Relaxation In Mesenteric Arteries Research Articles

IL‐10 Neither Causes Direct Vasorelaxation Nor Reduces Vasoconstriction in Rat Mesenteric Resistance Arteries

Perivascular adipose tissue (PVAT) is the fat that surrounds most of the peripheral blood vessels in the body. PVAT is protective and reduces contraction of blood vessels in health. We discovered that in health, PVAT around the rat small mesenteric resistance vessels (MRPVAT) contained a community of immune cells including T cells, B cells, macrophages, NK cells, neutrophils and mast cells. Interleukin (IL)‐10‐‐an anti‐inflammatory cytokine usually produced by T cells, B cells and macrophages‐‐was identified as one of the highly expressed (mRNA) cytokines in MRPVAT. One report suggests that IL‐10 causes relaxation of mouse mesenteric arteries. Hence, we hypothesized that IL‐10 directly causes vasorelaxation and/or reduces vasoconstriction in healthy rats. Male Sprague Dawley rats (12–14 weeks age) were used. Mesenteric resistance arteries expressed the receptor for IL‐10 (tunica intima and media) as determined by immunohistochemistry. Isolated tissue bath and third order mesenteric resistance arteries (without PVAT and with intact endothelium) were used for isometric contractility studies. Increasing concentrations [0.4–100 ng/mL] of recombinant rat (rr) IL‐10 or vehicle was directly added to half‐maximally constricted (phenylephrine PE or thromboxane A2 agonist U46619) vessels. IL‐10 did not reduce contraction, suggesting it was not a direct vasorelaxant. Further, the ability of rrIL‐10 to cause a rightward or downward shift of a vasoconstrictant‐response curve was tested. The vessels were incubated with rrIL‐10 [100 ng/mL or 10 ng/mL] or vehicle for 1.5 hours in the tissue bath followed by a cumulative PE [10−9–10−5 M] or U46619 [10−10–10−5 M] response curve. The maximal contractions or EC50 values (shown in table ) were not statistically different in rrIL‐10 incubated vessels vs vehicle. Thus, acute exposure of exogenous IL‐10 neither caused direct vasorelaxation nor reduced vasoconstriction. Further studies with neutralizing antibody for IL‐10 to vessels ± PVAT will help determine if IL‐10 produced in the PVAT causes vasorelaxation and/or reduction in local vasoconstriction indirectly, thus contributing to the anti‐contractile nature of PVAT.Support or Funding InformationNIH HL70687 −Log EC50 ± SEM (n=6–10 arteries/3–5 rats/curve) Vehicle rrIL‐10 (100 ng/mL) PE curve 5.7± 0.05 5.6±0.05 U46619 curve 7.6±0.28 7.2±0.59 Vehicle rrIL‐10 (10 ng/mL) PE curve 5.7±0.03 5.6±0.04 U46619 curve 7.1±0.12 7.2±0.13

Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea

IntroductionDe novo obstructive sleep apnea (OSA) in pregnancy, is associated with adverse gestational outcomes such as preeclampsia, gestational diabetes, and fetoplacental hypoxia. In addition, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction and enhanced vasoconstriction in pregnant mice. It is currently unknown whether gestational OSA has a long‐term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during late pregnancy will result in postpartum maternal vascular dysfunction.MethodsTimed pregnant female Long‐Evans rats were randomly assigned to two experimental groups: Normoxia (n=6) and CIH (n=6). The CIH group was exposed to five days (gestational days: 15–20) of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Gestational age at delivery was recorded and neonate’s crown‐rump length, abdominal girth, and body weights were measured within 12–16 hours from birth. At weaning (postnatal day 28), dams were euthanized and third‐order mesenteric arteries, and the main uterine artery, and uterine perivascular adipose tissue (PVAT) were excised. To assess endothelium‐dependent relaxation, we performed concentration‐response curves to acetylcholine (ACh: 10−9 – 3×10−5 M) using wire myography. In uterine arteries, we measured responses to ACh in the presence and absence of PVAT (0.1 g) based on our previous findings that uterine PVAT has vasoactive effects on these arteries.ResultsGestational CIH had no effect on the duration of gestation (Normoxia: 22.3 ± 0.21 days vs. CIH: 22.8 ± 1.16 days, p=0.24), litter size (Normoxia: 12.2 ± 1.64 vs. CIH: 10.3 ± 1.40, p=0.34), and neonatal weight at birth (Normoxia: 6.4 ± 0.24 g vs. CIH: 6.7 ± 0.19 g, p=0.16), while it increased neonatal crown‐rump length (Normoxia: 3.98 ± 0.07 cm vs. CIH: 4.19 ± 0.07 cm, p=0.05) and abdominal girth (Normoxia: 4.72 ± 0.10 cm vs. 4.94 ± 0.03 cm, p=0.07). At weaning, dams exposed to gestational CIH were lighter compared to dams exposed to normoxia (Normoxia: 319 ± 6.5 g vs. CIH: 298 ± 8.7 g, p=0.08). Exposure to gestational CIH had no effect on ACh‐induced relaxation in mesenteric resistance arteries (pEC50, Normoxia: 7.80 ± 0.03 vs. CIH: 7.89 ± 0.06, p=0.19). In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(−)PVAT/pEC50, Normoxia: 6.77 ± 0.08 vs. CIH: 7.13 ± 0.09, p<0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67 ± 0.08 vs. CIH: 6.70 ± 0.07, p=0.99]. The effects of PVAT were due to its anti‐dilatory influences on uterine arteries from CIH‐treated rats [CIH (−PVAT) vs. CIH (+PVAT), p = 0.003], whereas it had no effect on arteries from control rats [Normoxia (−PVAT) vs. Normoxia (+PVAT), p = 0.77].ConclusionExposure to CIH during gestation resulted in neonatal macrosomia, reduced maternal weight at weaning, and exaggerated postpartum uterine vascular smooth muscle relaxation responses, while it did not affect small resistance artery reactivity. Gestational OSA may impair maternal vascular recovery after birth.Support or Funding InformationUNTHSC Basic Seed Grant

