Effects of estradiol on renal cyclic guanosine monophosphate and oxidative stress in spontaneously hypertensive rats

Background: Evidence suggests that estradiol offers protection against the development of cardiovascular and renal pathologies, although the mechanisms involved are still under investigation. The nitric oxide (NO) pathway regulates blood pressure and kidney function, and estradiol is associated with increases in NO bioavailability. We hypothesized that in female spontaneously hypertensive rats (SHRs), estra-diol increases NO bioavailability, activates the NO synthase (NOS) pathway, and suppresses superoxide production compared with rats that underwent ovariectomy (OVX).Objective: The goal of this study was to determine whether estradiol regulates the NO/cyclic guanosine monophosphate (cGMP) pathway and superoxide levels in the kidneys of female SHR.Methods: Three types of SHRs were studied: gonad-intact females, OVX rats, and OVX rats with estra-diol replacement (OVX+E). Renal cortical cGMP levels were measured to assess NO bioavailability. NOS enzymatic activity, NOS protein expression, basal superoxide production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity were measured in the renal cortex.Results: Fifty-six SHRs were included in the study (17 intact females, 21 OVX rats, 18 OVX+E rats). Mean (SEM) cGMP levels were significantly lower in the renal cortex of OVX rats (0.03 [0.008] pmol/mg, n = 5) than in intact females (0.1 [0.02] pmol/mg, n = 6; P < 0.05), and estradiol restored cGMP levels to those seen in intact females (0.1 [0.01] pmol/mg, n = 5; P < 0.05). Despite a decrease in cGMP following OVX, renal cortical NOS activity, NOS1 and NOS3 protein expression, and the phosphorylation status of NOS3 were comparable among the 3 groups (n = 7–9 per group). However, mean basal superoxide production in the renal cortex was higher in OVX rats (3.2 [0.3] cpm/mg, n = 12) than in intact females (1.9 [0.3] cpm/mg, n = 8; P < 0.05) and lower in OVX+E rats (1.3 [0.3] cpm/mg, n = 9; P < 0.05). Mean NADPH oxidase activity was comparable in the renal cortex of intact females and OVX rats (81 [4] and 83 [12] cpm/35 μg, respectively [n = 5 per group]). OVX+E rats had significantly lower mean renal cortical NADPH oxidase activity than did rats in the other groups (45 [6] cpm/35 μg, n = 6; P < 0.05), and the decrease in activity was accompanied by a decrease in p22^phox protein expression.Conclusions: In vivo manipulations of estradiol levels influenced renal cortical NO bioavailability, as assessed indirectly by cGMP measurements. The decrease in cGMP following OVX was not due to alterations in the activity or expression of NOS.     

Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia.

L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury.     

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.     

A nitric oxide synthesis inhibitor decreased prostaglandin production in rat mesenteric vasculature

Endothelial cells synthesize and release nitric oxide (NO) and prostacyclin (PGI₂) which are involved in the regulation o f vascular tone and blood pressure. Our objective was to evaluate the effects of inhibiting NO synthesis on vascular prostaglandin (PG) and cyclic nucleotide production, as well as the pressor response to norepinephrine (NE). Isolated mesenteric arterial beds were perfused with Krebs-Henseleit solution containing 100 μM N^G-monomethyl-L-arginine (L-NMMA), 100 μM L-arginine (LA), or vehicle. After a 30 min equilibration 0.1, 0.5, 1, or 5 μM NE was infused into the superior mesenteric artery and the perfusion pressure was monitored. The basal perfusion pressure did not differ significantly between groups. The pressure-response curve was shifted to the right in the L-NMMA group vs. the LA and control groups. Perfusion was similarly performed with a Krebs-Henseleit solution containing 100 μM L-NMMA, LA, D-arginine, or vehicle. Perfusates were collected before and after NE infusion for the measurement of PGE₂, 6-keto-PGF_1α, TxB₂, cAMP, and cGMP. In the L-NMMA group the release of PGE₂ and 6-keto-PGF1α was decreased, and the release of cGMP was prevented. Production of cAMP did not differ between the four groups before NE infusion, and NE increased cAMP release in the L-NMMA group and controls. The results indicate that inhibition of NO synthesis by L-NMMA enhanced the pressor response to NE, possibly mediated by the decreased cGMP and PGI₂ production in resistance vessels.     

