Enalapril treatment corrects the reduced response to bradykinin in diabetes increasing the B2 protein expression.

Considering the growing importance of the interaction between components of kallikrein-kinin and renin-angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the effect of enalapril on the reduced response of bradykinin and on the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems, in an insulin-resistance model of diabetes. For the above purpose, the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. In n-STZ diabetic rats, enalapril treatment restored the reduced response to BK but not the potentiation of BK by Ang-(1-7) present in non-diabetic rats. The restorative effect of enalapril was observed at a dose that did not correct the altered parameters induced by diabetes such as hyperglycemia, glicosuria, insulin resistance but did reduce the high blood pressure levels of n-SZT diabetic rats. There was no difference in mRNA and protein expressions of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Enalapril treatment increased the B2 kinin receptor expression. From our data, we conclude that in diabetes enalapril corrects the impaired BK response probably by increasing the expression of B2 receptors. The lack of potentiation of BK by Ang-(1-7) is not corrected by this agent.

Lack of potentiation of bradykinin by angiotensin-(1-7) in a type 2 diabetes model: role of insulin.

Considering the growing importance of the interaction between components of kallikrein-kinin and renin-angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems in an insulin resistance model of diabetes, and the effect of insulin on it. For this the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. Though capable of potentiating BK in non-diabetic rats, Ang-(1-7) did not potentiate BK in n-STZ rats. Chronic but not acute insulin treatment restored the potentiation. This restorative effect of insulin was abolished by a K+ channel blocker (tetraethylammonium), by nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester) and by a cyclooxygenase inhibitor (indomethacin). On the other hand, Na(+)-,K(+)-ATPase inhibition (by ouabain) did not abolish the effect of insulin. There was no difference in mRNA and protein expression of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Insulin treatment did not alter the kinin receptor expression. Our data allow us to conclude that diabetes impaired the interaction between BK and Ang-(1-7) and that insulin restores it. The restoring effect of insulin depends on membrane hyperpolarization, nitric oxide release and cyclooxygenease metabolites but not Na+K+-ATPase. Alteration of kinin receptor expression might not be involved in the restoring effect of insulin on the potentiation of BK by Ang-(1-7).

Long-term effects of intrauterine malnutrition on vascular function in female offspring: implications of oxidative stress.

Maternal malnutrition is known to impair fetal growth and predispose to the development of hypertension and type 2 diabetes. Recently, studies have demonstrated that intrauterine malnutrition is followed later in male offspring by oxidative stress characterized by increased superoxide generation due to activation of NADPH oxidase and reduced antioxidant defenses. However, few studies have investigated the mechanisms involved in endothelial dysfunction in female offspring. We evaluated the effects of the exogenous application of superoxide scavengers on the endothelium-dependent vasorelaxation in the mesenteric microvessels of female offspring. In addition, we examined indicative parameters of oxidative stress by measuring superoxide anion concentration and the activity of superoxide dismutase (SOD) as a marker of antioxidant defenses. Pregnant female Wistar rats were fed either a normal diet or 50% of this, throughout gestation. Intrauterine malnutrition induced hypertension and increased superoxide production without affecting SOD activity. Topical application of MnTMPyP (SOD mimetic) and apocynin (NADPH oxidase inhibitor) significantly improved the altered arteriolar responses to acetylcholine and bradykinin. In addition, incubation with apocynin reduced superoxide generation in these female offspring. The data suggest that after exposure to intrauterine malnutrition, female offspring present an increased superoxide production that is, at least in part, responsible for an endothelial dysfunction observed in these animals. These effects may be mediated via modulation of enzyme systems that generate superoxide.

Enalapril and losartan restored blood pressure and vascular reactivity in intrauterine undernourished rats.