Deletion of murine slc29a4 modifies vascular responses to adenosine and 5‐hydroxytryptamine in a sexually dimorphic manner

Equilibrative nucleoside transporter 4 (ENT4), encoded by SLC29A4, mediates the flux of both 5‐hydroxytryptamine (5‐HT) and adenosine across cell membranes. We hypothesized that loss of ENT4 function in mice would modify the effects of these established regulators of vascular function. Male and female wild‐type (WT) and slc29a4‐null (ENT4‐KO) mice were compared with respect to their hemodynamics and mesenteric vascular function. Male ENT4‐KO mice had a complete loss of myogenic tone in their mesenteric resistance arteries. This was accompanied by a decrease in blood flow in the superior mesenteric artery in the male ENT4‐KO mice, and a reduced responsiveness to 5‐HT. In contrast, endothelium‐dependent relaxations of mesenteric arteries from female ENT4‐KO mice were more sensitive to Ca2+‐activated K+ (KCa) channel blockade than WT mice. Female ENT4‐KO mice also demonstrated an enhanced vasodilatory response to adenosine in vivo that was not seen in males. Ketanserin (5‐HT2A inhibitor) and GR55562 (5‐HT1B/1D inhibitor) decreased 5‐HT‐induced tone, but only ketanserin inhibited the relaxant effect of 5‐HT in mesenteric arteries. 5‐HT‐evoked increases in tone were elevated in arteries from ENT4‐KO mice upon block of endothelial relaxant pathways, with arteries from female ENT4‐KO mice showing the greatest increase. Adenosine A2b receptor expression was decreased, while other adenosine transporter subtypes, as well as adenosine deaminase and adenosine kinase were increased in mesenteric arteries from male, but not female, ENT4‐KO mice. These findings indicate that deletion of slc29a4 leads to sex‐specific changes in vascular function with significant consequences for regulation of blood flow and pressure by adenosine and 5‐HT.

5/6 nephrectomy induces different renal, cardiac and vascular consequences in 129/Sv and C57BL/6JRj mice

Experimental models of cardiovascular diseases largely depend on the genetic background. Subtotal 5/6 nephrectomy (5/6 Nx) is the most frequently used model of chronic kidney disease (CKD) in rodents. However, in mice, cardiovascular consequences of 5/6 Nx are rarely reported in details and comparative results between strains are scarce. The present study detailed and compared the outcomes of 5/6 Nx in the 2 main strains of mice used in cardiovascular and kidney research, 129/Sv and C57BL/6JRj. Twelve weeks after 5/6 Nx, CKD was demonstrated by a significant increase in plasma creatinine in both 129/Sv and C57BL/6JRj male mice. Polyuria and kidney histological lesions were more pronounced in 129/Sv than in C57BL/6JRj mice. Increase in albuminuria was significant in 129/Sv but not in C57BL/6JRj mice. Both strains exhibited an increase in systolic blood pressure after 8 weeks associated with decreases in cardiac systolic and diastolic function. Heart weight increased significantly only in 129/Sv mice. Endothelium-dependent mesenteric artery relaxation to acetylcholine was altered after 5/6 Nx in C57BL/6JRj mice. Marked reduction of endothelium-dependent vasodilation to increased intraluminal flow was demonstrated in both strains after 5/6 Nx. Cardiovascular and kidney consequences of 5/6 Nx were more pronounced in 129/Sv than in C57BL/6JRj mice.

Itaconimides derivatives induce relaxation in mesenteric artery and negative inotropism by inhibition of CA2+ influx.

