Mesenteric Rings Research Articles

Inhibitory effects of sertraline in rat isolated perfused kidneys and in isolated ring preparations of rat arteries

Sertraline is often prescribed to patients suffering with end stage renal disease, but its action on kidney has not been investigated. We aimed to investigate the pharmacological action of sertraline on rat kidney with emphasis on the underlying mechanisms involved in the vascular actions of the drug. The effects of sertraline were evaluated in rat isolated perfused kidneys and on ring preparations of mesenteric or segmental rat renal artery. In kidneys, sertraline prevented the effects of phenylephrine on perfusion pressure, glomerular filtration rate, urinary flow and renal vascular resistance. In mesenteric rings sertraline inhibited phenylephrine-induced contractions with potency 30-times lower than verapamil. Sertraline reversed sustained contractions induced by phenylephrine or 60mm K(+) within a similar concentration range. In segmental isolated rings, sertraline also reversed contractions induced by phenylephrine or 60mm K(+) with the same concentration range, but with higher potency compared with mesenteric preparations. Under Ca(2+) -free conditions, sertraline did not change the intracellularly-mediated phasic contractions induced by phenylephrine or caffeine. Sertraline was ineffective against contractions induced by extracellular Ca(2+) restoration after thapsigargin treatment and Ca(2+) store depletion with phenylephrine. Conversely, sertraline decreased the contractions induced by Ca(2+) addition in tissues under high K(+) solution or phenylephrine plus verapamil. In rat isolated kidneys and in rat ring preparations of mesenteric or renal vessels, sertraline had antispasmodic effects that appeared to be caused by a direct action on vascular smooth muscle cells. Its actions were ineffective against Ca(2+) -releasing intracellular pathways, but appeared to interfere with sarcolemmal Ca(2+) influx with reduced permeability of both receptor- and voltage-gated Ca(2+) channels.

Oxidative Stress Inhibits Vascular KATP Channels by S-Glutathionylation

The K(ATP) channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-glutathionylation was a modulation mechanism underlying oxidant-mediated vascular K(ATP) channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H(2)O(2)) impaired the K(ATP) channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H(2)O(2) or diamide caused a strong inhibition of the vascular K(ATP) channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H(2)O(2). These results suggest that S-glutathionylation is an important mechanism for the vascular K(ATP) channel modulation in oxidative stress.

Effects of extract, fractions and 2,3-dihydromyricetin-3-O-α-L-rhamnoside from Pradosia huberi (Ducke) Ducke on rat isolated mesenteric arteries

Pradosia huberi (Ducke) Ducke (Sapotaceae), an Amazonian species, is popularly known as casca-doce and used in the folk medicine for the treatment of gastritis. The ethanol extract of the bark contains mainly polyphenolic compounds, which are known to show a large number of activities, including cardioprotective and vasorelaxant effects. The aim of this study was to evaluate the pharmacological properties induced by P. huberi ethanol extract (PHEE) and fractions and 2-3-dihydromyricetin-3-O-α-L-rhamnoside derived from this extract, in isolated rat mesenteric arteries. PHEE was separated and the following fractions were obtained: CHCl3, CHCl3:AcOEt (1:1), AcOEt, AcOEt:MeOH (1:1) and MeOH. We isolated 2-3-dihydromyricetin-3-O-α-L-rhamnoside from the MeOH fraction, which was identified by 1H and 13C NMR spectra and compared with data in the literature. PHEE (1-100 µg/mL) induced concentration-dependent relaxations of 10 µM phenylephrine-induced tone (EC50=17,1±2,9 µg/mL; Emax=87.4±2.9 %, n=8). The MeOH fraction also relaxed mesenteric rings (EC50=31±2.0 µg/mL; Emax=54±12.5%, n=6) but less effectively when compared to PHEE. Both effects were completely abolished after removal of the vascular endothelium. The AcOEt:MeOH (1:1) fraction and the isolated flavonoid were ineffective in eliciting vasorelaxation. The study demonstrates that PHEE and MeOH fraction of Pradosia huberi possess a vasorelaxant effect, which may be completely dependent upon endothelium. The isolated flavonoid is not responsible for this vasorelaxant effect.

