Contractile Response Of Aortic Rings Research Articles

Acute copper overload induces vascular dysfunction in aortic rings due to endothelial oxidative stress and increased nitric oxide production

ABSTRACTThe mechanisms involved in vascular reactivity alterations promoted by copper (Cu) overload were investigated. Thoracic aorta obtained from male Wistar rats were cut into rings and exposed for 1 h to 10 µg/ml Cu. Exposure to Cu decreased the contractile responses of aortic rings to phenylephrine (PHE). Removal of endothelium and subsequent administration of N-nitro-L arginine methyl ester (L-NAME), tetrahydrobiopterin, aminoguanidine, diethyldithiocarbamic acid, catalase, or tetraethylammonium increased contractile responses. Incubation with apocinyn and tiron enhanced the sensitivity to PHE. Data demonstrated that high concentrations of Cu reduced PHE-mediated vascular reactivity which was associated with elevated production of nitric oxide (NO), which was attributed to activation of inducible NO synthase, and elevated levels of hydrogen peroxide probably related to a rise in superoxide dismutase activity and reactive oxygen species generation.

Smooth Muscle-Targeted Overexpression of Peroxisome Proliferator Activated Receptor-γ Disrupts Vascular Wall Structure and Function.

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.

Non-steroidal anti-inflammatory drugs attenuate the vascular responses in aging metabolic syndrome rats.

Metabolic syndrome (MS) and aging are low-grade systemic inflammatory conditions, and inflammation is a key component of endothelial dysfunction. The aim of this study was to investigate the effects of non-steroidal anti-inflammatory drugs (NSAIDs) upon the vascular reactivity in aging MS rats. MS was induced in young male rats by adding 30% sucrose in drinking water over 6, 12, and 18 months. When the treatment was finished, the blood samples were collected, and aortas were dissected out. The expression of COX isoenzymes and PLA2 in the aortas was analyzed using Western blot analysis. The contractile responses of aortic rings to norepinephrine (1 μmol/L) were measured in the presence or absence of different NSAIDs (10 μmol/L for each). Serum levels of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in control rats were remained stable during the aging process, whereas serum IL-6 in MS rats were significantly increased at 12 and 18 months. The levels of COX isoenzyme and PLA2 in aortas from control rats increased with the aging, whereas those in aortas from MS rats were irregularly increased with the highest levels at 6 months. Pretreatment with acetylsalicylic acid (a COX-1 preferential inhibitor), indomethacin (a non-selective COX inhibitor) or meloxicam (a COX-2 preferential inhibitor) decreased NE-induced contractions of aortic rings from MS rats at all the ages, with meloxicam being the most potent. Acetylsalicylic acid also significantly reduced the maximum responses of ACh-induced vasorelaxation of aortic rings from MS rats, but indomethacin and meloxicam had no effect. NSAIDs can directly affect vascular responses in aging MS rats. Understanding the effects of NSAIDs on blood vessels may improve the treatment of cardiovascular diseases and MS in the elders.

Increased Activation of the Rho-A/Rho-Kinase Pathway in the Renal Vascular System Is Responsible for the Enhanced Reactivity to Exogenous Vasopressin in Endotoxemic Rats*

We evaluated the role of the renal vascular system and the Rho-A/Rho-kinase pathway in the maintenance of the pressor effects of vasopressin in endotoxemic rats. In vitro and in vivo animal study. University research laboratory. Male Wistar rats (200-300 g). Rats received either saline or lipopolysaccharide (10 mg/kg, intraperitoneal) 6 or 24 hours before the experiments. The effects of vasopressin on isolated aortic rings, cardiac function, mean arterial pressure, and both the renal vascular perfusion pressure of perfused kidneys in vitro and renal blood flow in situ were evaluated. The role of Rho-kinase in the renal and systemic effects of vasopressin was investigated through administration of the selective inhibitor Y-27632 and Western blot analysis. The effect of vasopressin on mean arterial pressure was unaltered and that on renal vascular perfusion pressure enhanced in endotoxemic rats at both 6 and 24 hours after lipopolysaccharide, despite reduced contractile responses in aortic rings and the lack of effect on cardiac function. Vasopressin (3, 10, and 30 pmol/kg, IV) produced increased reduction in renal blood flow in endotoxemic rats. In perfused kidneys from lipopolysaccharide groups, administration of Y-27632 reverted the hyperreactivity to vasopressin. Treatment with Y-27632 partially inhibited the effects of vasopressin on mean arterial pressure and significantly reduced the effects of vasopressin on renal blood flow in control but not in endotoxemic rats. Although the protein levels of Rho-A and Rho-kinase I and II had not been impaired, the levels of phosphorylated myosin phosphatase-targeting subunit 1, the regulatory subunit of myosin phosphatase that is inhibited by Rho-kinase, were increased in both the renal cortex and the renal medulla of endotoxemic rats. Our data suggest that activation of Rho-kinase potentiates the vascular effects of vasopressin in the kidneys, contributing to the maintenance of the hypertensive effects of this agent during septic shock.

