Role Of Nitric Oxide Synthase Research Articles

Do nitric oxide synthase and cyclooxygenase contribute to the heat loss responses in older males exercising in the heat?

This study evaluated the separate and combined roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in forearm sweating and cutaneous vasodilatation in older adults during intermittent exercise in the heat. Twelve healthy older (62 ± 7 years) males performed two 30 min cycling bouts at a fixed rate of metabolic heat production (400 W) in the heat (35°C, 20% relative humidity). The exercise bouts were followed by 20 and 40 min of recovery, respectively. Forearm sweat rate (ventilated capsule) and cutaneous vascular conductance (CVC, laser Doppler perfusion units/mean arterial pressure) were evaluated at four skin sites that were continuously perfused via intradermal microdialysis with: (1) lactated Ringer solution (Control), (2) 10 mm ketorolac (non-selective COX inhibitor), (3) 10 mm N(G) -nitro-l-arginine methyl ester (l-NAME; non-selective NOS inhibitor) or (4) a combination of 10 mm ketorolac + 10 mm l-NAME. Sweating was not different between the four sites during either exercise bout (main effect P = 0.92) (average of last 5 min of second exercise, Control, 0.80 ± 0.06; ketorolac, 0.77 ± 0.09; l-NAME, 0.74 ± 0.07; ketorolac + l-NAME, 0.77 ± 0.09 mg min(-1) cm(-2) ). During both exercise bouts, relative to CVC evaluated at the Control site (average of last 5 min of second exercise, 69 ± 6%max), CVC was similar at the ketorolac site (P = 0.62; 66 ± 4%max) whereas it was attenuated to a similar extent at both the l-NAME (49 ± 8%max) and ketorolac + l-NAME (54 ± 8%max) sites (both P < 0.05). Thus, we demonstrate that NOS and COX are not functionally involved in forearm sweating whereas only NOS contributes to forearm cutaneous vasodilatation in older adults during intermittent exercise in the heat.

The ubiquitous ATP molecule: could it be the elusive thermal mediator igniting skin perfusion and sweating in the heat-stressed human?

Skin blood flow and sweating are two key thermoregulatory responses that help regulate body temperature in heat-stressed human beings. Adjustments in skin blood flow and sweating in response to thermal stimuli are mediated by local and reflex neural mechanisms (Johnson et al. 2014). Local thermosensitive mechanisms are important, as the application of heating to a particular area of the skin results in a localized elevation of blood flow and sweating in the heated skin. However, the precise pathways underpinning heat loss responses to heat stress, particularly those involving ATP as a mediator signal, are not fully established or understood. Heat stress-induced hyperthermia is associated with increases in intravascular ATP and accompanied by vasodilatation of the human skin and deep limb tissue vasculatures (Pearson et al. 2011; Heinonen et al. 2011). This supports a mechanistic coupling between the rise in intravascular ATP and the concomitant increase in local perfusion (Kalsi & Gonzalez-Alonso, 2012). However, sympathetic cholinergic nerves, which are known to modulate heat loss responses, co-release ATP with acetylcholine. These sensory nerves are therefore potential sources of ATP in the dermal interstitium. This raises the possibility that ATP may act as a thermal mediator signal, involved in the regulation of vasomotor and sudomotor activity in conditions of hyperthermia. In support of this idea, intradermal administration of ATP has been shown to induce cutaneous vasodilatation in human beings in vivo (Wingo et al. 2010), but the downstream mediators and its effect on sweating have not been investigated. Mechanistically, ATP can bind to P2Y purinergic receptors located in the cutaneous vasculature, and the sweat gland, and trigger cutaneous vasodilatation and increase sweating via at least three pathways: (i) activation of nitric oxide synthase (NOS), (ii) activation of cyclooxygenase (COX) and/or (iii) via the action of the ATP degradation compound adenosine. In this issue of The Journal of Physiology, Fujii et al. (2015) report the findings from an outstanding series of integrative physiology studies in human beings. The authors systematically investigated the roles of NOS and COX in the cutaneous vascular and sweating responses to intradermal administration of ATP, as well as the contribution of adenosine to ATP-induced skin vasodilatation in vivo. A major finding was that intradermal ATP infusion at a rate of 120 and 1200 nmol min−1 causes significant cutaneous vasodilatation. Co-infusion of the NOS inhibitor Nω–nitro-l-arginine (l-NNA) blunted about one-fourth of the ATP-evoked vasodilator response, implicating NOS as a mediator. The study also excluded two other potential regulatory pathways; blockade of COX or adenosine with co-infusion of ketorolac or theophylline did not alter the vasodilator response to intradermal ATP infusion. Lastly, none of the study protocols increased sweating. These observations collectively advance the basic physiological understanding of the control of skin blood flow and sweating. By showing that part of the vasodilator response to intradermal ATP administration is mediated by NOS and by providing conclusive evidence that ATP does not directly increase sweating in vivo, these data suggest that skin blood flow and sweating are not regulated by the same thermal mediator signal. The physiological relevance of interstitial ATP on thermal hyperaemia was not addressed in the study of Fujii et al. (2015). ATP needs to accumulate in the dermal interstitium for this ubiquitous molecule to exert its cutaneous vasodilator effects directly by binding to interstitial purinergic receptors. However, recent evidence suggests that local heating does not result in an accumulation of ATP in the interstitial space of human skin, and thus such accumulation is not necessary for local thermal hyperaemia to occur (Gifford et al. 2012). This observation and the additional finding by Fujii and coworkers that the intradermal administration of ATP at low infusion rates (0.12–12 nmol min−1) does not increase skin blood flow indicate that under physiological conditions ATP-mediated cutaneous vasodilatation is more likely to occur via the activation of purinergic receptors residing in the vascular endothelium. Although further research is needed to establish the physiological relevance of intravascular versus interstitial ATP in thermoeffector activity, the timely investigation of Fujii et al. (2015) highlights the capacity of ATP to be the elusive thermal mediator igniting skin perfusion in heat-stressed human beings, but not the signal that increases sweating.

