Sexual Dimorphism in Development of Kidney Damage in Aging Fischer-344 Rats

BackgroundAging kidneys exhibit slowly developing injury and women are usually protected compared with men, in association with maintained renal nitric oxide.ObjectivesOur purpose was to test 2 hypotheses: (1) that aging intact Fischer-344 (F344) female rats exhibit less glomerular damage than similarly aged males, and (2) that loss of female ovarian hormones would lead to greater structural injury and dysregulation of the nitric oxide synthase (NOS) system in aging F344 rat kidneys.MethodsWe compared renal injury in F344 rats in intact, ovariectomized, and ovariectomized with estrogen replaced young (6 month) and old (24 month) female rats with young and old intact male rats and measured renal protein abundance of NOS isoforms and oxidative stress.ResultsThere was no difference in age-dependent glomerular damage between young or old intact male and female F344 rats, and neither ovariectomy nor estrogen replacement affected renal injury; however, tubulointerstitial injury was greater in old males than in old females. These data suggest that ovarian hormones do not influence these aspects of kidney aging in F344 rats and that the greater tubulointerstitial injury is caused by male sex. Old males had greater kidney cortex NOS3 abundance than females, and NOS1 abundance (alpha and beta isoforms) was increased in old males compared with both young males and old females. NOS abundance was preserved with age in intact females, ovariectomy did not reduce NOS1 or NOS3 protein abundance, and estrogen replacement did not uniformly elevate NOS proteins, suggesting that estrogens are not primary regulators of renal NOS abundance in this strain. Nicotinamide adenine dinucleotide phosphate oxidase-dependent superoxide production and nitrotyrosine immunoreactivity were increased in aging male rat kidneys compared with females, which could compromise renal nitric oxide production and/or bioavailability.ConclusionsThe kidney damage expressed in aging F344 rats is fairly mild and is not related to loss of renal cortex NOS3 or NOS1 alpha.     

Gender differences in myogenic tone in superoxide dismutase knockout mouse: animal model of oxidative stress

Oxidative stress mediated by prooxidants has been implicated in the pathogenesis of vascular disorders. However, the effect of prooxidants on myogenic regulation of vascular function and the differential influence of gender is not known. SOD, an intracellular enzyme, restricts excess prooxidant levels and may limit vascular dysfunction. We therefore tested the effects of Cu,Zn SOD deficiency on vascular tone in both male and female SOD knockout (SOD-/-) mice. We hypothesized that myogenic tone would be enhanced in SOD-/- mice by excess prooxidants compared with wild-type control mice. Indeed, resistance-sized mesenteric arteries from SOD-/- mice exhibited enhanced myogenic tone compared with control mice. Myogenic tone was lower in female than male control mice. Interestingly, this gender effect was absent in SOD-/- mice, such that myogenic tone of mesenteric arteries from females was equated to that of arteries from males. Furthermore, the pathways that modulate myogenic tone were diverse. In both male and female control mice, inhibition of prostaglandin H synthase (PGHS) and nitric oxide synthase (NOS) pathways enhanced myogenic tone. In female SOD-/- mice, inhibition of PGHS and NOS pathways enhanced myogenic tone to a greater extent compared with control mice. Conversely, in male SOD-/- mice, NOS and PGHS inhibition did not alter tone and only inhibition of gap junctions enhanced myogenic tone. In conclusion, this study revealed enhanced myogenic tone in SOD-/- mice compared with control mice. Furthermore, Cu,Zn SOD deficiency particularly enhanced myogenic tone in female mice such that their vascular tone attained the level of male SOD-/- mice, possibly mediated by prooxidants.     

