NO Blockade Research Articles

23: Chronic nitric oxide inhibition in a rat model of preeclampsia (PE) leads to urinary proteomic biomarker signatures similar to humans

Urine proteomic profiling identified specific non-random N-terminus cleavage fragments of albumin as biomarkers characteristic of severe PE. Chronic NO inhibition in rats produces a PE-like syndrome (hypertension, proteinuria). We investigated whether NO blockade induces changes in the rat urine proteomic profile comparable to that observed in women with PE.

Flow-Induced Remodeling in Resistance Arteries From Obese Zucker Rats Is Associated With Endothelial Dysfunction

Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries. Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346+/-9 to 412+/-11 mum at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and l-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome.

Rosuvastatin improves basal nitric oxide activity of the renal vasculature in patients with hypercholesterolemia

Objective Impaired endothelium-dependent vasodilation represents an early manifestation of atherosclerosis. Prospective studies have demonstrated that impaired endothelial function in the peripheral circulation of hypercholesterolemic patients predicts CV events and can be restored by statin treatment. Whether this also holds true in the renal circulation has not yet been adequately addressed. Methods In a double-blind, randomized, placebo-controlled cross-over trial, 40 hypercholesterolemic patients were randomly assigned to receive rosuvastatin (10 mg/day) and matching placebo. The primary objective of the study was to assess the effect of 6-week treatment with rosuvastatin on basal NOS activity of the renal vasculature, as assessed by measuring renal plasma flow (RPF) both before and after blockade of NOS with systemic infusion of N G-monomethyl- l-arginine ( l-NMMA). In a subgroup of 20 patients we also studied the effects of a 3-day treatment regimen. Results Compared to placebo treatment, rosuvastatin decreased LDL-cholesterol levels both after 3 days and 6 weeks of treatment. The decrease in RPF in response to l-NMMA was significantly more pronounced after 6-week therapy with rosuvastatin compared to placebo (−13.7 ± 1.0% versus −11.3 ± 0.7%; p = 0.046), indicating increased basal NOS activity with rosuvastatin treatment. A trend towards improved basal NOS activity was already evident after 3-day treatment. Conclusion Treatment with rosuvastatin improved basal NOS activity in the renal circulation of hypercholesterolemic patients, suggestive of a nephroprotective effect. In view of the close relation between altered renal function and cardiovascular events, these nephroprotective effects may contribute to the improved CV prognosis associated with statin treatment.

Mechanisms underlying β2‐adrenoceptor‐mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l-arginine uptake, NOS activity (from l-arginine to l-citrulline conversion), membrane potential (using [(3)H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with betaAR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased l-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of l-arginine uptake by l-lysine inhibited NOS activity and, conversely, blockade of NOS using N(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by l-NAME, suggesting that the increase in l-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l-arginine uptake resulting from NO-mediated membrane hyperpolarization.

Effects of Aging and Hypertension on the Microcirculation

Alterations of structure and function of the microcirculation in hypertension in the elderly and changes with normotensive aging have not been fully clarified. We studied capillary pressure, density, and skin microvascular function in 46 subjects in 3 groups: elderly subjects (aged >60 years) with untreated hypertension (n=16), elderly normotensive subjects (n=16), and young normotensive subjects (age <45 years, n=14). In a subgroup of 19 subjects, we also studied resistance artery function in the isometric myograph. Capillary pressure was higher in both elderly groups (elderly hypertensives: 18.6±4.7 mm Hg, elderly normotensives: 17.6±4.0 mm Hg) compared with young normotensives (13.9±2.6 mm Hg, P <0.05), but capillary density did not differ between the groups. Skin vasodilating responses to acetylcholine were greater in young normotensives compared with both elderly groups ( P <0.05). In isolated resistance arteries, there was a greater inhibitory effect from blockade of the l -arginine-NO pathway in elderly normotensives ( P <0.05) and a reduction in the maximal inhibitory effect of combined blockade of NO, prostanoids, and endothelium-derived hyperpolarizing factor in elderly hypertensives ( P <0.05). This study has demonstrated a significant effect of aging but no additional effect of hypertension on capillary pressure and no effect of either on capillary density. Our findings with both in vivo and in vitro methods suggest that normotensive aging may depend on relative preservation of NO-dependent vasodilatation in resistance arteries at the expense of a rise in capillary pressure.