Cutaneous vascular and sweating responses to intradermal administration of ATP: a role for nitric oxide synthase and cyclooxygenase?

In humans in vivo, the mechanisms behind ATP-mediated cutaneous vasodilatation along with whether and how ATP increases sweating remains uncertain. Recent work has implicated nitric oxide synthase (NOS), cyclooxygenase (COX) and/or adenosine in the modulation of cutaneous vasodilatation and sweat production during both local (i.e. localized heating) and whole-body heat stress (i.e. exercise-induced heat stress). We evaluated whether ATP-mediated cutaneous vasodilatation and sweating is mediated via NOS, COX and/or adenosine. We show that in humans in vivo, intradermal administration of ATP induces pronounced vasodilatation which is partially mediated by NOS, but neither COX nor adenosine influences ATP-mediated vasodilatation, and ATP alone does not induce an increase in sweating. These findings advance our basic physiological knowledge regarding control of skin blood flow and sweating, and provide insight into the mechanisms governing thermoeffector activity, which has major implications for whole-body heat exchange and therefore core temperature regulation in humans during heat stress. In humans in vivo, the mechanisms behind ATP-mediated cutaneous vasodilatation and whether and how ATP increases sweating remain uncertain. We evaluated whether ATP-mediated cutaneous vasodilatation and sweating is mediated via nitric oxide synthase (NOS), cyclooxygenase (COX) and/or adenosine-dependent mechanisms. Cutaneous vascular conductance (CVC, laser Doppler perfusion units/mean arterial pressure) and sweat rate (ventilated capsule) were evaluated at intradermal microdialysis forearm skin sites, each receiving pharmacological agents (two separate protocols). In Protocol 1 (n = 12), sites were perfused with: (1) lactated Ringer solution (Control), (2) 10 mm N(ω) -nitro-l-arginine (l-NNA, a NOS inhibitor), (3) 10 mm ketorolac (Ketorolac, a COX inhibitor) or (4) a combination of 10 mm l-NNA + 10 mm ketorolac (l-NNA + Ketorolac). In Protocol 2 (n = 8), sites were perfused with: (1) lactated Ringer solution (Control) or (2) 4 mm theophylline (Theophylline, an adenosine receptor inhibitor). At all sites, ATP was simultaneously perfused at 0.12, 1.2, 12, 120 and 1200 nm min(-1) (each for 20 min). Relative to CVC at the Control site with ATP infused at 120 nm min(-1) (71 ± 9% of max CVC), CVC at the Ketorolac site was comparable (64 ± 13% of max CVC, P = 0.407), but lower at l-NNA (51 ± 15% of max CVC, P = 0.040) and l-NNA + Ketorolac (51 ± 13% of max CVC, P = 0.049) sites. Conversely, across the four skin sites at any other ATP infusion rate (all P > 0.174), no differences in CVC were observed. Theophylline did not influence CVC at any ATP infusion rate (all P > 0.234). Furthermore, no ATP infusion rate elicited an increase in sweating from baseline at any skin site (all P > 0.235). We show that NOS, but neither COX nor adenosine receptors, modulates ATP-mediated cutaneous vasodilatation, whereas ATP does not directly increase sweating.

