Mesenteric Vasorelaxation Research Articles

Zinc pyrithione induces endothelium-dependent hyperpolarization-mediated mesenteric vasorelaxation in healthy and colitic mice

BackgroundAlthough Zinc pyrithione (ZPT) could lower blood pressure by inducing vasorelaxation, it is unclear if it is able to induce vasorelaxation of mesenteric arterioles in health and ulcerative colitis (UC) to exert anti-colitic action. MethodsThe vasorelaxation of the second-order branch of the mesenteric artery from wide type (WT) mice, TRPV1−/−(KO) mice, and TRPV4−/−(KO) mice was determined using a Mulvany-style wire myograph. Calcium imaging and patch clamp were applied to analyze the actions of ZPT in human vascular endothelial cells. Mouse model of UC was used to evaluate the anti-colitic action of ZPT. ResultsZPT dose-dependently induced mesenteric vasorelaxation predominately through endothelium-dependent hyperpolarization (EDH), which could be attenuated by intracellular Zn2+ and Ca2+ chelators TPEN and BAPTA-AM. The ZPT/EDH-mediated vasorelaxation via TRPV1, TRPV4 and TRPA1 channels was verified by a combination of selective pharmacological inhibitors and TRPV1-KO and TRPV4-KO mice. Moreover. ZPT induced Ca2+ entry via vascular endothelial TRPV1/4 and TRPA1 channels and enhanced membrane non-selective currents through these channels. Notably, ZPT exerted anti-colitic effects by rescuing the impaired acetylcholine (ACh)-induced mesenteric vasorelaxation in colitic mice. ConclusionsZPT/Zn2+ induces EDH-mediated mesenteric vasorelaxation through activating endothelial multiple TRPV1/4 and TPPA1 channels in health, and rescues the impaired ACh-induced vasorelaxation to exert anti-colitic action. Our study may open a new avenue of potential vessel-specific targeted therapy for UC.

Nutrient-induced hyperosmosis evokes vasorelaxation via TRPV1 channel-mediated, endothelium-dependent, hyperpolarisation in healthy and colitis mice.

In humans, blood flow in the mesenteric circulation is greatly increased after meals, but the mechanisms underlying postprandial mesenteric vasorelaxation induced by nutrients and whether this process is involved in the pathogenesis of colitis, are not well understood. Here we have studied the direct actions of nutrients on mesenteric arterial tone and the underlying molecular mechanisms in healthy and colitis mice. Colitis in C57BL/6 mice was induced with dextran sodium sulphate. Nutrient-induced vasorelaxation of mesenteric arterioles from humans and mice was studied with wire myograph assays. Ca2+ and Na+ imaging were performed in human vascular endothelial cells and vascular smooth muscle cells, using selective pharmacological agents and shRNA knockdown of TRPV1 channels. Glucose, sodium and mannitol concentration-dependently induced endothelium-dependent relaxation of human and mouse mesenteric arterioles via hyperosmotic action,. Hyperosmosis-induced vasorelaxation was almost abolished by selective blockers for TRPV1, IKCa and SKCa channels. Glucose markedly stimulated Ca2+ influx through endothelial TRPV1 channels, an effect attenuated by selective blockers and shRNA knockdown of TRPV1 channels. Capsaicin synergised the glucose-induced vasorelaxation. Nutrient-induced hyperosmosis also activated Na+ /K+ -ATPase and the Na/Ca exchanger (NCX) to decrease [Ca2+ ]i in VSMCs. Glucose-induced vasorelaxation was impaired in mouse colitis. Nutrient-induced hyperosmosis evoked endothelium-dependent mesenteric vasorelaxation via the TRPV1/Ca2+ / endothelium-dependent hyperpolarisation pathway to increase normal mucosal perfusion, which is impaired in our model of colitis. The TRPV1/Ca2+ / endothelium-dependent hyperpolarisation pathway could provide novel drug targets for gastrointestinal diseases with hypoperfusion, such as chronic colitis and mesenteric ischaemia.