Curcumin attenuates fructose-induced vascular dysfunction of isolated rat thoracic aorta rings

Context: Consumption of high fructose is associated with metabolic abnormalities, insulin resistance, and hypertension. It is not known whether this hypertensive effect of fructose is related to metabolic abnormalities or due to the direct effect of fructose on blood vessels.Objective: Here, we investigated the direct effect of fructose on rat isolated aorta and the possible protective effect of curcumin.Materials and methods: The isolated rat thoracic aorta rings were used to measure the contractile responses to different concentrations of both phenylephrine and KCl, and the relaxant response to acetylcholine (Ach). The effect of curcumin (1 µM) alone or in combination with tempol (1 mM), a superoxide dismutase mimetic agent, and N-{[3(amino-methyl)-phenyl]-methyl} ethanimidamide dihydrochloride (1400 W), a specific inducible nitric oxide synthase (iNOS) inhibitor, (1 µM) on fructose-treated aorta was compared. The aortic rings were incubated with different treatments for 60 min before starting the experiment. Changes in the intracellular calcium in response to KCl and nitric oxide levels were also measured.Results and discussion: Fructose strongly increased the contractile response of aortic rings to both phenylephrine and KCl (Emax was increased by 147.3% and 150.5%, respectively) but it did not affect the relaxant response to Ach. Curcumin significantly decreased the hyper responsiveness of arterial rings to both vasopressors (for phenylephrine, Emax decreased from 147.3% in fructose incubated aorta to 81%, and for KCl, Emax decreased from 150.5% in fructose-incubated aorta to 77.24% respectively). Curcumin also reduces the intracellular calcium level (85% reduction in intracellular calcium). A 1400 W was the only agent that potentiates the effect of curcumin.Conclusion: Fructose has a direct deleterious effect on aortic vascular reactivity. Curcumin can partially protect against fructose-induced impairment in vascular contractility via an antioxidant effect and reduction of elevated intracellular calcium.

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K+ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K+ channels and Na+/K+-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4μg/100g, subsequent doses 0.05μg/100g, im, 7days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O2− production, and apocynin (0.3μM), superoxide dismutase (150U/mL) and catalase (1000U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K+-induced relaxation curves. Ouabain (100μM) plus L-NAME (100μM), aminoguanidine (50μM) or tetraethylammonium (TEA, 2mM) reduced the K+-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60mM/L) or preincubated with TEA (2mM), 4-aminopyridine (4-AP, 5mM), iberiotoxin (IbTX, 30nM), apamin (0.5μM) or charybdotoxin (0.1μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K+ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.

Isolated aortic rings from rats surviving sepsis present enhanced responsiveness to vasopressin and angiotensin II

Epidemiological studies reveal a high mortality rate among patients that are discharged after an episode of sepsis, when compared to age‐matched people. To investigate if changes in the functionality of cardiovascular system contribute to this high mortality rate, we assessed the responses of aortic rings from 3 groups: control, and sepsis induced by cecal ligation and puncture, 30 (S30), and 60 (S60) days after recovery. The effects of potassium, phenylephrine, angiotensin I, calcium chloride, caffeine, acetylcholine, and sodium nitroprusside were not changed in vessels from sepsis‐surviving rats. However, in S60 group angiotensin II (AII)‐ and vasopressin (AVP)‐induced vasoconstriction were increased by 70 and 78%, respectively, when compared to control. It was accompanied by reduced relaxation in response to Y‐27632, a Rho‐kinase inhibitor, with EC50 of 0.43 (0.33 – 0.55) and 1.03 (0.76 – 1.40) μM in control and S60 groups, respectively. AII and AVP induced augmented contractile responses in aortic rings from S60 rats kept in calcium‐free solution, an event reversed by thapsigargin (an inhibitor of SERCA). Our results suggest that aortic rings from sepsis‐surviving rats display increased reactivity to AII and AVP by mechanisms involving changes in calcium mobilization and sensitization.