The aim of this study was to evaluate the cardiovascular effects of N-phenyl-itaconimide (Imide-1), N-4-methyl-phenyl-itaconimide (Imide-2), N-4-methoxy-phenyl-itaconimide (Imide-3) and N-4-chloro-phenyl-itaconimide (Imide-4), and investigate the mechanisms of action involved in the observed responses. The relaxant effect was investigated in rat superior mesenteric arteries by using isometric tension measurements. Additionally, in isolated atria were evaluated the heart rate and force of cardiac contraction and in vivo experiments was evaluated blood pressure and heart rate. Cumulative administration of itaconimides (3 × 10-8 to 3 × 10-4M) in pre-contracted mesenteric artery rings with phenylephrine, 1μM, induced endothelium-independent vasorelaxation. The itaconimides showed similar maximum efficacies. Additionally, Imide-3 induced vasorelaxation in rings exposed to a depolarizing-tyrode solution containing 60mM KCl or 20mM KCl similar to the control, suggesting the non-participation of K+ channels. Imide-3 attenuated Ca2+ influx in a concentration-dependent manner. As well, imide-3 reduced CaCl2-induced contraction in nominally calcium-free medium, in the presence of cyclopiazonic acid (20μM), phenylephrine (1μM) and nifedipine (1μM), indicating a reduction of Ca2+ influx by receptor-operated channels (ROC) and store-operated channels (SOC). The presence of SKF 96365 (10-5M), SOC blocker, did not significantly alter the vasorelaxant effect induced by imide-3. Moreover, imide-3 induced a negative inotropic effect. In vivo studies, in non-anesthetized normotensive rats, imide-3 lowered blood pressure and induced bradycardia. These results suggest that itaconimides have concentration-dependent vascular effects and the vasorelaxation seems to be endothelium-independent. The vasodilatory effect induced by imide-3 may be due to a possible influence on the CaV and ROC. In addition, imide-3 is able to reduce force of cardiac contraction, blood pressure and promote bradycardia.

Moringa oleifera leaf extract induces vasorelaxation via endothelium-dependent hyperpolarization and calcium channel blockade in mesenteric arterial beds isolated from L-NAME hypertensive rats

ABSTRACT Background An aqueous extract of Moringa oleifera leaves (MOE) is known to cause relaxation of mesenteric resistance arteries of rats in which hypertension has been induced by the administration of L-NAME, but the mechanism(s) of action of MOE remains unclear. The purpose of this study was to investigate these mechanisms in mesenteric arterial beds isolated from L-NAME induced hypertensive rats. Methods: An investigation of vascular reactivity was conducted on isolated mesenteric arterial beds by measuring the changes in perfusion pressure using an in vitro system. Results MOE (0.001–3 mg in 0.1 ml injection volume) caused a dose-dependent relaxation in methoxamine (5 µM) pre-contracted arterial beds, which was partially abolished by endothelium removal. The endothelium-dependent component of vasorelaxation was insensitive to both L-NAME (100 µM) and indomethacin (10 µM), while completely inhibited in high KCl (45 mM)-induced contraction. MOE (1 and 3 mg/ml) showed a dose-dependent inhibitory effect on CaCl2-induced contractions of denuded preparations in Ca2+-free medium containing a high KCl (60 mM) or methoxamine (10 µM). In Ca2+-free medium, MOE (3 mg/ml) also inhibited phenylephrine-induced contractions of denuded preparations. Conclusion: These findings suggest that MOE relaxes mesenteric arterial beds of L-NAME hypertensive rats via both endothelium-dependent and endothelium-independent mechanisms. The endothelium-dependent action occurred via endothelium-derived hyperpolarizing factor-mediated hyperpolarization. The endothelium-independent action was related to blocking the entry of extracellular Ca2+ via voltage-operated and receptor-operated Ca2+ channels, and inhibiting mobilization of sarcolemmal Ca2+ via inositol trisphosphate receptor Ca2+ channels. MOE may be potentially useful as a natural vasodilator against hypertension.

Pharmacological properties of β-adrenoceptors mediating rat superior mesenteric artery relaxation and the effects of chemical sympathetic denervation