Vasorelaxant Action of N-p-Nitrophenylmaleimide in the Isolated Rat Mesenteric Artery

The vasorelaxant response of N-p-nitrophenylmaleimide (4-NO2-NPM) was evaluated. The mesenteric rings (1-2 mm i.d.) were suspended by cotton thread for isometric tension recordings in a Tyrode's solution at 37 degrees C and gassed with a mixture of 95% O2 and 5% CO2, under a resting tension of 0.75 g. 4-NO2-NPM induced relaxation in mesenteric rings pre-contracted with phenylephrine (Phe; 10 microM, pD2 = 6.7 +/- 0.3) or KCl (80 mM, pD2 = 3.9 +/- 0.2). This effect was significantly attenuated after removal of the vascular endothelium, N(G)-nitro L-arginine methyl ester (L-NAME; 100 microM), atropine (1 microM), indomethacin (10 microM), L-NAME + indomethacin or 1H-[1,2,3]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM). L-Arginine (1 mM) reversed the inhibitory effect of L-NAME. In endothelium-intact preparations pre-incubated with 20 mM KCl, tetraethylammonium bromide (TEA; 1 mM) or glibenclamide (Glib; 10 microM), the vasorelaxant effect was significantly attenuated when compared to controls (endothelium intact). In denuded rings, separate incubation with 20 mM KCl, TEA or Glib did not change the relaxation when compared with that obtained in denuded rings. 4-NO2-NPM inhibited in a concentration-dependent and non-competitive manner the concentration-response curves induced by CaCl2. In calcium-free medium, the transient contractions induced by Phe (10 microM) or caffeine (20 mM) were inhibited. The relaxant effect induced by 4-NO2-NPM appeared to be due to endothelial muscarinic receptors activation, NO and prostacyclin release and K(ATP) and BK(Ca) (Ca(2+)-activated K+ channels) endothelium-dependent activation. Inhibition of the Ca2+ influx and inhibition of the Ca2+ release from intracellular IP3- and caffeine-sensitive stores are also involved in the vasorelaxation.

Enhancement of mesenteric artery contraction to 5‐HT depends on Rho kinase and Src kinase pathways in the ob/ob mouse model of type 2 diabetes

Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identified the molecular mechanisms involved. Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay. Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups. These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase.

Reactive oxygen species inhibit vascular KATP channel via S‐glutathionylation

KATP channel is an important regulator of vascular tones. Its opening and closure associated with vasodilation and vasoconstriction are controlled by intra‐ and extracellular messengers. Such a subtle balance may be disrupted in oxidative stress known to be a major player in a variety of cardiovascular diseases. In the vascular diseases reactive oxygen species (ROS) are produced, which in turn contribute to the development of these diseases. To demonstrate how the vascular KATP channel is affected by ROS we performed the studies. In isolated mesenteric rings, the H2O2 treatment impaired vascular responses to vasodilators. To test whether the KATP channel is involved, we studied the cloned Kir6.1/SUR2B channel in HEK cells. H2O2 had only modest inhibitory effect in excised patches in the absence of cytosolic components. The administration of glutathione (GSH) drastically augmented the channel inhibition by micromolar range of H2O2, suggesting that H2O2 inhibits the channel via S‐glutathionylation. This finding was further supported by a variety of thiol modulating regents as well as biochemical experiments. Using mutagenesis screening, residue Cys176 was found to be critical for the channel inhibition. The mutations of Cys176 disrupted the channel responses to both oxidized glutathione and thiol modulating regents. Taken together, these data demonstrate a novel modulation mechanism of KATP channel by ROS.

