Acetylcholine-induced Dilation Research Articles

Flow- and acetylcholine-induced dilatation in small arteries from rats with renovascular hypertension — effect of tempol treatment

We investigated whether renovascular hypertension alters vasodilatation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) and the influence of the superoxide dismutase mimetic tempol on vasodilatation. One-kidney one-clip hypertensive Sprague–Dawley rats, treated with either vehicle or tempol (from weeks 5 to 10 after placement of the clip), and uninephrectomized control rats were investigated. In renal hypertensive rats systolic blood pressure increased to 171 ± 6 mmHg ( n = 10), while in tempol-treated rats systolic blood pressure remained normal (139 ± 7 mmHg, n = 5). In isolated pressurized mesenteric small arteries NO-mediated dilatation was obtained by increasing flow rate and EDHF-mediated dilatation by acetylcholine. In arteries from hypertensive rats, flow-induced dilatation was blunted, as compared to normotensive and tempol-treated rats, while acetylcholine-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol-treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads to increased generation of superoxide contributing to decreased NO bioavailability. Furthermore, the abnormal endothelium function can be corrected by tempol treatment, but this seems to involve mechanisms partly independent of NO.

Deletion of PTP1B Corrects Obesity‐Induced Mesenteric Endothelial Dysfunction

Obesity has been linked to endothelial dysfunction (ED) in humans. Obese mice show ED, but the mechanisms of this are unknown. We hypothesized that insulin resistance is the underlying mechanism responsible for obesity-induced ED. Obese Leptin receptor knockout mice (KH, n=12) and insulin sensitive Protein Tyrosine Phosphotase-1B knockout (HK, n=9) mice were bred to make insulin sensitive obese dual knockout mice (KK, n=7). Lean mice (HH, n=14) were used as a control. Small mesenteric arteries (SMA, 25–150 μm) were mounted in vitro, pressurized (60 mmHg) and ED was assessed by acetylcholine-induced dilation (Ach, 10−8–10−3 M). Passive mechanics were assessed in Ø-Ca2+-Krebs solution over 40–120 mmHg. HbA1C was increased in KH vs. HH (10.0±.1% vs. 5.0±.1%, p<0.05) but reduced in KK vs. KH (7.0±.4% vs. 10±.7%, p<0.05). KH were much less reactive to Ach than HH (max=50±4% vs. 70±3%, p<0.05). HK had a similar Ach response to HH (71±2% vs. 70±3%). KK reduced the ED shown in KH (64±4% vs. 50±4%, p<0.05). Passive mechanics were similar in all groups, thus ruling out structural changes as a reason for KH dilator deficits. We conclude that obesity induces ED in SMA secondary to insulin resistance. NIH,HL76533

Impaired Endothelium-Derived Hyperpolarizing Factor-Mediated Dilations and Increased Blood Pressure in Mice Deficient of the Intermediate-Conductance Ca 2+ -Activated K + Channel

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca(2+)-activated K(+) channels (K(Ca)), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K(Ca) (IK(Ca)/K(Ca)3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K(Ca)3.1. K(Ca)3.1-deficient mice (K(Ca)3.1(-/-)) were generated by conventional gene-targeting strategies. Endothelial K(Ca) currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K(Ca)3.1 gene abolished endothelial K(Ca)3.1 currents and significantly diminished overall current through K(Ca) channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K(Ca)3.1(-/-) mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K(Ca)3.1(-/-) mice. Moreover, the loss of K(Ca)3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K(Ca)3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Allicin in garlic protects against coronary endothelial dysfunction and right heart hypertrophy in pulmonary hypertensive rats