Inhibition of the neuronal nitric oxide synthase (nNOS) reduces cardiovascular responses elicited by acetylcholine (Ach) microinjection within the Nucleus of the Solitary Tract (NTS) of conscious rats

Ach seems to participate in baroreflex transmission modulating L-glutamate release in the NTS. Centrally released nitric oxide (NO) is involved in cardiovascular transmission within the NTS. We investigated whether inhibition of nNOS could affect cardiovascular responses elicited by Ach into the NTS of conscious Male Wistar rats. Five days before the experiments rats were anesthetized with Ketamin (50mg/kg) and Xylazin 2% and guide cannulae aiming at the NTS were implanted bilaterally. One day before the experiments, femoral artery was cannulated to record mean arterial pressure (MAP, mmHg) and heart rate (HR, bpm). Control unilateral microinjection of Ach (500pmol/100nl) elicited hypotension (Δ MAP: −50±6 mmHg) and bradycardia (Δ HR: −100±26 bpm). Ach responses were reduced 10 and 20 min after NOS blockade into the NTS (L-NAME, 10 nmol/100 nl):Δ MAP 10 min: −9±4 mmHg, and Δ MAP 20 min: − 6±4mmHg; Δ HR 10 min: −20±10 bpm, and Δ HR 20 min: −24± 9 bpm; p< .05; n=8. NTS microinjection of the selective nNOS inhibitor (TRIM 10 nmol/60 nl) also reduced Ach elicited hypotension (Δ MAP control: −42±4 mmHg vs after TRIM: Δ MAP 10 min: −26±7 mmHg; p< .05; n=7) and bradycardia (ΔHR control: −104±17 bpm vs after TRIM: Δ HR 10 min: −73±19 bpm). L-NAME and TRIM did not alter baseline MAP and HR, all responses were back to control 30–45 min after treatment. Ach responses were not affected after D-NAME or saline. We suggest that NO may modulate baroreflex transmission within the NTS through Ach release. Supported by Fapesp, CNPq-PIBIC, CNPq/PRONEX. ± Δ

Heterogeneous effects of chronic myocardial infarction on skeletal muscle arteriole functional dilation

Chronic myocardial infarction (MI) is known to diminish arteriolar responses to nitric oxide (NO)-dependent vasodilators. We investigated whether 1) MI leads to impaired arteriolar responses to muscle contraction, 2) MI affects different branching orders differently, and 3) NO is responsible for MI-related impairments. MI was induced by coronary artery ligation in female rats (n = 22; MI = 13, SHAM = 9). Intravital microscopy was used to measure the diameter and velocity responses of feed and arcade arterioles in the spinotrapezius muscle to electrically stimulated contraction before and after NOS blockade with L-NAME (30 mg/kg IV). Resting diameter and calculated flow through the feed arterioles was the same for the MI and SHAM groups. Post-contraction flow through the feed arterioles was diminished in the MI group compared to the SHAM group (p = 0.046), which was caused by a trend toward smaller post-contraction diameters in these arterioles. L-NAME had no effect on feed arterioles. In the arcade arterioles, resting and post-contraction diameter and flow were similar between MI and SHAM groups. L-NAME tended to reduce resting diameter (p = 0.054), and significantly reduced post-contraction diameter in arcade arterioles (p = 0.033). These results demonstrate that feed arterioles are a primary site of MI-induced derangements of microvascular function, while NO primarily affects the arcade arterioles. Support NHLBI HL18292, AHA-VA-0315362U, Jeffress Trust J-710