ALCOHOL DOES NOT MODULATE THE AUGMENTED ACETYLCHOLINE-INDUCED VASODILATORY RESPONSE IN HEMORRHAGED RODENTS

Our previous studies have shown that acute alcohol intoxication (AAI) decreases blood pressure, exacerbates hypotension after hemorrhagic shock, impairs the pressor response to fluid resuscitation, and blunts neuroendocrine activation. We hypothesized that impaired hemodynamic compensation during and after hemorrhagic shock in the acute alcohol-intoxicated host is the result of blunted neuroendocrine activation or, alternatively, of an impaired vascular responsiveness to vasoactive agents. The aim of this study was to examine the effects of AAI, AAI and hemorrhagic shock, and AAI and hemorrhagic shock and resuscitation on reactivity of isolated blood vessel rings to phenylephrine and acetylcholine. Chronically instrumented, conscious male Sprague-Dawley rats (300-350 g) received a primed continuous 15-h intragastric alcohol infusion (2.5 g x kg(-1) + 300 mg x kg(-1) x h(-1)), and time-matched controls received an isocaloric-isovolumic dextrose infusion. At completion of infusions, animals were randomized to sham, 60-min fixed-pressure hemorrhage, or hemorrhagic shock followed by resuscitation with lactated Ringer's solution. At the completion of the experimental protocols, animals were killed, and thoracic aorta and mesenteric artery ring segments (1-2 mm) were prepared and studied in myograph baths. Acute alcohol intoxication did not produce significant alterations in either pressor or dilator responses in aortic or mesenteric rings. These findings suggest that impaired hemodynamic counterregulation during hemorrhagic shock in AAI is not due to decreased vasopressor responsiveness. However, our results suggest a role for accentuated vasodilatory responses that may be central in progression to decompensatory shock.

Effect of L-carnitine supplementation on the sGC/cGMP pathway in vascular relaxing responses from exercised rats

Background It has been largely documented the beneficial effects of physical training on the endothelium-derived relaxing response by increasing nitric oxide production and/or its bioavailability to the smooth muscle [1]. L-carnitine (LCar) has been used as important supplement to regulate body composition associated with lipid metabolism. However, no studies exist investigating the effect of L-Car intake associated with dynamic exercise on the NO-independent sGC/cGMP pathway in the vascular responsiveness in rats. Thus, the aim of this work was to investigate the effect of L-Car supplementation on the NO-independent sGC/cGMP pathway in aortic and mesenteric rings in trained rats.

Hypotensive and endothelium-independent vasorelaxant effects of methanolic extract from Curcuma longa L. in rats

Ethnopharmacological relevance Curcuma longa L. (CL) is a yellow rhizome that is used in African traditional medicine to treat palpitation, hypertension or other related blood circulation disorders. Aim of the study To justify the use of CL in ethnomedicine, we investigated the vasorelaxant effect of methanolic extract of CL (CLME) and its underlying mechanisms in isolated rat mesenteric artery. Materials and methods The effect of CLME on the mean arterial pressure (MAP) and heart rate (HR) (pulse interval) were determined in vivo in non-anaesthetized rats. Superior mesenteric rings were isolated, suspended in organ baths containing Tyrode solution at 37 °C and gassed with 95% O 2 + 5% CO 2, under a resting tension of 0.75 g. The vasorelaxant effects of CLME were studied by means of isometric tension recording experiments. Results In normotensive rats, CLME (10, 20 and 30 mg/kg, i.v.) induced dose-dependent hypotension (2.0 ± 0.5%; 27.1 ± 5.0% and 26.7 ± 4.6%, respectively), and pronounced bradycardia (5.8 ± 1.2%, 19.3 ± 3.2% and 22.9 ± 4.6%, respectively). CLME (1–1000 μg/mL) induced concentration-dependent relaxation of tonic contractions evoked by phenylephrine (Phe) (10 μM) and KCl (80 mM) in rings with intact-endothelium ( E max = 82.3 ± 3.2% and 97.7 ± 0.7%) or denuded-endothelium ( E max = 91.4 ± 1.0% and 97.8 ± 1.1%). Also, in a depolarized, Ca 2+ free medium, CLME inhibited CaCl 2 (1 μM–30 mM)-induced contractions and caused a concentration-dependent rightward shift of the response curves, indicating that CLME inhibited the contractile mechanisms involving extracellular Ca 2+ influx. In addition, in Ca 2+ free media containing EGTA (1 mM), CLME inhibited the transient contraction of denuded rings constricted with Phe, but not those evoked by caffeine (20 mM). In contrast, neither glibenclamide, BaCl 2, tetraethylammonium nor 4-aminopyridine affected CLME-induced relaxation. Conclusions These results demonstrate the hypotensive and bradycardic effects of CLME, as well as its potent vasodilation of rat mesenteric arteries. These effects, may in part, be due to the inhibition of extracellular Ca 2+ influx and/or inhibition of intracellular Ca 2+ mobilization from Phe-sensitive stores.