Activation of K+ Channels and Na+, K+ ATPase Prevents Aortic Endothelial Dysfunction in 7 Day Lead‐treated Rats

We previously demonstrated that exposure for 7 days with a low lead concentration increased systolic blood pressure (SBP) and nitric oxide bioavailability in aortic rings as a compensatory mechanism in the initial stage of lead exposure. We investigated if lead treatment also alters the participation of K+ channels and Na+, K+‐ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent dose 0.05 μg/100 g, i.m. to cover daily loss) or vehicle. Lead treatment reduced contractile response of aortic rings to phenylephrine (PHE) but did not change vasodilator responses induced by acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, lead exposure increased basal O2− production and apocynin, superoxide dismutase and catalase reduced the response to PHE only in treated animals. Lead treatment also increased aortic NKA functional activity. When aortic rings were precontracted with high KCl or preincubated with tetraetilamonium, 4 aminopyridine (4AP), iberiotoxin (IbTX), apamin and charybdotoxin the ACh‐induced relaxation was more reduced in treated animals. 4‐AP and IbTX also reduced more the relaxation elicited by SNP in treated animals. Results suggest that lead‐treatment promoted NKA activation as well as of K+ channels and this effect might contribute to preserve aortic endothelial function against increased SBP and oxidative stress. (CAPES, FAPES/CNPq)

The hydrogen sulfide signaling system: changes during aging and the benefits of caloric restriction

Hydrogen sulfide gas (H(2)S) is a putative signaling molecule that causes diverse effects in mammalian tissues including relaxation of blood vessels and regulation of perfusion in the liver, but the effects of aging on H(2)S signaling are unknown. Aging has negative impacts on the cardiovascular system. However, the liver is more resilient with age. Caloric restriction (CR) attenuates affects of age in many tissues. We hypothesized that the H(2)S signaling system is negatively affected by age in the vasculature but not in the liver, which is typically more resilient to age, and that a CR diet minimizes the age affect in the vasculature. To investigate this, we determined protein and mRNA expression of the H(2)S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), H(2)S production rates in the aorta and liver, and the contractile response of aortic rings to exogenous H(2)S. Tissue was collected from Fisher 344 × Brown Norway rats from 8-38 months of age, which had been maintained on an ad libitum (AL) or CR diet. The results demonstrate that age and diet have differential effects on the H(2)S signaling system in aorta and liver. The aorta showed a sizeable effect of both age and diet, whereas the liver only showed a sizeable effect of diet. Aortic rings showed increased contractile sensitivity to H(2)S and increased protein expression of CSE and CBS with age, consistent with a decrease in H(2)S concentration with age. CR appears to benefit CSE and CBS protein in both aorta and liver, potentially by reducing oxidative stress and ameliorating the negative effect of age on H(2)S concentration. Therefore, CR may help maintain the H(2)S signaling system during aging.

