Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

Objectives

The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications.

Methods

Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O₂ ^.–), NA and ATP releases were also determined.

Results

EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O₂ ^.– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals.

Conclusion

Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

Diabetes alters neuronal nitric oxide release from rat mesenteric arteries. Role of protein kinase C

The objective of the present study was to assess the influence of diabetes in the neuronal nitric oxide (NO) release elicited by electrical field stimulation (EFS, 200 mA, 0.3 ms, 1-16 Hz, for 30 s, at 1 min interval) in endothelium-denuded mesenteric artery segments from control and streptozotocin-induced diabetic rats, assessing the influence of protein kinase C (PKC) in this release. N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 microM, a NO synthase inhibitor) enhanced EFS-elicited contractions in control, and specially in diabetic rats, whereas they were unaltered by AMT (5 nM, an inducible NO synthase inhibitor) and capsaicin (0.5 microM, a sensory neurone toxin). Calphostin C (0.1 microM, a PKC inhibitor) increased the contraction elicited by EFS in both types of arteries. This increase was further enhanced by calphostin C + L-NAME in diabetic rats. Phorbol 12,13-dibutyrate (PDBu, 1 microM) reduced and unaltered EFS-induced contractions in control and diabetic rats, respectively. The further addition of L-NAME reversed the reduction obtained in control rats, and enhanced the response observed in diabetic rats. These results suggest that the EFS-induced NO release from perivascular nitrergic nerves, that negatively modulates the contraction, which is synthesized by neuronal constitutive NO synthase. The NO synthesis is positively stimulated by PKC. This NO release is increased in diabetes, likely due to an increase in the activity of this enzyme. The sensory nerves of these arteries do not seem to be involved in the contractile response.     

Androgen deprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries.

This study examines the effects of male sex hormones on the vasoconstrictor response to electrical field stimulation (EFS), as well as neuronal NO modulation of this response. For this purpose, denuded superior mesenteric artery from orchidectomized and control male Sprague-Dawley rats was used. EFS induced similar frequency-dependent contractions in segments from both groups. The NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester strengthened EFS-elicited contractions more in arteries from orchidectomized than from control male rats. The expression of nNOS was more pronounced in segments from control than from orchidectomized animals. Basal and EFS-induced NO release was similar in segments from both groups. In noradrenaline (NA)-precontracted segments, sodium nitroprusside (SNP) induced a concentration-dependent relaxation, that was greater in segments from orchidectomized than control male rats. 8-Bromo-cGMP induced a similar concentration-dependent relaxation in NA-precontracted segments from either group, and the cGMP levels induced by SNP were also similar in the two groups. Superoxide dismutase (SOD), a superoxide anion scavenger, did not modify the relaxation in segments from control male rats. In contrast, SOD enhanced the relaxation induced by SNP in segments from orchidectomized rats, and the effect was reversed by preincubation with SOD plus catalase. The generation of superoxide anion and of peroxynitrite was greater in segments from orchidectomized than control rats. In NA-precontracted segments from control or orchidectomized rats, exogenous peroxynitrite and H(2)O(2) induced a concentration-dependent relaxation. These results suggest that EFS induces a similar nNOS-derived NO release in segments from orchidectomized and control male rats, despite the decrease in nNOS expression in orchidectomized rats. The NO metabolism is higher in segments from orchidectomized male rats due to the increases in anion superoxide generation and peroxynitrite formation. The vasodilator effects of the peroxynitrite and H(2)O(2)0 generated from the NO metabolism are what enhance the functional role of the nNOS-derived NO release in the orchidectomized rats.     