We recently reported that coronary endothelial cell (CEC) dysfunction may contribute to the development of right ventricular (RV) hypertrophy (RVH) in monocrotaline (MCT)-induced pulmonary hypertensive rats. This present study investigated whether preservation of CEC function with garlic and its active metabolite allicin could abrogate RVH. Rats were fed with 1% raw garlic (RG)-supplemented diet 1 day or 3 wk before and 1 day after MCT injection, and changes in RV pressure (RVP), RVH, and CEC function were assessed 3 wk after MCT administration. In all cases, RG feeding significantly inhibited the development of RVP and RVH in these MCT rats. However, similar treatments with either boiled garlic (BG) or aged garlic (AG), which do not contain the active allicin metabolite, were ineffective. CEC function, assessed with acetylcholine-induced dilation as well as N(omega)-nitro-l-arginine methyl ester-induced constriction, revealed marked attenuation in right, but not left, coronary arteries of the MCT rats. This is consistent with our earlier report. Feeding of RG, but not BG or AG, preserved the CEC function and prevented the exaggerated vasoconstrictory responses of the MCT coronary arteries. There was no change in the coronary dilatory responses to a nitric oxide donor sodium nitroprusside. Further testings of vasoactivity to garlic extracts showed that only RG, but not BG or AG, elicited a potent, dose-dependent dilation on the isolated coronaries. Taken together, these findings show that the protective effect of garlic against the development of RVP and RVH in MCT-treated rats is probably mediated via its active metabolite allicin action on coronary endothelial function and vasoreactivity.

Both endothelium and afferent nerve endings play a role in acetylcholine-induced renal vasodilation

We investigated the nature and signaling pathways of endothelium- and sensory-nerve ending-derived substances involved in acetylcholine-induced vasodilation in rat isolated perfused kidney. Endothelial denudation by Triton X-100 (0.2%, 0.1 ml) or depletion of afferent nerve endings by capsaicin (10 − 6 mol/l) attenuated acetylcholine-induced vasodilation. When these two agents were administered together, the response to acetylcholine was completely inhibited. CGRP1 receptor blocker CGRP 8–37 (10 − 7 mol/l) and adenosine A 2 receptor antagonist ZM 241 385 (10 − 7 mol/l) inhibited acetylcholine-induced dilation. When indomethacin (10 − 5 mol/l), a cyclooxygenase inhibitor, l-NOARG (10 − 4 mol/l), a nitric oxide (NO) synthase inhibitor, and potassium chloride (30 mmol/l), to test EDHF response, were perfused simultaneously, the inhibition was greater than that was observed with each agent alone. Guanylate cyclase inhibitor ODQ (10 − 5 mol/l) or protein kinase A inhibitor KT 5720 (5 × 10 − 7 mol/l) inhibited acetylcholine-induced dilation. Gap junction uncoupler 18α-glycyrrhetinic acid (10 − 4 mol/l) caused an uncontrollable increase in basal perfusion pressure making it impossible to test against acetylcholine-induced dilation. Our data suggest that NO, prostanoids, EDHF, and CGRP released from vascular endothelium and afferent nerve endings participate in acetylcholine-induced vasodilation and their signal transduction molecules include protein kinase A and guanylate cyclase.

Effects of extracellular superoxide dismutase on cerebrovascular function

Background Extracellular superoxide dismutase (ecSOD) is abundant in the wall of blood vessels. Effects of ecSOD deficiency on cerebrovascular function are not fully elucidated. The aim of this study was to examine the role of ecSOD in modulation of vasomotor function of cerebral arterioles. Methods Responses of cerebral arterioles (diameter 26±1 μm) in ecSOD knockout (KO) mice were examined using a cranial window in anesthetized, ventilated mice. Wild-type (WT) littermate mice were used as control. Results Total SOD activity of aorta in ecSOD KO mice was 23 % lower than in WT mice. Dilator responses to acetylcholine were similar in ecSOD WT and KO mice (34±5 [mean±SE] vs. 32±4 % to 10 μM of acetylcholine). During superfusion of angiotensin II (500 nM for 30 min), dilator responses to acetylcholine tended to be reduced in WT mice (to 28±6; n.s.). In ecSOD KO mice, acetylcholine-induced dilatation was impaired significantly (to 18±4; *p=0.01) (Figure). Nitroprusside-induced dilator responses were not affected by angiotensin II in ecSOD KO and WT mice. In the presence of the superoxide scavenger tempol (1 mM for 30 min) and angiotensin II, dilator responses to acetylcholine were restored in ecSOD KO mice (to 30±4 %). Conclusions These results indicate that lack of ecSOD does not impair endothelial function under baseline conditions. Endogenous ecSOD, however, protects cerebral arterioles against oxidative stress induced by angiotensin II, and helps to preserve endothelial function.