Participation of non-neuronal muscarinic receptors in the effect of carbachol with paclitaxel on human breast adenocarcinoma cells. Roles of nitric oxide synthase and arginase

Breast cancer is the most common type of cancer in women and represents a major issue in public health. The most frequent methods to treat these tumors are surgery and/or chemotherapy. The latter can exert not only beneficial effects by reducing tumor growth and metastasis, but also toxic actions on normal tissues. Metronomic therapy involves the use of low doses of cytotoxic drugs alone or in combination to improve efficacy and to reduce adverse effects. We have previously reported that breast tumors highly express functional muscarinic acetylcholine receptors (mAChRs) that regulate tumor progression. For this reason, mAChRs could be considered as therapeutic targets in breast cancer. In this paper, we investigated the ability of a combination of the cytotoxic drug paclitaxel plus carbachol, a cholinergic agonist, at low doses, to induce death in breast tumor MCF-7 cells, via mAChR activation, and the role of nitric oxide synthase (NOS) and arginase in this effect. We observed that the combination of carbachol plus paclitaxel at subthreshold doses significantly increased cytotoxicity in tumor cells without affecting MCF-10A cells, derived from human normal mammary gland. This effect was reduced in the presence of the muscarinic antagonist atropine. The combination also increased nitric oxide production by NOS1 and NOS3 via mAChR activation, concomitantly with an up-regulation of NOS3 expression. The latter effects were accompanied by a reduction in arginase II activity. In conclusion, our work demonstrates that mAChRs expressed in breast tumor cells could be considered as candidates to become targets for metronomic therapy in cancer treatment.

The multifaceted role of nitric oxide synthases in mitochondrial biogenesis and cell differentiation

Nitric oxide (NO) is physiologically synthetized by a family of enzymes called NO synthases (NOSs). NO is a pleiotropic second messenger having a fundamental role in several cellular processes including cell differentiation. Being a high reactive molecule, NO must be synthetized in close proximity to the effector/target. For this reason, the subcellular localization of NOSs is tightly regulated by different post-translation mechanisms. Recently, in murine C2C12 myoblasts, we have demonstrated that mitochondrial biogenesis, an essential event for cell differentiation, can be effective only if the site of NO production is located at nuclear level, where NO favors the CREB-dependent expression of PGC-1α gene. The increase of NO flux in nuclei is elicited by the up-regulation and redistribution of neuronal NOS (nNOS) toward nuclei.Herein we show that an upregulation of endothelial NOS (eNOS) occurs during adipocyte differentiation in 3T3-L1 cells. However, differently to differentiating myocytes, a concomitant redistribution of eNOS toward nuclei was not detected. We also observed that, upon treatment with the NO synthesis inhibitor L-NAME, mitochondrial biogenesis as well as triglyceride accumulation that normally occurs during adipogenesis were not impeded. The absence of eNOS in nuclei together with the ineffectiveness of L-NAME suggest that, at least during 3T3-L1 differentiation, NO is not fundamental for the induction of mitochondrial biogenesis and adipogenesis.

The role of nitric oxide synthase in an early phase Cd-induced acute cytotoxicity in MCF-7 cells.

Literature to date has confirmed that cadmium (Cd) can accomplish its toxic effects via the free radical-induced damage, but Cd itself cannot generate free radicals directly. Nitric oxide (NO) is a fundamental molecule that interplays with reactive oxygen species (ROS), which may be associated with the Cd-induced cytotoxicity. However, the role of nitric oxide synthase (NOS) in an early phase Cd-induced acute cytotoxicity and its interaction has not been studied. In this report, we provide data showing that CdCl2 (10 μM, 100 μM, 1 mM) could modulate NOS activity in terms of NO production which was first suppressed with the release of Ca(2+) and Zn(2+), then induced with the transcriptional and translational activation of the three NOS isoforms in a possible feedback manner. The ROS level in cells was increased after CdCl2 exposure. By using the free radical scavenger N-acetyl-L-cysteine (LNAC) or the NOS activity inhibitor N(G)-methyl-L-arginine (LNMMA), it was demonstrated that NOS played a critical role on the Cd-induced ROS generation. The Cd-induced cytotoxicity was associated with the NOS-mediated oxidative stress in MCF-7 cells.