Functional NOS 1 in the rat mesenteric arterial bed

Previously we have demonstrated functional nitric oxide synthase (NOS) 1 in large arteries. Because resistance arteries largely determine blood pressure, this study examined whether functional NOS 1 also exists in resistance arteries. Phenylephrine (PE) contraction was measured in the absence and presence of the NOS 1 inhibitor N(5)-(1-imino-3-butenyl)-L-ornithine (VNIO) in isolated mesenteric resistance arteries (endothelium intact and denuded) from Sprague-Dawley rats. For NOS 1 activity and expression, the mesenteric arterial bed was separated into cytosolic and particulate fractions. NOS activity was assayed by measuring the conversion of [(3)H]arginine to [(3)H]citrulline inhibited by a nonselective NOS inhibitor or VNIO. VNIO increased PE sensitivity in endothelium-intact and -denuded arteries. In cytosolic and particulate fractions of the arterial bed, approximately 40% of NOS activity was inhibited by VNIO. Immunoprecipitation and Western blot analysis revealed two NOS 1 immunoreactive bands. One band corresponded to the rat brain isoform, whereas the second was of a slightly lower molecular mass. The cytosolic fraction contained both isoforms; however, the particulate fraction had only the lower molecular mass form. These studies demonstrate the existence of functional NOS 1 in resistance arteries.     

Pregnancy-induced alterations of vascular function in mouse mesenteric and uterine arteries

Normal pregnancy involves dramatic changes to maternal vascular function, while abnormal vascular adaptations may contribute to pregnancy-associated diseases such as preeclampsia. Many genetic mouse models have recently emerged to study vascular pathologies of pregnancy. However, vascular adaptations to pregnancy in normal mice are not fully understood. Thus, we studied changes in vascular reactivity during normal mouse pregnancy. We hypothesized that pregnant mice will have enhanced endothelial-dependent vasodilation compared with nonpregnant mice, via an enhancement of the nitric oxide synthase (NOS) prostaglandin H synthase (PGHS), and other endothelial-derived hyperpolarizing pathways. Late pregnant (Day 17-18) C57BL/6J mice (n = 10) were compared with nonpregnant mice (n = 7). Uterine and mesenteric arteries were mounted on a wire myograph system and assessed for endothelium-dependent (methacholine) and -independent (sodium nitroprusside; SNP) relaxation responses. Endothelial-dependent relaxation was enhanced in pregnant uterine and mesenteric arteries, which was blunted after the addition of inhibitors of the PGHS or NOS pathways. In nonpregnant mice, these pathways had no effect in modulating relaxation in uterine arteries, whereas vasodilation in mesenteric arteries was reduced only by NOS inhibition. Both uterine and mesenteric vessels had nonnitric oxide- and nonprostaglandin-mediated relaxation, but this relaxation was not enhanced during pregnancy. Endothelial-independent relaxation was also enhanced in pregnant uterine but not mesenteric arteries. Our data indicate that uterine and mesenteric arteries from pregnant mice have enhanced vasodilation. Understanding vascular adaptations to normal mouse pregnancy is crucial for interpreting changes that may occur in genetic mouse models.     

The effect of diabetes and sex on nitric oxide-mediated cardiovascular dynamics

Diabetes is associated with impaired cardiovascular responses that are especially prominent in females. Since nitric oxide (NO)-mediated effects on cardiovascular dynamics are altered in diabetes, we evaluated the effect of L-NAME, a nitric oxide synthase (NOS) antagonist, on mean arterial pressure (MAP), heart rate (HR), and selective vascular flows in both male and female normal and diabetic rats as an index of NO activity. Rats were made diabetic using streptozotocin and maintained for 5-6 weeks. Following anesthesia with urethane/alpha-chloralose, the femoral artery and vein were cannulated for recording and sampling, and flow probes were placed on the iliac, renal, and superior mesenteric arteries. A bolus infusion of L-NAME (10mg/ kg) resulted in a rapid +52% and +68% increase in MAP in normal female and male rats, respectively. However, diabetic females' and males' responses were significantly lower (44% and 45%, respectively) when compared with their normal counterparts. The decreased HR in response to the peak pressor effect of L-NAME was more prominent in normal females compared with normal males (-14% vs 2%). The results in diabetic females and males were equivalent (-6% vs -9%, respectively). L-NAME decreased the conductance (flow/MAP) an average of 65% in all three vascular beds in normal female rats. In diabetic females, the iliac and superior mesenteric responses to L-NAME were less, and the renal conductance was contrastingly increased 23%. The response to L-NAME was comparable (-62%) in the renal and superior mesenteric and less (-40%) in the iliacs of normal versus diabetic males. We concluded that diabetes is associated with a decreased pressor response to NOS inhibition. And the impaired constriction response of the renal vessels noted in female diabetic rats may provide a basis for the increased renal pathology observed in diabetic humans.     

Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.     

Vascular endothelial dysfunction: does tetrahydrobiopterin play a role?

Tetrahydrobiopterin is one of the most potent naturally occurring reducing agents and an essential cofactor required for enzymatic activity of nitric oxide synthase (NOS). The exact role of tetrahydrobiopterin in the control of NOS catalytic activity is not completely understood. Existing evidence suggests that it can act as allosteric and redox cofactors. Suboptimal concentration of tetrahydrobiopterin reduces formation of nitric oxide and favors "uncoupling" of NOS leading to NOS-mediated reduction of oxygen and formation of superoxide anions and hydrogen peroxide. Recent findings suggest that accelerated catabolism of tetrahydrobiopterin in arteries exposed to oxidative stress may contribute to pathogenesis of endothelial dysfunction present in arteries exposed to hypertension, hypercholesterolemia, diabetes, smoking, and ischemia-reperfusion. Beneficial effects of acute and chronic tetrahydrobiopterin supplementation on endothelial function have been reported in experimental animals and humans. Furthermore, it appears that beneficial effects of some antioxidants (e.g., vitamin C) on vascular function could be mediated via increased intracellular concentration of tetrahydrobiopterin. In this review, the potential role of tetrahydrobiopterin in the pathogenesis of vascular endothelial dysfunction and mechanisms underlying beneficial vascular effects of tetrahydrobiopterin will be discussed.     

Microvascular versus macrovascular dysfunction in type 2 diabetes: differences in contractile responses to endothelin-1

Vascular dysfunction characterized by a hyperreactivity to vasoconstrictors and/or impaired vascular relaxation contributes to increased incidence of cardiovascular disease in diabetes. Endothelin (ET)-1, a potent vasoconstrictor, is chronically elevated in diabetes. However, the role of ET-1 in resistance versus larger vessel function in mild diabetes remains unknown. Accordingly, this study investigated vascular function of third-order mesenteric arteries and basilar arteries in control Wistar and Goto-Kakizaki (GK) rats, a model of mild Type 2 diabetes. Six weeks after the onset of diabetes, contractile responses to 0.1-100 nM ET-1 and relaxation responses to 1 nM-10 microM acetylcholine (ACh) in vessels preconstricted (baseline + 60%) with serotonin (5-HT) were assessed by myograph studies in the presence or absence of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (L-NNA). Maximum contractile response to ET-1 was augmented in mesenteric vessels (155 +/- 18% in GK vs. 81 +/- 6% in control; n = 5-7) but not in the basilar artery (134 +/- 29% in GK vs. 107 +/- 17% in control; n = 4 per group). However, vascular relaxation was impaired in the basilar arteries (22 +/- 4% in GK vs. 53 +/- 7% in control; n = 4 per group) but not in mesenteric arteries of GK rats. Inhibition of NOS decreased the relaxation response of basilar arteries to 15 +/- 8% and 42 +/- 5% in GK and control rats, respectively; whereas, in resistance vessels, corresponding values were 56 +/- 7% and 89 +/- 3% (vs. 109 +/- 2% and 112 +/- 3% without NOS blockade), indicating the involvement of different vasorelaxation-promoting pathways in these vascular beds. These findings provide evidence that the ET system is activated even under mild hyperglycemia and that it contributes to the hyperreactivity of resistance vessels, therefore, the ET system may play an important role in elevated blood pressure in Type 2 diabetes.     

