Vasodilation In Resistance Arteries Research Articles

Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-β- d -Ribofuranoside in the Muscle Microcirculation Increases Nitric Oxide Synthesis and Microvascular Perfusion

To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries ( approximately 100 mum) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19+/-4% at 2 mmol/L, P<0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-L-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (approximately 60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

Abstract 5039: Globular and Full-length Adiponectin Induce NO-Dependent Vasodilation in Resistance Arteries of ZL but Not ZDF Rats

Adiponectin increases NO-production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but it is not known whether adiponectin has a functional effect on small resistance arteries. In order to address this question, we assessed the direct vasodilatory response of mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rat and the Zucker lean (ZL) rats to globular (gAd) and full-length adiponectin (fAd) in a small vessel myograph after precontraction with norepinephrine. Serum adiponectin levels measured by ELISA were significantly higher in ZL than in ZDF rats (ZL 4.0±0.1 μ g/l vs ZDF 2.9±0.2 μ g/l; p&lt;0.001). Both gAd and fAd induced a relevant dose dependent vasodilation in ZL, but not ZDF rats (max. vasodilation to gAd: ZL 21.0±0.9% vs ZDF 2.9±0.9%; p&lt;0.001. max. vasodilation to fAd: ZL 23.8±1.2% vs ZDF 1.4±1.1%; p&lt;0.001). This vasodilatory effect in ZL rats could totally be blunted by preincubation with L-NAME (p&lt;0.001) indicating that it is exclusively mediated by NO. ZDF rats showed a significantly lower endothelium-dependent vasodilation response to acetylcholine (max. vasodilation: ZL 96.7±5.5% vs ZDF 60.1±6.0%; p&lt;0.001). The additional administration of low dose gAd or fAd could not improve the vasodilatory response to ACh. The gene expressions of AdipoR1 and AdipoR2 quantified by real-time RT-PCR were not significantly different in both animal models, AdipoR2 only tended to be higher expressed in ZDF rats (ZL 0.62±0.03 vs ZDF 0.68±0.06; p=0.061). But AdipoR1 was significantly more abundant expressed than AdipoR2 in resistance arteries of both ZL and ZDF rats (ZL: 1.06±0.04 vs 0.62±0.03; p&lt;0.001. ZDF: 1.06±0.08 vs 0.68±0.06; p&lt;0.001). In conclusion, we showed that adiponectin exerts a relevant NO-mediated vasodilation in resistance arteries of normal control rats. Reduced adiponectin serum levels and a blunted response to adiponectin in diabetic animals may contribute to endothelial dysfunction and thus play a role in the pathogenesis of atherosclerosis. In diabetes mellitus, adiponectin receptors are not downregulated in resistance arteries indicating a dysfunction downstream of the receptors.

Openers of SK Ca and IK Ca channels enhance agonist‐evoked endothelial nitric oxide synthesis and arteriolar vasodilation

Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.

L-Arginine is related to endothelium-dependent vasodilation in resistance and conduit arteries in divergent ways—The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study

Background Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the conversion of l-arginine to the endothelium-derived vasodilator nitric oxide. We evaluated if circulating levels of l-arginine and ADMA, and the ratio thereof, were related to endothelium-dependent vasodilation in both resistance and conductance arteries in the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study. Methods and results In this population-based study, vascular function of 1016 subjects aged 70 was evaluated by the invasive forearm technique with intra-brachial infusion of acetylcholine (EDV), by measurement of flow-mediated dilation (FMD) of the brachial artery, and by change in reflectance index (RI) by pulse wave analysis. After adjustment for resting forearm blood flow, gender, Framingham risk score, and glomerular filtration rate, the l-arginine/ADMA ratio was positively related to EDV ( P = 0.005). This association was mainly driven by l-arginine, because if both l-arginine and ADMA were entered as predictor variables in a single regression model, l-arginine was positively associated with EDV ( P = 0.001), whereas the negative association between ADMA and EDV was not significant. In contrast, after adjustment for baseline brachial diameter, gender, Framingham risk score, and glomerular filtration rate, the l-arginine/ADMA ratio was negatively related to FMD ( P = 0.004). Again, this association was mainly driven by l-arginine ( P = 0.002), whereas ADMA was not significantly related to FMD. The change in RI was not related to l-arginine, ADMA or their ratio. Conclusions In elderly subjects the l-arginine/ADMA ratio and l-arginine, but not ADMA, are positively related to endothelium-dependent vasodilation in resistance arteries, but negatively in a conduit artery.