Nitric oxide and thyroid hormone receptor alpha 1 contribute to ovarian follicular development in immature hyper- and hypo-thyroid rats

Thyroid dysfunction can cause ovarian cycle and ovulatory disturbances, however, the molecular link(s) between these two disorders remains largely unknown. In the current study, we examined the roles of nitric oxide synthase (NOS) and thyroid hormone receptor alpha 1 (TRα1) in these disorders using immature hyper-thyroid (hyper-T) and hypo-thyroid (hypo-T) rats. In comparison to controls, hyper-T rats had higher serum concentrations of triiodothyronine (T3) and thyroxine (T4), whereas hypo-T rats had lower serum T3 and T4. Serum estradiol (E2) level was decreased in both hyper-T and hypo-T animals and serum E2 in hyper-T rats were lower than in hypo-T rats. We found that neuronal NOS (nNOS) and TRα1 were present in oocytes, granulosa cells and theca cells of all examined rat groups. Ovarian nitric oxide (NO) content and the constitutive NOS (cNOS) activity in hyper-T rats were significantly decreased compared with control or hypo-T rats. Moreover, the number of large antral follicles was reduced in hyper-T rats, and number of primordial follicles was decreased in hypo-T rats compared with control rats. In conclusion, we observed an association between thyroid hormone and NO signaling pathways during the process of ovarian follicular development in immature rats. In hyperthyroidism, thyroid hormones induced an estrogen deficiency that inhibited the function of nNOS, resulting in the inhibition of NO synthesis and suppressed development of large antral follicles, while in hypothyroidism only development of primordial follicles was inhibited.

Involvement of nitric oxide in iodine deficiency-induced microvascular remodeling in the thyroid gland: role of nitric oxide synthase 3 and ryanodine receptors.

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10μM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.

To reheat, or to not reheat: that is the question: The efficacy of a local reheating protocol on mechanisms of cutaneous vasodilatation

The aim of this study is to determine the effect of repeated bouts of local skin heating on the roles of nitric oxide synthase (NOS) and sympathetic nerves in cutaneous vasodilatation. In 3 repeated-heating protocols skin blood flux of the forearm and leg was measured using laser-Doppler flowmetry and data are presented as cutaneous vascular conductance (CVC; flux/blood pressure). Local heating was performed from 33°C (thermoneutral) to 42°C at 0.5°C·10s−1, allowed to cool passively for ~60-min, then reheated at the same rate. In protocol 1, CVC was measured in response to repeated heating. In protocol 2, NOS was inhibited with NG-nitro-l-arginine methyl ester (L-NAME) and in protocol 3, sympathetic nerve blockade was achieved with bretylium tosylate (BT), both infused via intradermal microdialysis. In protocol 1, there were no differences (P>0.05) in CVC at either the forearm (88±4 vs. 86±4%max) or the leg (97±4 vs. 96±6%max) between heating bouts. In protocol 2, no differences (P>0.05) in CVC were observed between heating bouts at L-NAME treated sites at either the forearm (55±3 vs. 51±4%max) or the leg (71±3 vs. 70±4%max) . In protocol 3, there were differences (P<0.001) between BT treated sites when comparing the first and second bouts of heating for both the forearm (75±3 vs. 88±4%max) and the leg (79±3 vs. 97±4%max). The effect of sympathetic blockade on CVC responses to local heating was abolished following repeated bouts of heating. Consequently, it is our suggestion that when examining mechanisms of skin blood flow control, investigators use single bouts of local heating.