Enhanced acetylcholine-induced dilation in afferent arterioles in simvastatin-fed rats

Objective 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to increase renal blood flow and glomerular filtration rate independent of their lipid lowering effects. The purpose of this study was to determine the effect of simvastatin on acetylcholine-induced vasodilation in the renal microcirculation. The hypothesis of the study was that simvastatin would increase acetylcholine-induced vasodilation in the renal microcirculation. Methods The hydronephrotic kidney preparation was used. On the day of the experiment the kidney was prepared for videomicroscopy and dose–response curves were done with acetylcholine and sodium nitroprusside (10 − 10 M to10 − 5 M) in simvastatin-fed rats ??( n = 8) and control rats ( n = 13). The vasodilator responses of afferent and efferent arterioles were directly quantitated using videomicroscopy. l-NAME; ( N(omega)-nitro- l-arginine methyl ester) was also given in a group of simvastatin-fed rats (10 −5 M) to determine if it would block the acetylcholine (Ach)-induced vasodilation. Results Simvastatin enhanced Ach-induced vasodilation in the afferent arteriole compared to control rat responses to Ach. At 10 − 7 M, ACH caused a 31.6 ± 7.2% increase from baseline diameter in the afferent arteriole in the simvastatin-fed rats compared to a 23 ± 8.1% vasodilation in the control rats ( p < 0.05). There were no differences in the response to ACH in the efferent arteriole between the two groups. l-NAME completely abolished the ACH response in the simvastatin-fed rats. Conclusions: The increase in renal blood flow and glomerular filtration rate observed with simvastatin may be due to its preglomerular vasodilator effects. This may be due to an increase in nitric oxide production.

Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin.

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).

Bone medullary arterioles from ovariectomized rats have smaller baseline diameters but normal eNOS expression and NO-mediated dilation

This study was designed to test the hypothesis that endogenous estrogens decrease the expression of endothelial nitric oxide synthase (eNOS) in resistance-size bone arterioles, thereby reducing endothelium-dependent vasodilator function. Sexually mature female rats were ovariectomized to reduce endogenous estrogens. Age-matched female rats served as controls. Seven to ten days after ovariectomy, bone marrow tissue was collected from the femoral canal. Immuno-histochemistry was performed to detect expression of estrogen receptors, α and β and eNOS. eNOS protein content in medullary bone arterioles was compared using Western blot analysis. Endothelial cell function was assessed by quantitating the dilation of isolated, pressurized bone arterioles in response to acetylcholine. The results indicate that the endothelium of bone arterioles from ovariectomized and control rats express ER-α, ER-β and eNOS. eNOS protein content in the two groups of arterioles did not differ. However, the baseline diameter of arterioles from ovariectomized rats (63 ± 4 μm) was significantly smaller than the diameter of arterioles from control rats (75 ± 3 μm, p < 0.05). The two groups of arterioles dilated equally in response to acetylcholine. l-NAME, an inhibitor of eNOS, almost completely abolished the dilator responses to acetylcholine, but not to sodium nitroprusside. l-Arginine restored acetylcholine-induced dilation after l-NAME treatment. Thus, arteriole dilation to acetylcholine appears to be mediated almost exclusively by NO. The smaller diameter of arterioles from ovariectomized rats suggests that endogenous estrogens exert a significant dilator influence on bone arterioles. However, the dilator influence does not appear to be mediated by an increase in eNOS expression or enhanced NO-dependent vasodilation. These results indicate that estrogens do not decrease eNOS expression or diminish NO-mediated dilation of bone medullary arterioles.