E2 and not P4 increases NO release from NANC nerves of the gastrointestinal tract: implications in pregnancy

In women, during pregnancy, there is decreased motility of the gastrointestinal tract leading to a delay in gastric emptying and an increase in colonic transit time. Whether the rise in estradiol (E2) or progesterone (P4) is responsible for this effect is controversial. As the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves is responsible for modulating gastrointestinal motility in vivo, the purpose of this study was to evaluate whether the increased release of nitric oxide (NO) from the nitrergic component of the NANC nerves innervating the gastric fundus and colon that occurs during late pregnancy in rats is mediated by E2 or P4. Ovariectomized rats treated with E2 or P4 alone or in combination were used for our studies. We also wanted to assess the cellular and molecular mechanisms involved. The NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. Our studies indicated that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon after application of EFS to tissues obtained from animals treated with E2 alone or a combination of E2 + P4 but not from those treated with P4 alone. L-NAME attenuated relaxation responses in E2- and E2 + P4-treated animals. To elucidate whether the increased NO release may be due to an increase in neuronal NOS (nNOS) protein, we used both Western blot analysis and immunohistochemistry. We also used RT-PCR to determine whether there was an increase in nNOS mRNA after treatment with sex steroids. In nonpregnant animals, nNOS was detected by Western blot in the fundus and the colon and was barely detectable in the ileum. In pregnancy, there was an increase in nNOS in both the gastric fundus and the colon. The nNOS protein was also increased in ovariectomized animals treated with either E2 alone or E2 + P4 but not P4 alone when compared with ovariectomized animals receiving vehicle. Our results indicated that there was an increase in nNOS protein that was localized to the neurons of the myenteric plexus in the gastric fundus and colon in E2- and E2 + P4-treated animals, but this increase was not observed in animals treated with P4 alone. This increase in nNOS protein was accompanied by an increase in nNOS mRNA. These results suggest the possibility that E2, rather than P4, may be responsible for the delay in gastric emptying and increase in colonic transit time observed in pregnancy.     

Angiotensin II increases neurogenic nitric oxide metabolism in mesenteric arteries from hypertensive rats

We investigated, in mesenteric arteries from hypertensive rats (SHR), the possible changes in neurogenic nitric oxide (NO) release produced by angiotensin II (AII), and the possible mechanisms involved in this process. In deendothelialized segments the NO synthase inhibitor N(G)-nitro-L-arginine (L-NAME, 10 microM) increased the contractions caused by electrical field stimulation (EFS, 200 mA, 0.3 ms, 1-16 Hz, for 30 s). AII (0.1 nM) enhanced the response to EFS, which was unmodified by the subsequent addition of L-NAME. The AII antagonist receptor saralasine (0.1 microM) prevented the effect of AII, and the subsequent addition of L-NAME restored the contractile response. SOD (25 u/ml) decreased the reponse to EFS and the subsequent addition of L-NAME increased this response. AII did not modify the decrease in EFS response induced by SOD, and the addition of L-NAME increased the response. None of these drugs altered the response to exogenous noradrenaline (NA) or basal tone except SOD, which increased the basal tone, an effect blocked by phentolamine (1 microM). In arteries pre-incubated with [3H]-NA, AII did not modify the tritium efflux evoked by EFS, which was diminished by SOD. AII did not alter basal tritium efflux while SOD significantly increased it. These results suggest that EFS of SHR mesenteric arteries releases neurogenic NO, the metabolism of which is increased in the presence of AII by the generation of superoxide anions.     

Gastrointestinal motility during pregnancy: role of nitrergic component of NANC nerves

This study evaluated whether increased release of nitric oxide (NO) from the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves may be partly responsible for the decrease in gastrointestinal motility observed during pregnancy. Segments of fundal strip, ileum, and colon were obtained from nonpregnant rats, rats in midpregnancy (days 9-11), and rats in late pregnancy (days 18-20). NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. The magnitude of change in cGMP levels in the tissues after application of EFS was also assessed. Our studies indicate that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon, after application of EFS to tissues obtained only from animals in late pregnancy. These results paralleled the changes in cGMP levels in tissues. NOS activity in the gastric fundus was significantly increased in animals in late pregnancy compared with nonpregnant controls. Our studies suggest that the delay in gastric emptying and increase in colonic transit time observed in rats during pregnancy may be caused in part by increased activity of the nitrergic component of the NANC nerves innervating these organs.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.     

Role of neural NO synthase (nNOS) uncoupling in the dysfunctional nitrergic vasorelaxation of penile arteries from insulin-resistant obese Zucker rats

Objective

Erectile dysfunction (ED) is considered as an early sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors. Endothelial and neural dysfunction involving nitric oxide (NO) are usually implicated in the pathophysiology of the diabetic ED, but the underlying mechanisms are unclear. The present study assessed the role of oxidative stress in the dysfunctional neural vasodilator responses of penile arteries in the obese Zucker rat (OZR), an experimental model of metabolic syndrome/prediabetes.