Baicalein impairs vascular tone in normal rat aortas: Role of superoxide anions

Acute exposure to the flavonoid baicalein inhibited endothelium-dependent relaxation in physiological arteries, although the mechanisms are not fully understood. We investigated the effect of baicalein on vascular tone in Wistar–Kyoto (WKY) rat isolated aortic rings in the presence and absence of oxidative stress to further determine the underlying mechanisms. Exposure to baicalein (10 μM) completely abolished endothelium-dependent relaxation induced by acetylcholine and attenuated significantly the endothelium-independent relaxation induced by sodium nitroprusside. Baicalein, similar to Nω-nitro- l-arginine methyl ester ( l-NAME, 10 μM), potentiated significantly the contractile response of aortic rings to α 1-adrenoceptor agonist phenylephrine. In the presence of l-NAME the baicalein effect on phenylphrine contraction or acetylcholine relaxation was unaltered, suggesting that these effects of baicalein are (like l-NAME effect) endothelial nitric oxide synthase (eNOS)/endothelium-derived nitric oxide-dependent. Inhibition of cyclooxygenase activity with indomethacin (10 μM) or scavenging of superoxide anions with superoxide dismutase (150 units/ml), but not scavenging of hydrogen peroxide with catalase (800 units/ml), enhanced significantly by an essentially similar extent the relaxation to acetylcholine in baicalein-pretreated aortic rings. Relaxant effect to acetylcholine was significantly attenuated in control aortic rings, but was completely abolished in baicalein-pretreated aortic rings in the presence of reduced form of β-nicotinamide adenine di-nucleotide (β-NADH, 300 μM). Baicalein blocked β-NADH (300 μM)-induced transient contractions, suggesting that baicalein may have inhibited activity of NADH/NADPH-oxidase. Baicalein did not alter the failure of acetylcholine to induce relaxation in the presence of pyrogallol (300 μM). In summary, acute exposure to baicalein impairs eNOS/endothelium-derived nitric oxide-mediated vascular tone in rat aortas through the inhibition of endothelium-derived nitric oxide bioavailability coupled to reduced bioactivity of endothelium-derived nitric oxide and to cyclooxygenase-mediated release of superoxide anions.

Effect of Carvedilol on Cardiovascular Status of 2‐month‐old Syrian Cardiomyopathic Hamsters

Heart failure (HF) is a multifactorial and progressive disease that has been linked to activation of the renin-angiotensin (RAS) and the sympathetic systems. In recent years, beta-blockers have been shown to improve the status of HF patients, although the precise mechanisms are still unclear. The present study evaluates the effect of beta-blockade on cardiac and vascular function of 2-month-old Syrian cardiomyopathic hamsters (SCH) after treatment for a 4-week period with carvedilol (1mg/kg/d). At this stage cardiac function is preserved but vascular abnormalities exist, including endothelial dysfunction. Echocardiographic studies, which included stroke volume (SV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), ejection fraction (EF), cardiac output (CO), cardiac output index (COI), heart rate (HR), and left ventricular posterior wall thickness (LVPWT) were evaluated. Systolic blood pressure (SBP), and contractile responses of aortic rings to angiotensin II (Ang II) and norepinephrine (NE) were also determined. Age-matched golden hamsters were used as controls. In SCH, carvedilol administration decreases SBP from 107 to 77 mmHg, (n= 5, P<0.05), reduces body weight by 8 % (n=5, P<0.05), and increases the contractile response of aortic rings to Ang II from 0.83 to 1.23g (n=9, P<0.05). SV, CO, HR, COI, EF, LVESV, LVEDV, LVPWT, and the contractile response to NE, however, were unchanged. These results suggest that carvedilol improves vascular function in 2-month-old SCH without compromising cardiac function. Supported by Grant 2 SO6 GM08224 MBRS-SCORE.

Niveles plasmáticos de nitritos, PGF1α y nitrotirosina en ratas tratadas con LPS: efectos en aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.

Up-regulation of angiotensin II type 2 receptor in rat thoracic aorta by pressure-overload.

We have examined whether expression of angiotensin II (Ang II) type 1 (AT(1)) and/or type 2 (AT(2)) receptors are changed in thoracic aorta under pressure-overload by abdominal aortic banding in rats and determined whether their changes are accompanied by alteration in contractile response of thoracic aorta to Ang II. AT(2) receptor mRNA levels determined by reverse transcription-polymerase chain reaction or quantitative real-time polymerase chain reaction were increased by about 300% in aortas 4, 7, 14, and 28 days after banding without changes in AT(1) receptor mRNA levels. Contractile response of aortic rings to Ang II was decreased in thoracic aortas 7 days after banding, and AT(2) receptor antagonist PD123319 (1-[[4-(dimethulamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) (10(-6) M) increased the Ang II responsiveness in pressure-loaded but not in sham rings. After removal of the endothelium or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), no differences were observed in Ang II responsiveness between sham and pressure-loaded rings. Either losartan (1 mg/kg/day i.p.) or candesartan (2 mg/kg/day p.o.) for 7 days after banding not only abolished the up-regulation of AT(2) receptor mRNA in aortas but also recovered their Ang II responsiveness. Basal cGMP levels were 2 times higher in pressure-loaded than in sham rings; both levels were not affected by Ang II (10(-7) M; 5 min), but greatly decreased by L-NAME (10(-4) M, 30 min). These results suggest that pressure-overload induces the up-regulation of AT(2) receptor expression in aortas via AT(1) receptor and thereby negatively modulates the vasoconstrictor sensitivity to Ang II, probably mediated by the mechanisms independent of the nitric oxide-cGMP system.