Microvascular effects of corticotropin-releasing hormone in human skin vary in relation to estrogen concentration during the menstrual cycle

Females have a significantly greater life expectancy than males, which in part may be due to the cardio-protective effects of the female sex hormone, estrogen, on vascular function. However, the sex-specific mechanisms contributing to these differences are complex and not fully understood. Previously we have reported that corticotropin-releasing hormone (CRH) has potent dilator effects in the female skin circulation via mast cell degranulation. Furthermore the dilator response to CRH was more enhanced in females than in age-matched males, suggesting that estrogens may be involved. In this study we examined whether CRH-induced dilation and endothelial cell-dependent dilation in the skin circulation of pre-menopausal females were associated with changes in estrogen during the menstrual cycle. CRH-induced dilation (1 nM) was enhanced in the presence of high circulating concentrations of estrogen and a positive correlation was identified between CRH-induced dilation and plasma estrogen concentrations. Endothelial cell-dependent dilation was examined using acetylcholine. Acetylcholine-induced dilation (1 nM) was not correlated with circulating concentrations of estrogen. These data suggest the variation in CRH-induced dilation in the skin microvasculature during the menstrual cycle may be due to estrogenic effects on mast cell function and not due to direct changes in endothelial cell function.

Stiffening Our Resolve Against Adult Weight Gain

Increasing adiposity with a clinical endpoint of obesity is associated with a progressive increase in risk of cardiovascular diseases (CVD). However, we are only beginning to understand the physiological and pathophysiological mechanisms by which excessive body fat storage causes CVD. In this issue of Hypertension , the findings of Wildman et al1 extend and strengthen previous observations2–5 by showing that changes in arterial stiffness, as measured by aortic pulse-wave velocity (PWV), are related to changes in body weight over a 2-year follow-up period in healthy men and women aged 20 to 40 years. Weight gain was associated with an increase, whereas weight loss was associated with a decrease in aortic PWV, independent of changes in blood pressure. The greatest increases in aortic PWV were observed in those subjects who gained the most weight (≥4.5 kg). Importantly, the changes in aortic PWV were related to changes in body mass index (BMI), indicating that the associations with body weight were mediated by changes in adiposity (rather than fat-free mass). Changes in aortic PWV over time also were related to baseline body weight, BMI, and waist circumference (an indirect measure of abdominal adiposity), providing evidence that higher initial levels of total and abdominal body fatness are associated with greater future increases in arterial stiffness. The study included subjects with a broad range of BMI, suggesting that the findings are applicable to both nonobese and obese adults in the general population. An important finding was that black adults demonstrated greater increases in weight gain-adjusted and blood pressure-adjusted aortic PWV over time, suggesting that they undergo greater increases in arterial stiffness in response to the same age-associated weight gain compared with whites. These observations provide further support for the emerging concept that adiposity-driven weight gain during adulthood contributes significantly to the adverse changes …

CGMP-Dependent Protein Kinase Mediates NO- but not Acetylcholine-Induced Dilations in Resistance Vessels In Vivo

cGMP and cGMP-dependent protein kinase type I (cGKI) mediate the dilation of large vessels in response to NO and acetylcholine (ACh). However, the physiological significance of the NO/cGMP/cGKI pathway in resistance vessels is controversial. Here, we analyzed NO- and ACh-induced dilations of arterioles in cGKI-deficient (cGKI-/-) or endothelial NO synthase-deficient (eNOS-/-) mice. Mean arterial pressure was similar in cGKI-/- and wild-type mice (105 mm Hg). Pressure drops in response to intracarotid bolus application of the NO donor sodium nitroprusside (SNP) were almost abolished in cGKI-/- mice, whereas ACh-induced pressure decreases remained intact in cGKI-/- and eNOS-/- mice. The direct observation of arterioles in the cremaster muscle by intravital microscopy showed impaired SNP-induced dilations in cGKI-/- mice (by 80%) and normal ACh-induced dilations in cGKI-/- and eNOS-/- mice. ACh-induced dilations in eNOS-/- mice were attenuated by iberiotoxin (by 50%), indicating that they were mediated in part by Ca2+-activated K+ channels, but not by inhibitors of cyclooxygenase or p450-monooxygenases. We conclude that cGMP and cGKI are the major effectors of NO to induce acute dilations of murine resistance vessels. However, the NO/cGMP/cGKI pathway is not essential for ACh-induced dilation of arterioles and for basal blood pressure regulation in mice.