Estrogen Replacement Improves Mesenteric Arterial Relaxation in UC Davis Pre‐diabetic Rats: Possible Involvement of Estrogen Receptor Beta

The risk of cardiovascular diseases (CVD) is lower in premenopausal women compared to men, and this lower risk is related to the presumptive cardioprotective effects of female sex hormones, estrogen in particular. However, premenopausal women may lose this protection with the onset of menopause and/or when they become diabetic. Little is known about the vascular function in presence or absence of estrogen at the early stage of diabetes.The objective of this study was to investigate the effects of 17β‐estradiol (E2) replacement on mesenteric arterial (MA) function in ovariectomized (ovex) UC Davis type 2 diabetes mellitus (UCD‐T2DM) rats at the pre‐diabetic stage. MA were obtained from four groups of ovex rats implanted with a subcutaneous pellet for 45 days: (1) Control (Ctrl), ovex + placebo, (2) Ctrl, ovex + E2 (1.5mg/pellet), (3) pre‐diabetic, ovex + placebo, (4) pre‐diabetic, ovex + E2. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in MA were measured before and after indomethacin (Indo, 10μM), a cyclooxygenase (COX) inhibitor; followed by addition of L‐NAME (200μM), a nitric oxide synthase (NOS) inhibitor. The MA expression of estrogen receptors (ERs) and plasma E2 concentration were determined by Western blotting and ELISA assay, respectively.E2 treatment significantly increased plasma estradiol concentration in both Ctrl and Prediabetic groups. Analysis of ERs showed a higher expression of ER‐beta only in E2‐treated prediabetic group. ACh responses were similar in Ctrl groups, regardless of E2 replacement. ACh induced relaxation was, however, markedly reduced in MA of placebo treated pre‐diabetic group compared with other groups. Treatment of pre‐diabetic with E2 enhanced relaxation to the same level as those from Ctrl rats. Blocking of both COX and NOS completely abolished the relaxation responses to ACh in pre‐diabetic groups, regardless of E2 replacement, indicating no contribution of EDHF‐type relaxation in these groups. However, the added effect of L‐NAME, in presence of Indo, in blocking ACh‐mediated vasodilation was much more prominent in E2‐treated prediabetic group when compared with placebo treated group, indicating an enhanced role of NO. On the other hand, both NO and EDHF remained important for EDV in Ctrl groups. Addition of L‐NAME, however, caused a much smaller reduction of EDV in E2 treated than in placebo treated arteries, suggesting EDHF is the predominant mediator of mesenteric vasorelaxation in this groups.Here, we show that EDV is impaired at pre‐diabetic stage, may be due to loss of EDHF. E2 treatment restores EDV in MA of pre‐diabetic group, possibly via elevated contribution of NO‐mediated relaxation by increased E2 signaling (via ER beta) in this group. The role of ER alpha remains to be investigated.Support or Funding InformationSupported by NHLBI.

SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity.

Recent evidence implicates the critical role of Sirtuin 3 (SIRT3) in the development of many metabolic diseases, but the contribution of SIRT3 to vascular homeostasis remains largely unknown. The aim of this study was to investigate the role of SIRT3 in endothelial insulin resistance and vascular dysfunction in obesity. We found an impaired insulin-induced mesenteric vasorelaxation and concomitant reduced vascular SIRT3 expression in morbid obese human subjects compared with the non-obese subjects. Downregulation of SIRT3 in cultured human endothelial cells increased mitochondrial reactive oxygen species (mtROS) and impaired insulin signaling as evidenced by decreased phosphorylation of Akt and endothelial nitric oxide synthase and subsequent reduced nitric oxide (NO) release. In addition, obese mice induced by 24-week high-fat diet (HFD) displayed an impaired endothelium-dependent vasorelaxation to both insulin and acetylcholine, which was further exacerbated by the gene deletion of Sirt3. Scavenging of mtROS not only restored insulin-stimulated NO production in SIRT3 knockdown cells, but also improved insulin-induced vasorelaxation in SIRT3 knockout mice fed with HFD. Taken together, our findings suggest that SIRT3 positively regulates endothelial insulin sensitivity and show that SIRT3 deficiency and resultant increased mtROS contribute to vascular dysfunction in obesity.