Protective effects of Big-leaf mulberry and physiological roles of nitric oxide synthases in the testis of mice following water immersion and restraint stress

Big-leaf mulberry is a new hybrid plant from the application of cell engineering technology, but its effect in stress-induced testicular dysfunction is unknown. Nitric oxide (NO) is a tiny, highly reactive lipophilic molecule produced by nitric oxide synthases (NOS). Three isoforms of NOS (neuronal NOS, inducible NOS and endothelial NOS) have been identified. Our aim was to investigate the effect of water immersion and restraint stress (WIRS) on NOS in the testis, and the effect of Big-leaf mulberry to protect against WIRS. The activity and expression of NOS, and total antioxidant capacity (T-AOC) in the mouse testis of different treatment groups (non-WIRS, 3 h-WIRS, WIRS-recovery) were examined. Histological analysis of WIRS-induced testicular damage and immunohistochemical staining of NOS were also analyzed. Results demonstrated that WIRS-exposed mice produced several injuries and showed an increased iNOS and eNOS mRNA expression in testes, whereas pretreatment with Big-leaf mulberry down-regulated iNOS and eNOS mRNA expressions and up-regulated T-AOC activities. Immunohistochemical studies showed that both iNOS and eNOS were localized in germ cells, spermatozoa and blood vessels in addition to Leydig cells and Sertoli cells, but nNOS was not present in these areas. In conclusion, our results suggested that Big-leaf mulberry exerted a protective effect on WIRS-induced testicular dysfunction, and iNOS and eNOS appeared to exert an important action in mouse testes exposed to WIRS.

Nitric oxide synthases: three pieces to the puzzle?

Subarachnoid hemorrhage remains to be a devastating diagnosis in this day and age, with very few effective interventions. Rising evidence is now pointing towards the marked importance of secondary complications after the hemorrhage, and its active role in morbidity and mortality of this stroke. This review will focus on the role of Nitric Oxide Synthases (NOSes) the role they play in the pathogenesis of SAH.

Role of Nitric Oxide Synthases in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

The role of nitric oxide synthases (NOSs) in early blood-brain barrier (BBB) disruption was determined using a new mouse model of transient focal cerebral ischemia. Ischemia was induced by ligating the middle cerebral artery (MCA) at its M2 segment and reperfusion was induced by releasing the ligation. The diameter alteration of the MCA, arterial anastomoses and collateral arteries were imaged and measured in real time. BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. The reperfusion produced an extensive vasodilation and a sustained hyperemia. Although expression of NOSs was not altered at 3 hours of reperfusion, L-NAME (a non-specific NOS inhibitor) abolished reperfusion-induced vasodilation/hyperemia and significantly reduced EB and Na-F extravasation. L-NIO (an endothelial NOS (eNOS) inhibitor) significantly attenuated cerebral vasodilation but not BBB disruption, whereas L-NPA and 7-NI (neuronal NOS (nNOS) inhibitors) significantly reduced BBB disruption but not cerebral vasodilation. In contrast, aminoguanidine (AG) (an inducible NOS (iNOS) inhibitor) had less effect on either cerebral vasodilation or BBB disruption. On the other hand, papaverine (PV) not only increased the vasodilation/hyperemia but also significantly reduced BBB disruption. Combined treatment with L-NAME and PV preserved the vasodilation/hyperemia and significantly reduced BBB disruption. Our findings suggest that nNOS may play a major role in early BBB disruption following transient focal cerebral ischemia via a hyperemia-independent mechanism.

Role of nitric oxide synthases in the pathogenesis of cardiovascular disease

Role of nitric oxide synthases in the pathogenesis of cardiovascular disease

Roles of Nitric Oxide Synthases in Arteriosclerotic Vascular Disease:Insights from Murine Genetic Models

Nitric Oxide (NO) exerts a variety of biological actions under both physiological and pathological conditions. NO is synthesized by three distinct NO synthase (NOS) isoforms, encoded by three distinct NOS genes, including neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS), all of which are expressed in the human vascular system. Although the roles of the NOSs in arteriosclerotic vascular diseases have been described in pharmacological studies with selective and non-selective NOS inhibitors, the selectivity and specificity of the NOS inhibitors continue to be an issue of debate. To solve this issue, genetically altered animals have been established. All types of NOS gene-deficient animals have been developed, including singly, doubly, and triply NOS-deficient mice and various types of NOS Gene-Transgenic (TG) animals have also been generated, including conditional and non-conditional TG mice bearing site-specific overexpression of each NOS gene. The roles of individual NOS isoforms as well as the entire NOSs system in arteriosclerotic vascular diseases have been extensively investigated in those mice, providing pivotal insights into an understanding of the pathophysiological significance of the NOSs in human arteriosclerotic vascular diseases. The present review, which is based on studies with the murine NOS genetic models, summarizes the latest knowledge about the NOSs and arteriosclerotic vascular diseases.

