Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: role of NO and prostanoids.

BACKGROUND AND AIM: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. METHODS AND RESULTS: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. CONCLUSION: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations.

Maternal diabetes affects specific extracellular matrix components during placentation.

During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring.

The influence of improved glycaemic control with chlorpropamide on microvascular reactivity and nitric oxide synthase activity in diabetic rats.

Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to reduced production of nitric oxide. The aim of this study was to verify the influence of improved glycaemic control with chlorpropamide on microvascular reactivity, endothelial nitric oxide synthase (e-NOS) expression, and NOS activity in neonatal streptozotocin-induced diabetic rats (n-STZ). Diabetes was induced by STZ injection into neonates Wistar rats. n-STZ diabetic rats were treated with chlorpropamide (200 mg kg(-1), 15 days, by gavage). The changes in mesenteric arteriolar and venular diameters were determined in anaesthetized control and n-STZ diabetic rats, before and after topical application of acetylcholine, bradykinin and sodium nitroprusside (SNP). We also assessed e-NOS expression (using polymerase chain reaction after reverse transcription of mRNAs into cDNAs) and NOS activity (conversion of L-arginine to citrulline) in the mesenteric vascular bed of chlorpropamide-treated n-STZ, vehicle-treated n-STZ, and control rats. In n-STZ, chlorpropamide treatment reduced high glycaemic levels, improved glucose tolerance and homoeostatic model assessment (HOMA-beta), and restored NOS activity. Impaired vasodilator responses of arterioles and venules to acetylcholine, bradykinin and SNP were partially corrected by chlorpropamide treatment in n-STZ. We concluded that improved metabolic control and restored NOS activity might be collaborating with improved microvascular reactivity found in chlorpropamide-treated n-STZ.

Direct vital microscopic study of defective leukocyte-endothelial interaction in diabetes mellitus.

The number of leukocytes rolling along the venular endothelium of the vascular network of the internal spermatic fascia was determined in nondiabetic control rats and diabetic rats with television microscopy. A marked decrease in the number of rolling cells was observed in animals rendered diabetic by the injection of alloxan 10, 30, or 180 days before relative to matching controls. Blood leukocyte counts, however, were equivalent in both control and diabetic rats. Under the influence of a local inflammatory stimulus, cells emerged into the perivascular tissue in control animals, and this was accompanied by a reduction in the number of rolling leukocytes. In diabetic rats, the number of rolling leukocytes remained unaltered, and only a few cells accumulated in the connective tissue adjacent to the vessel. Reversal of the defective leukocyte-endothelial interaction was attained by treatment of diabetic animals with insulin. Inhibitors of arachidonic acid metabolism were ineffective to improve leukocyte-endothelial interactions in diabetic animals. Control rats injected intravenously with lyophilized plasma constituents, obtained after dialysis of diabetic rat plasma with 12,000-Mr retention dialysis tubing, behaved as diabetic animals in that they exhibited a reduced number of leukocytes rolling along the venular endothelium. In contrast, material obtained from control rat plasma produced no effect. Heating of active samples for 1 h at 56 degrees C resulted in the complete loss of the inhibitory effect. We conclude that a substance or substances present in diabetic plasma induce a defective leukocyte-endothelial interaction that further impairs resistance to infection.

Endothelin-1 contributes to the sexual differences in renal damage in DOCA-salt rats.

We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats.

AIMS: Hypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. MAIN METHODS: SHR and Wistar rats were treated with anti-TLR4 antibody (1µg/day) or unspecific IgG for 15days (i.p.). KEY FINDINGS: Anti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SIGNIFICANCE: Together, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Influence of verapamil and diclofenac on leukocyte migration in rats.