Abstract 180: Downregulation of GRK2 by Long-Term Exercise Improves Vascular Insulin Sensitivity and Delays the Development of Hypertension in SHRs

Aims: Exercise training, a recommended nonpharmacological strategy for hypertensive patients, exerts beneficial effect on blood pressure modulation, but the underlying mechanisms remain elusive. Vascular insulin resistance is an early pathologic alteration in hypertensive vascular injury and plays a critical role in the development of hypertension. This study aimed to investigate whether long-term physical exercise starting at prehypertensive period prevents the development of hypertension via improving vascular insulin sensitivity. Methods: Young (4-week-old) prehypertensive spontaneously hypertensive rats (SHRs) and their normotensive Wistar-Kyoto controls (WKY) were subjected to a 10-week free-of-loading swim training session (60 min/day, 5 days/week). Insulin-induced mesenteric arteriolar vasorelaxation were determined. Results: SHRs showed higher systolic blood pressure (SBP) and decreased insulin sensitivity of the whole body and resistance vessels compared with those of WKY rats. SBP in the exercised SHRs was significantly lower than that in sedentary ones (180.2±5.1 vs. 205.7±3.7 mmHg, n=6, P<0.05). Vascular insulin sensitivity in mesenteric arteries was improved after exercise training as evidenced by increased vasodilation response to insulin (29.0%±2.5% vs. 18.4%±5.9% to 1 μmol/L insulin, n=6, P<0.05). In addition, exercise downregulated vascular GRK2 expression and activity, which further increased insulin-stimulated vascular Akt/eNOS activation in exercised SHRs. More importantly, suppression of GRK2 with siRNA mimicked the effect of exercise-enhanced vascular insulin sensitivity, while upregulation of GRK2 by Chariot-mediated delivery reversed the exercise-induced vascular insulin sensitization. Conclusions: Long-term regular physical exercise beginning at prehypertensive stage improves vascular insulin sensitivity via downregulating GRK2 and consequently delays the development of hypertension.

Mesenteric endothelial dysfunction in a cardiopulmonary bypass rat model: The effect of diabetes

Diabetes is a risk factor for perioperative complications after cardiac surgery. We studied its effects on mesenteric endothelial function in a cardiopulmonary bypass (CPB) model. Forty Wistar rats were divided into four groups: sham (D-CPB-), cardiopulmonary bypass (D-CPB+), diabetic (D+CPB-) and diabetic that have undergone CPB (D+CPB+). Two samples of mesenteric artery were used for nitric oxide synthase (NOS) Western blot analysis, and two others for assessing contractile response and endothelium relaxations. Nitrite products and tumour necrosis factor-alpha (TNF-α) were assessed as markers of inflammatory response. We observed an enhanced contractile response to the α-adrenergic agonist associated with impairment of mesenteric vasorelaxation in D+CPB+ rats. Western immunoblot analysis of D+CPB+ highlighted an additive effect of hyper-expression of inducible NOS. A significantly increased inflammatory response was observed after CPB in diabetic animals. This work confirms the potential deleterious impact of diabetes on the mesenteric endothelium during CPB in cardiac surgery.

The Endogenous Brain ConstituentN-Arachidonoyl l-Serine Is an Activator of Large Conductance Ca2+-Activated K+Channels

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.

Role of Ca2+‐dependent potassium channels in in vitro anandamide‐mediated mesenteric vasorelaxation in rats with biliary cirrhosis