Induction of PDE5 attenuates NO mediated vasodilation in resistance arteries under chronic high and low flow

In other studies we have observed the induction of NOS and PDE5 isozymes in the mesenteric artery (MA1) under conditions of sustained high and low flow (HF/LF). In this study we used pharmacological methods to test the role of NO and PDE5 in regulating vascular tone in HF/LF. Alternating pairs of second order MAs are ligated, resulting in chronic LF in the upstream MA1 and chronic HF in the adjacent MA1. Vascular reactivity was studied in a wire myograph system 4 days later, when NOS and PDE5 expression are increased. HF/LF Day4 MA1s were less sensitive than control to the α‐agonist phenylephrine (PE). Pre‐treatment with L‐NAME nearly normalized the sensitivity to PE in HF/LF MA1s. The PDE5 inhibitor Sildenafil at 1 μM without L‐NAME induced more complete relaxation of PE pre‐constricted HF/LF MA1 vs control (HF:76.7±8.0%; LF 90.3±4.7% ; control 46.3±11.1%; P&lt;0.05). This difference could reflect the increased release of NO and/or increased activity of PDE5. To distinguish these, MA1s were pre‐treated with L‐NAME and Sildenafil and dose‐response to DEA/NO measured. The HF/LF MA1s were more sensitive to DEA/NO only in the presence of Sildenafil (EC50: HF 27±4; LF 24±4; Con 45 ± 6 nM; P&lt;0.01). The responses to the PDE1 inhibitor Vinpocetine did not differ. We conclude that induction of PDE5 serves to de‐sensitize the vascular smooth muscle to chronic NO signaling under sustained HF/LF conditions.

Key role of α1β1-integrin in the activation of PI3-kinase-Akt by flow (shear stress) in resistance arteries

Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow (shear stress) is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established, and alterations in FMD are early markers of cardiovascular disorders. alpha(1)-Integrin, which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of alpha(1)-integrins in FMD was tested with selective antibodies and mice lacking the gene encoding for alpha(1)-integrins. Both anti-alpha(1) blocking antibodies and genetic deficiency in alpha(1)-integrin in mice (alpha(1)(-/-)) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium-independent dilation (sodium nitroprusside). Similarly, vasoconstrictor tone (myogenic tone and phenylephrine-induced contraction) was not affected. In MRA phosphorylated Akt and phosphatidylinositol 3-kinase (PI3-kinase) were significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice, although total Akt and endothelial nitric oxide synthase (eNOS) were not affected. Pharmacological blockade of PI3-kinase-Akt pathway with LY-294002 inhibited FMD. This inhibitory effect of LY-294002 was significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice. Thus alpha(1)-integrin has a key role in flow (shear stress)-dependent vasodilation in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Because of the central role of flow (shear stress) activation of the endothelium in vascular disorders, this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.