Effects of Aging and Hypertension on the Microcirculation

Alterations of structure and function of the microcirculation in hypertension in the elderly and changes with normotensive aging have not been fully clarified. We studied capillary pressure, density, and skin microvascular function in 46 subjects in 3 groups: elderly subjects (aged >60 years) with untreated hypertension (n=16), elderly normotensive subjects (n=16), and young normotensive subjects (age <45 years, n=14). In a subgroup of 19 subjects, we also studied resistance artery function in the isometric myograph. Capillary pressure was higher in both elderly groups (elderly hypertensives: 18.6+/-4.7 mm Hg, elderly normotensives: 17.6+/-4.0 mm Hg) compared with young normotensives (13.9+/-2.6 mm Hg, P<0.05), but capillary density did not differ between the groups. Skin vasodilating responses to acetylcholine were greater in young normotensives compared with both elderly groups (P<0.05). In isolated resistance arteries, there was a greater inhibitory effect from blockade of the l-arginine-NO pathway in elderly normotensives (P<0.05) and a reduction in the maximal inhibitory effect of combined blockade of NO, prostanoids, and endothelium-derived hyperpolarizing factor in elderly hypertensives (P<0.05). This study has demonstrated a significant effect of aging but no additional effect of hypertension on capillary pressure and no effect of either on capillary density. Our findings with both in vivo and in vitro methods suggest that normotensive aging may depend on relative preservation of NO-dependent vasodilatation in resistance arteries at the expense of a rise in capillary pressure.

Role of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) in the regulation of blood pressure by leptin in lean and obese rats

We investigated the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in hemodynamic action of leptin. The effect of leptin (1 mg/kg i.p.) on systolic blood pressure (SBP) was examined in lean rats and in rats made obese by feeding highly palatable diet for either 1 or 3 months. Separate groups received NO synthase inhibitor, l-NAME, or EDHF inhibitors, the mixture of apamin + charybdotoxin or sulfaphenazole, before leptin administration. Leptin increased NO production, as evidenced by increase in plasma and urinary NO metabolites and cyclic GMP. This effect was impaired in both obese groups. In lean rats either leptin or EDHF inhibitors had no effect on blood pressure. l-NAME increased blood pressure in lean animals and this effect was prevented by leptin. However, when leptin was administered to animals pretreated with both l-NAME and EDHF inhibitors, blood pressure increased even more than after l-NAME alone. In the 1-month obese group leptin had no effect on SBP, however, pressor effect of leptin was observed in animals pretreated with EDHF inhibitors. In the 3-month obese group leptin alone increased SBP, and EDHF inhibitors did not augment its pressor effect. The results suggest that leptin may stimulate EDHF when NO becomes deficient, e.g. after NOS blockade or in short-term obesity. Although the effect of leptin on NO production is impaired in the 1-month obese group, BP does not increase, probably because EDHF compensates for NO deficiency. In contrast, leptin increases BP in 3-month obesity because its effect on EDHF is also attenuated.

Mechanisms involved in the swelling of erythrocytes caused by Pacific and Caribbean ciguatoxins

The mechanisms underlying the swelling of frog red blood cells (RBC), induced by Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins (CTXs), were investigated by measuring the length, width and surface of their elliptic shape. P-CTX-1 (0.5 to 5 nM) and C-CTX-1 (1 nM) induced RBC swelling within 60 min. The CTXs-induced RBC swelling was blocked by apamin (1 μM) and by Sr 2+ (1 mM). P-CTX-1-induced RBC swelling was prevented and inhibited by H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (27 μM), an inhibitor of soluble guanylate cyclase (sGC), and NOS blockade by NG methyl- l-arginine ( l-NMA; 10 μM). Cytochalasin D (cytD, 10 μM) increased RBC surface and mimicked CTX effect but did not prevent the P-CTX-1-induced l-NMA-sensitive extra increase. Calculations revealed that P-CTX-1 and cytD increase RBC total surface envelop and volume. These data strongly suggest that the molecular mechanisms underlying CTXs-induced RBC swelling involve the NO pathway by an activation of the inducible NOS, leading to sGC activation which modulates intracellular cGMP and regulates L-type Ca 2+ channels. The resulting increase in intracellular Ca 2+ content, in turn, disrupts the actin cytoskeleton, which causes a water influx and triggers a Ca 2+-activated K + current through SK2 isoform channels.

