Mechanisms Of Vascular Relaxation Research Articles

Prunus yedoensis Bark Downregulates the Expression of Cell Adhesion Molecules in Human Endothelial Cell Lines and Relaxes Blood Vessels in Rat Aortic Rings.

The incidence of cardiovascular diseases, such as high blood pressure, is increasing worldwide, owing to population aging and irregular lifestyle habits. Previous studies have reported the vasorelaxant effects of Prunus yedoensis bark methanol extract. However, various solvent extracts of P. yedoensis bark and their vascular relaxation mechanisms have not been sufficiently studied. We prepared extracts of P. yedoensis bark using various solvents (water, 30% ethanol, and 70% ethanol). P. yedoensis bark 30% ethanol extract (PYB-30E) decreased the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in human umbilical vein endothelial cells (HUVECs) activated with 200 ng/mL TNF-α. Additionally, PYB-30E showed vasodilatory effects on isolated rat aortic rings. This was confirmed to be the result of the activation of the NO/cGMP pathway, regulation of non-selective calcium-activated K+ channels, and calcium channel blockade. Additionally, PYB-30E significantly reduced systolic and diastolic blood pressure in spontaneously hypertensive rats (SHR). Taken together, our results indicated that PYB-30E is a candidate functional material with preventive and therapeutic effects against hypertension.

A new oxime synthesized from Senecio nutans SCh. Bip (chachacoma) reduces calcium influx in the vascular contractile response in rat aorta.

Senecio nutans Sch. Bip is an endemic plant commonly employed in the Andes culture to counteract the effects of mountain sickness, and its bioactive molecules could provide new drugs for treating hypertension. The purpose was to determine whether the vascular response of the plant bioactive molecules, such as (5-acetyl-6-hydroxy-2-isopropenyl-2,3-dihydrobenzofurane; Sn-I), could be improved by a simple structural modification to synthesize oximes (Ox-Sn-I). We characterized both compounds using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). We investigated vascular relaxation mechanisms in response to Sn-I and Ox-Sn-I using rat aorta and vascular smooth muscle cells (A7r5) as experimental models. Preincubation of aortic rings with Sn-I (10-5 M) significantly (p < 0.001) decreased the contractile effect in response to phenylephrine (PE) and potassium chloride (KCl). The sensitivity (EC50) to PE significantly (p < 0.01) decreased in the presence of Sn-I (10-5 M), but not with Ox-Sn-I. Sn-I significantly (p < 0.001) reduced the PE-induced contraction under calcium-free conditions. When A7r5 cells were preincubated with Sn-I and Ox-Sn-I (10-5 M), both compounds blunted the increase in intracellular Ca2+ induced by KCl. 2,3-Dihydrobenzofurane derived from S. nutans (Sn-I) reduces the contractile response probably by blocking Ca2+ entry through voltage-gated Ca2+ channels (VGCC) in vascular smooth cells. This effect also causes relaxation in rat aorta mediated by reduction of intracellular Ca2+ concentration, rather than an increase of NO generation in endothelial vascular cells.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy increases blood pressure and impairs vascular relaxation mechanisms in the adult offspring

Perfluorooctanesulfonate (PFOS) is a persistent environmental agent. We examined whether PFOS exposure during pregnancy alters blood pressure in male and female offspring, and if this is related to sex-specific changes in vascular mechanisms. PFOS was administered through drinking water (50 μg/mL) to pregnant Sprague-Dawley rats from gestational day 4 until delivery. PFOS-exposure decreased maternal weight gain but did not significantly alter feed and water intake in dams. The male and female pups born to PFOS mothers were smaller in weight by 29 % and 27 %, respectively. The male PFOS offspring remained smaller through adulthood, but the female PFOS offspring exhibited catch-up growth. The blood pressure at 12 and 16 weeks of age was elevated at similar magnitude in PFOS males and females than controls. Mesenteric arterial relaxation to acetylcholine was reduced in both PFOS males and females, but the extent of decrease was greater in females. Relaxation to sodium-nitroprusside was reduced in PFOS females but unaffected in PFOS males. Vascular eNOS expression was not changed, but phospho(Ser1177)-eNOS was decreased in PFOS males. In PFOS females, both total eNOS and phospho(Ser1177)-eNOS expression were reduced. In conclusion, PFOS exposure during prenatal life (1) caused low birth weight followed by catch-up growth only in females (2) lead to hypertension of similar magnitude in both males and females; (2) decreased endothelium-dependent vascular relaxation in males but suppressed both endothelium-dependent and -independent relaxation in females. The endothelial dysfunction is associated with reduced activity of eNOS in males and decreased expression and activity of eNOS in females.