The Rho-A/Rho-kinase pathway is up-regulated but remains inhibited by cyclic guanosine monophosphate–dependent mechanisms during endotoxemia in small mesenteric arteries*

We investigated whether a reduced activity in the Rho-A/Rho-kinase pathway could be involved in the impaired vascular reactivity observed in septic shock. Ex vivo animal study. University research laboratory. Male Wistar rats. Rats received an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg) either 6 or 24 hours before the onset of our experiments. The effects of Y-27632 (a Rho-kinase inhibitor) were assessed in first-order mesenteric rings taken from these animals using wire myograph. The expression of Rho-A, Rho-kinases I and II, and the total and phosphorylated myosin phosphatase targeting subunit 1 (MYPT1) were assessed by Western blotting. The EC50 to Y-27632 was reduced from 2.10 microM (1.22-3.66 microM) (control) to 0.21 microM (0.09-0.44 microM), and 9.54 (0.82-110.30) nM in LPS-treated groups 6 and 24 hours, respectively. The increased potency of Y-27632 was partially reversed by endothelium removal at both 6 and 24 hours. Incubation of Nomega-nitro-l-arginine methyl ester hydrochloride or 1400W (a nonselective and an inducible nitric oxide synthase inhibitor, respectively) normalized the responses to Y-27632 seen 6 hours after LPS. However, 1400W had no effect, whereas Nomega-nitro-l-arginine methyl ester hydrochloride caused a partial reduction in the enhanced potency of Y-27632 found 24 hours after LPS. The soluble guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one was able to bring the Y-27632 response back to normal both 6 and 24 hours after LPS. Rho-A, Rho-kinase I, Rho-kinase II, and MYPT1 were increased in mesenteric arteries from endotoxemic rats, but the phosphorylated MYPT1 was significantly reduced. However, incubation with oxadiazolo[4,3-alpha]quinoxalin-1-one circumvented the inhibition of MYPT1 phosphorylation found in preparations from LPS-treated animals. Our findings revealed an impaired Rho-A/Rho-kinase-mediated phosphorylation of MYPT1 in vessels from endotoxemic animals in a cyclic guanosine monophosphate-dependent manner, suggesting that changes in mechanisms involved in calcium sensitization play a pivotal role in cardiovascular changes observed in septic shock.

Endothelium-Derived Nitric Oxide Contributes to the Vasorelaxant Response Induced by Mesoionic 2-(4-Chlorophenyl)-3-methyl-4-(4-methoxyphenyl)-1;3-thiazolium-5-thyolate (CMMTT) in Rats