Polymorphisms in the estrogen receptor alpha gene and endothelial function in resistance and conduit arteries in the elderly

Background Prior evidence suggests an important role for estrogen in the regulation of endothelium-dependent vasodilation, but the mechanisms modulating this are not known. Our aim was to examine the relations of single nucleotide polymorphisms (SNPs) in the estrogen receptor alpha gene ( ESR1) to endothelium-dependent vasodilation. Methods We evaluated 959 70-year-old participants (51% men) of the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study, using invasive forearm technique with intra-brachial infusion of acetylcholine (EDV; reflecting vasodilation in resistance arteries), and brachial artery ultrasound to assess flow-mediated vasodilation (FMD; reflecting vasodilation in conduit arteries). We genotyped 25 common SNPs in the ESR1 gene, and related them to EDV and FMD using multivariable linear regression, adjusting for sex and other potential confounders, such as major cardiovascular risk factors and medications. Haplotypes were estimated using the PHASE software. Results We observed an association between rs1709183 in the ESR1 gene and EDV (nominal P = 0.0012), with a lower EDV in carriers of the minor allele (C). This association remained significant after adjustment for multiple testing (empirical P = 0.031, obtained using bootstrap re-sampling). Two ESR1 haplotypes in the block containing rs1709183 were associated with EDV (individual haplotype P = 0.0015 and P = 0.025); the directions of effect were consistent with individual SNP analyses. FMD was not associated with any of the ESR1 SNPs. Conclusions In our community-based study of elderly, a polymorphism in the estrogen receptor alpha gene was associated with endothelium-dependent vasodilation in resistance, but not conduit arteries. Our findings should stimulate further exploration in other settings.

Abstract 1437: Polymorphisms in the Estrogen Receptor Genes and Endothelium-Dependent Vasodilation in Elderly

Background: Single nucleotide polymorphisms (SNPs) in the estrogen receptor 1 ( ESR1 ) and 2 ( ESR2 ) genes have been associated with blood pressure, subclinical and overt cardiovascular disease, but their relation to endothelium-dependent vasodilation is unknown. Methods: We evaluated 959 70-year-old participants of the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study, using invasive forearm technique with intra-brachial infusion of acetylcholine (EDV; reflecting vasodilation in resistance arteries), and brachial artery ultrasound to assess flow-mediated vasodilation (FMD; reflecting vasodilation in conduit arteries). We analyzed 25 common SNPs in the ESR1 gene and 5 in the ESR2 gene by minisequencing, and related them to EDV and FMD using multivariable linear regression, adjusting for sex, systolic blood pressure, diastolic blood pressure, pulse rate, anti-hypertensive treatment, total cholesterol, high-density cholesterol, lipid-lowering medication, fasting glucose, anti-diabetic medication, body mass index, current smoking, and prior diagnosis of cardiovascular disease. Haplotypes were estimated using the PHASE software. Results: We observed an association between rs1709183 in the ESR1 gene and EDV (nominal P=0.0012), with a lower EDV in carriers of the minor allele (C). This association remained significant after adjustment for multiple testing (empirical P=0.031, obtained using bootstrap re-sampling). Two ESR1 haplotypes in the block containing rs1709183 were associated with EDV (individual haplotype P=0.0015 and P=0.025); the directions of effect were consistent with the individual SNP analyses. There were no associations between SNPs in the ESR2 gene and EDV with a nominal P=0.01. FMD was not associated with either ESR1 or ESR2 . Conclusions: In our community-based study of elderly, a polymorphism in the ESR1 gene was associated with endothelium-dependent vasodilation in resistance arteries, but not in conduit arteries. Our findings should stimulate to replication and further exploration in other settings.

Angiotensin Type 2 Receptor in Resistance Arteries of Type 2 Diabetic Hypertensive Patients