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

The time-of-day variation in vascular smooth muscle contractility depends on a nitric oxide signalling pathway

The dip in blood pressure during the resting-period is paradoxically associated with an increase in total peripheral resistance and occurs at a time when the vascular response to vasoconstrictor compounds is heightened, and to vasodilators reduced. However, the cellular mechanisms responsible for this time-of-day variation are not well defined. We have investigated the role of nitric oxide synthase (NOS) signalling in the control of contraction in mesenteric resistance arteries using wire myography, combined with quantitative PCR analysis of gene transcription and western blot analysis of protein. Small rings of mesenteric arteries, isolated from rats at two opposing time-points corresponding to the animal's active and resting-period, were mounted in a wire myograph. Vessels exhibited a time-of-day variation in their contractile-response to phenylephrine, with a reduced maximal contraction during the active- versus the resting-period (11.8±0.8 versus 18.6±1.2mN P<0.001). Vessels preconstricted with phenylephrine were also more responsive to vasodilation with acetylcholine during the active-period, with an EC50 of 58.6±11 versus 232±31nM in resting-period vessels (P<0.0001). These differences were abolished in the presence of l-NAME. Quantitative RT-PCR reveals a functioning peripheral circadian clock in mesenteric arteries and a 3.3-fold increase in endothelial NO synthase mRNA levels in active- versus resting-period vessels (P<0.001), which translated to a 1.7-fold increase in total eNOS protein (P<0.05). The time-of-day variation in the response of mesenteric resistance vessels to phenylephrine and acetylcholine is dependent on NOS signalling.

The regional differences in the contribution of nitric oxide synthase to skin blood flow at forearm and lower leg sites in response to local skin warming

We investigated the role of nitric oxide synthase (NOS) on regional cutaneous vascular function at rest (thermoneutral conditions) and during the vasodilator response to increased local skin temperature (Tloc). Dorsal forearm and lateral leg sites were instrumented with microdialysis fibers, local heaters, and laser-Doppler probes. All sites were heated from 33°C to 42°C. Each limb had 1 skin site treated with l-NAME to inhibit NOS, and 1 site infused with lactated Ringer's to serve as a control. Basal cutaneous vascular conductance (CVC) was measured at 33°C, forearm sites averaged 14±1%max and 17±1%max at l-NAME and control sites, respectively (P=0.26). CVC sites in the leg were different between l-NAME (17±1%max) and control (27±2%max) (P=0.04). CVC between the forearm and the leg across control sites differed (P<0.05). In contrast, at l-NAME treated sites, there was no difference between the forearm and leg sites (P=0.23). When Tloc was increased to 42°C, CVC at the control sites differed between the forearm 93±1%max and leg 98±1%max (P=0.02). There were no differences between the arm and leg at l-NAME treated sites at 42°C (P=0.45). The findings of the current study were that the contribution of nitric oxide (NO) to the vasodilator response to an elevated Tloc is consistent between the arm and the leg, and, under thermoneutral conditions (33°C), NO plays a larger role in the basal vascular function in the legs than that of the forearm. Accordingly, these data suggest, in part, that the differences in basal CVC between the forearm and leg are due to NOS activity.

Abstract 105: The nitroso-redox-induced SR Ca 2 + leak is temperature-sensitive: Role of Nitric Oxide Synthase 1

Sarcomeric reticulum (SR) Ca 2+ leak contributes to an impaired Ca 2+ cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modifications of RyR2 associated with nitroso-redox (NO/redox) imbalance. Ca 2+ cycling is affected by both NO signaling and temperature. We hypothesized that NO/redox balance modulates SR Ca 2+ leak in a temperature–dependent manner. We used tetracaine to investigate SR Ca 2+ leak in cardiomyocytes (CMs) using a model of NO/redox imbalance (NOS1 −/− mice) or WT mice. Reactive oxygen species (ROS) were measured by H 2 DCF. The expression of NOS isoforms was assessed by real time PCR. Experiments were carried out at 23°C - 37°C. The SR Ca 2+ leak increased as the temperature decreased from 34°C to 23°C in WT CMs whereas, in NOS1 −/− CMs, the leak suddenly increased when the temperature surpassed 30°C (Fig. A). SR Ca 2+ content in NOS1 −/− CMs was reduced at 37°C compared to 23°C (42.7 mol/L vs. 115.6 mol/L, respectively; p =0.001), whereas it was unaffected in WT CMs. Differences in Ca 2+ leak between WT and NOS1 −/− were magnified according the SR Ca 2+ load increased. Contrarily to WT, there was a trend to rise in ROS production at temperatures over 30°C in NOS1 −/− CMs (Fig. B). There was a linear association between ROS and leak in both strains. The NOS isoforms were unaffected by temperature in WT. Our observations show that Ca 2+ leak increases when the temperature drops below 34°C; however, in the absence of NOS1 activity, the leak rises at temperatures greater than 30°C, and is associated with more ROS production. These results suggest that Ca 2+ leak from the SR is regulated by a temperature-dependent balance between NO and ROS.