NADPH-diaphorase activity in the nociceptive pathways of land snail Megalobulimus abbreviatus: the involvement of pedal ganglia

NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS), widely used to identify nitric oxide (NO) producing cells in the nervous system of both vertebrates and invertebrates. Using NADPH-d histochemistry and semi-quantitative optical densitometry, we characterized the NO-producing neurons in the pedal ganglia of young and adult Megalobulimus abbreviatus, subjected to aversive thermal stimulus. The animals were killed at different times (3, 6, 12 and 24 h) following stimulus. The enzymatic activity was detected in different cellular subsets and neuronal processes. In all the studied pedal ganglia subregions, the optical density of positive neurons (P < 0.05) and neuropilar area 1 (P < 0.01) was significantly different in treated animals when compared to controls. The increase in nitrergic activity induced by nociceptive stimulus suggests the involvement of NO in the nociceptive circuit of M. abbreviatus, which is well maintained throughout evolution, and could be helpful in drawing cellular homologies with other gastropods.     

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS^–/–) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS^–/– cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N^G-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS^–/– cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS^–/– hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development     

Does nitric oxide buffer arterial blood pressure variability in humans?

Animal studies suggest that nitric oxide (NO) plays an important role in buffering short-term arterial pressure variability, but data from humans addressing this hypothesis are scarce. We evaluated the effects of NO synthase (NOS) inhibition on arterial blood pressure (BP) variability in eight healthy subjects in the supine position and during 60 degrees head-up tilt (HUT). Systemic NOS was blocked by intravenous infusion of N(G)-monomethyl-L-arginine (L-NMMA). Electrocardiogram and beat-by-beat BP in the finger (Finapres) were recorded continuously for 6 min, and brachial cuff BP was recorded before and after L-NMMA in each body position. BP and R-R variability and their transfer functions were quantified by power spectral analysis in the low-frequency (LF; 0.05-0.15 Hz) and high-frequency (HF; 0.15-0.35 Hz) ranges. L-NMMA infusion increased supine BP (systolic, 109 +/- 4 vs. 122 +/- 3 mmHg, P = 0.03; diastolic, 68 +/- 2 vs. 78 +/- 3 mmHg, P = 0.002), but it did not affect supine R-R interval or BP variability. Before L-NMMA, HUT decreased HF R-R variability (P = 0.03), decreased transfer function gain (LF, 12 +/- 2 vs. 5 +/- 1 ms/mmHg, P = 0.007; HF, 18 +/- 3 vs. 3 +/- 1 ms/mmHg, P = 0.002), and increased LF BP variability (P < 0.0001). After L-NMMA, HUT resulted in similar changes in BP and R-R variability compared with tilt without L-NMMA. Increased supine BP after L-NMMA with no effect on BP variability during HUT suggests that tonic release of NO is important for systemic vascular tone and thus steady-state arterial pressure, but NO does not buffer dynamic BP oscillations in humans.     

Roles of cytokines and progesterone in the regulation of the nitric oxide generating system in bovine luteal endothelial cells

Nitric oxide (NO) produced by luteal endothelial cells (LECs) plays important roles in regulating corpus luteum (CL) function, yet the local mechanism regulating NO generation in bovine CL remains unclear. The purpose of the present study was to elucidate if tumor necrosis factor‐α (TNF), interferon γ (IFNG), and/or progesterone (P4) play roles in regulating NO generating system in LECs. Cultured bovine LECs obtained from the CL at the mid‐luteal stage (Days 8–12 of the cycle) were treated for 24 hr with TNF (2.9 nM), IFNG (2.5 nM), or P4 (0.032–32 µM). NO production was increased by TNF and IFNG, but decreased by P4 (P < 0.05). TNF and IFNG stimulated the relative steady‐state amounts of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein expression (P < 0.05), whereas P4 inhibited relative steady‐state amounts of iNOS mRNA and iNOS protein expression (P < 0.05). In contrast, endothelial nitric oxide synthase (eNOS) expression was not affected by any treatment. TNF and IFNG stimulated NOS activity (P < 0.05) and 1400W, a specific inhibitor of iNOS, reduced NO production stimulated by TNF and IFNG in LECs (P < 0.05). Onapristone, a specific P4 receptor antagonist, blocked the inhibitory effect of P4 on NO production in LECs (P < 0.05). The overall findings suggest that TNF and IFNG accelerate luteolysis by increasing NO production via stimulation of iNOS expression and NOS activity in bovine LECs. P4, on the other hand, may act in maintaining CL function by suppressing iNOS expression in bovine LECs. Mol. Reprod. Dev. 79: 689–696, 2012. © 2012 Wiley Periodicals, Inc.     