The role of angiotensin type 2 receptor (AT(2)R) on vascular responses to angiotensin II in humans remains unclear. In this study we explored whether AT(2)R is expressed and functionally active on peripheral resistance arteries of hypertensive diabetic patients treated for 1 year with either the angiotensin receptor blocker valsartan or the beta-blocker atenolol. Twenty-six hypertensive type 2 diabetic patients treated with oral hypoglycemic and antihypertensive agents (not receiving angiotensin receptor blockers or beta-blockers) were randomly assigned to double-blind treatment for 1 year with valsartan or atenolol once daily added to their previous therapy in a clinical trial that we reported recently and compared with 10 normal subjects. Resistance arteries dissected from gluteal subcutaneous tissues were assessed on a pressurized myograph. Vasomotor response curves to angiotensin II (1 nmol/L to 1 micromol/L) were performed on norepinephrine precontracted vessels in the presence of valsartan (10 micromol/L) with or without the AT(2)R inhibitor PD123319 (1 micromol/L). AT(2)R expression was evaluated by confocal microscopy. After 1 year of treatment, systolic and diastolic blood pressure was controlled and comparable in the valsartan and atenolol groups. Angiotensin II evoked a significant vasodilatory response only on resistance arteries from patients treated with valsartan, effect blocked by PD123319. AT(2)R expression was 4-fold higher in small arteries of valsartan-treated patients. In conclusion, AT(2)Rs are upregulated and contribute to angiotensin II-induced vasodilation in resistance arteries of hypertensive diabetic patients treated with angiotensin type 1 receptor blockers and may mediate, in part, vascular actions of these drugs in high cardiovascular risk patients.

Effect of testosterone on ex vivo vascular reactivity in man

Testosterone is reported to have an acute vasodilating action in vitro, an effect that may impart a favourable haemodynamic response in patients with chronic heart failure. However, the effect of chronic testosterone exposure on general vascular reactivity is poorly described. In the present study, fresh subcutaneous resistance arteries were obtained from patients with heart failure (n=10), healthy controls (n=9) and men with androgen-deficiency (n=17). All arteries were studied using a wire myograph to examine the effect of cumulative additions of testosterone (1 nmol/l-100 micromol/l) compared with vehicle control following maximal pre-constriction with KCl (1-100 micromol/l). The vascular reactivity of arteries from androgen-deficient patients was examined further by recording tension concentration curves to cumulative additions of noradrenaline (1 nmol/l-100 micromol/l) and U46619 (1-300 nmol/l), followed by relaxation concentration curves to additions of ACh (acetylcholine; 10 nmol/l-30 micromol/l) and SNP (sodium nitroprusside; 10 nmol-30 micromol/l) respectively. In all cases, statistical analysis was performed by ANOVA. Patients with proven androgen-deficiency were treated according to clinical recommendations for a minimum of 3 months and further arteries (n=19) were taken for experimentation using the same protocol. In all groups, testosterone was confirmed to be an acute concentration-dependent vasodilator at concentrations > or =1 micromol/l (P=0.0001). The dilating effect of testosterone was augmented in patients with androgen-deficiency prior to treatment, and this effect was abrogated following appropriate testosterone replacement. Testosterone therapy significantly reduced the normal vascular dilating response to ACh and SNP (P<0.01) and significantly increased the contractile response to noradrenaline (P<0.01), but not U46619. Testosterone is an acute dose-dependent vasodilator of resistance arteries. Physiological testosterone replacement attenuates general vascular reactivity in androgen-deficient subjects. The numerous perceived benefits of testosterone replacement may be offset by a decline in vascular reactivity and, therefore, further studies and careful monitoring of patients is recommended.

Differential effects of ouabain on the vasodilator actions of nitric oxide and S-nitrosothiols in vivo: Relevance to the identity of EDRF/EDHF