This study was performed to investigate the mechanisms involved in the vasorelaxation induced by mesoionic 2-(4-chlorophenyl)-3-methyl-4-(4-methoxyphenyl)-1;3-thiazolium-5-thyolate (CMMTT), a newly synthesized mesoionic compound, in rat superior mesenteric arteries. In phenylephrine (10 microM)-pre-contracted mesenteric rings, CMMTT (10(-14) - 10(-6) M) induced a concentration-dependent relaxation [pD(2) = 10.26 +/- 0.05, E(max) = 80.8 +/- 5.8%], and this effect was almost abolished after either removal of the vascular endothelium [E(max) = 17.7 +/- 4.2%, P<0.001], removal of the vascular endothelium plus100 microM N(omega)-nitro-L-arginine methyl esther (L-NAME) [E(max) = 21.0 +/- 2.0 %, P<0.001], or after pre-treatment of the rings with 100 microM L-NAME [E(max) = 13.3 +/- 2.4%, P<0.001] or 10 microM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) [E(max) = 13.6 +/- 4.8%, P<0.001]. However, endothelium-dependent relaxation induced by CMMTT was not significantly modified after 1 microM indomethacin plus 1 nM atropine [pD(2) = 11.12 +/- 0.08, E(max) = 73.8 +/- 5.15%] or 100 nM charybdotoxin (ChTX) plus 100 nM apamin [pD(2) = 10.89 +/- 0.08, E(max) = 58.91 +/- 9.8%]. In mesenteric rings, CMMTT (10(-6) M) was able to increase nitric oxide (NO)(x) levels, and this effect was abolished after removal of the vascular endothelium. In conclusion, the present study, using combined functional and biochemical approaches, demonstrated that CMMTT induced a significant vasorelaxant effect, almost completely mediated by the endothelium, likely via NO release and activation of the NO-cGMP pathway.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

BackgroundObesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings.MethodsMale Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting.ResultsHigh caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels.ConclusionThe improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.

Influence of acute pancreatitis on the in vitro responsiveness of rat mesenteric and pulmonary arteries

BackgroundAcute pancreatitis is an inflammatory disease characterized by local tissue injury and systemic inflammatory response leading to massive nitric oxide (NO) production and haemodynamic disturbances. Therefore, the aim of this work was to evaluate the vascular reactivity of pulmonary and mesenteric artery rings from rats submitted to experimental pancreatitis.Male Wistar rats were divided into three groups: saline (SAL); tauracholate (TAU) and phospholipase A2 (PLA2). Pancreatitis was induced by administration of TAU or PLA2 from Naja mocambique mocambique into the common bile duct of rats, and after 4 h of duct injection the animals were sacrificed. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PHE) in isolated mesenteric and pulmonary arteries were obtained. Potency (pEC50) and maximal responses (EMAX) were determined. Blood samples were collected for biochemical analysis.ResultsIn mesenteric rings, the potency for ACh was significantly decreased from animals treated with TAU (about 4.2-fold) or PLA2 (about 6.9-fold) compared to saline group without changes in the maximal responses. Neither pEC50 nor EMAX values for Ach were altered in pulmonary rings in any group. Similarly, the pEC50 and the EMAX values for SNP were not changed in both preparations in any group. The potency for PHE was significantly decreased in rat mesenteric and pulmonary rings from TAU group compared to SAL group (about 2.2- and 2.69-fold, for mesenteric and pulmonary rings, respectively). No changes were seen in the EMAX for PHE. The nitrite/nitrate (NOx-) levels were markedly increased in animals submitted to acute pancreatitis as compared to SAL group, approximately 76 and 68% in TAU and PLA2 protocol, respectively.ConclusionAcute pancreatitis provoked deleterious effects in endothelium-dependent relaxing response for ACh in mesenteric rings that were strongly associated with high plasma NOx- levels as consequence of intense inflammatory responses. Furthermore, the subsensitivity of contractile response to PHE in both mesenteric and pulmonary rings might be due to the complications of this pathological condition in the early stage of pancreatitis.