Epidemiological studies suggest that intrauterine undernutrition plays an important role in the development of arterial hypertension and endothelial dysfunction in adulthood. We have evaluated the effect of the Renin Angiotensin System inhibition on the blood pressure and the mesenteric arteriolar reactivity of the intrauterine undernourished rats. Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. In this study only the male offspring was used. At 16 weeks of age, the rats were used for the study of blood pressure, microvascular reactivity studied in vivo-in situ to Angiotensin II (Ang II), Bradykinin (Bk) and Acetylcholine (Ach) before and after either losartan (10 mg/kg/15 days) or enalapril (15 mg/kg/21 days) treatment. We also evaluated the mesenteric and plasmatic Angiotensin Converting Enzyme (ACE), renal function, lipid plasmatic content, and insulin and glucose metabolism. Intrauterine undernutrition induced hypertension and increased response of mesenteric arterioles to Ang II and decreased vasodilation to Bk and Ach. The treatments with losartan or enalapril normalized the blood pressure levels and significantly improved the arteriolar responses to Bk, Ach and reduced the response to Ang II. No differences have been detected to ACE activity, renal function, lipid content and insulin and glucose metabolism. This study shows for the first time that Renin Angiotensin System inhibitors can normalize the cardiovascular alterations induced by intrauterine undernutrition.

Mechanisms involved in the reduced leukocyte migration in intrauterine undernourishment.

OBJECTIVE: We investigated factors that may be involved in the reduced leukocyte migration observed in intrauterine undernourished rats. METHODS: Male Wistar rat offspring (8-9 wk of age) of dams fed during pregnancy with 50% less food than control dams were used to measure L-selectin expression (by flow cytometry), bone marrow cell count, blood cell count, laminin and type IV collagen in the basal membrane of venules of the spermatic fascia (by immunohistochemistry), total protein level and serum albumin, and the production of leukotriene B4 after stimulation with tumor necrosis factor-alpha and corticosterone plasma levels (by enzyme-linked immunosorbent assay). RESULTS: Hypocellularity in bone marrow and peripheral blood and reduced L-selectin expression were found in the undernourished rat offspring (UR) compared with nourished offspring (NR; P < 0.05). Type IV collagen in the basal membrane of the venules of the spermatic fascia was less in UR than in NR (P < 0.05). The total protein levels and serum albumin did not differ between the two groups. Leukotriene B4 production after stimulation with tumor necrosis factor-alpha was lower in UR (P < 0.05). These differences could not be attributed to circulating glucocorticoids levels, which were not different in the NR and UR groups. CONCLUSION: Our data suggest that all observed differences contribute to reduced leukocyte migration in undernourishment.

Role of the kallikrein-kinin system in Ang-(1-7)-induced vasodilation in mesenteric arterioles of Wistar rats studied in vivo-in situ.

Angiotensin-(1-7) [Ang-(1-7)], exerts a variety of actions in the cardiovascular system, with an important effect being vasodilation. In this work, we investigated the relationship between the vasodilatory activity of Ang-(1-7) and the kallikrein-kinin system. Intravital microscopy was used to study the vasodilation caused by Ang-(1-7) in the mesenteric vascular bed of anesthetized Wistar rats. The topical application of Ang-(1-7) caused vasodilation of mesenteric arterioles that was reduced by A-779, JE 049 and peptidase inhibitors (aprotinin, SBTI, PKSI 527, E-64, PMSF). These results indicated that the vasodilation induced by Ang-(1-7) in the mesenteric arterioles of Wistar rats was heavily dependent on the activation of kallikrein and subsequent kinin formation.

Differential effects of chloral hydrate- and ketamine/xylazine-induced anesthesia by the s.c. route.