Aimsβ-Adrenoceptors (β-ADRs) mediating the relaxation of rat superior mesenteric arteries (SMAs) were pharmacologically identified, and the effects of chemical sympathetic denervation on β-ADR-mediated relaxation were examined. Main methodsThe tension changes of endothelium-denuded SMAs were isometrically recorded and the mRNA of endothelium-denuded SMA β-ADR was detected using RT-PCR. Key findingsIn endothelium-denuded SMAs contracted with ≥10−7 M phenylephrine (an α1-ADR agonist), isoprenaline (a β-ADR agonist)-induced relaxation was competitively inhibited by 3 × 10−9–10−8 M propranolol (a β1,2-ADR antagonist), but not further affected by ≥10−8 M propranolol. Although isoprenaline-induced relaxation was not affected by ICI-118,551 (10−9–10−8 M; a β2-ADR antagonist), it was competitively inhibited by atenolol (10−7–3 × 10−7 M; a β1-ADR antagonist) in the presence of ICI-118,551. In the presence of 10−7 M propranolol, isoprenaline- and CGP-12177A (a β3-ADR partial agonist)-induced relaxation was competitively inhibited by high concentrations of bupranolol (a β1,2,3-ADR antagonist), with pA2 values of 6.49 and 5.76, respectively. We detected the mRNA of β1- and β3-ADRs in endothelium-denuded SMAs. Treatment with 6-hydroxydopamine (a catecholaminergic neurotoxin) reduced maximal isoprenaline-induced relaxation in the presence and absence of 10−7 M propranolol, but not CGP-12177A-induced relaxation. SignificanceIsoprenaline-induced relaxation of rat SMAs is mediated by β1- and β3-ADRs. β-ADR-mediated relaxation of rat SMAs is shown to be attenuated by chemical sympathetic denervation. The differences in the effects of bupranolol and chemical sympathetic denervation on the responses to isoprenaline and CGP-12177A in rat SMAs might be explained by the possible presence of multiple β3-ADRs with different pharmacological properties.

An in vivo examination of the differences between rapid cardiovascular collapse and prolonged hypotension induced by snake venom

We investigated the cardiovascular effects of venoms from seven medically important species of snakes: Australian Eastern Brown snake (Pseudonaja textilis), Sri Lankan Russell’s viper (Daboia russelii), Javanese Russell’s viper (D. siamensis), Gaboon viper (Bitis gabonica), Uracoan rattlesnake (Crotalus vegrandis), Carpet viper (Echis ocellatus) and Puff adder (Bitis arietans), and identified two distinct patterns of effects: i.e. rapid cardiovascular collapse and prolonged hypotension. P. textilis (5 µg/kg, i.v.) and E. ocellatus (50 µg/kg, i.v.) venoms induced rapid (i.e. within 2 min) cardiovascular collapse in anaesthetised rats. P. textilis (20 mg/kg, i.m.) caused collapse within 10 min. D. russelii (100 µg/kg, i.v.) and D. siamensis (100 µg/kg, i.v.) venoms caused ‘prolonged hypotension’, characterised by a persistent decrease in blood pressure with recovery. D. russelii venom (50 mg/kg and 100 mg/kg, i.m.) also caused prolonged hypotension. A priming dose of P. textilis venom (2 µg/kg, i.v.) prevented collapse by E. ocellatus venom (50 µg/kg, i.v.), but had no significant effect on subsequent addition of D. russelii venom (1 mg/kg, i.v). Two priming doses (1 µg/kg, i.v.) of E. ocellatus venom prevented collapse by E. ocellatus venom (50 µg/kg, i.v.). B. gabonica, C. vegrandis and B. arietans (all at 200 µg/kg, i.v.) induced mild transient hypotension. Artificial respiration prevented D. russelii venom induced prolonged hypotension but not rapid cardiovascular collapse from E. ocellatus venom. D. russelii venom (0.001–1 μg/ml) caused concentration-dependent relaxation (EC50 = 82.2 ± 15.3 ng/ml, Rmax = 91 ± 1%) in pre-contracted mesenteric arteries. In contrast, E. ocellatus venom (1 µg/ml) only produced a maximum relaxant effect of 27 ± 14%, suggesting that rapid cardiovascular collapse is unlikely to be due to peripheral vasodilation. The prevention of rapid cardiovascular collapse, by ‘priming’ doses of venom, supports a role for depletable endogenous mediators in this phenomenon.

Vascular adenosine monophosphate-activated protein kinase: Enhancer, brake or both?

Adenosine monophosphate-activated protein kinase (AMPK), expressed/present ubiquitously in the body, contributes to metabolic regulation. In the vasculature, activation of AMPK is associated with several beneficial biological effects including enhancement of vasodilatation, reduction of oxidative stress and inhibition of inflammatory reactions. The vascular protective effects of certain anti-diabetic (metformin and sitagliptin) or lipid-lowering (simvastatin and fenofibrate) therapeutic agents, of active components of Chinese medicinal herbs (resveratrol and berberine) and of pharmacological agents (AICAR, A769662 and PT1) have been attributed to the activation of AMPK (in endothelial cells, vascular smooth muscle cells and/or perivascular adipocytes), independently of changes in the metabolic profile (eg glucose tolerance and/or plasma lipoprotein levels), leading to improved endothelium-derived nitric oxide-mediated vasodilatation and attenuated endothelium-derived cyclooxygenase-dependent vasoconstriction. By contrast, endothelial AMPK activation with pharmacological agents or by genetic modification is associated with reduced endothelium-dependent relaxations in small blood vessels and elevated systolic blood pressure. Indeed, AMPK activators inhibit endothelium-dependent hyperpolarization (EDH)-type relaxations in superior mesenteric arteries, partly by inhibiting endothelial calcium-activated potassium channel signalling. Therefore, AMPK activation is not necessarily beneficial in terms of endothelial function. The contribution of endothelial AMPK in the regulation of vascular tone, in particular in the microvasculature where EDH plays a more important role, remains to be characterized.