Methods and Results

Electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions evoked relaxations that were significantly reduced in penile arteries of OZR compared with those of lean Zucker rats (LZR). Blockade of NO synthase (NOS) inhibited neural relaxations in both LZR and OZR, while saturating concentrations of the NOS substrate L-arginine reversed the inhibition and restored relaxations in OZR to levels in arteries from LZR. nNOS expression was unchanged in arteries from OZR compared to LZR and nNOS selective inhibition decreased the EFS relaxations in LZR but not in OZR, while endothelium removal did not alter these responses in either strain. Superoxide anion production and nitro-tyrosine immunostaining were elevated in the erectile tissue from OZR. Treatment with the NADPH oxidase inhibitor apocynin or acute incubation with the NOS cofactor tetrahydrobiopterin (BH4) restored neural relaxations in OZR to levels in control arteries, while inhibition of the enzyme of BH4 synthesis GTP-cyclohydrolase (GCH) reduced neural relaxations in arteries from LZR but not OZR. The NO donor SNAP induced decreases in intracellular calcium that were impaired in arteries from OZR compared to controls.

Conclusions

The present study demonstrates nitrergic dysfunction and impaired neural NO signalling due to oxidative stress and nNOS uncoupling in penile arteries under conditions of insulin resistance. This dysfunction likely contributes to the metabolic syndrome-associated ED, along with the endothelial dysfunction also involving altered NO signalling.     

Effects of pregnancy and female sex steroid hormones on calcitonin gene-related peptide content of mesenteric artery in rats

Calcitonin gene-related peptide (CGRP) levels in plasma and the dorsal root ganglia (DRG) are increased during pregnancy and in ovariectomized rats injected with ovarian hormones. Vasodilatory responses to CGRP are also increased in these animals. In the present study, we hypothesized that pregnancy and ovarian hormones elevate the contents of CGRP in perivascular nerves. We assessed CGRP-dependent mesenteric vascular relaxation induced by electrical field stimulation (EFS) and arterial content of CGRP. Because the mesenteric artery represents resistance vessels, segments of mesenteric arteries collected from female rats at different stages of the estrous cycle, pregnancy, or postpartum and from male rats were used in this study. The EFS-induced relaxation in the presence and absence of CGRP(8-37), an antagonist of CGRP, was used to measure CGRP-dependent relaxation, and radioimmunoassay (RIA) of tissue homogenates was used to assess changes in CGRP content in mesenteric branch arteries. The results show that CGRP-dependent, EFS-induced relaxation response was lower in female rats at diestrus and proestrus than in male rats, and no statistically significant differences were observed between Gestational Day 20 and Postpartum Day 2. The RIA revealed significantly lower mesenteric artery CGRP levels in female rats at proestrus, gestation, and postpartum than in female rats at diestrus or in male rats. We conclude that no correlation exists between CGRP-dependent, EFS-induced relaxation and CGRP content in the mesenteric arteries of these animal groups. Because both CGRP levels in DRG and serum are reported to be elevated, the reduced CGRP content in the vasculature during pregnancy and proestrus implicate enhanced basal release of CGRP at the nerve terminal in these animals.     

Nitric oxide synthase inhibition decreases tolerance to hyperoxia in newborn rats

We evaluated the effects of sustained perinatal inhibition of NO synthase (NOS) on hyperoxia induced lung injury in newborn rats. N(G)-nitro-Larginine-methyl-ester (L-NAME) or untreated water was administered to pregnant rats for the final 7 days of gestation and during lactation; followed by postnatal exposure to hyperoxia (>95% O(2)) or room air. The survival rate of L-NAME treated pups when placed in > 95% O(2) at birth was significantly lower than controls from day 4 (L-NAME, 87%; control pups, 100%, p < 0.05) to 14 (L-NAME, 0%; control pups, 53%, p < 0.05). Foetal pulmonary artery vasoconstriction was induced by L-NAME with a decrease in internal diameter from 0.88 +/- 0.03 mm to 0.64 +/- 0.01 mm in control vs. L-NAME groups (p < 0.05), respectively. We conclude that perinatal NOS inhibition results in pulmonary artery vasoconstriction and a decreased tolerance to hyperoxia induced lung injury in newborn rats.     