The proper use of anesthetics in animal experimentation has been intensively studied. In this study we compared the use of chloral hydrate (500 mg kg(-1)) and ketamine (167 mg kg(-1)) combined with xylazine (33 mg kg(-1)) by the s.c. route in male Wistar rats. Chloral hydrate and ketamine/xylazine produced a depth of anesthesia and analgesia sufficient for surgical procedures. The decrease of systolic and diastolic blood pressure was of a higher magnitude in rats anesthetized with chloral hydrate than with ketamine/xylazine. The initial microvascular diameter and blood flow velocity did not differ between both agents. On the other hand, ketamine/xylazine reduced the heart rate more intensively than chloral hydrate. Both anesthetics promoted an increase in arterial pCO(2) and a decrease in pH levels compared to unanesthetized animals. The blood glucose levels were of a higher magnitude in rats after ketamine/xylazine anesthesia than after chloral hydrate. In mesenteric arterioles studied in vivo, ketamine/xylazine anesthesia reduced the constrictive effect of noradrenaline and the dilator effect of bradykinin. However, both anesthetics did not modify the vasodilator effect promoted by acetylcholine. Based on our data, we concluded that both anesthetics alter metabolic and hemodynamic parameters, however the use of chloral hydrate in studies of microvascular reactivity in vivo is more appropriate since ketamine/xylazine reduces the responses to vasoactive agents and increases blood glucose levels.

Differential effect of losartan in female and male spontaneously hypertensive rats.

We demonstrated that the decreased response to acetylcholine observed in aorta of male and female spontaneously hypertensive rats is corrected after sustained (15 days) reduction of blood pressure levels by losartan. In order to verify if the same occurs in resistance vessels, vascular diameter changes induced by topical application of acetylcholine and bradykinin (endothelium-dependent vasodilators) and sodium nitroprusside (endothelium-independent vasodilator) to mesenteric arterioles studied in vivo, in situ were determined in rats treated with losartan for 24 h (acute) or 15 days (chronic). Rats that presented similar reduction (in %) of the blood pressure levels after losartan treatment were chosen. Sodium nitroprusside induced similar responses in losartan-treated and untreated male or female SHR. Whereas in female SHR, losartan corrected the diminished arteriolar response to endothelium-dependent vasodilators after acute and chronic treatment, in male SHR this correction only occurred after chronic treatment. Thus, losartan corrected the endothelial dysfunction more easily in female than in male SHR and independently of the normalization or the magnitude of the reduction of the blood pressure levels. In an attempt to explain the difference, we evaluated the losartan effect on nitric-oxide synthase (NOS) activity and angiotensin II AT1 and AT2 receptor gene expression in these animals. In male and female SHR, NOS activity and AT1 receptor expression were not altered by acute or chronic treatment. On the other hand, AT2 receptor expression was augmented only in female SHR by these treatments. Therefore, augmented AT2 receptor expression, but not alteration of NOS activity or AT1 receptor expression, might explain the difference observed.

Angiotensin II-induced venoconstriction involves both AT1 and AT2 receptors and is counterbalanced by nitric oxide.

The venoconstrictor effect of Angiotensin II (Ang II) was investigated in the rat mesenteric venules and portal vein. Mesenteric venules were perfused at a constant rate and reactivity to Ang II (0.1 nmol) was evaluated as changes in the perfusion pressure. Rings of portal vein were mounted in organ baths and curves to Ang II (0.1-100 nmol/L) were generated. In venules, Ang II-contraction (10.6+/-1.1 mmHg) was abolished by losartan (0.9+/-0.3 mmHg*), reduced by PD 123,319 (5.8+/-0.9 mmHg*), increased by L-NAME (16.5+/-1.8 mmHg*) and not altered by indomethacin. In portal veins, curves to Ang II (-logEC50: 8.9+/-0.1 mol/L) were shifted to the right by losartan (-log EC50: 7.5+/-0.1 mol/L*) and by PD 123,319 (-logEC50: 8.0+/-0.1 mol/L*). L-NAME increased the maximal response to Ang II (Emax: 0.91+/-0.1g versus 1.62+/-0.3g*) and indomethacin had no effect. In conclusion, Ang II induces venoconstriction by activating AT1 and AT2 receptors. Data obtained with L-NAME provide evidence that the basal nitric oxide release from the endothelium of the venous system can modulate the Ang II-induced venoconstriction.

Role of PGI2 and effects of ACE inhibition on the bradykinin potentiation by angiotensin-(1-7) in resistance vessels of SHR.