Mechanisms underlying the vascular relaxation activities of Zingiber officinale var. rubrum in thoracic aorta of spontaneously hypertensive rats

ObjectiveTo evaluate vasorelaxant and vasoconstriction effects of Zingiber officinale var. rubrum (ZOVR) on live rats and isolated aortic rings of spontaneously hypertensive rats (SHRs). MethodsExtracts of ZOVR were subjected to in-vivo antihypertensive screening using noninvasive blood pressures in SHRs. The most potent extract, ZOVR petroleum ether extract (ZOP) was then fractionated using n-hexane, chloroform and water. Isolated thoracic aortic rings were harvested and subjected to vascular relaxation studies of n-hexane fraction of ZOP (HFZOP) with incubation of different antagonists such as Nω-nitro-l-arginine methyl ester (L-NAME, 10 µmol/L), indomethacin (10 µmol/L), methylene blue (10 µmol/L), atropine (1 µmol/L), glibenclamide (10 µmol/L), prazosin (0.01 µmol/L), and propranolol (1 µmol/L). ResultsDuring the screening of various ZOVR extracts, ZOP produced the most reduction in blood pressures of SHRs and so did HFZOP. HFZOP significantly decreased phenylephrine-induced contraction and enhanced acetylcholine-induced relaxation. L-NAME, indomethacin, methylene blue, atropine, and glibenclamide significantly potentiated the vasorelaxant effects of HFZOP. Propranolol and prazosin did not alter the vasorelaxant effects of HFZOP. HFZOP significantly suppressed the Ca2+-dependent contraction and influenced the ratio of the responses to phenylephrine in Ca2+-free medium. ConclusionThis study demonstrates that ZOP may exert an antihypertensive effect in the SHR model. Its possible vascular relaxation mechanisms involve nitric oxide and prostacyclin release, activation of cGMP-KATP channels, stimulation of muscarinic receptors, and transmembrane calcium channel or Ca2+ release from intracellular stores. Possible active compounds that contribute to the vasorelaxant effects are 6-gingerol, 8-gingerol and 6-shogaol.

Role of CSE-Produced H2S on Cerebrovascular Relaxation via RhoA-ROCK Inhibition and Cerebral Ischemia-Reperfusion Injury in Mice.

The role of CSE-produced H2S on cerebrovascular relaxation and cerebral ischemia-reperfusion (I/R) injury was investigated using CSE knockout (CSE-/-) and wild-type (CSE+/+) mice. The relaxation of the cerebral basilar artery (BA) to CSE-produced H2S and its mechanism were detected. The results revealed that both NaHS, a donor of exogenous H2S, and ROCK inhibitor Y27632 could induce significant relaxation of the BA, but the relaxation of the BA to NaHS was significantly attenuated by Y27632. In addition, removal of endothelium could reduce the relaxation of the BA to Y27632; CSE knockout also significantly attenuated Y27632-induced BA relaxation with endothelium rather than without endothelium. By contrast, the contraction of the BA from CSE-/- mice to RhoA agonist LPA or U46619 was stronger than that from CSE+/+ mice. Furthermore, RhoA activity and ROCK protein expression remarkably increased in the BA vascular smooth muscle cells (VSMCs) from CSE-/- mouse, which were inhibited by NaHS pretreatment. These findings revealed that the CSE-produced H2S induced cerebrovascular relaxation is generated from endothelial cells and the mechanism of vascular relaxation may relate to inhibition of RhoA-ROCK pathway. We next sought to confirm the protective effect of CSE-produced H2S on cerebral I/R injury produced by middle cerebral artery occlusion and bilateral common carotid artery occlusion in mice. We investigated the changes of neurological deficit, cerebral infarct, brain water content, LDH decrease, MDA increase as well as impairment of learning and memory function. The results showed that the cerebral injury became more grievous in CSE-/-mice than that in CSE+/+mice, which could be remarkably alleviated by NaHS pretreatment.