Enhanced concentrations of relevant markers of nitric oxide formation after exercise training in patients with metabolic syndrome

Metabolic syndrome (MetS) denotes a clustering of risk factors that may affect nitric oxide (NO) bioavailability and predispose to cardiovascular diseases, which are delayed by exercise training. However, no previous study has examined how MetS affects markers of NO formation, and whether exercise training increases NO formation in MetS patients. Here, we tested these two hypotheses. We studied 48 sedentary individuals: 20 healthy controls and 28 MetS patients. Eighteen MetS patients were subjected to a 3-month exercise training (E + group), while the remaining 10 MetS patients remained sedentary (E−group). The plasma concentrations of nitrite, cGMP, and ADMA (asymmetrical dimethylarginine; an endogenous nitric oxide synthase inhibitor), and the whole blood nitrite concentrations were determined at baseline and after exercise training using an ozone-based chemiluminescence assay, and commercial enzyme immunoassays. Thiobarbituric acid reactive species (TBA-RS) were measured in the plasma to assess oxidative stress using a fluorometric method. We found that, compared with healthy subjects, patients with MetS have lower concentrations of markers of NO formation, including whole blood nitrite, plasma nitrite, and plasma cGMP, and increased oxidative stress (all P < 0.05). Exercise training increased the concentrations of whole blood nitrite and cGMP, and decreased both oxidative stress and the circulating concentrations of ADMA (both P < 0.05). These findings show clinical evidence for lower endogenous NO formation in patients with MetS, and for improvements in NO formation associated with exercise training in MetS patients.     

Nitric oxide stimulates embryonic somatotroph differentiation and growth hormone mRNA and protein expression through a cyclic guanosine monophosphate-independent mechanism

In the pituitary gland, NO is locally synthesized by gonadotroph and folliculo-stellate cells. Many reports have shown that NO can modulate the growth hormone (GH) secretion. However, its role on mice embryo GH regulation remains unclear. In addition, it is unknown whether the regulation is associated with the proliferation of pituitary cells. In this study, we have investigated the regulatory effects of NO on somatotroph differentiation, proliferation and GH mRNA and protein expression using primary cell cultures of mice fetal pituitaries (embryonic days 16.5, ED 16.5). Our results show that incubation of pituitary cells in the presence of sodium nitroprusside (SNP; 1 mM), a NO donor, for 4.5 h resulted in a significant increase in GH mRNA and protein expression (P < 0.05) and the stimulation of SNP can be inhibited by hemoglobin, a NO scavenger. But the addition of cyclic guanosine monophosphate (cGMP; 3.0 mM), the second messenger of multiple NO actions cannot influence GH mRNA and protein expression. The cyclic nucleotide cellular efflux pumps existed in the pituitary cells can transport the majority of de novo-produced cGMP and effectively block cGMP accumulation. For maintaining intracellular concentration of cGMP, probenecid (0.5 mM), a blocker of cGMP efflux pump, combined with cGMP (3.0 mM) was used to treat the pituitary cells. This also cannot influence GH mRNA and protein expression. In addition, the ratio of GH-positive cells is increased significantly after the stimulation of SNP (P < 0.05). However, SNP cannot modulate the pituitary cell proliferation. From these results we conclude that NO can increase GH mRNA and protein expression in fetal pituitary cells and cGMP is not involved in this hormonal regulation. Stimulation of NO on the somatotroph differentiation does not occur due to pituitary cell proliferation.     