Impaired nitric oxide‐ and endothelium‐derived hyperpolarizing factor‐dependent dilation of renal afferent arteriole in Dahl salt‐sensitive rats

We previously demonstrated that acetylcholine elicited nitric oxide-dependent sustained and endothelium-derived hyperpolarizing factor (EDHF)-dependent transient dilation of afferent arterioles. The present study examined whether free radicals interacted with nitric oxide-dependent and EDHF-dependent vasodilator mechanisms in renal microvessels of salt-sensitive hypertension, using the isolated perfused hydronephrotic kidney. Following the pretreatment with indomethacin (100 micromol/L) with or without nitro- l-arginine methylester (100 micromol/L), the effect of acetylcholine on noradrenaline (0.3 micromol/L)-induced constriction was evaluated in kidneys from Dahl salt-sensitive and salt-resistant rats. Although acetylcholine (0.01-10 micromol/L) caused dose-dependent and sustained vasodilation of afferent arterioles, attenuated dilation was observed in Dahl salt-sensitive rats, compared with that in salt-resistant rats (58 +/- 4 vs 101 +/- 11% reversal at 10 micromol/L acetylcholine). In the presence of nitro- l-arginine methylester, acetylcholine elicited only transient dilation, with vasodilator response blunted in Dahl salt-sensitive rats (64 +/- 4 vs 100 +/- 9% reversal at 10 micromol/L acetylcholine). Furthermore, chronic (8-10 weeks) treatment with tempol caused partial restoration of acetylcholine (10 micromol/L)-induced sustained arteriolar dilation (71 +/- 3% reversal), but complete reversal of transient dilation (92 +/- 4% reversal). Finally, acute treatment with tempol not only improved the sustained component of the acetylcholine-induced dilation but also restored the impaired responsiveness of transient dilation in Dahl salt-sensitive rats. Both sustained (nitric oxide-mediated) and transient (EDHF-mediated) components of acetylcholine-induced afferent arteriolar dilation were attenuated in Dahl salt-sensitive rats, which was attributed, in part, to enhanced free radical activity. A reversal of the sustained and transient vasodilation by the acute tempol treatment suggests possible interaction between free radicals and EDHF as well as increased bioavailability of nitric oxide.

Influence of exercise on dilatation of the basilar artery during diabetes mellitus

Our goal was to examine whether exercise training alleviates impaired nitric oxide synthase (NOS)-dependent dilatation of the basilar artery in Type 1 diabetic rats. To test this hypothesis, we measured in vivo diameter of the basilar artery in sedentary and exercised nondiabetic and diabetic rats in response to NOS-dependent (acetylcholine) and -independent (nitroglycerin) agonists. To determine the potential role for nitric oxide in vasodilatation in sedentary and exercised nondiabetic and diabetic rats, we examined responses after NG-monomethyl-l-arginine (l-NMMA). We found that acetylcholine produced dilatation of the basilar artery that was similar in sedentary and exercised nondiabetic rats. Acetylcholine produced only minimal vasodilatation in sedentary diabetic rats. However, exercise alleviated impaired acetylcholine-induced vasodilatation in diabetic rats. Nitroglycerin produced dilatation of the basilar artery that was similar in sedentary and exercised nondiabetic and diabetic rats. l-NMMA produced similar inhibition of acetylcholine-induced dilatation of the basilar artery in sedentary and exercised nondiabetic and diabetic rats. Finally, we found that endothelial NOS (eNOS) protein in the basilar artery was higher in diabetic compared with nondiabetic rats and that exercise increased eNOS protein in the basilar artery of nondiabetic and diabetic rats. We conclude that 1) exercise can alleviate impaired NOS-dependent dilatation of the basilar artery during diabetes mellitus, 2) the synthesis and release of nitric oxide accounts for dilatation of the basilar artery to acetylcholine in sedentary and exercised nondiabetic and diabetic rats, and 3) exercise may exert its affect on cerebrovascular reactivity during diabetes by altering levels of eNOS protein in the basilar artery.