Characterization of potassium channels involved in the mechanisms of vascular relaxation induced by omega‐3 polyunsaturated fatty acids in rat arteries; a critical role for KATP channels

IntroductionFish oils are involved in the vasodilation of arteries and studies indicate that the main components involved are omega‐3 polyunsaturated fatty acids (n‐3 PUFA) that include docosahexaenoic acid (DHA) and eicosapentaenoic acids (EPA) (1). We previously demonstrated that inhibition of KCa channels partially attenuates fish oil mediated relaxation in rat mesenteric arteries (2). Since a considerable proportion of the relaxation to n‐3 PUFA was residual to blockade of KCa, vasodilation pathways involving other types of potassium channels could be involved. Studies report that the predominant vasodilation mechanisms can differ depending upon the type of artery an (3). Therefore, our main objective was to investigate these pathways in n‐3 PUFA‐induced relaxation of both large muscular (aorta) and resistance rat arteries (mesenteric).Methods2mm segments of rat arteries were mounted on a wire myograph and cumulative concentration response curves to DHA or EPA (10nM–30μM) mediated relaxation were examined following pre‐constriction with U46619 (10–500nM) in rat aorta and mesenteric artery. In certain experiments isotonic Krebs solution containing high [KCl] (final bath concentration 30mM; inhibits hyperpolarization mediated by potassium channels) or PNU37883A (3μM), a KATP blocker were administered prior to assessing relaxation. Relaxation was expressed as mean % relaxation of U46619‐induced constriction ± SEM of n animals. Data was analysed using one‐way ANOVA with Bonferroni's post‐test. P‐value of ≤ 0.05 was considered as being statistically significant.ResultsNon‐selective inhibition of potassium channels with high [KCl] Krebs virtually abolished both DHA‐ and EPA‐induced relaxation in rat aorta. Furthermore, High [KCl] Krebs also partially inhibited relaxation mediated by both DHA and EPA in rat mesenteric artery. Inhibition of KATP with PNU37883A attenuated fish oil‐induced relaxation in rat aorta. Consistently, in rat mesenteric artery PNU37883A also inhibited both DHA‐ and EPA‐induced relaxation.ConclusionsWe demonstrate that the non‐selective blockade of potassium channels abolished both DHA‐ and EPA‐mediated relaxation of rat aorta. Furthermore, inhibition of potassium channels in the resistance mesenteric artery also inhibited relaxation induced by DHA and EPA. We further characterised the potassium channels involved; blockade of KATP channels inhibited both DHA and EPA induced relaxation in rat aorta and mesenteric artery. This is the first study indicating that DHA and EPA evoke relaxation through KATP in both rat large muscular and resistance arteries. We will incorporate patch clamp in our future studies to investigate whether n‐3 PUFA directly regulate KATP channels.Support or Funding InformationThis work was supported by British Heart Foundation (PhD Studentship no. FS/14/33/30799)

Egg white protein hydrolysate reduces blood pressure, improves vascular relaxation and modifies aortic angiotensin II receptors expression in spontaneously hypertensive rats

Angiotensin converting enzyme (ACE) inhibitory peptides from egg have demonstrated reduction of blood pressure (BP) in vivo. In this study we evaluated the effects of egg white hydrolysate (EWH) prepared by thermolysin and pepsin in spontaneously hypertensive rats (SHRs). Fourteen to sixteen week old SHRs were implanted with telemetry devices. After recovery, the rats were divided randomly into three groups: untreated, EWH low dose (250 mg/kg BW), and EWH high dose (1000 mg/kg BW) for 12 days. BP showed a significant reduction in the EWH high dose group compared to untreated controls. BP reduction was associated with enhanced ex vivo vasodilation reduced oxidative/nitrosative stress, reduced angiotensin converting enzyme and angiotensin II type 1 receptor expression, while enhanced angiotensin II type 2 receptor expression. Circulating level of angiotensin II was unaffected. Thus, EWH exerted anti-hypertensive effects in SHRs through multiple mechanisms of vascular relaxation and RAS modulation.