Nonsteroidal anti-inflammatory drugs and calcium channel blockers can reduce inflammatory responses. Leukocytes play an important role in these responses. An increased expression of adhesion molecules may increase leukocyte migration. Verapamil and diclofenac are known to reduce leukocyte-endothelium interaction. To investigate a possible synergism between these drugs that could be beneficial in cardiovascular diseases, we studied leukocyte behavior by using intravital microscopy. Venules of the spermatic fascia of anesthetized Wistar rats were observed with a closed-circuit TV coupled to an optical microscope. The number of leukocytes rolling along the venular endothelium ("rollers"), sticking after application of a stimulus such as leukotriene B(4) or zymozan-activated plasma ("stickers"), or migrating after a carrageenan stimulus was reduced by verapamil at the dose of 10 mg/kg IP and by diclofenac at the dose of 2.5 mg/kg IP. The combination of both did not augment the effect of each agent alone. Verapamil, diclofenac, or their combination did not interfere with vessel diameter, number of circulating leukocytes, blood pressure levels, or heart rate. Verapamil alone or together with diclofenac reduced venular blood flow velocity and in consequence, the venular shear rate. Our data allow us to suggest that these drugs might interfere with the expression of adhesion cell molecules to reduce cell migration in inflammation. The lack of synergism between the drugs might be explained by the reduction in venular shear rate induced by verapamil, which might not be sufficient to hinder the effect of verapamil alone but hindered the summation effects of both.

Spontaneously hypertensive versus control rat aorta response to neutrophil-derived factors.

We designed experiments to study the interaction of activated rat peritoneal neutrophils with aortas from spontaneously hypertensive rats (SHR) compared with those from normotensive rats. In aortic rings precontracted with phenylephrine, neutrophils obtained from normotensive rats caused a cell number-dependent relaxation of normotensive rat aorta with or without endothelium, whereas relaxation (at lower concentrations) followed by contraction (at higher concentrations) was observed in SHR aorta with endothelium. In SHR aortic rings denuded of endothelium, neutrophils did not induce contraction. The relaxation might be due to a factor indistinguishable from nitric oxide. The contraction might be due to prostaglandin H2 because it was blocked by indomethacin, a cyclooxygenase inhibitor, and ridogrel, a thromboxane A2 synthetase inhibitor/thromboxane A2-prostaglandin H2 antagonist, but not by superoxide dismutase, a superoxide anion scavenger, or dazoxiben, a thromboxane A2 synthetase inhibitor. SHR neutrophils caused a cell number-dependent relaxation of normotensive rat aorta with or without endothelium, whereas relaxation followed by contraction was observed in SHR aorta with endothelium. In SHR aortic rings denuded of endothelium, neutrophils did not induce contraction. The relaxation might be due to a factor indistinguishable from nitric oxide. The contraction seems to be due to superoxide anion because it was inhibitable by indomethacin and superoxide dismutase but not by dazoxiben and ridogrel. Equivalent amounts of superoxide anion were produced by unstimulated and phorbol myristate acetate-stimulated neutrophils obtained from either SHR or normotensive rats. Therefore, increased production of this anion could not explain the contraction observed in hypertensive aortas.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of female sex hormones on endothelium-derived vasoconstrictor prostanoid generation in microvessels of spontaneously hypertensive rats.