Objectives This study examined the role of Na +/K +-ATPase in the vasodilator actions of nitric oxide (NO), S-nitrosothiols and the endothelium-dependent agonist, acetylcholine. Methods The vasodilator responses elicited by intravenous injections of (i) the NO-donors, sodium nitroprusside and MAHMA NONOate, (ii) the S-nitrosothiols, l- S-nitrosocysteine and S-nitrosocoenzyme A, and (iii) acetylcholine, in urethane-anesthetized rats. Results The NO-donors, S-nitrosothiols and acetylcholine elicited dose-dependent depressor responses and reductions in hindquarter (HQR) and mesenteric (MR) vascular resistances. The depressor responses and associated reductions in HQR elicited by NO-donors were markedly attenuated after injection of ouabain. In contrast, the depressor responses and reductions in HQR elicited by the S-nitrosothiols and acetylcholine were not affected. The reductions in MR elicited by all vasodilator agents were exaggerated after injection of ouabain. Finally, the decomposition of sodium nitroprusside, MAHMA NONOate, l- S-nitrosocysteine and S-nitrosocoenzyme A to NO upon addition to rat blood or vascular preparations was not affected by ouabain. Conclusion This study demonstrates that ouabain has opposing effects on NO-mediated vasodilation in resistance arteries in the hindquarter and mesenteric beds of the rat. The similarity of effects of ouabain on the vasodilator actions of acetylcholine, l- S-nitrosocysteine and S-nitrosocoenzyme A as opposed to the NO-donors supports the possibility that endothelium-derived relaxing factor released by acetylcholine in resistance arteries is an S-nitrosothiol.

Vasodilation in resistance arteries is related to the apolipoprotein B/A1 ratio in the elderly – The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study

Background Recent studies have shown the apolipoprotein B to apolipoprotein A1 ratio (apoB/A1) to be superior to LDL-cholesterol measurements to predict cardiovascular events. The present study aims to relate apoB/A1 to endothelium-dependent vasodilation, an early marker of atherosclerosis, in the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS) study. Methods and results In this population-based study, 1016 subjects aged 70 years were evaluated by the invasive forearm technique with acetylcholine (EDV), brachial artery ultrasound to assess flow-mediated vasodilation (FMD) and pulse wave analysis with a beta-2 receptor agonist challenge, terbutaline. EDV and the pulse wave response, but not FMD, were related to apoB/A1 levels ( r = −0.11, p = 0.0038 for EDV, r = −0.16, p < 0.0001 for the pulse wave analysis and r = 0.01, p = 0.65 for FMD). Neither LDL-cholesterol, nor non-HDL-cholesterol, was significantly related to the measurements of endothelium-dependent vasodilation. Also endothelium-independent vasodilation (EIDV) evaluated by the invasive forearm technique with sodium nitroprusside was related to apoB/A1 levels ( r = −0.12, p < 0.0016). Conclusion The apoB/A1 levels, but not LDL-cholesterol, were inversely related to endothelium-dependent vasodilation evaluated by EDV and pulse wave analysis, but not by FMD. Also EIDV showed the same pattern, suggesting a general deterioration in vasoreactivity mainly in resistance arteries in elderly subjects with high apoB/A1 levels.

Involvement of the alpha1‐beta1 integrin and PI3Kinase‐Akt in flow (shear stress) ‐mediated vasodilation in resistance arteries

Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established and alterations in FMD are early markers of cardiovascular disorders. Integrin (a1-b1), which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of a1-b1 integrins in FMD was tested using selective antibodies and mice lacking the gene encoding for a1 integrins. Both anti-b1 blocking antibodies and the genetic deficiency in a1-integrin in mice (a1-/-) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium independent dilation. Similarly, vasoconstrictor tone (myogenic tone and phenylephrine contraction) was not affected. In MRA phosphorylated-Akt and PI3-kinase were significantly lower in a1-/- mice than in a1-/- mice, although Akt and eNOS were not affected. Pharmacological blockade of the PI3kinase-Akt pathway with LY294002 inhibited FMD. This inhibitory effect of LY294002 was significantly lower in a1-/- mice than in a1+/+ mice. Thus, a1 integrin has a key role in FMD in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Due to the central role of flow activation of the endothelium in vascular disorders this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.