Mechanism Underlying the Protective Effect of Selenium on NO-Induced Oxidative Damage in Bovine Mammary Epithelial Cells

This experiment was conducted to investigate the effects and mechanism of selenium (Se) on antioxidant and immune function of bovine mammary epithelial cells (BMEC) damaged by nitric oxide (NO). The third-generation BMEC was randomly divided into eight treatments with six replicates. The BMEC in the control group was cultured in the medium without Se and diethylenetriamine/NO (DETA/NO) for 30 h. For the DETA/NO group and Se protection group BMEC were exposed to different concentrations of Se (0, 10, 20, 50, 100, 150, and 200 nmol/L) for 24 h, followed by treatment with DETA/NO (1000 μmol/L) for 6 h. Compared with the control group, DETA/NO decreased proliferation rate and activity of thioredoxin reductase (TrxR; P < 0.05). Additionally, DETA/NO decreased the gene expression of both nuclear factor-E2-related factor 2 (Nrf2) and TrxR, as well as the protein expression level of TrxR. However, the activity, and expression levels of inducible nitric oxide synthase (iNOS), as well as the concentration and gene expression level of interleukin-1β (IL-1β) and the concentration of NO significantly increased (P < 0.05). The gene expression levels of indexes related to the mitogen-activated protein kinase (MAPK) signaling pathway showed similar changes. Treatment of BMEC with Se significantly reversed DETA/NO-induced changes in a linear or quadratic dose-dependent manner (P < 0.05), with greatest benefit at 50 nmol/L. These data suggests that Se improves the antioxidant function of BMEC, and protects cells from DETA/NO-induced oxidative damage, primarily by enhancing the activity of TrxR and decreasing the concentration of NO through modulation of Nrf2 and MAPK signaling pathways.

     

“Immunonutrition” Has Failed to Improve Peritonitis-Induced Septic Shock in Rodents

Background

Immunonutrition in sepsis, including n-3 poly-unsaturated fatty acids (PUFAs) or L-arginine supplementation, is a controversial issue that has yielded a great number of studies for the last thirty-five years, and the conclusions regarding the quantity and quality of this support in patients are deceiving. The aim of the present experimental study is to investigate the effects of a pretreatment with enteral nutrition enriched with n-3 PUFAs or L-arginine on vascular dysfunctions, inflammation and oxidative stress during septic shock in rats.

Design

Rats were fed with enteral Peptamen^® HN (HN group), Peptamen^® AF containing n-3 PUFAs (AF group) or Peptamen^® AF enriched with L-arginine (AFA group). On day 4, peritonitis by cecal ligation and puncture (CLP) was performed. Rats were resuscitated (H18) once septic shock was established. After a 4-hour resuscitation, vessels and organs were harvested to assess inflammation, superoxide anion, nitric oxide and prostacyclin levels. Ex-vivo vascular reactivity was also performed.

Results

Compared to CLP-AF or CLP-HN groups, 47.6% of CLP-AFA rats died before the beginning of hemodynamic measurements (vs. 8.0% and 20.0% respectively, p<0.05). AF and AFA rats required significantly increased norepinephrine infusion rates to reach the mean arterial pressure objective, compared to CLP-HN rats. Both CLP-AF and CLP-AFA reduced mesenteric resistance arterial contractility, decreased vascular oxidative stress, but increased NF-κB (0.40±0.15 in CLP-AF and 0.69±0.06 in CLP-AFA vs. 0.09±0.03 in SHAM rats and 0.30±0.06 in CLP-HN, ß-actin ratio, p<0.05) and pIκB expression (0.60±0.03 in CLP-AF and 0.94±0.15 in CLP-AFA vs. 0.04±0.01 in SHAM rats and 0.56±0.07 in CLP-HN, ß-actin ratio, p<0.05), nitric oxide and prostacyclin production in septic rats.

Conclusions

Although n-3 PUFAs or L-arginine supplementation exhibited an antioxidant effect, it worsened the septic shock-induced vascular dysfunction. Furthermore, mortality was higher after L-arginine supplementation.     

Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function

Background

The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.

Methods and Results

Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r² = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.

Conclusions

Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.     