Exploring the role of orexin B-sirtuin 1-HIF-1α in diabetes-mellitus induced vascular endothelial dysfunction and associated myocardial injury in rats

AimThe present study explored the role and possible interrelationship between orexin B-sirtuin 1-HIF-1α signaling pathways in diabetes-mellitus induced vascular dysfunction and enhancement in myocardial injury. Material and methodsStreptozotocin (60 mg/kg) was employed to induce diabetes mellitus in male Wistar albino rats, which were kept for eight weeks. The vascular function was noted by assessing acetylcholine-induced relaxation in norepinephrine precontracted mesenteric arteries. The hearts were subjected to ischemia-reperfusion injury on the Langendorff apparatus. Myocardial injury was assessed by noting the release of CK-MB, cardiac troponin and measuring myocardial infarction. The levels of orexin B, sirtuin 1 and HIF-1α were measured. YNT-185 (orexin B type 2 receptor agonist), STR2104 (sirtuin 1 agonist) and EX527 (sirtuin 1 antagonist) were employed as pharmacological tools. ResultsDiabetes led to significant development of vascular dysfunction and enhanced ischemia-reperfusion injury in isolated hearts. There was a significant decrease in the levels of orexin B, sirtuin 1 and HIF-1α in diabetic animals. Treatment with YNT-185 and/or STR2104 significantly attenuated the diabetes-induced increase in myocardial injury and vascular dysfunction. Co-administration of EX527 abolished the effects of YNT-185 suggesting orexin B-mediated effects may be through activation of sirtuin 1. Moreover, YNT-185-induced increase in the expression of sirtuin 1 and HIF-1α was also abolished in the presence of EX527. ConclusionDiabetes-induced significant decline in orexin B levels in the plasma along with a decrease in the expression of sirtuin 1 and HIF-1α in the heart following ischemia-reperfusion injury may possibly contribute in exacerbating the myocardial injury and vascular dysfunction.

Associations of inflammatory markers and vascular cell adhesion molecule-1 with endothelial dysfunction in collagen-induced arthritis

We determined the role of high-grade inflammation on endothelial function and its association with biomarkers of endothelial dysfunction in collagen-induced arthritis. Sprague-Dawley rats were divided into a control (n = 12) or collagen-induced arthritis (CIA; n = 21) group. To induce arthritis, Bovine-type-II collagen emulsified in incomplete Freund's adjuvant was injected at the base of the tail. Nine-weeks after the primary immunisation, vascular reactivity in mesenteric and saphenous arteries was assessed using a wire-myograph. Serum concentrations of inflammatory markers (tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), C-reactive protein (CRP)) and biomarkers of endothelial dysfunction (vascular cell adhesion molecule-1 (VCAM-1) and asymmetric dimethylarginine (ADMA)) were measured by ELISA. Acetylcholine-induced relaxation in mesenteric and saphenous arteries was impaired in CIA compared to controls (P < 0.05). Responses to sodium nitroprusside were similar between controls and CIA in mesenteric arteries and marginally impaired in saphenous arteries of CIA rats. Compared to controls, TNF-α, IL-6, IL-1β, CRP (all P < 0.00001) and VCAM-1 (P = 0.02) were elevated in CIA. TNF-α (std β(SE) = 0.39(0.16); P = 0.03), IL-6 (std β(SE) = 0.37(0.17); P = 0.03), IL-1β (std β(SE) = 0.41(0.16); P = 0.02) and CRP (std β(SE) = 0.36(0.17); P = 0.04) were associated with VCAM-1. Associations between inflammatory markers and the maximal relaxation (Emax) to acetylcholine in mesenteric arteries were no longer significant after adjusting for VCAM-1 (except for IL-1β). VCAM-1 was inversely associated with the Emax to acetylcholine in mesenteric (std β(SE) = -0.49(0.16); P = 0.01) but not in saphenous arteries (std β(SE) = -0.06(0.18); P = 0.76). In conclusion, exposure to high-grade inflammation impairs endothelial-dependent relaxation. The inflammation-induced increase in VCAM-1 concentrations may contribute to the impaired endothelium-dependent relaxation in mesenteric arteries of CIA rats.

Loss-of-Function Mutations in Human Regulator of G Protein Signaling RGS2 Differentially Regulate Pharmacological Reactivity of Resistance Vasculature.