Response to “Does Angiotensin-Dependent Superoxide Production Help to Prevent Salt-Induced Endothelial Dysfunction in 2 Kidney–1 Clip Hypertensive Rats?”

To the Editor: We recently reported the surprising finding that the salt-induced endothelial dysfunction and downregulation of copper (Cu)/zinc (Zn) superoxide dismutase (SOD) that occurred in cerebral arteries of sham-operated animals were absent in 2 kidney–1 clip (2K1C) hypertensive rats, which exhibit prolonged elevations of plasma angiotensin II (ANG II) levels, and that AT1 receptor blockade with losartan eliminated endothelium-dependent vasodilation in 2K1C rats.1 We concluded that the elevated ANG II levels in the 2K1C rats played a key role in protecting against salt-induced vascular dysfunction and suppression of Cu/Zn SOD by upregulating antioxidant defense mechanisms in the arteries. In a letter to the editor entitled “Could AT1 Receptor Activation Increase Antioxidant Defense to Prevent Salt-Induced Vascular Dysfunction of 2 Kidney-1 Clip Hypertensive Rats?”, Dr Valdir A. Braga stated that it is difficult to draw conclusions about the vascular actions of ANG II without looking at oxidant enzymes and downstream production of reactive oxygen species. Braga cites a list of well-known and indisputable findings showing that ANG II, acting via AT1 receptors, activates NADPH oxidase to produce superoxide (O2 −) radicals and that ANG II has been linked to oxidative stress in the context of vascular dysfunction and hypertension. Based on those conclusions, Braga suggests that the upregulation of Cu/Zn SOD occurs in response to increases in O2 − rather than a possible antioxidant effect of ANG II. In our estimation, Braga’s conclusion that O2 − production in response to the elevated ANG II levels in the 2K1C rats may play an important role in preventing salt-induced endothelial dysfunction through upregulation of Cu/Zn SOD is quite plausible and could provide an explanation for the surprising findings in our study (i.e., the “antioxidant effect” of ANG II to upregulate Cu/Zn SOD in 2K1C rats). In support of that hypothesis, Papaiahgari et al.2 reported that O2 − produced by NADPH oxidase was essential to activate the ERK 1/2 pathway in C10 alveolar epithelial cells exposed to hyperoxia and that this led to upregulation of the nuclear transcription factor Nrf2, which upregulates multiple antioxidant and cell protective enzymes, including Cu/Zn SOD. Consistent with those findings, McEwen et al.3 showed that the protective effect of low-dose ANG II infusion to restore vascular relaxation mechanisms in cerebral arteries of salt-fed Sprague-Dawley rats is eliminated by AT1 receptor blockade and by inhibiting the ERK 1/2 pathway. Further emphasizing the importance of physiological levels of ANG II in maintaining normal vascular function is our recent study,4 which showed that diet-induced obesity, which is known to elevate plasma ANG II levels, restores normal endothelial function in a model of chronically low levels of plasma ANG II, the Dahl salt-sensitive rat.The precise mechanisms by which O2 − radicals formed by NADPH oxidase in response to elevated ANG II levels contribute to maintenance of antioxidant defense mechanisms (including Cu/Zn SOD) in 2K1C hypertensive rats remain to be determined. However, investigation of the relationship between ANG II levels, vascular O2 −, and activation of the Nrf2 antioxidant defense system appears to be a promising avenue of investigation.