Effects of nitric oxide synthase inhibition on cutaneous vasodilation during body heating in humans

We sought toexamine further the potential role of nitric oxide (NO) in the neurallymediated cutaneous vasodilation in nonacral skin during body heating inhumans. Six subjects were heated with a water-perfused suit whilecutaneous blood flow was measured by using laser-Doppler flowmetersplaced on both forearms. The NO synthase inhibitorN^G-monomethyl-L-arginine(L-NMMA) was given selectivelyto one forearm via a brachial artery catheter after marked cutaneousvasodilation had been established. During body heating, oraltemperature increased by 1.1 ± 0.1°C while heart rate increasedby 30 ± 6 beats/min. Mean arterial pressure stayed constant at 84 ± 2 mmHg. In the experimental forearm, cutaneous vascularconductance (CVC; laser-Doppler) decreased to 86 ± 5% of the peakresponse to heating (P < 0.05 vs.pre-L-NMMA values) afterL-NMMA infusion. In somesubjects, L-NMMA caused CVC tofall by ~30%; in others, it had little impact on the cutaneouscirculation. CVC in the control arm showed a similar increase withheating, then stayed constant whileL-NMMA was given to thecontralateral side. These results demonstrate that NO contributesmodestly, but not consistently, to cutaneous vasodilation during bodyheating in humans. They also indicate that NO is not the only factorresponsible for the dilation.

     

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes

Background aimsObesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3′,5′-cyclic monophosphate (cGMP) as well as adenosine 3′,5′-cyclic monophosphate (cAMP) on preadipocytes in vitro.MethodsPreadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30–150 μm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated.ResultsDETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion.ConclusionsOur results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.     

Nitric oxide-endothelin-1 interaction in humans

Ahlborg, Gunvor, and Jan M. Lundberg. Nitricoxide-endothelin-1 interaction in humans. J. Appl.Physiol. 82(5): 1593-1600, 1997.Healthy menreceived N^G-monomethyl-L-arginine(L-NMMA) intravenously to studycardiovascular and metabolic effects of nitric oxide synthase blockadeand whether this alters the response to endothelin-1 (ET-1) infusion.Controls only received ET-1.L-NMMA effects were that heartrate (17%), cardiac output (17%), and splanchnic and renal blood flow(both 33%) fell promptly (all P < 0.01). Mean arterial blood pressure (6%), and systemic (28%) andpulmonary (40%) vascular resistances increased(P < 0.05 to 0.001). Arterial ET-1levels (21%) increased due to a pulmonary net ET-1 release(P < 0.05 to 0.01). Splanchnic glucose output (SGO) fell (26%, P < 0.01). Arterial insulin and glucagon were unchanged. Subsequent ET-1infusion caused no change in mean arterial pressure, heart rate, orcardiac output, as found in the present controls, or in splanchnic andrenal blood flow or splanchnic glucose output as previously found withET-1 infusion (G. Ahlborg, E. Weitzberg, and J. M. Lundberg.J. Appl. Physiol. 79: 141-145,1995). In conclusion, L-NMMAlike ET-1, induces prolonged cardiovascular effects and suppresses SGO.L-NMMA causes pulmonary ET-1release and blocks responses to ET-1 infusion. The results indicatethat nitric oxide inhibits ET-1 production and thereby interacts withET-1 regarding increase in vascular tone and reduction of SGO inhumans.

     

Acute Kidney Injury in Severe Sepsis and Septic Shock in Patients with and without Diabetes Mellitus: A Multicenter Study

IntroductionWhether diabetes mellitus increases the risk of acute kidney injury (AKI) during sepsis is controversial.ResultsFirst, we compared 451 patients with severe sepsis or septic shock and diabetes to 3,277 controls with severe sepsis or septic shock and without diabetes. Then, we compared 318 cases (with diabetes) to 746 matched controls (without diabetes). Diabetic patients did not have a higher frequency of AKI (hazard ratio [HR], 1.18; P = 0.05]) or RRT (HR, 1.09; P = 0.6). However, at discharge, diabetic patients with severe sepsis or septic shock who experienced acute kidney injury during the ICU stay and were discharged alive more often required RRT (9.5% vs. 4.8%; P = 0.02), had higher serum creatinine values (134 vs. 103 µmoL/L; P<0.001) and had less often recovered a creatinine level less than 1.25 fold the basal creatinine (41.1% vs. 60.5%; P<0.001).ConclusionsIn patients with severe sepsis or septic shock, diabetes is not associated with occurrence of AKI or need for RRT but is an independent risk factor for persistent renal dysfunction in patients who experience AKI during their ICU stay.     