Regulator of G protein signaling 2 (RGS2) plays a role in reducing vascular contraction and promoting relaxation due to its GTPase accelerating protein activity toward Gαq. Previously, we identified four human loss-of-function (LOF) mutations in RGS2 (Q2L, D40Y, R44H, and R188H). This study aimed to investigate whether those RGS2 LOF mutations disrupt the ability of RGS2 to regulate vascular reactivity. Isolated mesenteric arteries (MAs) from RGS2-/- mice showed an elevated contractile response to 5 nM angiotensin II and a loss of acetylcholine (ACh)-mediated vasodilation. Reintroduction of a wild-type (WT) RGS2-GFP plasmid into RGS2-/- MAs suppressed the vasoconstrictor response to angiotensin II. RGS2 LOF mutants failed to suppress the angiotensin II constriction response compared with RGS2 WT. In contrast, ACh-mediated vasoconstriction was restored by expression of RGS2 WT, D40Y, and R44H but not by RGS2 Q2L or R188H. Phosphorylation of RGS2 D40Y and R44H by protein kinase G (PKG) may explain their maintained function to support relaxation in MAs. This is supported by phosphomimetic mutants and suppression of vasorelaxation mediated by RGS2 D40Y by a PKG inhibitor. These results demonstrate that RGS2 attenuates vasoconstriction in MAs and that RGS2 LOF mutations cannot carry out this effect. Among them, the Q2L and R188H mutants supported less relaxation to ACh, whereas relaxation mediated by the D40Y and R44H mutant proteins was equal to that with WT protein. Phosphorylation of RGS2 by PKG appears to contribute to this vasorelaxation. These results provide insights for precision medicine targeting the rare individuals carrying these RGS2 mutations. SIGNIFICANCE STATEMENT: Regulator of G protein signaling 2 (RGS2) has been implicated in the control of blood pressure; rare mutations in the RGS2 gene have been identified in large-scale human gene sequencing studies. Four human mutations in RGS2 that cause loss of function (LOF) in cell-based assays were examined in isolated mouse arteries for effects on both vasoconstriction and vasodilation. All mutants showed the expected LOF effects in suppressing vasoconstriction. Surprisingly, the D40Y and R44H mutant RGS2 showed normal control of vasodilation. We propose that this is due to rescue of the mislocalization phenotype of these two mutants by nitric oxide-mediated/protein kinase G-dependent phosphorylation. These mechanisms may guide drug discovery or drug repurposing efforts for hypertension by enhancing RGS2 function.

ENOS-NO-induced small blood vessel relaxation requires EHD2-dependent caveolae stabilization.

Endothelial nitric oxide synthase (eNOS)-related vessel relaxation is a highly coordinated process that regulates blood flow and pressure and is dependent on caveolae. Here, we investigated the role of caveolar plasma membrane stabilization by the dynamin-related ATPase EHD2 on eNOS-nitric oxide (NO)-dependent vessel relaxation. Loss of EHD2 in small arteries led to increased numbers of caveolae that were detached from the plasma membrane. Concomitantly, impaired relaxation of mesenteric arteries and reduced running wheel activity were observed in EHD2 knockout mice. EHD2 deletion or knockdown led to decreased production of nitric oxide (NO) although eNOS expression levels were not changed. Super-resolution imaging revealed that eNOS was redistributed from the plasma membrane to internalized detached caveolae in EHD2-lacking tissue or cells. Following an ATP stimulus, reduced cytosolic Ca2+ peaks were recorded in human umbilical vein endothelial cells (HUVECs) lacking EHD2. Our data suggest that EHD2-controlled caveolar dynamics orchestrates the activity and regulation of eNOS/NO and Ca2+ channel localization at the plasma membrane.

Abstract P122: Downregulation and Inhibition of PKCα Causes Increased Ca 2+ -Sensing Receptor-Mediated Vascular Relaxation in Aged Dahl Salt-Sensitive Rats

The G protein-coupled Ca 2+ -Sensing Receptor (CaSR) mediates extracellular calcium (Ca 2+ e ) - induced relaxation of mesenteric arteries under tension and may therefore play a role in blood pressure regulation. This presents CaSR as promising target for therapeutic drug design for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is an important regulator of CaSR signaling and can phosphorylate the receptor leading to desensitization. In this study, we investigated the effects of CaSR mutation and downregulation of PKCα in CaSR-mediated relaxation of phenylephrine (PE)-contracted mesenteric arteries from aged Dahl salt-sensitive rats, fed a low salt (0.1% NaCl) diet, using automated wire myography. We have observed in our laboratory, that there is significant decrease in relaxation response of PE-contracted mesenteric arteries to Ca 2+ e . as animals get older. The results show minimal relaxation responses of arteries to Ca 2+ e and that downregulation of CaSR expression had no significant effect. PKCα downregulation (siRNA) and inhibition of PKC (Gö 6976) showed greater than 80% increase in maximum Ca 2+ -induced relaxation, with significant decreases in EC 50 values compared to untreated controls. The results indicate that PKCα plays an important role in the regulation of CaSR -mediated relaxation of mesenteric arteries. Therefore, inhibition of PKC may lead to increased CaSR sensitivity to Ca 2+ indicating a possible therapeutic mechanism for intervention in age-related vascular dysfunction.