Development of progressive aortic vasculopathy in a rat model of aging

Recent studies have established that age is the major risk factor for vascular disease. Numerous aberrant changes occur in vascular structure and function during aging, and animal models are the primary means to determine the underlying mechanisms of age-mediated vascular pathology. The Fischer 344/Brown Norway F1 hybrid (F344xBN) rat thoracic aorta has been shown to display age-related pathology similar to what occurs in humans. This study utilized the F344xBN rat aorta and both morphometric and global gene expression analyses to identify appropriate time points to study vascular aging and to identify molecules associated with the development and progression of vascular pathology. In contrast to some previous studies that indicated age-related abrupt changes, a progressive increase in intimal and medial thickness, as well as smooth muscle cell-containing intimal protrusions, was observed in thoracic aorta. This structural vascular pathology was associated with a progressive, but nonlinear, increase in global differential gene expression. Gene products with altered mRNA and protein expression included inflammation-related molecules: specifically, the adhesion molecules ICAM-1 and VCAM-1 and the bone morphogenic proteins osteopontin and bone sialoprotein-1. Intimal-associated macrophages were found to increase significantly in number with age. Both systemic and tissue markers of oxidant stress, serum 8-isoprostane and 3-nitrotyrosine, respectively, were also found to increase during aging. The results demonstrate that major structural abnormalities and altered gene expression develop after 6 mo and that the progressive pathological development is associated with increased inflammation and oxidant stress.     

Changing standard chow diet promotes vascular NOS dysfunction in Dahl S rats

We hypothesized that vascular nitric oxide synthase (NOS) function and expression is differentially regulated in adult Dahl salt-sensitive rats maintained on Teklad or American Institutes of Nutrition (AIN)-76A standard chow diets from 3 to 16 wk old. At 16 wk old, acetylcholine (ACh)-mediated vasorelaxation and phenylephrine (PE)-mediated vasoconstriction in the presence and absence of NOS inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), was assessed in small-resistance mesenteric arteries and aortas. Rats maintained on either diet throughout the study had similar responses to ACh and PE in the presence or absence of L-NAME in both vascular preparations. We reasoned that changing from one diet to another as adults may induce vascular NOS dysfunction. In the absence of L-NAME, small arteries from Teklad-fed rats switched to AIN-76 diet and vice versa had similar responses to ACh and PE. Small-arterial NOS function was maintained in rats switched to AIN-76A from Teklad diet, whereas NOS function in response to ACh and PE was lost in the small arteries from rats changed to Teklad from AIN-76A diet. This loss of NOS function was echoed by reduced expression of NOS3, as well as phosphorylated NOS3. The change in NOS phenotype in the small arteries was observed without changes in blood pressure. Aortic responses to ACh or PE in the presence or absence of L-NAME were similar in all diet groups. These data indicate that changing standard chow diets leads to small arterial NOS dysfunction and reduced NOS signaling, predisposing Dahl salt-sensitive rats to vascular disease.     

Gain of function mutation in the mineralocorticoid receptor of the Brown Norway rat