Reduced angiotensin II levels cause generalized vascular dysfunction via oxidant stress in hamster cheek pouch arterioles

ObjectivesWe investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. MethodsArteriolar diameters were measured via television microscopy during short-term (3–6days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100mg/kg/day). ResultsACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogs carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. ConclusionsThese findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

Abstract 531: Role of Insulin Resistance in the Altered Vascular Relaxation Mechanisms and Hypertension in Caveolin-1 Deficient Mice

Insulin resistance (IR) and endothelial dysfunction are often associated with hypertension; however, the specific causality and genetic underpinnings of these associations are unclear. Caveolin-1 (cav-1) is a transmembrane anchoring protein and potential modulator of vascular function and insulin sensitivity of the vascular and metabolic pathways. Our recent findings point to a role for cav-1 in IR, endothelial dysfunction and hypertension. In humans, cav-1 gene variants are associated with IR in hypertensive individuals. In mice, cav-1 deficiency leads to changes in endothelial function, IR and blood pressure (BP). To test whether the vascular changes associated with cav-1 deficiency are related to or are independent of IR, we assessed BP, blood glucose and vascular function in WT and cav-1 KO mice treated with placebo or metformin 400 mg/Kg/day for 21 days. BP was greater in cav-1 KO vs WT (140.1±2.1 vs 116.5±1.3 mmHg), and metformin treatment did not change BP in cav-1 KO (140.7±1.4) or WT (112.5±1.6 mmHg). Fasting blood glucose was greater in cav-1 KO vs WT (112±3.9 vs. 83.1±4.4 mg/dl), supporting a link between cav-1 deficiency and IR. Metformin did not change blood glucose levels in cav-1 KO (110.4±4.1) or WT (86.7±8.2 mg/dl). Acetylcholine (ACh)-induced maximal aortic relaxation was greater in cav-1 KO vs WT (85.3±5.7 vs. 17.5±4.4%), and was abolished by endothelium removal or the NOS inhibitor L-NAME, supporting changes in endothelial NO production/signaling. Metformin reduced ACh relaxation in cav-1 KO to 60.7±6.6%, but had no effect in WT. The exogenous NO donor sodium nitroprusside (SNP) produced similar maximal relaxation in cav-1 KO and WT (98.4±1.6 and 98.2±1.1%), but was more potent in cav-1 KO vs WT (pEC50, 8.6 vs 8.1). Metformin reduced SNP relaxation in cav-1 KO (84.9±6.4%) but not in WT, and reversed the enhanced sensitivity to SNP in cav-1 KO mice. The metformin-induced decrease in vascular relaxation and sensitivity to NO in cav-1 KO mice suggests a role for vascular IR in the changes in endothelial function. The lack of effect of metformin on fasting blood glucose in cav-1 KO mice suggests recalcitrant IR of the metabolic pathways. Together, these data support partial independence of vascular function from systemic IR in cav-1 deficiency states.

Protein Kinase G-I Deficiency Induces Pulmonary Hypertension through Rho A/Rho Kinase Activation

Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.

Impaired endothelium-dependent and -independent relaxation of aorta from diabetic rats

Vascular complication in diabetes has been reported to be due to the effects of chronic high blood glucose on the vascular system. Different relaxation mechanisms exist in the vasculature and effect of chronic high glucose on vascular relaxation mechanisms is not clearly understood. We assessed the effect of streptozotocin (STZ, 70 mg/kg, for 12 wks)-induced diabetes on vascular reactivity to isoproterenol (Isop, 10-9-10-5 M), a cAMP-dependent agent, acetylcholine (ACh, 10-9-10-5 M), a stimulant of NO (nitric oxide) synthase, sodium nitroprusside (SNP, 10-10-10-5 M), NO donor, or bradykinin (BK, 10-9-10-5 M) in the rat isolated aortic ring. Isop, ACh, SNP, or BK dose-dependently relaxed phenylephrine (PE, 10-7 M) pre-constricted ring producing a maximum relaxation of 82 % for Isop (10-5 M), 85 % for ACh (10-5 M), 100 % for SNP (10-6 M), and 30 % for BK (10-5 M) respectively. STZ attenuated Isop, ACh, and BK-induced relaxation by 45 % (n=7, pn (Fig. 5, Ref. 24).