Role of the endothelium in the vascular effects of vitamin C in rats

The present study was undertaken to determine whether vascular responsiveness and endothelial function were altered in rats after 8 weeks of vitamin C treatment. Thoracic aortae were isolated from control and vitamin C-treated rats and analysed for changes in vascular reactivity. Vitamin C treatment attenuated the contractile response of aortic rings to noradrenaline and KCl. Removal of the endothelium increased the sensitivity of control rings but did not alter the effect of vitamin C. Endothelium-dependent relaxation to acetylcholine was significantly ( P<0.05) enhanced by vitamin C, but the endothelium-independent relaxation response to sodium nitroprusside was not affected by vitamin C. The results suggest that the endothelium is not involved in the reduction in vascular sensitivity to contractile agonists caused by vitamin C. In addition, the enhancement of endothelium-dependent relaxation may be due to protection of nitric oxide against inactivation by oxygen free radicals.

Evidence for a role of nuclear factor-κB in acute hypovolemic hemorrhagic shock

Background. In acute hypovolemic shock, a rapid systemic release of the inflammatory cytokine tumor necrosis factor (TNF-α) contributes to vascular failure. Nuclear factor κB (NF-κB) is an ubiquitous rapid-response transcription factor involved in inflammatory reactions and exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. The purpose of this study was to evaluate the role of NF-κB in acute hypovolemic hemorrhagic shock. Methods. Hemorrhagic shock was induced in anesthetized male rats by intermittently withdrawing blood from an iliac catheter for 20 minutes (bleeding period) until mean arterial blood pressure (MAP) decreased and stabilized within the range of 20 to 30 mm Hg. Two minutes after bleeding was discontinued the rats received tacrolimus (100 μg/kg), an inhibitor of NF-κB activation, or its vehicle. We then evaluated survival rate and survival time, liver NF-κB activation by means of electrophoretic mobility shift assay, liver IκBα protein in the cytoplasm, hepatic TNF-α messenger RNA expression, plasma TNF-α, arterial blood pressure, and the contractile response of aortic rings to phenylephrine. Results. Rats that underwent hemorrhagic shock died 28 ± 2 minutes after bleeding was discontinued, experienced marked hypotension (MAP, 20-30 mm Hg), and had enhanced plasma levels of TNF-α (218 ± 28 pg/mL 20 minutes after bleeding was discontinued). Aortas taken 20 minutes after bleeding was discontinued in rats that underwent hemorrhagic shock showed marked hyporeactivity to phenylephrine (1 nmol/L-10 μmol/L) compared with aortas harvested from sham shocked rats. Rats that underwent hemorrhagic shock also had increased levels of TNF-α messenger RNA in the liver. Furthermore, electrophoretic mobility shift assay showed that liver NF-κB binding activity increased in the nucleus, and Western blot analysis suggested that the levels of inhibitory IκBα protein in the cytoplasm decreased. Tacrolimus (100 μg/kg, administered 2 minutes after bleeding was discontinued) inhibited the loss of IκBα protein from the cytoplasm and prevented NF-κB binding activity in the nucleus. Moreover, tacrolimus increased survival time (118 ± 7 minutes; P <.01) and survival rate (vehicle = 0 and tacrolimus = 90% 240 minutes after bleeding was discontinued), reverted the marked hypotension, decreased liver messenger RNA for TNF-α reduced plasma TNF-α (35 ± 6 pg/mL), and restored the hyporeactivity to phenylephrine to control values. Conclusions. Our results suggest that acute blood loss (50% of the estimated total blood volume during a 20-minute period) causes activation of NF-κB and that tacrolimus, by inhibiting this transcription factor, protects against acute hypovolemic shock. (Surgery 2002;131:50-8.)