The effects of alcohol binge on vessel reactivity

Alcohol (ALC) consumption is associated with alterations in blood pressure and changes in vascular function. Chronic and excessive ALC consumption increases the risk for hypertension and stroke, while moderate daily intake has been shown to reduce incidence of coronary artery disease leading to a reduction in the risk of myocardial infarction. The underlying mechanism has been suggested to be altered endothelial function, with moderate ALC consumption enhancing endothelial function, while chronic excessive consumption impairs function. Previous studies have shown that acute ALC intoxication (AAI) decreases blood pressure, enhances hypotension following hemorrhagic shock, and impairs pressor response to fluid resuscitation. This study examined the effects of AAI on blood vessel reactivity to phenylephrine (PE), acetylcholine (Ach), and nitroprusside (NP) ex‐vivo. Chronically instrumented, conscious male Sprague‐Dawley rats (300–350 g) received a primed continuous 15 hour intragastric ALC infusion (1.75g/kg + 300 mg/kg/hr). Thirty minutes after discontinuing ALC, animals were sacrificed for isolation of thoracic aorta and mesenteric arteries. Aortic and mesenteric ring segments (1–2 mm) were suspended in myograph baths containing Krebs‐Henseleit bicarbonate buffer, pH 7.4, gassed with 95% O2: 5% CO2. Arterial rings from AAI rats had decreased PE‐induced tension (aorta: 2.28 ± 0.09 vs. 2.6 ± 0.13 g; mesenteric artery: 1.70 ± 0.06 vs. 1.91 ± 0.10), greater (21%) Ach‐mediated relaxation and similar NP‐mediated relaxation. These results indicate that AAI favors vasodilatation and are consistent with enhanced endothelial dilator function. (Supported by DOD PR‐054196).

Mechanisms involved in the vasodilator effect induced by diosgenin in rat superior mesenteric artery

The aim of this study was to investigate the vasorelaxant effect induced by diosgenin in superior mesenteric rings. In rings pre-contracted with phenylephrine (10 microM), diosgenin caused concentration-dependent relaxations [EC(50) = (3.3 +/- 1.2) x 10(- 4)M, E(max) = 94.2 +/- 2.6 %]. Vascular relaxation induced by diosgenin was significantly inhibited after removal of the endothelium (E(max) = 46 +/- 8.8%, p < 0.001) or after pre-treatment of the rings with N-nitro-L-arginine methyl esther (l-NAME) 100 or 300 microM (E(max) = 35.3 +/- 4%; 28.1 +/- 3.3%, respectively, p < 0.001), atropine 1 microM (E(max) = 24.6 +/- 3.4%, p < 0.001), hydroxocobalamin 30 microM (E(max) = 54.0 +/- 9.6%, p < 0.001), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) 10 microM (E(max) = 46.0 +/- 8.0%, p < 0.001) or indomethacin 1 microM (E(max) = 22.6 +/- 11.8%, p < 0.001). Vasorelaxation evoked by diosgenin was significantly inhibited after pre-treatment of preparations with both selective and non-selective inhibitors of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, iberiotoxin 100 nM or tetraethylammonium (TEA) 1mM, respectively (E(max) = 62.5 +/- 9.1%; 65.7 +/- 1.1%, p < 0.001). Conversely, in endothelium-denuded vessels, none of BK(Ca) channel blockers modified the relaxant effect induced by diosgenin. In mesenteric endothelial cells loaded with FURA-2 diosgenin was able to increase intracellular calcium concentrations, which were significantly decreased by atropine 1 microM. In addition, in isolated mesenteric rings, diosgenin induced marked increase in nitric oxide (NO) levels, which was completely abolished after functional endothelium removal. The results obtained here demonstrated that diosgenin-induced relaxation appears to involve endothelial muscarinic receptor activation with increase in intracellular calcium concentrations and consequent release of endothelium-derived relaxing factors (EDRFs), mainly NO and cyclooxygenase derivatives, which activate BK(Ca) channels. Nevertheless, further studies are necessary to clearly elucidate residual endothelium-independent relaxation induced by diosgenin.

Vascular effects of long-term propranolol administration after chronic nitric oxide blockade