C-Kit deficiency impairs nitric oxide signaling in smooth muscle cells

IntroductionReceptor tyrosine kinases have been implicated in various vascular remodeling processes and cardiovascular disease. However, their role in the regulation of vascular tone is poorly understood. Herein, we evaluate the contribution of c-Kit signaling to vasoactive responses. MethodsThe vascular reactivity of mesenteric arteries was assessed under isobaric conditions in c-Kit deficient (KitW/W−v) and littermate control mice (Kit+/+) using pressure myography. Protein levels of soluble guanylyl cyclase beta 1 (sGCβ1) were quantified by Western blot. Mean arterial pressure was measured after high salt (8% NaCl) diet treatment using the tail-cuff method. ResultsSmooth muscle cells (SMCs) from c-Kit deficient mice showed a 5-fold downregulation of sGCβ1 compared to controls. Endothelium-dependent relaxation of mesenteric arteries demonstrated a predominance of prostanoid vs. nitric oxide (NO) signaling in both animal groups. The dependence on prostanoid-induced dilation was higher in c-Kit mutant mice than in controls, as indicated by a significant impairment in vasorelaxation with indomethacin with respect to the latter. Endothelium-independent relaxation showed significant dysfunction of NO signaling in c-Kit deficient SMCs compared to controls. Mesenteric artery dilation was rescued by addition of a cGMP analog, but not with a NO donor, indicating a deficiency in cGMP production in c-Kit deficient SMCs. Finally, c-Kit deficient mice developed higher blood pressure on an 8% NaCl diet compared to their control littermates. Conclusionc-Kit deficiency inhibits NO signaling in SMCs. The existence of this c-Kit/sGC signaling axis may be relevant for vascular reactivity and remodeling.

Regulation of YAP by Mammalian Target of Rapamycin Complex 1 in Endothelial Cells Controls Blood Pressure Through COX-2/mPGES-1/PGE2 Cascade.

Endothelial cells regulate vascular tone by producing both relaxing and contracting factors to control the local blood flow. Hypertension is a common side effect of mTORC1 (mammalian target of rapamycin complex 1) inhibitors. However, the role of endothelial mTORC1 in hypertension remains elusive. The present study aimed to determine the role of endothelial mTORC1 in Ang II (angiotensin II)-induced hypertension and the underlying mechanism. Endothelial mTORC1 activity was increased by Ang II both in vitro and in vivo. Blood pressure was higher in Tie-2-Cre-mediated regulatory associated protein of mTOR (mammalian target of rapamycin; Raptor) heterozygous-deficient (Tie2Cre-RaptorKD) mice than control mice both before and after Ang II infusion. Acetylcholine-evoked endothelium-dependent relaxation of mesenteric arteries was impaired in Tie2Cre-RaptorKD mice. Treatment with indomethacin or a specific COX (cyclooxygenase)-2 inhibitor, NS-398, but not L-NG-nitroarginine methyl ester reduced endothelium-dependent relaxation in Raptorflox/- mice to a similar extent as in Tie2Cre-RaptorKD mice. Metabolomic profiling revealed that the plasma content of prostaglandin E2 was reduced in Tie2Cre-RaptorKD mice with or without Ang II infusion. In endothelial cells, reduction of the protein level of YAP (yes-associated protein) with siRNA-mediated RPTOR deficiency was autophagy dependent and transcriptionally regulated the expression of COX-2 and mPGES-1 (microsomal prostaglandin E synthase-1). Hence, overexpression of YAP in endothelial cells enhanced the mRNA and protein levels of COX-2 and mPGES-1 and reversed the endothelial dysfunction and hypertension in Tie2Cre-RaptorKD mice. The present results demonstrate that suppression of mTORC1 activity in endothelial cells reduces prostaglandin E2 production and causes hypertension by reducing YAP-mediated COX-2/mPGES-1 expression.

Acute activation of endothelial AMPK surprisingly inhibits endothelium-dependent hyperpolarization-like relaxations in rat mesenteric arteries.