The link between the insecticide heptachlor epoxide, estradiol, and breast cancer

Given the suspected effects of estrogens on breast cancer, xenoestrogenic insecticides may be a risk factor. Studies of the weak xenoestrogen, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), have failed to demonstrate a causal relationship, though another estrogenic organochlorine insecticide, dieldrin, belonging to the cyclodiene family, has recently been linked to breast cancer. Other cyclodienes such as heptachlor epoxide (HE) and oxychlordane (OC) present in breast tissue have not been evaluated as rigorously, presumably due to their lower concentration and lower recovery using solvent extraction procedures. We used sparging extraction coupled with gas chromatography to determine the levels of HE, OC, and DDE in adipose tissue within breast biopsies in a series of 34 women evaluated for breast abnormality. Of the three insecticides tested, only HE (p=0.007) was positively associated with prevalence of breast cancer in the biopsies. In rapid, non-genomic studies using isolated human leukocytes, flow cytometric methods were used to measure HE-induced oxidants and DNA damage. These studies indicated that HE, at concentrations similar to those in breast biopsies, induced an inverted-U increase in intracellular oxidants and DNA strand breaks [both blocked by specific nitric oxide- (NO-) synthesis blockade withL: -NMMA] in human polymorphonuclear leukocytes (PMNs). HE-treated PMNs also induced damage to surrounding lymphocytes in mixed-leukocyte incubations (also inhibited by NO blockade). The HE-induced changes in NO were inhibited by 17beta-estradiol-(17beta-E2) receptor antagonists and were mimicked by similar concentrations of 17beta-E2. The addition of tumor necrosis factor-alpha (TNF-alpha) increased intracellular oxidants and DNA damage and shifted the responses to lower HE concentrations. This study, along with others, suggests that HE-induced NO production may contribute to initiation, promotion, and progression of cancer.

Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development

During early embryonic (E12) development almost all dorsal root ganglion (DRG) neurons express the neuronal isoform of nitric oxide synthase (nNOS). At this stage, the axons of these neurons are rudimentary and have not made contact with peripheral tissue targets. As their axons establish contact with peripheral targets such as the skin, the number of neurons expressing nNOS decrease that correspond to increased immunoreactivity for nerve growth factor (NGF) in the skin, and its high affinity receptor, tyrosine kinase A ( trkA) in both skin and DRG neurons. During late postnatal development, very few DRG neurons express nNOS; however, axotomy or NGF deprivation of cultured DRG neurons induce nNOS and NOS blockade causes neuronal death. In contrast, NGF-deprived embryonic and neonatal DRG neurons die by apoptosis, while NOS blockade has no effect. Overall, these observations suggest that NGF and nitric oxide (NO) interact during embryonic and postnatal development to facilitate neuronal selection and survival. The roles of NO, NGF and its receptor trkA in DRG neurons during different stages of development are discussed.

Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway via neuron-glial communication in the peripheral nervous system.

The NO-cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)-deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co-culture of neonatal DRG neurons with either Schwann cells pre-treated with an NO donor or a membrane-permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO-sGC-cGMP-mediated NGF and NT3 synthesis by Schwann cells protect neurons.

Renal cortex remodeling in nitric oxide deficient rats treated with enalapril.

The kidney NO synthase is one of the most important renal controlling systems. This paper aims the quantification of renal cortical components involved in blood pressure regulation under NOs blockade. Spontaneous hypertensive rats (SHRs) are submitted to chronic blockade of NOs by L-nitro-arginine-methyl-ester (L-NAME) and an ACE inhibitor (enalapril) in comparison with the normotensive Wistar rats. Twenty SHRs and 5 Wistar rats were divided in 5 groups and observed for 21 days for blood pressure (BP) and serum creatinine: control Wistar (5) (C-W), control SHR (5) (C-SHR), L-SHR (5)--received L-NAME 30 mg/kg/day, L+E-SHR (5)--received L-NAME and Enalapril maleate 15 mg/kg/day, E-SHR (5)--received Enalapril maleate. A quantitative morphometric study (glomerular density, QA[gl], interstitium volume density, Vv[i], tubular surface and length densities, Sv[t] and Lv[t]) were performed at the end. The BP reached 226+/-15 mmHg in L-SHR group. The BP difference between the L-SHR and the C-SHR groups was significant from the first week while the E-SHR group became significant from the second week. At the end of the experiment the BP of the E-SHR group was similar to the BP in the C-W group. The QA[gl] was similar among C-SHR, L-SHR and L+E-SHR groups and no difference was found between E-SHR and C-W groups. In the L-SHRs serum creatinine was greatly increased, and microscopy showed thickening of arteriolar tunica media with an increase of the wall-to-lumen ratio, perivascular fibrosis, inflammatory infiltrated, tubular atrophy and interstitial fibrosis with focal segmental glomerulosclerosis. The use of enalapril was not completely efficient in reducing BP and morphological injury when the hypertension of SHRs was increased with the NOs blockade suggesting that NO deficiency-induced hypertension is not entirely mediated by the RAAS.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice.