The present study determined the participation of PGI2 in the angiotensin-(1-7) [Ang-(1-7)]/bradykinin (BK) interaction, in the presence and absence of Angiotensin Converting Enzyme (ACE) inhibition, trying to correlate it with tissue levels of both peptides. The isolated mesenteric arteriolar bed of Spontaneously Hypertensive Rats (SHR) was perfused with Krebs or Krebs plus enalaprilat (10 nM), and drugs were injected alone or in association. BK (10 ng)-induced relaxation was potentiated by Ang-(1-7) (2.2 microg) in the presence or absence of enalaprilat. Ang-(1-7) receptor blockade [A-779 (4.8 microg)] did not interfere with the BK effect in preparations perfused with normal Krebs, but reversed the increased BK relaxation observed after ACE inhibition. PGI2 release by mesenteric vessels was not altered by BK or Ang-(1-7) alone, but was increased when both peptides were injected in association, in the absence or in the presence of enalaprilat. ACE inhibition caused a 2-fold increase in the BK tissue levels, and a significant decrease in the Ang-(1-7) values. We conclude that endogenous Ang-(1-7) has an important contribution to the effect of ACE inhibitors participating in the enhancement of BK response. The mechanism of Ang-(1-7) potentiating effect probably involves an increased production of PGI2. Our results suggest that a different enzymatic pathway (non-related to ACE) is involved in the local Ang-(1-7) metabolism.

Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression.

Abnormalities in vascular function are well recognized in diabetes. Hyperglycemia may be central to the pathogenesis of vascular dysfunction but is not certain whether improvements in glycaemic control will improve vascular function. The effects of metformin, an antidiabetic agent that improves insulin sensitivity and glycaemic control, on the microvascular reactivity have not been reported in neonatal streptozotocin-induced (n-STZ) diabetes. Diabetes was induced by STZ injection (160 mg/kg, ip) in neonates (2-day-old) Wistar rats. n-STZ diabetic rats were treated with metformin (300 mg/kg, 15 d, by gavage). Using intravital microscopy the changes in mesenteric arteriolar and venular diameters were determined in anesthetized control and n-STZ diabetic rats, before and after topical application of endothelium-dependent vasodilator agents, mediators or not of the inflammatory response, and endothelium-independent vasodilator agent. We also determined the total nitric oxide synthase (NOS) activity (conversion of L-arginine to citrulline) and endothelial(e), inducible(i), and neuronal(n) NOS expression (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs) in the mesentery of metformin-treated n-STZ diabetic and vehicle-treated n-STZ diabetic and control rats. Although metformin treatment did not correct the high glycaemic levels and the impaired glucose tolerance, the reduced vasodilator responses and total NOS activity in n-STZ diabetic rats were corrected by the treatment. Neither diabetes nor metformin treatment altered the expression of the three NOS isoforms. We concluded that metformin restores the reduced response to vasodilator agents, independently of the correction of the metabolic alterations. Improvement of total NOS activity might be in part responsible for the correction.

A lower ratio of AT1/AT2 receptors of angiotensin II is found in female than in male spontaneously hypertensive rats.

OBJECTIVE: Sexual dimorphism has been observed in arterial hypertension. Blood pressure levels are lower in female than in male spontaneously hypertensive rats (SHR). Angiotensin II (Ang II) plays a major role in the regulation of blood pressure. The aim of this study was to compare Ang II vascular reactivity and AT(1) and AT(2) receptor gene expression in female and male SHR. METHODS: SHR animals were divided into four groups: (I) male, (II) female in physiological estrus, (III) ovariectomized and (IV) ovariectomized treated with estrogen. Arterial blood pressure, AT(1) and AT(2) mRNA expression were determined. Ang II responses in aorta and mesenteric vessels were also evaluated. RESULTS: In female SHR, aorta and mesenteric microvessels were hyporeactive to Ang II in comparison to male SHR. In ovariectomized females, Ang II vasoconstriction was similar to that of males. Estrogen treatment abolished this difference. The mRNA expression for AT(1) was higher in aorta and mesenteric vessels from males than in females. In ovariectomized SHR, mRNA expression for AT(1) was comparable to that of males. Treatment with estrogen reversed the over expression observed. Whereas AT(2) gene expression did not differ, a lower ratio AT(1)/AT(2) was found in female than in male vessels. A higher mRNA expression for AT(1) was observed in kidney from male than in female. Ovariectomy resulted in up-regulation of this subtype receptor. Treatment with estrogen reversed the overexpression. AT(2) gene expression was higher in kidney from female than male SHR. Ovariectomy reduced AT(2) gene expression and estrogen treatment reversed the alteration observed in kidney. CONCLUSION: There is sexual dimorphism in vascular reactivity and in receptor gene expression to Ang II in SHR. We conclude that estrogen modulates AT(1) and AT(2) receptor gene expression and that this might explain at least partially the lower blood pressure observed in female SHR.