Long-term propranolol treatment reduces arterial blood pressure in hypertensive individuals mainly by reducing peripheral vascular resistance, but mechanisms underlying their vasodilatory effect remain poorly investigated. This study aimed to investigate whether long-term propranolol administration ameliorates the impairment of relaxing responses of aorta and mesenteric artery from rats made hypertensive by chronic nitric oxide (NO) deficiency, and underlying mechanisms mediating this phenomenon. Male Wistar rats were treated with N(omega)-Nitro-L-arginine methyl ester (L-NAME; 20 mg/rat/day) for four weeks. DL-Propranolol (30 mg/rat/day) was given concomitantly to L-NAME in the drinking water. Treatment with L-NAME markedly increased blood pressure, an effect largely attenuated by DL-propranolol. In phenylephrine-precontracted aortic rings, the reduction of relaxing responses for acetylcholine (0.001-10 microM) in L-NAME group was not modified by DL-propranolol, whereas in mesenteric rings the impairment of acetylcholine-induced relaxation by L-NAME was significantly attenuated by DL-propranolol. In mesenteric rings precontracted with KCl (80 mM), DL-propranolol failed to attenuate the impairment of acetylcholine-induced relaxation by L-NAME. The contractile responses to extracellular CaCl2 (1-10 mM) were increased in L-NAME group, and co-treatment with DL-propranolol reduced this response in both preparations in most Ca2+ concentrations used. The NO2/NO3 plasma levels and superoxide dismutase (SOD) activity were reduced in L-NAME-treated rats, both of which were significantly prevented by DL-propranolol. In conclusion, propranolol-induced amplification of the relaxation to acetylcholine in mesenteric arteries from L-NAME-treated rats is sensitive to depolarization. Additional mechanisms involving blockade of Ca2+ entry in the vascular smooth muscle and increase in NO bioavailability contributes to beneficial effects of long-term propranolol treatment.

Adiponectin is a novel humoral vasodilator

Perivascular adipose tissue secretes an adipocyte-derived relaxing factor(s) (ADRF) that opens K(v) channels in rat arteries. Visceral fat accumulation causes adipocyte dysfunction, including hyposecretion of adiponectin. We tested the hypothesis that ADRF might be adiponectin and that adiponectin plays a role in the paracrine control of vascular tone by perivascular adipose tissue. We studied Sprague-Dawley rats, wild-type and adiponectin gene-deficient (Apn 1-/-) mice, and New Zealand obese (NZO) mice. In rat aortas, recombinant adiponectin at serum levels (2-5 microg/ml) inhibited serotonin-induced contractions. The effects were abolished by K(v) channel inhibition with 4-aminopyridine (4-AP, 2 mM). Similar effects were observed in NZO mouse mesenteric arteries. To study vascular function in Apn 1-/- mice, the mesenteric vascular bed was isolated, cannulated, and perfused at a constant 4-5-ml/min flow in the absence and presence of serotonin. 4-AP (2 mM) induced a similar increase in perfusion pressure in the Apn 1-/- perfused isolated mesenteric vascular bed, compared to wild-type mice. Removal of perivascular fat increased the vasoconstrictor responses, but abolished the 4-AP effects. The anti-contractile effects of perivascular fat were similar in mesenteric artery and aortic rings from Apn 1-/- and wild-type mice. Despite high adiponectin levels, the anti-contractile effects of perivascular fat were diminished in mesenteric arteries of NZO mice with age. Adiponectin is a novel humoral vasodilator that relaxes aortic and mesenteric rings by opening K(v) channels. Similar to the rat, perivascular adipose tissue of the mouse harbors an ADRF, which is malfunctional in NZO mice and is not adiponectin.

Endothelium-dependent vasodilation induced by Hancornia speciosa in rat superior mesenteric artery

The vasodilator effect of the ethanolic extract of leaves from Hancornia speciosa Gomes (HSE) was evaluated in superior mesenteric artery rings. HSE produced a concentration-dependent vasodilation (IC50 = 10.8 +/- 4.0 microg/mL) in arterial rings pre-contracted with phenylephrine, which was completely abolished in endothelium-denuded vessels. Endothelium-dependent vasodilation induced by HSE was strongly reduced by L-NAME (100 microM), a nitric oxide (NO) synthase inhibitor, but neither by atropine, a muscarinic receptor antagonist (1 microM), nor by indomethacin (10 microM), a cyclooxygenase inhibitor. In rings pre-contracted with 80 mM KCl, the vasodilator effect of HSE was shifted to the right and was completely abolished in the presence of L-NAME (100 microM). Similar effects were obtained in mesenteric rings pre-contracted with phenylephrine in the presence of KCl 25 mM alone or in addition to 100 microM L-NAME. In addition, BaCl2 (1 mM) dramatically reduced the vasodilation induced by HSE. Together, these findings led us to conclude that HSE induces an endothelium-dependent vasodilation in rat mesenteric artery, by a mechanism dependent on NO, on the activation of potassium channels and endothelium-derived hyperpolarizing factor release. Rutin, identified as a major peak in the HPLC fingerprint obtained for HSE, might contribute for the observed vasodilator effect, since it was able to induce an endothelium-dependent vasodilation in rat superior mesenteric arteries.