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Metabolic control of muscle blood flow during exercise in humans.

During muscle contraction, several mechanisms regulate blood flow to ensure a close coupling between muscle oxygen delivery and metabolic demand. No single factor has been identified to constitute the primary metabolic regulator, yet there are signal transduction pathways between skeletal muscle and the vasculature that induce vasodilation. A link between muscle metabolic events and microvascular control of blood flow is illustrated by local dilation of terminal arterioles during contraction of muscle fibers and conduction of vasodilation upstream. Endothelial-derived vasodilator mechanisms are known to exert control of muscle vasodilation. Adenosine, nitric oxide (NO), prostacyclin (PGI2), and endothelial-derived hyperpolarization factor (EDHF) are possible mediators of muscle vasodilation during exercise. In humans, adenosine has been shown to contribute to functional hyperemia as blood flow is reduced under nonselective adenosine-receptor blockade. No clear role has been demonstrated for either NO or PGI2(2), based on studies employing selective inhibition of these substances individually, suggesting a redundancy of vasodilator mechanisms. This is supported by recent work demonstrating that combined blockade of NOS and PGI2, and NOS and cytochrome P450, both attenuate exercise-induced hyperemia in humans. Combined vasodilator blockade studies offer the potential to uncover important interactions and compensatory vasodilator responses. The signaling pathways that link metabolic events evoked by muscle contraction to vasodilatory signals in the local vascular bed remains an important area of study.

Beneficial effect of enalapril in spontaneously hypertensive rats cardiac remodeling with nitric oxide synthesis blockade.

To study the efficiency of an angiotensin converting enzyme inhibitor on the blood pressure (BP) and the myocardium remodeling when spontaneously hypertensive rats (SHRs) are submitted to nitric oxide synthesis (NOs) blockade (with L-NAME) and simultaneously treated. Young adult male SHRs were separated in four groups (n = 5) and treated for 20 days: Control, L-NAME, L-NAME+Enalapril, and Enalapril. The alterations of the BP, heart mass/body mass ratio and stereological parameters for myocytes, connective tissue and intramyocardial vessels were studied among the groups. The SHRs with NOs blockade showed a great modification of the myocardium with extensive areas of reparative and interstitial fibrosis and accentuated hypertrophy of the cardiac myocytes (cross sectional area 60% higher in animals taking L-NAME than in Control SHRs). Comparing the SHRs with NO deficiency (L-NAME group), the Control SHRs and the Enalapril treated SHRs significant differences were found in the BP and in all stereological parameters. The NO deficiency caused an important BP increment in SHRs that was partially attenuated by Enalapril. This Enalapril effect was more pronounced in Control SHRs. A significant increment of the intramyocardial vessels was observed in NO deficient SHRs and Control SHRs treated with Enalapril demonstrated by the stereology (greater microvascular densities in treated SHRs). Enalapril administration showed a beneficial effect on vascular remodeling and myocardial hypertrophy in SHRs. In SHRs with NO blockade, however, the beneficial effect of Enalapril occurred only in vascular remodeling.

Thromboxane A2 in vasomotor effects of phenylephrine, acetylcholine, and bradykinin in rat mesenteric bed.