Gender differences in superoxide generation in microvessels of hypertensive rats: role of NAD(P)H-oxidase.

OBJECTIVE: This study is aimed to explore whether gender plays a role in the generation of nitric oxide (NO) and superoxide anion (O(2)(-)) in microvessels of hypertensive rats (SHR), as well as the potential mechanisms involved in these effects. METHODS AND RESULTS: NO generation in mesenteric arterioles was evaluated by measuring NO synthase (NOS) activity and protein expression. Oxidative stress was studied in vivo in mesenteric arterioles from male and female SHR by hydroethidine microfluorography. Although we did not observe any sex-related differences in NO generation, we found that hydroethitine oxidation is markedly increased (30.9+/-2.4%) in male compared to female (12.3+/-2.5%; p<0.05), demonstrating a gender difference in O(2)(-) production. The treatment of mesenteries with DPI (NAD(P)H-oxidase inhibitor) and treatment of SHR with losartan [Angiotensin-II type 1 (AT-1) receptor antagonist] markedly reduced O(2)(-) production in male, while produced a minor effect in female, suggesting that overexpression/activity of AT-1 receptor and NAD(P)H-oxidase contribute for the sexual dimorphism in superoxide generation. Immunoblot analyses provide evidences of overexpression of the NAD(P)H-oxidase components p22(phox), gp91(phox), p47(phox) and p67(phox) in arterioles from male in comparison to female. Losartan treatment inhibited the overexpression of these subunits in male, without affecting the responses in female. CONCLUSION: Taken together, our findings demonstrate that male SHR presents higher superoxide anion concentration under basal condition than does female. An AT-1-dependent overexpression of the NAD(P)H-oxidase components may account for the sexual dimorphism in oxidative stress, and may play an important role in the noted gender differences on incidence of cardiovascular disease.

Intrauterine undernutrition--renal and vascular origin of hypertension.

OBJECTIVE: A large number of clinical and experimental studies supports the hypothesis that intrauterine undernutrition is an important determinant of hypertension, coronary heart disease and non-insulin-dependent diabetes in the adult offspring. In this review, the renal and vascular repercussions of maternal undernutrition are emphasized, and the physiopatologic mechanisms discussed. The origin of hypertension is detailed based upon the findings of kidney functional parameters and endothelium function studies. A working model linking hypertension to intrauterine undernutrition is proposed.

NADPH oxidase and enhanced superoxide generation in intrauterine undernourished rats: involvement of the renin-angiotensin system.