Downstream mechanisms of nitric oxide-mediated skeletal muscle glucose uptake during contraction

There is evidence that nitric oxide (NO) is required for the normal increases in skeletal muscle glucose uptake during contraction, but the mechanisms involved have not been elucidated. We examined whether NO regulates glucose uptake during skeletal muscle contractions via cGMP-dependent or cGMP-independent pathways. Isolated extensor digitorum longus (EDL) muscles from mice were stimulated to contract ex vivo, and potential NO signaling pathways were blocked by the addition of inhibitors to the incubation medium. Contraction increased (P < 0.05) NO synthase (NOS) activity (～40%) and dichlorofluorescein (DCF) fluorescence (a marker of oxidant levels; ～95%), which was prevented with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), and antioxidants [nonspecific antioxidant, N-acetylcysteine (NAC); thiol-reducing agent, DTT], respectively. L-NMMA and NAC both attenuated glucose uptake during contraction by ～50% (P < 0.05), and their effects were not additive. Neither the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, which prevents the formation of cGMP, the cGMP-dependent protein (PKG) inhibitor Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3',5'-cyclic monophosphorothioate sodium salt nor white light, which breaks S-nitrosylated bonds, affects glucose uptake during contraction; however, DTT attenuated (P < 0.05) contraction-stimulated glucose uptake (by 70%). NOS inhibition and antioxidant treatment reduced contraction-stimulated increases in protein S-glutathionylation and tyrosine nitration (P < 0.05), without affecting AMPK or p38 MAPK phosphorylation. In conclusion, we provide evidence to suggest that NOS-derived oxidants regulate skeletal muscle glucose uptake during ex vivo contractions via a cGMP/PKG-, AMPK-, and p38 MAPK-independent pathway. In addition, it appears that NO and ROS may regulate skeletal muscle glucose uptake during contraction through a similar pathway.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

High Glucose-enhanced Acetylcholine Stimulated CGMP Masks Impaired Vascular Reactivity in Tail Arteries from Short-Term Hyperglycemic Rats

Impaired vascular endothelium-dependent relaxation and augmented contractile responses have been reported in several models of long-term hyperglycemia. However, the effects of short-term ambient hyperglycemia are poorly understood. Since oxidative stress has been implicated as a contributor to impaired vascular function, we investigated the following:Aims: (1) the effects of high glucose exposure in vitro (7 – 10 days) on vascular relaxation to acetylcholine (Ach) and contractility to norepinephrine (NE) and KCl; (2) if NO-dependent cGMP generation is affected under these conditions; and (3) aortic redox status.Methods: Non-diabetic rat tail artery rings were incubated in normal (5mM) (control NG) or high (20mM) glucose buffer (control HG). Vascular responses to Ach, NE and KCl were compared to those of streptozotocin (SZ) diabetic animals in the same buffers (diabetic NG, diabetic HG). Ach stimulated cGMP levels were quantitated as an indirect assessment of endothelial nitric oxide (NO) production and oxidative stress evaluated by measuring vascular glutathione and oxidized glutathione.Results: Rings from diabetic rats in NG showed impaired relaxation to Ach (P = 0.002) but relaxed normally, when maintained in HG. Similarly, contractile responses to NE were attenuated in diabetic rings in NG but similar to controls in HG. HG markedly augmented maximal contraction to KCl compared to control and diabetic vessels in NG (P < 0.0001). Diabetic vessels in a hyperosmolar, but normoglycemic, milieu respond like those in HG. in vitro, HG for 2 hours changed neither relaxation nor contractile responses to NE and KCl in control rings. Basal cGMP levels were lower in aortae from diabetic animals pre-incubated in NG than in HG/LG or in control rings in NG (P < 0.05). cGMP responses to Ach were exaggerated in diabetic vessels in HG (P = 0.035 vs. control NG, P = 0.043 vs. diabetic NG) but not different between control and diabetic rings in NG. Vessels from diabetic animals had lower levels of GISH (P < 0.0001) and higher levels of GSSG (P < 0.0001) indicating oxidative stress.Conclusions: Our data indicate that endothelium dependent relaxation is altered early in the diabetic state and that increased NO responses may compensate for augmented oxidative stress but the lack of effect of short-term exposure of normal vessels to HG suggests that short-term hyperglycemia per se does not cause abnormal vascular responses.