Changes in endoneurial blood flow, motor nerve conduction velocity and vascular relaxation of epineurial arterioles of the sciatic nerve in ZDF-obese diabetic rats.

We have previously reported that in streptozotocin-induced diabetic rats, reduction in endoneurial blood flow (EBF) and impairment of acetylcholine-mediated vascular relaxation of arterioles that provide circulation to the sciatic nerve precedes slowing of motor nerve conduction velocity (MNCV). However, in animal models of type 2 diabetes it is unknown whether slowing of MNCV is accompanied by vascular dysfunction. Using ZDF-lean and ZDF-obese diabetic rats we examined whether diabetes-induced slowing in MNCV was associated with a reduction in EBF and impaired vascular relaxation in epineurial arterioles of the sciatic nerve. We measured MNCV in the sciatic nerve using a non-invasive procedure, and sciatic nerve nutritive blood flow using microelectrode polarography. In vitro videomicroscopy was employed to quantify arteriolar diameter responses to acetylcholine in arterioles overlying the sciatic nerve. MNCV and EBF in hyperglycemic (4-week duration) ZDF-obese diabetic rats were significantly decreased by 30% and 63%, respectively, compared to age-matched ZDF-lean rats. Acetylcholine elicited a dose-dependent dilation of epineurial vessels from ZDF-lean and ZDF-obese diabetic rats, although acetylcholine-induced dilation was significantly reduced in ZDF-obese diabetic rats. Determination of markers of oxidative stress provided ambiguous results. Superoxide levels were increased in epineurial vessels from ZDF-obese diabetic rats. Lens glutathione levels were decreased and serum thiobarbituric acid reactive substances increased in ZDF-obese diabetic rats but sciatic nerve conjugated diene and glutathione levels were not significantly different compared to ZDF-lean rats. Diabetes causes a reduction in EBF and impairment in vascular relaxation in epineurial vessels in ZDF-obese diabetic rats. This impaired vascular response is associated with neural dysfunction.

Impaired hyperpolarization in regenerated endothelium after balloon catheter injury.

Ca(2+)-activated K(+) (K(Ca)) channels control endothelial Ca(2+) homeostasis and the formation of vasodilators. After angioplasty, dysfunction of the regenerated endothelium leads to abnormal vasoregulation. In this study, we tested the expression and function of K(Ca) channels in regenerated endothelium at 6 weeks after balloon catheter injury of rat carotid arteries (CAs) by using single-cell reverse transcription-polymerase chain reaction, patch-clamp techniques, and analysis of vasoreactivity. In single regenerated endothelial cells (ECs), the percentage of ECs expressing the K(Ca) genes, rSK3 (12+/-8%) and rIK1 (22+/-9%), was significantly lower compared with the percentage of native ECs expressing these genes (rSK3 58+/-8%, rIK1 64+/-10%). In patch-clamp experiments, K(Ca) currents and acetylcholine-induced hyperpolarization were markedly reduced in regenerated ECs (shift of membrane potential -6+/-3 mV) compared with those in native ECs (shift of membrane potential -21+/-5 mV). In pressure myograph experiments, acetylcholine-induced dilation was impaired in reendothelialized CAs compared with normal CAs. Intraluminal application of the K(Ca) blocker apamin and charybdotoxin inhibited dilation by 30% in normal CAs but was without effect in reendothelialized CAs. Intraluminal application of 1-ethyl-2-benzimidazolinone (100 micromol/L), an opener of K(Ca) channels, evoked dilation by 29% in normal CAs but had no effect in reendothelialized CAs. In conclusion, the impaired expression of K(Ca) channels in regenerated endothelium results in defective hyperpolarization and impaired dilation. Thus, the impaired K(Ca) channel function contributes to functional alterations of regenerated endothelium after angioplasty.