Cardiovascular autonomic modulation by nitric oxide synthases accounts for the augmented enalapril-evoked hypotension in ethanol-fed female rats

In this study, we investigated the role of nitric oxide synthase (NOS) isoforms in the enhanced enalapril-evoked hypotension in ethanol-fed female rats by examining the effect of the selective inhibitors of eNOS [N5-(1-iminoethyl)-l-ornithine; l-NIO], nNOS (Nω-propyl-l-arginine; NPLA), or iNOS (1400W) inhibition on the cardiovascular effects of enalapril in ethanol- (5% w/v) fed rats and in their pair-fed controls. In liquid diet-fed control rats, enalapril- (10 mg/kg) evoked hypotension was abolished by l-NIO (20 mg/kg), but not by NPLA (1 mg/kg) or 1400W (5 mg/kg), suggesting a preferential role for eNOS in this response. Enalapril had no effect on spectral indices of hemodynamic variability or +dP/dtmax (myocardial contractility). However, in ethanol-fed rats, the greater enalapril-evoked hypotension was associated with reductions in (i) +dP/dtmax, (ii) low-frequency/high-frequency ratio of interbeat intervals (IBILF/HF), suggesting cardiac parasympathetic dominance, and (iii) low-frequency spectral band of systolic blood pressure (BP), a marker of vasomotor sympathetic tone. While NPLA or 1400W attenuated the enalapril-evoked hemodynamic and autonomic responses in ethanol-fed rats, l-NIO virtually abolished the hypotensive response and was more efficacious in rectifying autonomic responses to enalapril. Together, these findings implicate NOS isoforms, particularly eNOS, in the altered cardiovascular autonomic control that leads to the augmented enalapril-evoked hypotension in ethanol-fed female rats.

Role of neuronal nitric oxide in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats

Isoform-specific nitric oxide (NO) synthase (NOS) contributions to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle are incompletely understood. The purpose of the present study was to investigate the role of neuronal NOS (nNOS) in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. We hypothesized that acute pharmacological inhibition of nNOS would augment sympathetic vasoconstriction in resting and contracting skeletal muscle, demonstrating that nNOS is primarily responsible for NO-mediated inhibition of sympathetic vasoconstriction. Sprague-Dawley rats (n = 13) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and lumbar sympathetic chain stimulating electrodes. Triceps surae muscles were stimulated to contract rhythmically at 60% of maximal contractile force. In series 1 (n = 9), the percent change in femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction before and after selective nNOS blockade with S-methyl-l-thiocitrulline (SMTC, 0.6 mg/kg iv) and subsequent nonselective NOS blockade with N(ω)-nitro-l-arginine methyl ester (l-NAME, 5 mg/kg iv). In series 2 (n = 4), l-NAME was injected first, and then SMTC was injected to determine if the effect of l-NAME on constrictor responses was influenced by selective nNOS inhibition. Sympathetic stimulation decreased FVC at rest (-25 ± 7 and -44 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-7 ± 3 and -19 ± 5%FVC at 2 and 5 Hz, respectively). The decrease in FVC in response to sympathetic stimulation was greater in the presence of SMTC at rest (-32 ± 6 and -49 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-21 ± 4 and -28 ± 4%FVC at 2 and 5 Hz, respectively). l-NAME further increased (P < 0.05) the sympathetic vasoconstrictor response at rest (-47 ± 4 and -60 ± 6%FVC at 2 and 5 Hz, respectively) and during muscle contraction (-33 ± 3 and -40 ± 6%FVC at 2 and 5 Hz, respectively). The effect of l-NAME was not altered by the order of nNOS inhibition. These data demonstrate that NO derived from nNOS and endothelial NOS contribute to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle.