Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

鞘内注射NOS反义寡核苷酸抑制吗啡戒断大鼠脊髓和脑干NMDA1A受体mRNA表达的影响

运用逆转录－多聚酶联反应（RT－PCR）、鞘内注射和反义技术,研究脊髓水平一氧化氮（NO）对大鼠吗啡戒断反应和脊髓及脑干NMDA1A受体mRNA(NMDA1AR mRNA)表达的影响。结果表明,鞘内注射NOS反义寡核苷酸能明显减轻吗啡戒断反应,且脑型NOS（nNOS）反义寡苷酸的作用强于内皮型NOS(eNOS）反义寡核苷酸,吗啡依赖大鼠脊髓和脑干NMDA1AR mRNA表达增加,纳洛酮催促戒断,使其进一步增加;鞘内注射nNOS反义寡核苷酸,能明显抑制吗啡戒断大鼠脊髓和脑干NMDA1AR mRNA表达的增加;eNOS反义寡核苷酸也可抑制吗戒断大鼠脊髓NMDA1AR mRNA表达的增加,但作用弱于nNOS反义寡核苷酸,对脑干NMDA1AR mRNA表达无明显影响,上述结果提示：脊髓水平NO参与介导吗啡戒断反庆和NMDA受体表达的调控。     

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.     

Extremely low frequency magnetic field induces hyperalgesia in mice modulated by nitric oxide synthesis

We investigated an effect of extremely low frequency magnetic field (ELF-MF, 60 Hz) on hyperalgesia using hot plate test. The level of nitric oxide (NO) and the expression of nitric oxide synthase (NOS) were measured to determine if ELF-MF is engaged in NO mediated pain mechanism. Additionally, the involvement of Ca2+-dependent NO pathway in ELF-MF induced hyperalgesia was evaluated by blocking Ca2+ sources with NMDA receptor antagonist and Ca2+ channel blocker. The exposure of mice to ELF-MF lowered pain threshold and elevated NO synthesis in brain and spinal cord. An NOS inhibitor blocked these effects of ELF-MF with attenuating the reduction of pain threshold and the rise of NO level in brain and spine by the exposure of ELF-MF. The hyperalgesic effects of ELF-MF were also blocked by a Ca2+ channel blocker, nimodipine, but not by a NMDA receptor antagonist, MK-801. The expression of Ca2+ -dependent nNOS and eNOS and Ca2+ -independent iNOS were not changed by ELF-MF. These results indicated that the exposure of ELF-MF might cause Ca2+ -dependent NOS activation, which then induces hyperalgesia with the increase in NO synthesis. In conclusion, ELF-MF may produce hyperalgesia by modulating NO synthesis via Ca2+ -dependent NOS.     

MEK抑制剂抑制吗啡依赖及戒断大鼠脊髓神经元NOS的表达

目的：观察鞘内注射丝裂原活化蛋白激酶抑制剂U0126对吗啡依赖及戒断大鼠戒断症状和痛敏行为以及脊髓神经元NOS表达的影响。方法：采用吗啡依赖及戒断模型,分为正常对照组、依赖组、戒断组（戒断1h）、U0126组,分别作行为学评分（n=8）、免疫组织化学（n=6）和免疫印迹检测（n=4）。结果：①鞘内注射U0126可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28．6±4．89,U0126组为22．5±4．09（P〈0．05）;戒断组TEA评分为13．5±2．55,U0126组为10．0±2．76（P〈0．05）;②鞘内注射U0126可明显减少L5节段脊髓背角Fos阳性神经元的数目,U0126组为287±54,低于戒断组（380±71,P〈0．05）;③U0126组nNOS和iNOS阳性神经元的数目分别为180±32、10．8±2．8,均低于戒断组（239±45,16．8±5．1,P〈0．05）,两给药组脊髓NOS蛋白的表达也显著减少。结论：MEK抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制NOS的表达,表明ERK可能参与调控NOS的表达。     

Nitric oxide synthase, an essential factor in peripheral nerve regeneration.