Pregnancy-associated increase in rat systemic arteries endothelial nitric oxide production diminishes vasoconstrictor but does not enhance vasodilator responses

Late pregnancy in rats is characterized by a decrease in arterial pressure and in isolated arterial vessels response to vasoconstrictors. In uterine arteries the pregnancy-associated attenuation of the response to vasoconstrictors has been attributed to an increase in basal and agonist-induced endothelial NO production. However, the role of NO in pregnancy-associated changes of systemic arteries reactivity to vasoactive agents remains to be fully elucidated. We examined whether pregnancy influences the reactivity of systemic arteries to vasodilator or vasoconstrictor agents through NO-dependent mechanisms. Thoracic aortic rings and mesenteric arterial bed of late pregnant rats showed refractoriness to phenylephrine-induced vasoconstriction that was abolished by NO synthase inhibition. The potency of L-NNA to enhance tension of aortic rings preconstricted with phenylephrine (10–20% of their maximal response) was significantly lower in preparations from pregnant animals. In phenylephrine-contracted aortas and mesenteric bed, the effects of the endothelium-dependent vasodilators acetylcholine, A23187 and bradykinin, were not influenced by pregnancy. Similarly, pregnancy did not affect the vasodilator responses of adenosine, isoproterenol, capsaicin, nitroprusside, forskolin, and Hoe234 in the mesenteric bed. NO synthase activity measured by determining the conversion of L−[³H]-arginine to L−[³H]-citrulline in aorta and mesenteric arteries homogenates was not altered by pregnancy. These findings show that endothelial-dependent and -independent vasodilators action as well as NO synthase activity in systemic arteries is uninfluenced by pregnancy, whereas pregnancy-associated hyporeactivity of systemic arteries to vasoconstrictors is related to an enhanced endothelial NO production either spontaneous or elicited directly or indirectly by vasoconstrictor agents. This interpretation implies that the enhanced NO production observed in systemic arteries during late pregnancy involves cellular pathways other than the ones involved in the response to endothelium-dependent vasodilators such as acetylcholine.     

Nitric oxide-mediated nonadrenergic noncholinergic relaxation of piglet pulmonary arteries decreases with postnatal age.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of adult pulmonary and systemic vascular tone. However, their actions in the neonatal circulation have not been studied. We aimed to investigate NANC vasorelaxation in neonatal and 2-week-old piglet pulmonary and mesenteric arteries and to examine the potential role of nitric oxide (NO) in this phenomenon. Responses to electric field stimulation (EFS, 50V, 0.25-32 Hz) were investigated in pulmonary and mesenteric artery rings (external diameter 150-200 microm) precontracted with the thromboxane A2 mimetic U46619, in the presence of guanethidine (10 microM) and atropine (10 microM). Under these conditions, EFS resulted in a frequency dependent relaxation of newborn pulmonary (maximal relaxation of 53+/-9.1%), mesenteric (68.8.2+/-7.1%) and 2-wk-old mesenteric (46 6.3%) arteries but this relaxation was significantly reduced (4.5+/-2.2%) in 2-week-old pulmonary arteries. In neonatal pulmonary arteries, the neurotoxin tetrodotoxin (0.3 muM), the NO synthase inhibitor L-NAME (0.1 mM), and the guanylyl cyclase inhibitor ODQ (10 microM) abolished EFS-induced relaxations, suggesting that NANC relaxation of porcine neonatal pulmonary arteries is mediated by NO, which is probably neuronal in origin. However, The expression in pulmonary arteries of the neuronal NO synthase (nNOS), as determined by Western-blot analysis, increased with postnatal age whereas the expression of the endothelial NOS (eNOS) did not change. In conclusion, NANC relaxation is present in neonatal pulmonary and mesenteric arteries and it is, at least partially, mediated through NO. NANC relaxation of porcine pulmonary and mesenteric arteries decreases with postnatal maturation.     