The aim of this research was to identify the molecular bases of differences in sensitivity to corticosteroid hormones between Brown Norway and Fischer 344 rats. We previously showed an apparent insensitivity to adrenalectomy in Brown Norway rats. Based on our first hypothesis of a different activity/reactivity of the mineralocorticoid signaling pathway between the two rat strains, we sequenced Brown Norway and Fischer 344 mineralocorticoid receptor cDNA and identified a tyrosine to cysteine substitution (Y73C) in the N-terminal part of the Brown Norway mineralocorticoid receptor. As a first step, this substitution gave us a means to distinguish the Brown Norway allele from the Fischer 344 at the mineralocorticoid receptor locus in an F2 population. We showed a strong genetic linkage between the mineralocorticoid receptor genotype and sensitivity to adrenalectomy. A subsequent genome-wide linkage analysis confirmed the involvement of the mineralocorticoid receptor locus and implicated other loci, including one on chromosome 4, which collectively explain a large part of the strain differences in corticosteroid receptor responses. In vitro studies further revealed that the Y73C substitution induces greater transactivation of the mineralocorticoid receptor by aldosterone, and surprisingly by progesterone as well, which could substitute for aldosterone after adrenalectomy in Brown Norway rats. We challenged this hypothesis in vivo and showed that plasma progesterone is higher in Brown Norway male rats and partially compensates for aldosterone after adrenalectomy. This work illustrates the interest of a pluristrategic approach to explore the mineralocorticoid receptor signaling pathway and its implication in the regulation of hydroelectrolytic homeostasis and blood pressure.     

Vascular aging in the longest-living rodent, the naked mole rat

The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known [maximum lifespan potential (MLSP): >28 yr] and is a unique model of successful aging showing attenuated declines in most physiological function. This study addresses age-related changes in endothelial function and production of reactive oxygen species in NMR arteries and vessels of shorter-living Fischer 344 rats (MLSP: approximately 3 yr). Rats exhibit a significant age-dependent decline in acetylcholine-induced responses in carotid arteries over a 2-yr age range. In contrast, over a 10-yr age range nitric oxide (NO)-mediated relaxation responses to acetylcholine and to the NO donor S-nitrosopencillamine (SNAP) were unaltered in NMRs. Cellular superoxide anion (O(2)(*-)) and H(2)O(2) production significantly increased with age in rat arteries, whereas they did not change substantially with age in NMR vessels. Indicators of apoptotic cell death (DNA fragmentation rate, caspase 3/7 activity) were significantly enhanced ( approximately 250-300%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a approximately 50% increase in apoptotic cell death. In the hearts of NMRs (2 to 26 yr old), expression of endothelial NO synthase, antioxidant enzymes (Cu,Zn-SOD, Mn-SOD, catalase, and glutathione peroxidase), the NAD(P)H oxidase subunit gp91(phox), and mitochondrial proteins (COX-IV, ATP synthase, and porin, an indicator of mitochondrial mass) did not change significantly with age. Thus long-living NMRs can maintain a youthful vascular function and cellular oxidant-antioxidant phenotype relatively longer and are better protected against aging-induced oxidative stress than shorter-living rats.     

Changes of renal function and structure in rats exposed to cold

The kidney appears to play a crucial role in both initiating and maintaining the high blood pressure in cold-induced hypertension (CIH). The aim of the present study was to evaluate the changes of renal function and structure in rats exposed to cold for 2, 4 and 6 weeks. Systolic blood pressure increased significantly after 2 weeks of cold exposure and was maintained throughout the whole experiment. Upregulation of angiotensin type 1 receptor (AT₁R) expression was seen in the vascular zone and distal tubule after 4 and 6 weeks of cold exposure. This was accompanied by an increase in malondialdehyde (MDA) levels and decreases in superoxide dismutase (SOD), nitric oxide synthase (NOS) activities and nitric oxide (NO) content in kidney. Structural changes were also observed in glomeruli, tubules and arteries in cold-treated rats. These results suggest that upregulation of kidney AT₁R plays a critical role in the development of CIH, and its interaction with oxidative stress, NO and NOS may be involved in changes of renal function and structure.     