In vessels, pharmacological agents displace the balance between relaxing and contracting factors. A cross-talk between those factors has been shown in some vascular beds. To examine whether NO may regulate the vascular tone by modulating prostanoid synthesis or release, we analyzed the response of resistance rat mesenteric arterial bed to vasoactive agents. Phenylephrine, bradykinin (BK), and acetylcholine (ACH) were administered in the absence or in the presence of either NO synthesis inhibition (N(G)-nitro-L-arginine methyl ester [L-NAME]), cyclo-oxygenase inhibition (indomethacin), and/or a thromboxane A2 (TXA2) receptor antagonist (S18886), or a combination thereof. In the presence of L-NAME, the response to phenylephrine was markedly increased. Indomethacin limited these changes, which are attributed to TXA2 release since they were abolished by S18886 and a marked increase in TXB2 release (stable metabolite of TXA2) was found during phenylephrine infusion under NO blockade. Similarly, BK response under NO blockade was markedly attenuated with an improved response with indomethacin and a restoration of vasorelaxation with S18886. In contrast, indomethacin decreased further the response to ACH during L-NAME treatment, and TXA2 inhibition had no effect. Thus, in pathophysiological conditions where an endothelial dysfunction is present, TXA2 stimulation induced by NO release impairment may contribute to an altered response to phenylephrine or BK.

Prior exercise training produces NO-dependent increases in collateral blood flow after acute arterial occlusion

We previously reported that prior training improves collateral blood flow (BF) to the calf muscles after acute-onset occlusion of the femoral artery (Yang HT et al. Am J Physiol Heart Circ Physiol 279: H1890-H1897, 2000). The purpose of this study was to test the hypothesis that increased release of nitric oxide (NO) by NO synthase (likely endothelial NOS) contributes to the increased BF to calf muscles of trained rats after acute femoral artery occlusion. Adult male Sprague-Dawley rats (~325 g) were limited to cage activity and were sedentary (SED; n = 28) or exercise trained (TR; n = 30) for 6 wk by treadmill running. On the day of the investigation, rats were anesthetized with ketamine-acepromazine and instrumented for determination of BF (using (141)Ce- and (85)Sr-labeled microspheres) and distal limb arterial pressure, and femoral arteries were occluded bilaterally. Four hours after surgery, collateral BF was determined twice during treadmill running: first at a demanding speed (20 m/min, 15% grade) and second, after a brief rest and at a faster running speed (25 m/min, 15% grade). The fact that BF did not increase further at the higher running speed indicated that maximal collateral BF was measured. Approximately half of the rats in each group received 20 mg/kg body wt N(G)-nitro-L-arginine methyl ester (L-NAME) intra-arterially 30 min before treadmill exercise and BF measurement to block production of NO by NOS. Results indicate that prior training improved collateral-dependent BF to the skeletal muscle of rats after acute femoral artery occlusion due primarily to an increase in the conductance of the upstream collateral circuit. Blockade of NOS with L-NAME produced decreased vascular conductance, both in the upstream collateral circuit and in the distal skeletal muscle microcirculation, and the difference between collateral vascular conductance in TR and SED rats was abolished. Our results indicate that the primary determinant of the increased collateral BF with prior training is the resistance of the upstream collateral circuit and imply that enhanced endothelium-mediated dilation induced by training serves to increase collateral BF following acute arterial occlusion.

Myocardial contractile function and heart rate in mice with myocyte-specific overexpression of endothelial nitric oxide synthase.

The major source of nitric oxide (NO) in the heart is the constitutive form of NO synthases (eNOS, NOS III) that is expressed in vascular endothelium and cardiac myocytes. NO mediates endothelium-dependent vasodilation and may modulate cardiac function. We examined the role of NO in hearts from transgenic (TG) mice overexpressing eNOS exclusively in cardiac myocytes. Three independent TG lines with varying levels of NOS activity were selected, and the hearts were isolated and retrogradely perfused at constant flow. We found that NO is positively inotropic in spontaneously beating hearts from wild-type (WT) mice, whereas hearts overexpressing eNOS had reduced basal contractility that was partially reversed by NOS blockade. Heart rate was not altered. Acetylcholine (10 to 1000 nmol/L) increased contractility in unstimulated hearts and decreased contractility after beta-adrenergic stimulation with norepinephrine, and these responses were identical in WT and TG hearts. Finally, resting systolic intracellular calcium (Ca(2+)(i)) tended to be lower in TG than in WT hearts, and the beat-to-beat responsiveness to Ca(2+)(i) was reduced in hearts with eNOS overexpression. High levels of endogenous myocyte-derived NO blunt myofilament Ca(2+) sensitivity. The similar effects of acetylcholine on contractility and heart rate, as well as the identical basal intrinsic heart rate in WT and TG hearts, provide a solid argument against NO as an important modulator of neurohormonal control of myocardial function.