Endothelium-dependent hyperpolarizations (EDHs) contribute to the regulation of peripheral resistance. They are initiated through opening of endothelial calcium-activated potassium channels (KCa ); the potassium ions released then diffuse to the underlying smooth muscle cells, causing hyperpolarization and thus relaxation. The present study aimed to examine whether or not AMPK modulates EDH-like relaxations in rat mesenteric arteries. Arterial rings were isolated for isometric tension recording. AMPK activity and protein level were measured by ELISA and western blotting respectively. The AMPK activator, AICAR, reduced ACh-induced EDH-like relaxations and increased AMPK activity in preparations with endothelium; these responses were prevented by compound C, an AMPK inhibitor. AICAR inhibited relaxations induced by SKA-31 (opener of endothelial KCa ) but did not affect potassium-induced, hyperpolarization-attributable relaxations or increase AMPK activity in preparations without endothelium. A769662, another AMPK activator, not only caused a similar inhibition of relaxations to ACh and SKA-31 in preparations with endothelium but also inhibited hyperpolarization-attributable relaxations and augmented AMPK activity in rings without endothelium. Protein levels of total AMPKα, AMPKα1, or AMPKβ1/2 were comparable between preparations with and without endothelium. Activation of endothelial AMPK, by either AICAR or A769662, acutely inhibits EDH-like relaxations of rat mesenteric arteries. Furthermore, A769662 inhibits signalling downstream of smooth muscle hyperpolarization. In view of the major blunting effect of AMPK activation on EDH-like relaxations, caution should be applied when administering therapeutic agents that activate AMPK in patients with endothelial dysfunction characterized by reduced production and/or bioavailability of NO.

Direct Impairment of the Endothelial Function by Acute Indoxyl Sulfate through Declined Nitric Oxide and Not Endothelium-Derived Hyperpolarizing Factor or Vasodilator Prostaglandins in the Rat Superior Mesenteric Artery.

Upon stimulation, endothelial cells release various factors to regulate the vascular tone. In particular, vasorelaxing factors, called endothelium-derived relaxing factors (EDRFs), are altered in the production and/or release, as well as their signaling every vessel and under pathophysiological states, including cardiovascular, kidney, and metabolic diseases. Although indoxyl sulfate is known as a protein-bound uremic toxin and circulating levels are elevated in the impaired kidney functions, direct impact on the vascular function, especially EDRF's signaling, remains unclear. In this study, we hypothesize that acute exposure to indoxyl sulfate could alter vascular relaxation in the rat superior mesenteric artery. Accordingly, we measured acetylcholine (ACh)-induced endothelium-dependent relaxation in the absence and presence of several inhibitors to divide into each EDRF, including nitric oxide (NO), vasodilator prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF). Indoxyl sulfate reduced the sensitivity to ACh but not sodium nitroprusside. Under cyclooxygenase (COX) inhibition or inhibitions of COX plus source of EDHF, such as small (SKCa)- and intermediate (IKCa)-conductance calcium-activated K+ channels, the decreased sensitivity to ACh in indoxyl sulfate exposed vessel was still preserved. However, under inhibition of NO synthase (NOS) or inhibitions of NOS and COX, the difference of sensitivity to ACh between vehicle and indoxyl sulfate was eliminated. These findings indicated that acute exposure of indoxyl sulfate in the rat superior mesenteric artery specifically explicitly impaired NO signaling but not EDHF or vasodilator PGs.

Antihypertensive activity of diethyl-4,4ʹ-dihydroxy-8,3ʹ-neolign-7,7ʹ-dien-9,9ʹ-dionate: A continuation study in L-NAME treated wistar rats

In the present study, we report that neolignan1 (Diethyl-4,4ʹ-dihydroxy-8,3ʹ-neolign-7,7ʹ-dien-9,9ʹ-dionate) relaxes the superior mesenteric artery in a concentration dependent manner (pD2 value 5.392 ± 0.04; n = 8 for endothelium intact and 5.204 ± 0.03; n = 8 for endothelium denuded mesenteric rings, respectively). The relaxation response of neolignan1 was found to be endothelium independent and sensitive to 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-on (ODQ; 1 μM) and tetraethyl ammonium (TEA; 1 mM). In-silico studies showed good LibDock score (92.66) of neolignan1 with BKCa channel and are in well corroboration with ex-vivo study. Further, neolignan1 significantly decreased the systolic blood pressure, diastolic blood pressure and mean arterial pressure in the Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 50 mg/kg) treated Wistar rats at the dose of 30 and 100 mg/kg given once orally for 15 days. In addition, neolignan1 is well tolerated up to 100 mg/kg when given as a repeated dose, once orally for 28 days in Swiss albino mice. Neolignan1 was well absorbed from oral route, reached peak at 4 h and eliminated below detection level by 12 h after administration. Our present study concludes that neolignan1 produced relaxation in superior mesenteric artery by opening of BKCa channel and produced significant antihypertensive activity in L-NAME treated Wistar rats and was well tolerated by the experimental animal.

Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats.

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive Gӧ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.