Endothelial and Neuronal Nitric Oxide Activate Distinct Pathways on Sympathetic Neurotransmission in Rat Tail and Mesenteric Arteries

Nitric oxide (NO) seems to contribute to vascular homeostasis regulating neurotransmission. This work aimed at assessing the influence of NO from different sources and respective intracellular pathways on sympathetic neurotransmission, in two vascular beds. Electrically-evoked [³H]-noradrenaline release was assessed in rat mesenteric and tail arteries in the presence of NO donors or endothelial/neuronal nitric oxide synthase (NOS) inhibitors. The influence of NO on adenosine-mediated effects was also studied using selective antagonists for adenosine receptors subtypes. Location of neuronal NOS (nNOS) was investigated by immunohistochemistry (with specific antibodies for nNOS and for Schwann cells) and Confocal Microscopy. Results indicated that: 1) in mesenteric arteries, noradrenaline release was reduced by NO donors and it was increased by nNOS inhibitors; the effect of NO donors was only abolished by the adenosine A₁ receptors antagonist; 2) in tail arteries, noradrenaline release was increased by NO donors and it was reduced by eNOS inhibitors; adenosine receptors antagonists were devoid of effect; 3) confocal microscopy showed nNOS staining in adventitial cells, some co-localized with Schwann cells. nNOS staining and its co-localization with Schwann cells were significantly lower in tail compared to mesenteric arteries. In conclusion, in mesenteric arteries, nNOS, mainly located in Schwann cells, seems to be the main source of NO influencing perivascular sympathetic neurotransmission with an inhibitory effect, mediated by adenosine A₁ receptors activation. Instead, in tail arteries endothelial NO seems to play a more relevant role and has a facilitatory effect, independent of adenosine receptors activation.     

Flow-mediated vasodilation is impaired in adult rat offspring exposed to prenatal hypoxia

There is now a demonstrated association between low birth weight and increased mortality later in life. Changes in fetal development may program the cardiovascular system and lead to an increased risk of cardiovascular diseases later in life. In addition, aging is a risk factor for vascular endothelial-dependent dysfunction. However, the impact of being born intrauterine growth restricted (IUGR) on the normal aging mechanisms of vascular dysfunction is not clear. We hypothesized that IUGR would cause changes in vascular function that would affect the mechanisms of flow-induced vasodilation later in life in an age- or sex-dependent manner. To create an IUGR model, pregnant Sprague-Dawley rats were placed in a hypoxic (11.5% O?) or control (room air, 21% O?) environment from days 15 to 21 of pregnancy. Both male and female offspring were investigated at 4 or 12 mo of age. Vascular function was assessed in small mesenteric arteries using flow-induced vasodilation, a physiological stimuli of vasodilation, in a pressure myograph. Flow-induced vasodilation was unaffected at a young age, but was significantly reduced in aging IUGR compared with aging controls (P < 0.05). Underlying vasodilator mechanisms were altered such that nitric oxide-mediated vasodilation was abolished in both young adult and aging IUGR males and females and in aging control females (P > 0.05). Endothelium-derived hyperpolarizing factor-mediated vasodilation was maintained in all groups (P < 0.01). A change in the mechanisms of vasodilation occurring at an earlier age in IUGR offspring may predispose them to develop cardiovascular diseases as an aging adult.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Oxidative Stress-Dependent Coronary Endothelial Dysfunction in Obese Mice

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A₂ synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.     

Increased myogenic tone in 7-month-old adult male but not female offspring from rat dams exposed to hypoxia during pregnancy

Intrauterine growth restriction (IUGR) increases the risk of cardiovascular disease later in life. Vascular dysfunction occurs in adult offspring from animal models of IUGR including maternal undernutrition, but the influence of reduced fetal oxygen supply on adult vascular function is unclear. Myogenic responses, essential for vascular tone regulation, have not been evaluated in these offspring. We hypothesized that 7-mo-old offspring from hypoxic (12% O(2); H) or nutrient-restricted (40% of control; NR) rat dams would show greater myogenic responses than their 4-mo-old littermates or control (C) offspring through impaired modulation by vasodilators. Growth restriction occurred in male H (P < 0.01), male NR (P < 0.01), and female NR (P < 0.02), but not female H, offspring. Myogenic responses in mesenteric arteries from males but not females were increased at 7 mo in H (P < 0.01) and NR (P < 0.05) vs. C offspring. There was less modulation of myogenic responses after inhibition of nitric oxide synthase (P < 0.05), prostaglandin H synthase (P < 0.005), or both enzymes (P < 0.001) in arteries from 7-mo male H vs. C offspring. Thus reduced vasodilator modulation may explain elevated myogenic responses in 7-mo male H offspring. In contrast, there was increased modulation of myogenic responses in arteries from 7-mo female H vs. C or NR offspring after inhibition of both enzymes (P < 0.05). Thus increased vasodilator modulation may maintain myogenic responses in female H offspring at control levels. In summary, vascular responses in adult offspring from adverse intrauterine environments are impaired in a gender-specific, age-dependent, and maternal insult-dependent manner, with males more profoundly affected.     