Asymmetrical Dimethylarginine Plasma Concentrations Are Related to Basal Nitric Oxide Release but Not Endothelium-Dependent Vasodilation of Resistance Arteries in Peritoneal Dialysis Patients

Vascular dysfunction in chronic renal failure may be linked to reduced nitric oxide (NO) bioactivity and increased circulating concentrations of the endogenous NO synthase inhibitor asymmetrical dimethyl L-arginine (ADMA). The association between ADMA and basal endothelial NO release and endothelium-dependent vasodilation in resistance arteries of chronic renal failure patients is unknown. Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine, the endothelium-independent vasodilator nitroglycerine, and the endothelium-dependent vasoconstrictor N(G)-monomethyl-L-arginine (L-NMMA) were assessed in 37 peritoneal dialysis patients. L-arginine and ADMA plasma concentrations were measured by HPLC. ADMA (mean +/- SEM: 0.68 +/- 0.02 micromol/L) was associated with basal forearm blood flow (r = -0.33; P < 0.05) and L-NMMA induced vasoconstriction (r = -0.55; P < 0.0005), but not with dilator effects of acetylcholine or nitroglycerine. L-arginine (68 +/- 3 micromol/L) tended to correlate with acetylcholine-induced vasodilation (r = 0.32; P = 0.05) but was not associated with other parameters. ADMA is related to basal but not to acetylcholine-stimulated NO bioactivity in patients on peritoneal dialysis. Impaired endothelium-dependent vasodilation found in chronic renal failure is not explained by elevated circulating NO synthase inhibitors in renal failure.

Novel PPARγ Agonists GI 262570, GW 7845, GW 1929, and Pioglitazone Decrease Calcium Channel Function and Myogenic Tone in Rat Mesenteric Arteries

Novel non-thiazolidinedione, tyrosine-derived peroxisome proliferator-activated receptor γ agonists, GI 262570, GW 7845, GW 1929, developed by GlaxoSmithKline (GSK) along with pioglitazone and nisoldipine, were studied on currents through L-type voltage-dependent calcium channels (VDCC) in freshly isolated smooth muscle cells from mesenteric arteries, and on the diameter of pressurized mesenteric arteries in vitro. Using Ba²⁺ (10 mmol/l) as the charge carrier through VDCC, the half-inhibition constants (IC₅₀) for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.0 ± 0.5, 3.0 ± 0.5, 5.0 ± 0.7, and 10.0 ± 0.8 µmol/l, respectively. For arterial diameter measurements the IC₅₀ values for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.4, 4.1, 6.3, and 13.9 µmol/l, respectively. Each GSK compound and pioglitazone was effective at inhibiting VDCC and relaxing pressurized arteries, suggesting that the vasodilation of resistance arteries could be explained by the inhibition of calcium entry through VDCC.

Voluntary physical exercise-induced vascular effects in spontaneously hypertensive rats

Forced training has been shown to have beneficial vascular effects in various animal exercise models. In the present study, we explored possible physiological and molecular effects of voluntary physical exercise on various vascular beds. SHR (spontaneously hypertensive rats) performed voluntary exercise for 5 weeks in a computerized wheel cage facility. Ex vivo myograph studies revealed an increased sensitivity of the ACh (acetylcholine)-mediated vasodilation in resistance arteries of the exercised animals (ED50=15.0+/-3.5 nmol/l) compared with the controls (ED50=37.0+/-8.8 nmol/l; P=0.05). The exercise/control difference was abolished after scavenging reactive oxygen radicals. In conduit arteries, ACh induced a similar vasodilatory response in both groups. The in vivo aortic wall stiffness, assessed by means of Doppler tissue echography, was significantly lower in the exercising animals than in controls. This was demonstrated by significantly increased peak systolic aortic wall velocity (P=0.03) and the velocity time integral (P=0.01) in exercising animals compared with controls. The relative gene expression of eNOS (endothelial nitric oxide synthase) was similar in both groups of animals, whereas Cu/ZnSOD (copper/zinc superoxide dismutase) gene expression was significantly increased (+111%; P=0.0007) in the exercising animal compared with controls. In conclusion, voluntary physical exercise differentially improves vascular function in various vascular beds. Increased vascular compliance and antioxidative capacity may contribute to the atheroprotective effects associated with physical exercise in conduit vessels.