Nitric oxide (NO) exerts both, pro-apoptotic and anti-apoptotic actions and appears to be acritical factor inneuronal degenerative and regenerative processes. NO is synthesized from L-arginine by NO synthase occurring in three isoforms of (neuronal, nNOS; endothelial, eNOS; inducible, iNOS). In a mice sciatic nerve model the regenerative outcome was assessed when the endogenous NO supply was deficient by knocking out the respective NOS isoform and compared to that of wild type mice after nerve transection. In nNOS knock-out mice a delay in regeneration, preceded by slowedWallerian degeneration and a disturbed pruning of uncontrolled sprouts, was observed. This was associated with a delayed recovery of sensory and motor function. Additionally, deficiency of nNOS led after nerve cut to a substantial loss of small and medium-sized dorsal root ganglia neurons, spinal cord interneurons and, to a lesser extent, spinal cord motor neurons. A lack of iNOS resulted in a delayed Wallerian degeneration and impaired regenerative outcome without consequences for neuronal survival. A lack of eNOS was well tolerated, although a delay in nerve revascularization was observed. Thus, after peripheral nerve lesion, regular NOS activity is essential for cell survival and recovery with reference to the nNOS isoform.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Renal actions of atrial natriuretic peptide in spontaneously hypertensive rats: the role of nitric oxide as a key mediator

Atrial natriuretic peptide (ANP) is an important regulator of blood pressure (BP). One of the mechanisms whereby ANP impacts BP is by stimulation of nitric oxide (NO) production in different tissues involved in BP control. We hypothesized that ANP-stimulated NO is impaired in the kidneys of spontaneously hypertensive rats (SHR) and this contributes to the development and/or maintenance of high levels of BP. We investigated the effects of ANP on the NO system in SHR, studying the changes in renal nitric oxide synthase (NOS) activity and expression in response to peptide infusion, the signaling pathways implicated in the signaling cascade that activates NOS, and identifying the natriuretic peptide receptors (NPR), guanylyl cyclase receptors (NPR-A and NPR-B) and/or NPR-C, and NOS isoforms involved. In vivo, SHR and Wistar-Kyoto rats (WKY) were infused with saline (0.05 ml/min) or ANP (0.2 μg·kg(-1)·min(-1)). NOS activity and endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS expression were measured in the renal cortex and medulla. In vitro, ANP-induced renal NOS activity was determined in the presence of iNOS and nNOS inhibitors, NPR-A/B blockers, guanine nucleotide-regulatory (G(i)) protein, and calmodulin inhibitors. Renal NOS activity was higher in SHR than in WKY. ANP increased NOS activity, but activation was lower in SHR than in WKY. ANP had no effect on expression of NOS isoforms. ANP-induced NOS activity was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in kidney. The renal NOS response to ANP was reduced by G(i) protein and calmodulin inhibitors. We conclude that ANP interacts with NPR-C, activating Ca-calmodulin eNOS through G(i) protein. NOS activation also involves NPR-A/B. The NOS response to ANP was diminished in kidneys of SHR. The impaired NO system response to ANP in SHR participates in the maintenance of high blood pressure.     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

Expression profiles of NOS isoforms in gingiva of nNOS knockout mice

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from l-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20–25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.     

Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.     

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6–16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.     

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.     

Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.     

Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to approximately 480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.     

Regulation of diaphragmatic nitric oxide synthase expression during hypobaric hypoxia

Nitric oxide (NO) is normally synthesized inside skeletal muscle fibers by both endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases. In this study, we evaluated the influence of hypobaric hypoxia on the expression of NOS isoforms, argininosuccinate synthetase (AS), argininosuccinate lyase (AL), and manganese superoxide dismutase (Mn SOD) in the ventilatory muscles. Rats were exposed to hypobaric hypoxia ( approximately 95 mmHg) from birth for 60 days or 9-11 mo. Age-matched control groups of rats also were examined. Sixty days of hypoxia elicited approximately two- and ninefold increases in diaphragmatic eNOS and nNOS protein expression (evaluated by immunoblotting), respectively, and about a 50% rise in diaphragmatic NOS activity. In contrast, NOS activity and the expression of these proteins declined significantly in response to 9 mo of hypoxia. Hypoxia elicited no significant alterations in AS, AL and Mn SOD protein expression. Moreover, the inducible NOS (iNOS) was not detected in normoxic and hypoxic diaphragmatic samples. We conclude that diaphragmatic NOS expression and activity undergo significant adaptations to hypobaric hypoxia and that iNOS does not participate in this response.     

Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity

The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced trinitrobenzenesulfonic acid colitis in eNOS, nNOS, and iNOS knockout (KO) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS KO mice developed a more severe colitis compared with wild-type mice. During colitis, iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells, whereas eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS KO mice, but not in wild-type, iNOS, or nNOS KO mice. Furthermore, eNOS KO mice had fewer colonic goblet cells, impaired mucin production, and exhibited increased susceptibility to an inflammatory stimulus that was subthreshold to other mice. This susceptibility was reversible, because the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS KO mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.