Although female sex hormones may attenuate endothelial dysfunction in spontaneously hypertensive rats (SHR) by increasing endothelium-derived relaxing factors (EDRFs), the influence of ovarian hormones on the generation of endothelium-derived contracting factors (EDCFs) remains unknown. The aim of this study was to evaluate the effect of estrogen and progesterone on the generation of vasoconstrictor prostanoids and superoxide anion (O2(-)) by microvessels from SHR. Vascular reactivity to norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) were evaluated in the mesenteric arteriolar bed from estrous (OE) and ovariectomized (OVX) SHR. OVX-SHR were treated for 24 hours or 15 days with estradiol and for 15 days with estradiol+progesterone. The vascular reactivity was evaluated in the absence or presence of indomethacin (INDO, 10 micromol/L) and sodium diclofenac (DIC, 10 micromol/L), ridogrel (RID, 50 micromol/L), dazoxiben (DAZ, 10 micromol/L), or superoxide dismutase (SOD, 100 U/mL). Prostanoid levels in the arteriolar perfusate of mesenteries with or without endothelium were measured by enzyme immunoassay. An increased reactivity to NE and reduced sensitivity to ACh were observed in microvessels from OVX-SHR compared with OE-SHR. There were no differences in the responses to SNP. Treatments with estradiol and estradiol+progesterone similarly restored these altered responses. INDO, DIC, RID, and SOD also restored the NE and ACh responses in OVX-SHR. DAZ had no effect on the vascular reactivities. The release of PGF(2alpha), but not of TXB(2) and 6-keto-PGF(1alpha), was greater in OVX-SHR than in OE-SHR microvessels with endothelium when stimulated by NE. This response was normalized by hormonal treatments. Neither NE nor ACh stimulated prostanoid production by microvessels without endothelium. These results suggest that estrogen may protect female SHR against severe hypertension partly by decreasing the synthesis of EDCFs such as PGH(2)/PGF(2alpha) and O2(-).

Comparison of the effect of endothelin on microvessels and macrovessels in Goldblatt II and deoxycorticosterone acetate-salt hypertensive rats.

The response to endothelin, a novel 21-amino acid peptide, is investigated in isolated aortas with and without endothelium and in mesenteric microvessels in vivo-in situ, in Goldblatt II (GII) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Median effective concentrations and maximal responses to endothelin did not differ in aortas with endothelium isolated from GII, DOCA-salt hypertensive, and control rats. After removal of the endothelium, the potentiation of the aorta responses to endothelin was of the same magnitude in hypertensive and control rats. A closed-circuit television system was used to observe the microvascular bed of the exteriorized mesentery of anesthetized GII, DOCA-salt hypertensive, and control rats. The time necessary to induce a vasoconstrictor response was determined after the topical application of endothelin. Vessel diameters at rest and after endothelin application were also estimated. At the microcirculatory level, a greater reactivity to endothelin was observed in both hypertensive rat groups, whereas higher sensitivity to endothelin was recorded in the GII hypertensive microvessel preparations alone. It is suggested that the increased response to endothelin observed in hypertensive rats might be due to abnormal sensitivity or reactivity of the microvessels of these rats reflecting an alteration of the contractile sequence possibly at the plasma membrane level, or due to both. Endothelial dysfunction at the microcirculatory level, however, cannot be dismissed.

Indirect evidence for an endothelium-derived contracting factor released in arterioles of deoxycorticosterone acetate salt hypertensive rats.

In order to investigate if endothelium-derived contracting factor (EDCF) is involved in the altered reactivity of microvessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats, mesenteric arterioles, either perfused in vitro or studied in vivo in situ, were used. The responses to norepinephrine, acetylcholine, sodium nitroprusside and papaverine were studied in animals treated with indomethacin. Norepinephrine was equally effective in evoking a constrictor response in the in vitro perfused arteriolar bed of DOCA-salt hypertensive and control rats. A potentiation to this agent was, however, observed in preparations tested in vivo in situ. A decreased response to acetylcholine, an endothelium-dependent vasodilator, was observed in perfused in vitro and in vivo in situ preparations. The responses to sodium nitroprusside, an endothelium-independent agent, in microvessel preparations of hypertensive rats, and the response to papaverine, an agent partially dependent on endothelium, were unaltered. Indomethacin treatment did not correct the altered response to norepinephrine in mesenteric preparations studied in vivo in situ. Thus, the involvement of an EDCF which is sensitive to indomethacin blockade could be discarded. Since indomethacin treatment corrected the decreased response to acetylcholine observed in both mesenteric arterioles perfused in vitro or tested in vivo in situ, it is suggested that in arterioles of DOCA-salt hypertensive rats, an EDCF is involved in the decreased response to acetylcholine. Smooth muscle vasodilating capability appears to be unaltered.