OBJECTIVE: We previously reported that intrauterine undernutrition increased the oxidative stress by decreasing superoxide dismutase activity. In the present study, we tested whether NADPH oxidase, xanthine oxidase, cyclooxygenase or nitric oxide synthase are responsible for the increased O(2)(-) generation observed in rats submitted to intrauterine undernutrition. In addition, we investigated the effect of angiotensin II (ANG II) on O(2)(-) production via activation of NADPH oxidase. METHODS: Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 16 weeks of age, the rats were used for the study of intravital fluorescence microscopy; microvascular reactivity, local ANG II concentration and AT(1), p22(phox) and gp91(phox) gene expression. In this study only the male offspring was used. RESULTS: Treatment of mesenteric arterioles with the xanthine oxidase inhibitor oxypurinol, the nitric oxide synthase inhibitor L-NAME or the cyclooxygenase inhibitor diclofenac did not significantly change superoxide production. Thus, these vascular sources of superoxide were not responsible for the increased superoxide concentration. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased superoxide generation and improved vascular function. On the other hand, intrauterine undernutrition did not alter the gene expression for p22(phox) and gp91(phox). The fact that the local ANG II concentration was increased and the attenuation of oxidative stress by blocking AT(1) receptor with losartan, led us to suggest that ANG II induces O(2)(-) generation in intrauterine undernourished rats. CONCLUSION: Our study shows that NADPH oxidase inhibition attenuated superoxide anion generation and ameliorated vascular function in rats submitted to intrauterine undernutrition. Although it is not clear which mechanisms are responsible for the increase in NADPH oxidase activity, a role for ANG II-mediated superoxide production via activation of NADPH oxidase is suggested.

Intrauterine undernutrition: expression and activity of the endothelial nitric oxide synthase in male and female adult offspring.

OBJECTIVE: Epidemiological studies suggest that intrauterine undernutrition plays an important role in the development of arterial hypertension in adulthood. In an attempt to define the mechanisms whereby blood pressure may be raised, we have hypothesized that arteries from offspring of nutritionally restricted dams exhibit abnormalities in the endothelial function and in nitric oxide synthesis. In order to investigate the existence of potential gender differences on the effects of intrauterine undernutrition, both male and female offspring of pregnant Wistar rats on normal and restricted diets were studied in adulthood. METHODS: Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 14 weeks of age, the rats were used for the study of vascular reactivity, eNOS and iNOS gene expression, eNOS activity and, in the case of females, estrogen levels. RESULTS: Intrauterine undernutrition induced hypertension in both male and female offspring, but hypertension was more severe in male rats. Endothelium-intact aortic rings from male and female rats in the restricted diet group exhibited increased responses to norepinephrine, decreased vasodilation to acetylcholine and unaltered responses to sodium nitroprusside in comparison to aortic rings from control rats. No gender-related differences were observed in the vascular reactivity studies. Intrauterine undernutrition promoted decreased gene expression for eNOS in aorta isolated from male, but not female, offspring, reduction in eNOS activity in both male and female offspring and impairment in synthesis of estrogen in female offspring. CONCLUSION: Our data show that intrauterine undernutrition: (1) induces hypertension both in the male and female offspring, hypertension being more severe in male than in female rats; (2) alters endothelium-dependent responses in aortas from the resulting offspring. The endothelial dysfunction is associated with a decrease in activity/expression of eNOS in aortas from male offspring. The mechanism involved in altered response to ACh in female offspring might be a consequence of reduction in estrogen levels leading to reduced eNOS activity.

Elevated glucose blocks angiotensin-(1-7) and bradykinin interaction: the role of cyclooxygenase products.

The interaction between angiotensin-(1-7) [Ang-(1-7)] and bradykinin (BK) was studied in the isolated mesenteric arteriolar bed of control and diabetic rats perfused with either 5.5 or 22 mM of glucose. Prostanoids release after the administration of BK, Ang-(1-7) and Ang-(1-7)+BK was also studied. In control and diabetic preparations perfused with Krebs Henseleit solution with 5.5mM of glucose, Ang-(1-7) potentiates BK-induced vasodilation. On the other hand, the potentiating effect disappeared in control and diabetic preparations perfused with 22 mM of glucose. Prostaglandin F(2alpha) (PGF(2alpha)) release induced by BK and Ang-(1-7)+BK was increased in perfusates of diabetic preparations containing 22 mM of glucose. The release of thromboxane A(2) (TXA(2)) (measured as TXB(2)) or prostaglandin I(2) (PGI(2)) (measured as 6-keto-PGF(1alpha)) did not differ in control and diabetic preparations perfused with 5.5 and 22 mM of glucose. Our data allow us to suggest that hyperglycemia may be involved in the lack of potentiation in control and diabetic preparations; increase in PGF(2alpha) release, but not other cyclooxygenase products, may explain the absence of potentiation in diabetic preparations.