Anandamide can activate potassium (K(+)) channels to induce an endothelium-dependent vasorelaxation in normal rat mesenteric arteries. Cannabinoids contribute partly to the splanchnic vasodilation in cirrhosis. This study investigated the roles of vascular K(+) channels in anandamide-induced mesenteric vasorelaxation in isolated rat cirrhotic vessels. The effects of the pretreatment of AM251, a specific CB(1) receptor antagonist, were assessed on the vascular reactivity to phenylephrine (PE), potassium chloride (KCl), acetylcholine (ACh) and sodium nitroprusside (SNP). Additionally, cannabinoid (CB(1) and CB(2)) receptors' protein expression and the effects of different K(+) channel blockers on vascular reactivity to anandamide were also studied. Cirrhotic mesenteric arteries showed an overexpression of CB(1) receptor associated with hyporeactivity to PE and KCl, and hyper-response to ACh, SNP and anandamide. Pretreatment with AM251 significantly improved the hyporeactivity to KCl and ameliorated the hyper-response to ACh in cirrhotic vessels. Increased relaxation response to anandamide was suppressed by combinations of vascular Ca(2+)-dependent K(+) channel blockers (including apamin+charybdotoxin+iberiotoxin or apamin+TRAM-34+iberiotoxin) (TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole). In cirrhotic mesenteric arteries, vascular CB(1) receptor and anandamide contribute to the in vitro hyporeactivity to KCl. In addition, hyper-response to ACh may probably act through the modulation of vascular Ca(2+)-dependent K(+) channels.

Duplex ultrasound for assessment of superior mesenteric artery blood flow.

Duplex ultrasound (DU) is recognised as a valuable tool for the assessment of blood flow in many vascular territories. The application of this technique to the superior mesenteric artery (SMA) has increased rapidly throughout the last decade. The purpose of this review is to collate currently available information on the utility of SMA DU, both in terms of research and clinical practice. Research investigations have revealed low intra- and interobserver variability in the estimation of Doppler variables, while reliable evaluation of B-mode dimensions requires repeated measurements. SMA blood flow velocity has been found to be dependent upon changes in central haemodynamics and in peripheral resistance, which was documented in studies with hypotension, medication and post-prandially. Food intake induces mesenteric vasorelaxation reflected by a 10-fold increase in the diastolic velocity. This feature has been utilised in studies on mesenteric physiology, which confirmed parasympathetic activity during hypovolaemia, and showed that exercise increases splanchnic resistance and reduces its blood flow following a 50% reduction in the hepato-splenic and a 25% reduction in the mesenteric blood flow. Clinical studies have documented high sensitivity and specificity of DU in detection of disease in splanchnic arteries. Diastolic velocity was found to be the most accurate indicator of SMA stenosis, while an absent Doppler signal from a well visualised vessel has been found to be a reliable predictor of occlusion. The high predictive value of DU in the detection of mesenteric artery disease, together with its simplicity and non-invasiveness, suggests that DU should take precedence over arteriography in both clinical practice and laboratory investigations.

The impact of biochemical methods for single muscle fibre analysis.

Biochemical methods for single muscle fibre analysis provide sensitive measures for elucidating muscle fibre heterogeneity. The understanding of the complexity of skeletal muscle fibres, initially based on qualitative histochemistry and immunohistochemistry, has been greatly expanded by quantitative micromethods, such as microphotometry and microbiochemical assays. Assessment of metabolic enzyme activity levels has revealed pronounced scattering within and between different fibre types and has highlighted the use of specific enzyme activity ratios as discriminative measures. With the exception of type I fibres, metabolic properties are loosely coupled with molecular properties of the myofibrillar apparatus. As such, myosin heavy chain (MHC) isoforms appear to be the best choice for fibre type delineation. Among the two available methods for MHC-based fibre type distinction, single fibre electrophoresis appears to be superior to immunohistochemistry. The electrophoretic separation of MHC isoforms in single fibres is quantitative and, as opposed to immunohistochemistry, yields important information on MHC isoform proportions in hybrid fibres. Histochemical staining for myofibrillar ATPase activity can, thus, be correlated in most cases with specific MHC isoform profiles. Single fibre studies have demonstrated a relationship between ATP phosphorylation potential and MHC isoform complement. This relationship corresponds to different tension costs and provides an additional rationale for the MHC-based fibre type diversity and transitions. The combination of reverse transcriptase (RT) with polymerase chain reaction (PCR) has proved to be a highly sensitive tool and has extended single fibre analysis to the level of MHC mRNA isoforms. Application of RT-PCR techniques to single fibre fragments identified by their MHC protein isoform profile, provides insights at two levels of expression and, thus, has extended our knowledge on the plasticity of muscle and the dynamical state of muscle fibres.