two constitutive isoforms of NO-synthase were discovered: endothelial and neuronal. Both isoforms were observed in various cells, however, endothelial NO-synthase is predominantly present in the endothelium. Injury of the endothelium disturbs the balance between vasodilation and vasoconstriction and triggers different pathological alterations. In addition, whereas the intact endothelium protects vascular smooth muscle from oxidative attack, intervention in the vascular wall integrity increases the concentration of vascular superoxides, thus disturbing the effects of NO. To preserve NO-mediated vasorelaxation, different reserve mechanisms have developed. In case of damage of some endothelial receptor type, vasodilation could be ensured by activation of some other type of the present receptors. Moreover, morphological evidence demonstrated that both isoforms of NO-synthase were expressed also in smooth muscle cells and functional studies revealed that different pathological interventions in endothelial function (such as oxidative stress or hypertension) were associated with NO generation in the vascular media. In this case, the generation of NO by vascular smooth muscle may represent a physiologically relevant compensation of endothelial NO deficiency. Whereas long-term inhibition of endothelial NO-synthase resulted in an unequivocal pattern of cardiovascular changes, inhibition of neuronal NO-synthase led to opposite effects, suggesting a specific position of neuronal NO-synthase in the regulation of cardiovascular tone. The specificity of endothelial or neuronal NO function seems to be related to a particular circulatory area and it is presumably determined by mutual interactions with other regulatory systems (sympathoadrenergic, renin-angiotensin, etc.).
Neuronal NOS-dependent dilation to flow in coronary arteries of male eNOS-KO mice
American journal of physiology. Heart and circulatory physiology, 2002
Flow-induced dilation was examined in isolated coronary arteries of endothelial nitric oxide (NO) synthase knockout mice (eNOS-KO) and wild-type (WT) mice. The basal tone of arteries (percentage of passive diameter) was significantly greater in eNOS-KO than in WT mice; their flow-induced dilations, however, were similar. Endothelial removal eliminated the dilations in vessels of both strains of mice. In arteries of WT mice, N(omega)-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) or indomethacin (10(-5) M) alone, inhibited flow-induced dilation by approximately 50%, whereas their simultaneous administration abolished the responses. In arteries of eNOS-KO mice, flow-induced dilation was inhibited by approximately 40% with L-NAME. The residual portion (60%) of the response was eliminated by the additional administration of indomethacin. 7-Nitroindazole (10(-4) M) attenuated flow-induced dilation by approximately 40% in arteries of eNOS-KO mice, but did not affect responses in those ...
British Journal of Pharmacology, 1994
1 We aimed to determine whether nitric oxide (NO) and/or the endothelium is involved in cholinergic neurogenic vasodilatation in the rat isolated hindquarters. 2 The abdominal aorta was cannulated for perfusion of the rat hindquarters with Krebs bicarbonate solution containing phenylephrine, to induce basal constrictor tone. In the presence of noradrenergic neurone blockade with guanethidine (200 mg kg', i.p.) electrical stimulation of peri-aortic nerves induced frequency-dependent decreases in hindquarters perfusion pressure, indicating vasodilatation. Both the endothelium-dependent vasodilator, acetylcholine (ACh) and the endothelium-independent vasodilator, sodium nitroprusside (SNP) induced dose-dependent decreases in perfusion pressure. In each experiment, responses to either nerve stimulation, ACh or SNP were recorded before and after treatment with saline vehicle, atropine (1 gLM), NG-nitro-L-arginine (L-NOARG, 100 JM), L-arginine (1 mM), L-arginine plus L-NOARG, or 3-3 cholamidopropyl dimethylammonio 1-propanesulphonate (CHAPS, 30 mg). Hindquarters dilatation after each treatment was expressed as a percentage of the control response. 3 Following treatment with saline, responses to nerve stimulation and ACh were 99 ± 9% and 107 ± 10% of control, respectively demonstrating the reproducibility of these responses. Nerve stimulation-induced dilatation was abolished by atropine (0 ± 0% of control, P <0.05) or reduced to 14 ± 10% of control by NO synthase inhibition with L-NOARG (P <0.05). Dilator responses to ACh were also abolished by atropine (0 ± 0% of control, P<0.05) or inhibited by L-NOARG (59 ± 10% of control, P<0.05), indicating that the neurogenic dilatation is cholinergic and is mediated by NO. The administration of the NO precursor, L-arginine, prevented the inhibitory effect of L-NOARG on dilator responses to nerve stimulation and ACh (L-arginine plus L-NOARG: 89 ± 13% and 122 ± 24% of control, respectively). In addition CHAPS, which removes endothelial cells, inhibited responses to both nerve stimulation (0 ± 0% of control, P <0.05) and ACh (33 ± 8% of control, P <0.05). In contrast, no treatment significantly reduced the vasodilator responses to SNP. 4 These observations suggest that cholinergic neurogenic vasodilatation in the rat isolated hindquarters requires the synthesis and release of NO from the endothelium.