Role of Na +/H + exchanger in dilator responses of rat basilar artery in vivo

We tested the hypothesis that activation of Na +/H + exchanger is involved in dilator responses of the basilar artery to endothelium-dependent vasodilators in vivo. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to acetylcholine and bradykinin. Topical application of acetylcholine and bradykinin increased diameter of the basilar artery in a concentration-related manner. Because N G-nitro- l-arginine, an inhibitor of nitric oxide synthase, almost abolished vasodilator responses to acetylcholine and bradykinin, vasodilatation produced by the agonists appears to be mediated primarily by nitric oxide. 5- N, N-Hexamethyleneamiloride, an inhibitor of Na +/H + exchanger, did not affect baseline diameter of the basilar artery, but inhibited vasodilatation in response to acetylcholine and bradykinin, without affecting vasodilatation produced by sodium nitroprusside. FR183998, another inhibitor of Na +/H + exchanger, also attenuated acetylcholine-induced dilatation of the basilar artery without affecting vasodilatation in response to sodium nitroprusside. Monomethylamine hydrochloride, which produces intracellular alkalinization, enhanced acetylcholine-induced dilatation of the basilar artery in the presence of 5- N, N-hexamethyleneamiloride. These results suggest that intracellular alkalinization produced by activation of Na +/H + exchanger may enhance nitric oxide production in the basilar arterial endothelium and thereby contribute to dilator responses of the artery in vivo.

Sepsis Increases NOS-2 Activity and Decreases Non-NOS-Mediated Acetylcholine-Induced Dilation in Rat Aorta

Introduction. Acetylcholine (Ach) is frequently used to assess endothelium-dependent vasodilation during sepsis. However, the effects of sepsis on constitutive nitric oxide synthase activity (NOS-1 and -3) and other non-NOS effects of Ach are unclear.Methods. Sepsis was induced in rats by inoculation of an implanted sponge with Escherichia coli and Bacteroides fragilis (109 CFU each). Thoracic aortic rings (2 mm) were harvested at 24 h from septic (N = 9) and control (N = 9) rats and were suspended in physiological salt solution (PSS), PSS + l-N6-(1-iminoethyl)lysine (l-NIL: NOS-2 inhibitor, 10 μM), or PSS + l-NG-monomethylarginine (l-NMMA: NOS-1, -2, and -3 inhibitor, 60 μM). Rings were set at 1-g preload and precontracted with phenlyephrine (10−8 M). Relaxation dose–response curves were generated with six doses of Ach (3 × 10−8 to 10−5 M).Results. Sepsis increased the maximal relaxation to Ach under basal conditions. NOS 2 inhibition with l-NIL decreased Ach-induced relaxation in controls (66% vs 84%, P < 0.05, two-way ANOVA) and more so in septic rats (44% vs 93%, P < 0.05). Total NOS inhibition with l-NMMA decreased Ach-induced relaxation to 45% (P < 0.05) in controls and to 30% (P < 0.05) in septic animals.Conclusions. Inhibition of NOS-1, -2, and -3 failed to abolish Ach-induced relaxation, suggesting the presence of other Ach-induced vasodilator mechanisms. NOS-2 inhibition reduced Ach-induced relaxation by 20–25% in the normal thoracic aorta, but by 50% in septic animals. The remaining Ach-induced non-NOS vasodilation (after inhibition of NOS-1 + NOS-2 + NOS-3) was reduced from 45% in normals to 30% in septic animals. Vascular dysregulation in sepsis is a complex event involving increased NOS-2, decreased NOS-1 + NOS-3, and decreased Ach-induced non-NOS vasodilator mechanisms.