Effect of NOS inhibition on central response to atrial distension during pregnancy

Atrial distension increases c-fos expression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either D- or N(G)-nitro-L-arginine methyl ester (L-NAME) (120 mg/2 ml at 10 microg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c-fos expression. In the virgin rats, and in the pregnant rats treated with L-NAME, atrial distension significantly increased hypothalamic c-fos expression. In the pregnant animals treated with D-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.     

Cortisol-mediated potentiation of uterine artery contractility: effect of pregnancy

During pregnancy, maternal plasma cortisol concentrations approximately double. Given that cortisol plays an important role in the regulation of vascular reactivity, the present study investigated the potential role of cortisol in potentiation of uterine artery (UA) contractility and tested the hypothesis that pregnancy downregulated the cortisol-mediated potentiation. In vitro cortisol treatment (3, 10, or 30 ng/ml for 24 h) produced a dose-dependent increase in norepinephrine (NE)-induced contractions in both nonpregnant and pregnant (138-143 days gestation) sheep UA. However, this cortisol-mediated response was significantly attenuated by approximately 50% in pregnant UA. The 11 beta-hydroxysteroid dehydrogenase (11-beta HSD) inhibitor carbenoxolone did not change the effect of cortisol in nonpregnant UA but abolished its effect in pregnant UA by increasing the NE pD(2) in control tissues from 6.20 +/- 0.05 to 6.59 +/- 0.11. The apparent dissociation constant value of NE alpha(1)-adrenoceptors was not changed by cortisol in pregnant UA but was decreased in nonpregnant UA. There was no difference in glucocorticoid receptor density between nonpregnant and pregnant UA. Cortisol significantly decreased endothelial nitric oxide (NO) synthase protein levels and NO release in both nonpregnant and pregnant UA, but the effect of cortisol was attenuated in pregnant UA by approximately 50%. Carbenoxolone alone had no effects on NO release in nonpregnant UA but was decreased in pregnant UA. These results suggest that cortisol potentiates NE-mediated contractions by decreasing NO release and increasing NE-binding affinity to alpha(1)-adrenoceptors in nonpregnant UA. Pregnancy attenuates UA sensitivity to cortisol, which may be mediated by increasing type-2 11-beta HSD activity in UA.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

Deregulation of Hepatic Insulin Sensitivity Induced by Central Lipid Infusion in Rats Is Mediated by Nitric Oxide

Background

Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis.

Methodology

We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats).

Principal Findings

Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid).

Conclusions/Significance

We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.     

Endogenous estrogen mediates vascular reactivity and distensibility in pregnant rat mesenteric arteries

The role of estrogen in the maternal systemic cardiovascular adaptations during pregnancy is still controversial. Female Sprague-Dawley rats were implanted at day 14 of pregnancy with either a 50-mg tamoxifen pellet (estrogen receptor blocker, n = 10) or placebo pellet (n = 10). Virgin female rats were a nonpregnant control (n = 7). At days 20-22 of pregnancy, resistance-sized mesenteric arteries were mounted onto a dual-chamber arteriograph system. Pregnancy significantly blunted the pressor response to phenylephrine [measurement of the effective concentration that yielded 50% maximum response (EC(50)) values were 1.5 +/- 0.22 vs. 0.69 +/- 0.16 microM (P < 0.05)] and enhanced vasodilation to ACh [EC(50) = 1.13 +/- 2.53 vs. 3.13 +/- 6.04 nM (P < 0.05)] compared with nonpregnant rats. However, tamoxifen treatment during pregnancy reversed these effects. Inhibition of nitric oxide (NO) synthase with N(G)-monomethyl-L-arginine (250 microM) shifted only the responses of the placebo-treated pregnant group to both phenylephrine and ACh. Arterial distensibility in the placebo-treated pregnant group was also significantly increased (P < 0.05) compared with nonpregnant and tamoxifen-treated pregnant animals. In summary, endogenous estrogen during pregnancy increases NO-dependent modulation of vessel tone and arterial distensibility.     

Nitric oxide production in the ventilatory muscles in response to acute resistive loading

The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.