Angiotensin II maintains cerebral vascular relaxation via EGF receptor transactivation and ERK1/2

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT(1)) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10(-9)-10(-5) M), iloprost (10(-16)-10(-11) g/ml), and reduced PO(2) was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ngxkg(-1)xmin(-1)) or epidermal growth factor (EGF; 2 microg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 microg/h), the ERK1/2 inhibitor PD-98059 (10 microg/h), or the protein synthesis inhibitor cycloheximide (5 microg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO(2), and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO(2) and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.

Endothelium negatively modulates the vascular relaxation induced by nitric oxide donor, due to uncoupling NO synthase

Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO](3+)-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD(2)) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD(2): 6.17+/-0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD(2): 6.65+/-0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD(2): 6.46+/-0.10), by the superoxide anion (O(2)(-)) scavenger TIRON (pD(2): 6.49+/-0.08), and by an NOS cofactor BH(4) (pD(2): 6.80+/-0.10). The selective dye for O(2)(-) (DHE) shows that TERPY enhances O(2)(-) concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00+/-317.75) compared with the basal concentration (IF: 7760.67+/-381.50), and this enhancement is blocked by L-NAME (IF: 8892.33+/-1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63+/-0.17% and after TERPY+L-NAME: -4.63+/-0.14%). Considering that TERPY could induce uncoupling NOS, thus producing O(2)(-), we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA(2)) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD(2) INDO: 6.80+/-0.17 and SQ29548: 6.85+/-0.15, respectively). However, a selective prostaglandin F(2alpha) receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA(2) stable metabolite (TXB(2)), but this effect is blocked by L-NAME and TIRON. The present findings indicate that TERPY induces uncoupling of eNOS, enhancing O(2)(-) concentration. This enhancement in O(2)(-) concentration induces COX activation, producing TXA(2), which negatively modulates the rat aorta relaxation induced by the NO donor TERPY.

Nitric oxide‐induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI‐17 and MYPT1

Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats

Normal pregnancy is associated with reduced blood pressure (BP) and decreased pressor response to vasoconstrictors, even though the renin-angiotensin system is upregulated. Angiotensin II (ANG II) activates both angiotensin type 1 receptors (AT(1)Rs) and angiotensin type 2 receptors (AT(2)Rs). Although the role of the AT(1)R in vascular contraction is well documented, the role of the AT(2)R in vascular relaxation, particularly during pregnancy, is less clear. It was hypothesized that the decreased BP and vasoconstriction during pregnancy was, at least in part, due to changes in AT(2)R amount, distribution, and/or postreceptor mechanisms of vascular relaxation. To test this hypothesis, systolic BP was measured in virgin and pregnant (day 19) Sprague-Dawley rats. Isometric contraction/relaxation was measured in isolated aortic rings, and nitric oxide (NO) production was measured using 4-amino-5-methylamino-2',7'-difluorescein fluorescence. AT(1)R and AT(2)R mRNA expression and protein amount were measured in tissue homogenates using real-time RT-PCR and Western blots, and their local distribution was visualized in cryosections using immunohistochemistry and immunofluorescence. BP was lower in pregnant than virgin rats. Phenylephrine (Phe) caused concentration-dependent contraction that was reduced in the aorta of pregnant compared with virgin rats. Treatment with the AT(2)R antagonist PD-123319 caused greater enhancement of Phe contraction, and the AT(2)R agonist CGP-42112A caused greater relaxation of Phe contraction in the aorta of pregnant than virgin rats. ANG II plus the AT(1)R blocker losartan induced greater NO production in the aorta of pregnant than virgin rats. RT-PCR revealed increased mRNA expression of vascular endothelial NO synthase (eNOS), little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Western blots revealed an increased protein amount of activated phospho-eNOS, little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Immunohistochemistry and immunofluorescence analysis in aortic sections of virgin rats revealed abundant AT(1)R staining in tunica media that largely colocalized with actin in vascular smooth muscle and less AT(2)Rs mainly in the tunica intima and endothelium. In pregnant rats, AT(1)R staining in the smooth muscle layer and adventitia was reduced, and endothelial AT(2)R staining was enhanced. These data suggest an enhanced AT(2)R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT(2)Rs and increased postreceptor activated phospho-eNOS, which may contribute to the decreased BP during pregnancy.