Reactivity of mesenteric and aortic rings from trained rats fed with high caloric diet

A strong association between the benefits of physical exercise on the cardiovascular disease with an improvement of the endothelium-derived relaxing factor production has been consistently shown. The purpose of this study was to evaluate the effect of exercise training associated with high caloric diet in the reactivity of rat mesenteric and aortic rings. Experimental protocol consisted of 4 weeks of high caloric diet consumption previous to 4 weeks of run training (1.2 km/h, 0% grade, in sessions of 60 min, 5 days/week). Concentrations of triglycerides, glucose, insulin and nitrite/nitrate levels were measured and atherogenic index was calculated. Concentration-response curves to acetylcholine (10 nM-100 microM), sodium nitroprusside (100 pM-100 nM) and phenylephrine (1 nM-3 microM) were obtained. Exercise training reduced body mass (6%) and triglyceride levels (about 54%), without changes in glucose and insulin concentrations. An improvement of endothelium-dependent relaxation responses to acetylcholine in mesenteric and aortic rings was observed in trained group. No changes were seen for sodium nitroprusside and phenylephrine. In conclusion, our study is the first to show clearly that run training promotes an improvement of the endothelium-dependent relaxing response in aorta and mesenteric rings from rats fed with high caloric diet and that is associated with increase of NO production.

VASORELAXING EFFECTS OF PROPRANOLOL IN RAT AORTA AND MESENTERIC ARTERY: A ROLE FOR NITRIC OXIDE AND CALCIUM ENTRY BLOCKADE

1. Propranolol has been prescribed successfully to patients with cardiovascular diseases, but the exact mechanisms by which it reduces peripheral vascular resistance have been poorly investigated. 2. The present study was designed to investigate the relaxing effects of propranolol in the rat isolated aorta and mesenteric artery, focusing on the contribution of the nitric oxide (NO)-cGMP pathway and calcium entry blockade. Relaxation responses to propranolol were obtained in precontracted rat aortic and mesenteric artery rings. 3. DL-Propranolol (10-100 micromol/L) produced concentration-dependent relaxations in the aorta and mesenteric artery rings with intact endothelium. The isomers D- and L-propranolol produced relaxation responses that were equipotent to the racemic mixture. 4. Metoprolol (10-100 micromol/L) produced slight relaxations, whereas atenolol (10-100 micromol/L) had no relaxant activity. 5. The NO inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1 micromol/L), as well as removal of the endothelium, significantly reduced the relaxation responses induced by the lower concentrations of propranolol without affecting maximal responses. In addition, DL-propranolol markedly increased cGMP levels in endothelium-intact preparations. 6. In Ca(2+)-free Krebs' solution, DL-propranolol (10-100 micromol/L) caused marked rightward shift in the concentration-response curves to CaCl(2), with a decrease of maximal responses in tissues with either intact or denuded endothelium. Nifedipine (1 micromol/L) in combination with DL-propranolol virtually abolished the CaCl(2)-induced contractile responses. 7. The relaxation responses induced by DL-propranolol were significantly reduced in aortic and mesenteric rings precontracted with phorbol-12,13-dibutyrate (1 micromol/L). 8. In conclusion, DL-propranolol relaxes arterial smooth muscle by mechanisms involving activation of the NO-cGMP pathway and calcium influx blockade, independent of beta-adrenoceptor blockade.