Inhibitory effects of melatonin on vascular reactivity: possible role of vasoactive mediators

Melatonin (MEL), the principal hormone of the vertebral pineal gland, elicits several neurobiological effects. However, the effects of MEL on vascular tissues are still vague. The first goal of this study was to investigate the effect of MEL on isolated rabbit aortic rings and its role in the vascular reactivity to contractile agents, noradrenaline (NA) and phenylephrine (PHE) and relaxant agents (acetylcholine and sodium nitroprusside). In addition, the levels of nitric oxide (NO), cGMP, total calcium, lipid peroxides, superoxide dismutase (SOD) and glutathione (GSH) were also investigated in tissue homogenates of rabbit aortic rings preincubated (20 min) in MEL with and without contractile agents. Our results revealed that MEL has an endothelium-dependent vaso-relaxant effect and potentiated significantly the vaso-relaxant effect of acetylcholine. Moreover, MEL (10 −4 M) had a significant inhibitory effect on the contractile responses of aortic rings to both NA and PHE. In comparison with control tissue rings, the levels of lipid peroxides were significantly increased while the levels of GSH, and SOD activities were significantly decreased in tissue homogenates of aortic rings pre-incubated (20 min) in NA or PHE. In addition, the levels of NO and cGMP were significantly lower in tissue rings pre-treated with NA and PHE, respectively. Also, the levels of total calcium were significantly increased only in tissue rings pre-treated with NA. The levels of lipid peroxides were significantly decreased, while the levels of GSH, NO and cGMP and SOD activities were significantly increased in tissue homogenates of aortic rings incubated (20 min) in MEL (10 −4 M) in comparison to ring tissues incubated in NA or PHE alone. In aortic rings incubated in MEL+PHE, the levels of lipid peroxides were significantly lower while the levels of GSH and cGMP and SOD activities were significantly higher than their levels in ring tissues incubated in PHE. In aortic rings incubated in MEL+NA, the levels of lipid peroxides and total calcium were significantly lower while the levels of NO were significantly higher than their levels in ring tissues incubated in NA alone. We conclude that MEL has an endothelium dependent vasorelaxant effect and potentiates the endothelium dependent vasorelaxation induced by acetylcholine. MEL inhibits the contractile responses of aortic rings to NA and PHE. These effects may be, in part, due to re-balancing the pro-oxidant/antioxidants system, lowered calcium content and elevated NO and cGMP levels in vascular tissue.

Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha (1E, 3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7- oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.

Mechanisms underlying attenuated contractile response of aortic rings to noradrenaline in fructose-fed mice

We hypothesized that an impairment of endothelial dysfunction and an increased response to α-adrenoceptor agonists may occur in fructose-fed, insulin-resistant mice. The aim of the present study was to assess the relationship between endothelial dysfunction and agonist-induced contractile responses in such mice. The acetylcholine-induced relaxation was significantly attenuated in streptozotocin-diabetic and fructose-fed mice. The contractile response to noradrenaline was significantly weaker than the control in fructose-fed but not in streptozotocin-diabetic mice; treatment with N G-nitro- l-arginine effectively restored this response. Incubating aortic rings with noradrenaline increased the NO x [nitrite (NO 2 −) and nitrate (NO 3 −)] level and this level was significantly higher in fructose-fed mice than in control mice. Clonidine induced a dose-dependent relaxation in aortic rings pre-contracted with prostaglandin F 2α that was completely abolished by N G-nitro- l-arginine; this relaxation was markedly enhanced in fructose-fed mice. In both control and fructose-fed mice, the clonidine-induced relaxation was significantly attenuated and the noradrenaline-induced contraction augmented by pertussis toxin. These results suggest that endothelial function is attenuated in both fructose-fed and streptozotocin-diabetic mice. It is suggested that the decreased noradrenaline contractile response in fructose-fed mice (compared to both controls and streptozotocin-diabetic mice) may be due to an increase in nitric oxide formation mediated by endothelial GTP-binding-coupled α 2-adrenoceptors.