NO- activates soluble guanylate cyclase and Kv channels to vasodilate resistance arteries.

Nitric oxide (NO) plays an important role in the control of vascular tone. Traditionally, its vasorelaxant activity has been attributed to the free radical form of NO (NO*), yet the reduced form of NO (NO-) is also produced endogenously and is a potent vasodilator of large conduit arteries. The effects of NO- in the resistance vasculature remain unknown. This study examines the activity of NO- in rat small isolated mesenteric resistance-like arteries and characterizes its mechanism(s) of action. With the use of standard myographic techniques, the vasorelaxant properties of NO* (NO gas solution), NO- (Angeli's salt), and the NO donor sodium nitroprusside were compared. Relaxation responses to Angeli's salt (pEC50=7.51+/-0.13, Rmax=95.5+/-1.5%) were unchanged in the presence of carboxy-PTIO (NO* scavenger) but those to NO* and sodium nitroprusside were inhibited. l-Cysteine (NO- scavenger) decreased the sensitivity to Angeli's salt (P<0.01) and sodium nitroprusside (P<0.01) but not to NO*. The soluble guanylate cyclase inhibitor ODQ (3 and 10 micromol/L) concentration-dependently inhibited relaxation responses to Angeli's salt (41.0+/-6.0% versus control 93.4+/-1.9% at 10 micromol/L). The voltage-dependent K+ channel inhibitor 4-aminopyridine (1 mmol/L) caused a 9-fold (P<0.01) decrease in sensitivity to Angeli's salt, whereas glibenclamide, iberiotoxin, charybdotoxin, and apamin were without effect. In combination, ODQ and 4-aminopyridine abolished the response to Angeli's salt. In conclusion, NO- functions as a potent vasodilator of resistance arteries, mediating its response independently of NO* and through the activation of soluble guanylate cyclase and voltage-dependent K+ channels. NO- donors may represent a novel class of nitrovasodilator relevant for the treatment of cardiovascular disorders such as angina.

3638 Testosterone is a vasodilator of resistance arteries in men with heart failure

3638 Testosterone is a vasodilator of resistance arteries in men with heart failure

Effects of sildenafil citrate on human hemodynamics

Nitric oxide (NO) induces the formation of intracellular cyclic guanosine monophosphate (cGMP) by guanylate cyclase. Sildenafil, which selectively inhibits phosphodiesterase type 5 (PDE5) found predominantly in the corpora cavernosa of the penis, effectively blocks the degradation of cGMP and enhances erectile function in men with erectile dysfunction. The NO–cGMP pathway also plays an important role in mediating blood pressure. It is, therefore, possible that the therapeutic doses of sildenafil used to treat erectile dysfunction may have clinically significant effects on human hemodynamics. Three studies were undertaken to assess the effects of intravenously, intra-arterially, and orally administered doses of sildenafil on blood pressure, heart rate, cardiac output, and forearm blood flow and venous compliance in healthy men. A fourth study evaluated the hemodynamic effects of intravenous sildenafil in men with stable ischemic heart disease. In healthy men, significant (p <0.01) decreases in supine systolic and diastolic blood pressures were observed with intravenous sildenafil (20, 40, and 80 mg) at the end of the infusion period when plasma levels of sildenafil were highest (mean decreases from baseline of 7.0/6.9 and 9.2/6.7 mm Hg, for the 40- and 80-mg doses, respectively). These changes were transient and not dose related. Modest reductions in systemic vascular resistance also were observed (maximum decrease 16%), although heart rate was not affected by sildenafil administration when compared with placebo. Single oral doses of sildenafil (100, 150, and 200 mg) produced no significant changes in cardiac index from 1–12 hours postdose between placebo- and sildenafil-treated subjects. The approved dosage strengths of sildenafil citrate are 25 mg, 50 mg, and 100 mg. The 80-mg intravenous dose and the 200-mg oral dose of sildenafil produced comparable plasma levels at twice the maximum therapeutic dose (recommended range, 25–100 mg). After brachial artery infusion of sildenafil (up to 300 μg/min), there was a modest vasodilation of resistance arteries and a reversal of norepinephrine-induced preconstriction of forearm veins. These hemodynamic effects were similar to but smaller in magnitude than those of nitrates. In a small pilot study of men with ischemic heart disease, decreases from baseline in pulmonary arterial pressure (−27% at rest and −19% during exercise) and cardiac output (−7% at rest and −11% during exercise) were observed after 40-mg intravenous doses of sildenafil. Sildenafil was well tolerated by subjects and patients in all studies, with headache and other symptoms of vasodilation the most commonly reported adverse effects of treatment. Modest, transient hemodynamic changes were observed in healthy men after single intravenous or oral doses of sildenafil even at supratherapeutic doses. In men with stable ischemic heart disease, sildenafil produced modest effects on hemodynamic parameters at rest and during exercise.