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca 2+ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca 2+ mobilization study shows that l-NAME inhibited the reduction of Ca 2+-concentration in VSM cells and reduced the increase in Ca 2+-concentration in endothelial cells induced by SNP. This enhancement in Ca 2+-concentration in the endothelial cells is due to a voltage-dependent Ca 2+ channels activation. The present findings indicate that the relaxation and [Ca 2+] i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.

EDHF Redux: EETs, TRPV4, and Ca 2+ Sparks

See related article, pages 1270–1279 Over the past two decades the complexity of the molecular processes underlying endothelial-dependent vascular relaxation has gradually become apparent. After the ground breaking discovery of the role of NO as a major endothelium-derived relaxant factor (EDRF), evidence emerged indicating the existence of a hyperpolarizing factor (endothelium-derived hyperpolarizing factor [EDHF]) with actions distinct from the cyclase-dependent relaxations associated with endothelial production of NO and prostacyclin.1–3 The specific molecules and vascular relaxation mechanisms underlying this intensely studied phenomenon have been difficult to establish, however. H2O2, K ions, and epoxygenase products have all been implicated as the EDHF, and evidence has suggested in turn that the mechanism by which EDHF relaxes arteries involves diffusion of the substance to smooth muscle and direct gating effects on calcium-activated K+ (maxiK) channels,4,5 actions mediated through endothelial small and intermediate conductance Ca2+-activated K+ (SK and IK) channels,6–8 and passive ionic effects associated with an extracellular accumulation of K+ ions.9 To further muddy these dark waters, recent evidence has implicated TRPV4 channels in EDHF signaling in several vascular preparations. In this issue of Circulation Research , Earley and colleagues10 identify a new putative mechanism and one that links EDHF to the regulation of Ca2+ release by ryanodine receptors (RYR), thereby forging a mechanistic link between EDHF and the well described local gating of large conductance Ca2+-activated K+ …

Chronic At1 Receptor Blockade Alters the Mechanisms Mediating Hypoxic Dilation in Middle Cerebral Arteries

The purpose of this study was to determine whether chronic blockade of the angiotensin II (ANG II) AT1 receptor under normal physiological conditions impairs vascular relaxation mechanisms in isolated middle cerebral arteries (MCA). Male Sprague-Dawley rats on a standard diet were given losartan (1 mg/mL) in the drinking water or normal water ad libitum for 7 days. Vessel diameters were measured by television microscopy before and during exposure to various vasodilator agonists and reductions in PO2 from 140 mm Hg to 35-45 mm Hg. Dilations to acetylcholine (1 microM), the stable prostacyclin analogue iloprost (10 pg/mL), and the Gs protein activator cholera toxin (1 ng/mL) were completely eliminated in vessels from losartan-treated animals. However, middle cerebral arteries from control and losartan-treated rats still demonstrated significant dilations in response to reduced PO2. Hypoxic dilation of middle cerebral arteries from control rats was eliminated by indomethacin (1 microM) and unaffected by the NOS inhibitor L-NAME (100 microM) whereas dilation in response to reduced PO2 in middle cerebral arteries from losartan-treated rats was eliminated by L-NAME and unaffected by indomethacin. Middle cerebral arteries from control and losartan-treated animals exhibited similar dilations in response to the NO-donor sodium nitroprusside (1 microM). These data suggest that AT1 receptor activation is important in maintaining normal vascular relaxation mechanisms in cerebral resistance arteries during normal physiological conditions, and that AT1 receptor blockade causes a shift in the mechanisms of hypoxic dilation of middle cerebral arteries from cyclooxygenase metabolites to NO.