Chronic administration of losartan plus hydrochlorothiazide improves vascular status in young cardiomyopathic hamsters

The combination of an angiotensin II receptor antagonist and a thiazide has been used extensively in the treatment of patients with overt heart failure. The effect of this combination on the vascular wall early in the disease, however, has not been investigated. To evaluate this effect, the vascular status of 3-month-old cardiomyopathic hamsters was assessed after daily administration of a combination of losartan (25 mg/kg, p.o.) and hydrochlorothiazide (6.5 mg/kg, p.o.) over an 8-week period. Age-matched golden hamsters were used as healthy controls. The contractile response of aortic rings to endothelin-1 was significantly higher in cardiomyopathic hamsters than in control animals. Concentration–response curves for the endothelin-1-induced contraction were displaced to the right after hydrochlorothiazide+losartan treatment (toward the curves for healthy controls); however, E max from treated hamsters was significantly reduced when compared to E max from untreated cardiomyopathic animals (1.016±0.073 vs. 1.346±0.153 g, P<0.05, n=6). No significant differences in the EC 50 values from these curves were observed between hydrochlorothiazide+losartan treated and untreated cardiomyopathic animals (2.90±0.95 vs. 1.10±0.85 nM, P>0.05). The acetylcholine-induced relaxation observed in cardiomyopathic animals was not improved after treatment with hydrochlorothiazide+losartan or hydrochlorothiazide alone, but the combination of these drugs increased significantly the basal production of nitric oxide (NO). Angiotensin-converting enzyme activity increased in plasma (from 29.9±1.23 to 41.16±1.82 nmol mg −1 min −1, n=8, P<0.05) but decreased in the aorta (from 0.33±0.02 to 0.25±0.017 nmol mg −1 min −1, n=6, P<0.05) after treatment with hydrochlorothiazide+losartan. In addition, the combination of these drugs reduced the heart-to-body mass ratio (3.96±0.07 for treated vs. 5.01±0.20 mg/g for untreated animals, n=7, P<0.05), and the thickness of the aortic media (0.076±0.003 for treated vs. 0.149±0.009 mm for untreated animals, n=8, P<0.05). Although hydrochlorothiazide alone lowered systolic blood pressure to the same level achieved with both drugs in combination (from 166±10 for untreated cardiomyopathic animals to 84±1 mm Hg for hydrochlorothiazide+losartan, and 80±5 mm Hg for hydrochlorothiazide alone, P<0.05), no significant reduction in heart-to-body mass ratio was observed in animals treated with the diuretic alone ( P>0.05). In conclusion, in this model of heart failure, chronic hydrochlorothiazide+losartan administration normalizes the vascular responses to endothelin-1, improves basal vascular tone, and prevents the development of cardiac and vascular hypertrophy.

Effect of Nabumetone Treatment on Vascular Responses of the Thoracic Aorta in Rat Experimental Arthritis

Nabumetone is a nonsteroidal anti-inflammatory (NSAI) drug which is known to cause less gastrointestinal damage than other NSAI drugs. This study was performed to evaluate whether nabumetone treatment might alter the vascular aberrations related to inflammation in a rat model of adjuvant-induced arthritis. Nabumetone treatment (120 or 240 mg·kg^–1·day^–1, orally) was initiated on the 15th day of adjuvant inoculation and continued for 14 days. Arthritic lesions, vascular contractile and relaxant responses and gastroduodenal histopathological preparations were evaluated 29 days after adjuvant inoculation. The contractile responses of aortic rings to phenylephrine and KCl were increased in grade 2 arthritic rats. In grade 3 arthritis only the phenylephrine contractility was decreased. The relaxant responses to acetylcholine and sodium nitroprusside were decreased in grades 2 and 3. In healthy rats, nabumetone did not change the vascular responses. After treatment of arthritic rats with nabumetone, both the contractile and relaxant response of the aortic rings returned to normal, and arthritic score and paw swelling were reduced. Gastroduodenal histopathology did not show erosions or ulcers in any of the groups. In conclusion, nabumetone improved the systemic signs and vascular alterations in experimental arthritis without showing any gastrointestinal side effects.