Angiotensin-(1-7) and bradykinin interaction in diabetes mellitus: in vivo study.

The interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK) was determined in the mesentery of anesthetized Wistar alloxan-diabetic and non-diabetic rats using intravital microscopy. Impaired BK vasodilation observed in arterioles of diabetic rats was restored by acute and chronic insulin treatment as well as by enalapril. Though capable of potentiating BK in non-diabetic rats, Ang-(1-7) did not potentiate BK in diabetic rats. Chronic but not acute insulin treatment restored the potentiation, whereas enalapril did not. Potassium channel blockade (by tetraethylammonium (TEA)) but not nitric oxide (NO) synthase inhibition (by N-omega-nitro-L-arginine-methyl-esther (L-NAME)) abolished the restorative effect of insulin. Our data allow us to suggest that the alteration observed is restored by insulin by a mechanism involving membrane hyperpolarization but not NO release. The beneficial effect of enalapril in diabetes might not involve the potentiation of BK by Ang-(1-7).

Amlodipine reduces the antimigratory effect of diclofenac in spontaneously hypertensive rats.

Amlodipine, an antihypertensive drug, and diclofenac, an antiinflammatory drug, may generally be combined, particularly in elderly patients; therefore, the potential for their interaction is high. We aim to determine if amlodipine interferes with the antimigratory effect of diclofenac. For this, male spontaneously hypertensive rats (SHRs) were treated with either diclofenac (1 mg.kg.d, 15 d) alone or combined with amlodipine (10 mg.kg.d, 15 d). Leukocyte rolling, adherence, and migration were studied by intravital microscopy. Diclofenac did not change (180.0 +/- 2.3), whereas amlodipine combined (163.4 +/- 5.1) or not (156.3 +/- 4.3) with diclofenac reduced the blood pressure (BP) levels in SHR (183.1 +/- 4.4). Diclofenac and amlodipine reduced leukocyte adherence, migration, and ICAM-1 expression, whereas only diclofenac reduced rolling leukocytes as well. Combined with amlodipine, the effect of the diclofenac was reduced. Neither treatment tested increased the venular shear rate or modified the venular diameters, number of circulating leukocytes, P-selectin, PECAM-1, L-selectin, or CD-18 expressions. No difference could be found in plasma concentrations of both drugs given alone or in association. In conclusion, amlodipine reduces leukocyte migration in SHR, reducing endothelial cell ICAM-1 expression. Amlodipine reduces the effect of the diclofenac, possibly by the same mechanism. A pharmacokinetic interaction as well as an effect on the other adhesion molecules tested could be discarded.

Constrictor responses to noradrenaline, hemodynamic profile, and superoxide levels measured by hydroethidine oxidation in diabetic rats.

The aim of this study was to test the hypothesis that vascular dysfunction in neonatal streptozotocin (n-STZ)-induced diabetic rats could be associated with alterations in blood pressure, hemodynamic profile, and levels of superoxide anion. Diabetes was induced by STZ injection (160 mg/kg, i.p.) in neonate (2-d-old) Wistar rats. Using intravital microscopy the changes in mesenteric arteriolar diameters to vasoconstrictor agent noradrenaline (NA) and the levels of superoxide anion, measured by hydroethidine microfluorography, were determined in anaesthetized control and n-STZ rats. Blood pressure (BP) was determined in anaesthetized and unanaesthetized animals. Heart rate, shear rate, and blood flow velocity were also assessed. n-STZ rats showed, after 8 weeks of STZ injection, increased BP (unanaesthetized animals), hyperactivity to NA, and increased superoxide anion levels. However, heart rate, arteriolar shear rate, and blood flow velocity were unchanged in n-STZ. In conclusion, the results of the current study describe a significant increase in blood pressure, hyperactivity to NA-mediated vasoconstriction, and increased superoxide levels measured by hydroethidine oxidation. Taken together, our findings demonstrate that the compromised ability of mesenteric microvessels to respond properly in n-STZ diabetic rats is associated with several vascular alterations.