Insulin reverses impaired acetylcholine-induced dilatation of the rat basilar artery during diabetes mellitus

The goal of this study was to determine whether treatment of diabetic rats with insulin reverses impaired nitric oxide synthase-dependent reactivity of the basilar artery in vivo. We measured the diameter of the basilar artery in non-diabetic rats, diabetic (streptozotocin; 50–60 mg/kg i.p.) rats, and diabetic rats treated with insulin implants in response to acetylcholine and nitroglycerin before and following topical application of N G-monomethyl- l-arginine ( l-NMMA). Reactivity of the basilar artery was measured 2–3 months after injection of streptozotocin. We found that topical application of acetylcholine (1.0 and 10 μM) produced dose-related dilatation of the basilar artery in non-diabetic rats, which was inhibited (>80%) by topical application of l-NMMA (10 μM). In diabetic rats, the magnitude of vasodilation produced by acetylcholine was significantly less than in non-diabetic rats. Further, topical application of l-NMMA did not affect dilatation of the basilar artery in diabetic rats in response to acetylcholine. In insulin treated diabetic rats, dilatation of the basilar artery in response to acetylcholine was significantly greater than that observed in either non-diabetic or diabetic rats. In contrast, dilatation of the basilar artery in response to nitroglycerin was similar in insulin treated diabetic rats, non-diabetic rats and diabetic rats. Topical application of l-NMMA only partially inhibited dilatation of the basilar artery in response to acetylcholine in diabetic rats treated with insulin. Thus, the findings of this study suggest that impaired acetylcholine-induced dilatation of the basilar artery during diabetes mellitus can be restored by treatment with insulin. Further, it appears that the contribution of cellular pathways, in addition to nitric oxide synthase, may contribute to dilatation of the basilar artery in response to acetylcholine in rats treated with insulin.

Role of calcium-sensitive K(+) channels and nitric oxide in in vivo coronary vasodilation from enhanced perfusion pulsatility.

In vitro studies support K(+)(Ca) channel-induced smooth muscle hyperpolarization as underlying acetylcholine-mediated (or bradykinin-mediated) vasodilation that persists despite combined nitric oxide (NO) and PGI(2) inhibition. We tested the hypothesis that these channels are activated by enhanced pulsatile perfusion in vivo and contribute substantially to vasodilation from this stimulus. The canine left descending coronary artery was perfused with whole blood at constant mean pressure, and physiological flow pulsatility was set at 40 or 100 mm Hg by computer servo-pump. Cyclooxygenase was inhibited by indomethacin. Mean flow increased +18+/-2% (P:<0.0001) with enhanced pulsatility. This response declined approximately 50% by blocking NO synthase (L-NMMA) or K(+)(Ca) [charybdotoxin (CbTX)+apamin (AP)]. Combining both inhibitors virtually eliminated the flow rise. Inhibiting either or both pathways minimally altered basal coronary flow, whereas agonist-stimulated flow was blocked. Bradykinin-induced dilation declined more with CbTX+AP than with L-NMMA (-66% versus -46%, P:=0.03) and was fully blocked by their combination. In contrast, acetylcholine-induced dilation was more blunted by L-NMMA than by CbTX+AP (-71% versus -44%, P:<0.002) and was not fully prevented by the combination. Substituting iberiotoxin (IbTX) for CbTX greatly diminished inhibition of pulse pressure and agonist flow responses (with or without NOS inhibition). Furthermore, blockade by IbTX+AP was identical to that by AP alone, supporting a minimal role of IbTX-sensitive large-conductance K(+)(Ca) channels. K(+)(Ca) activation and NO comodulate in vivo pulsatility-stimulated coronary flow, supporting an important role of a hyperpolarization pathway in enhanced mechanovascular signaling. Small- and intermediate-conductance K(+)(Ca) channels are the dominant species involved in modulating both pulse pressure- and bradykinin-induced in vivo coronary dilation.