Modulation of cerebral vascular tone by activated glia: involvement of nitric oxide

The ability of activated glia to affect cerebral vascular tone has been evaluated using an in vitro experimental system in which basilar arteries were incubated with glial cultures activated by treatment with lipopolysaccharide (LPS). Vascular tone was measured with an isometric myograph. Contraction in response to high KCl and serotonin was reduced in arteries co-incubated for 24 h with LPS-activated glia, whereas the response to acetylcholine was not modified. The reduced contraction was prevented when the nitric oxide synthase (NOS) inhibitor L-N-nitro-arginine (L-NNA) was added throughout the whole incubation time (activation of glial cells with LPS + co-incubation of glial cells with cerebral arteries). Under these conditions, nitrite levels were drastically reduced. A reduced contraction to KCl was also observed after treatment of the cerebral vessel with sodium nitroprusside. In contrast, L-NNA added to the vessel did not modify the response to contracting stimuli and the expression of endothelial NOS was not modified in cerebral arteries pre-incubated with activated glia. These results suggest that activated glia, which finds an in vivo correlate in several neuropathological conditions, can contribute to changes of vascular tone by modifying the levels of nitric oxide (NO) to which the vessel is exposed.     

Prenatal lipopolysaccharide exposure causes mesenteric vascular dysfunction through the nitric oxide and cyclic guanosine monophosphate pathway in offspring

Cardiovascular diseases, such as hypertension, could be programmed in fetal life. Prenatal lipopolysaccharide (LPS) exposure in utero results in increased blood pressure in offspring, but the vascular mechanisms involved are unclear. Pregnant Sprague–Dawley rats were intraperitoneally injected with LPS (0.79 mg/kg) or saline (0.5 ml) on gestation days 8, 10, and 12. The offspring of LPS-treated dams had higher blood pressure and decreased acetylcholine (ACh)-induced relaxation and increased phenylephrine (PE)-induced contraction in endothelium-intact mesenteric arteries. Endothelium removal significantly enhanced the PE-induced contraction in offspring of control but not LPS-treated dams. The arteries pretreated with l-NAME to inhibit nitric oxide synthase (eNOS) in the endothelium or ODQ to inhibit cGMP production in the vascular smooth muscle had attenuated ACh-induced relaxation but augmented PE-induced contraction to a larger extent in arteries from offspring of control than those from LPS-treated dams. In addition, the endothelium-independent relaxation caused by sodium nitroprusside was also decreased in arteries from offspring of LPS-treated dams. The functional results were accompanied by a reduction in the expressions of eNOS and soluble guanylate cyclase (sGC) and production of NO and cGMP in arteries from offspring of LPS-treated dams. Furthermore, LPS-treated dam’s offspring arteries had increased oxidative stress and decreased antioxidant capacity. Three-week treatment with TEMPOL, a reactive oxygen species (ROS) scavenger, normalized the alterations in the levels of ROS, eNOS, and sGC, as well as in the production of NO and cGMP and vascular function in the arteries of the offspring of LPS-treated dams. In conclusion, prenatal LPS exposure programs vascular dysfunction of mesenteric arteries through increased oxidative stress and impaired NO–cGMP signaling pathway.