Differences in nitric oxide production in porcine resistance arteries and epicardial conduit coronary arteries.

The purpose of this study was to test the hypothesis that endothelial cells from resistance arteries and epicardial conduit coronary arteries differ in their expression of nitric oxide synthase (NOS) and calcium metabolism, and that these differences contribute to the mechanism underlying disparate physiological vasodilator responses observed between the two populations of vessels. The functional vasodilator responses of isolated resistance arteries and epicardial conduit coronary arteries were compared in vitro using both the receptor-independent agonist A23187 ionophore to increase intracellular calcium and the receptor-dependent agonist bradykinin. Constitutive NOS (cNOS) activity in monocultures of endothelial cells derived from resistance arteries and conduit arteries was assayed using a fibroblast-reporter cell method. Intracellular calcium concentration was assessed using fura-2 microfluorometry. Nitric oxide production was determined using a chemiluminescence technique, while cNOS protein was quantitated by Western blot analysis. A23187 was a less potent vasodilator of resistance arteries studied in vitro, compared to epicardial conduit arteries (EC50 = 1.6 microM, resistance artery vs. EC50 = 0.03 microM, conduit artery); however, bradykinin was more potent in resistance arteries (EC50 = 0.3 nM, resistance artery vs. EC50 = 2 nM, conduit artery). In pure monocultures of endothelium, nitric oxide production measured by chemiluminescence both basally and in response to A23187 was significantly less in resistance arteries (6.1 +/- 0.5, basal vs. 10.80 +/- 0.55, stimulated nmol/microgram protein), compared to conduit arteries (7.7 +/- 0.5, basal vs. 17.00 +/- 1.52, stimulated nmol/microgram protein; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). cNOS enzyme activity assessed by cGMP production in reporter cell fibroblasts was also lower in resistance arteries compared to conduit arteries (0.17 +/- 0.03 vs. 0.33 +/- 0.05 fmol cGMP/microgram protein, respectively; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). Conduit arteries expressed 2.1 x more cNOS protein than resistance arteries, as assessed by Western blotting of cellular homogenates. No significant differences were found with microfluorimetry in either basal or ionophore-stimulated intracellular calcium concentrations. The results signified that porcine resistance arteries expressed less NOS and produced less nitric oxide than epicardial conduit arteries both basally and in response to an increase in intracellular calcium. This difference was reflected functionally as a decreased vasodilatory response to increased intracellular calcium in resistance arteries that could not be explained on the basis of differences in the metabolism of intracellular calcium. In contrast, the functional vasodilator response of intact vessels to a receptor-mediated agonist was enhanced in resistance arteries compared to conduit arteries, suggesting an important role of signal transduction mechanisms in specific physiological responses. Thus, the ability of the endothelium to regulate on a regional basis the expression of NOS and integrate receptor-mediated responses with these differences may provide a mechanism for diverse vasomotor responses in different populations of vessels.