Reactivity of aorta and mesenteric microvessels to drugs in spontaneously hypertensive rats: role of the endothelium.

The response to vasoactive agents of microvessels in situ and aortae in vitro was studied in normal and spontaneously hypertensive rats (SHR). Noradrenaline (NA) was equally effective in evoking a constrictor response of mesenteric microvessels in normal rats and SHR. The vasodilator response to acetylcholine (ACH), as endothelium-dependent relaxing agent, was lower in SHR microvessels whereas isoproterenol, papaverine, agents which are partially dependent on endothelium, and sodium nitroprusside, an endothelium-independent vasodilator, induced similar responses in control rats and SHR. Median effective concentrations and maximal responses to NA obtained in isolated SHR aortae, with or without endothelium, were similar to those obtained in their respective controls. NA-precontracted aortae with intact endothelium were less responsive to ACH in SHR than in controls. The relaxant response of the preparations was lost after endothelial cell removal in both groups. Sodium nitroprusside evoked similar relaxing effect in SHR and control NA-precontracted aortae. Isoproterenol-induced responses were potentiated in SHR-precontracted aortae, with or without endothelium. Removal of the endothelium diminished isoproterenol-induced relaxation, both in controls and SHR. With submaximal concentration of papaverine there was no difference between SHR aortae with or without endothelium and control aortae with endothelium. Control aortae without endothelium relaxed less than control aortae with endothelium and SHR aortae with or without endothelium. The rate of relaxation after papaverine was altered in aortae without endothelium isolated from SHR or control rats. These results indicate that the endothelium of SHR is altered. This could explain its decreased response to ACH. It is suggested that smooth muscle cells develop a compensatory mechanism that increases the response of agents that mobilize cAMP, such as papaverine and isoproterenol.

Indirect evidence for an endothelium-derived contracting factor release in aorta of deoxycorticosterone acetate-salt hypertensive rats.

In order to investigate the involvement of endothelium-derived vasoactive substances in deoxycorticosterone acetate (DOCA)-salt hypertension, the responses to noradrenaline, acetylcholine, sodium nitroprusside and papaverine were studied in the absence and presence of indomethacin. Noradrenaline was equally effective in evoking a constrictor response of aorta, with or without endothelium, isolated from DOCA-salt hypertensive rats, while in controls, noradrenaline induced higher submaximal responses in rubbed than in unrubbed preparations. A decreased response to acetylcholine, an endothelium-dependent vasodilator, was observed in aorta with endothelium which had been precontracted with noradrenaline isolated from hypertensive rats. The relaxant response was lost after removal of the endothelium in both control and DOCA-salt hypertensive groups. The response to sodium nitroprusside, an endothelium-independent agent, in aorta isolated from hypertensive rats as well as the response to papaverine, an agent partially dependent on the endothelium, was not altered. Indomethacin treatment altered the response to noradrenaline only in unrubbed aorta of hypertensive rats. In these preparations, a biphasic response to noradrenaline was observed. At lower concentrations noradrenaline induced the characteristic constrictor response, while at higher concentrations a relaxant response was obtained that was abolished by methylene blue, a guanylate cyclase inhibitor. This could indicate that noradrenaline induced the release of endothelium-derived relaxing factor (EDRF) in aorta of hypertensive rats. Furthermore, indomethacin treatment restored the decreased response to acetylcholine in aorta isolated from DOCA-salt hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular reactivity in diabetes mellitus: role of the endothelial cell.

The response to vasoactive agents of microvessels in situ and large arteries in vitro was compared in normal and alloxan-diabetic rats. Noradrenaline was equally effective in evoking a constrictor response of mesenteric microvessels in normal and diabetic animals. The constrictor response to a standard amount of noradrenaline in such vessels was fully antagonized by acetylcholine or papaverine, the minimum effective doses being equivalent in normal and diabetic animals. In contrast, the minimum doses of histamine or bradykinin, effective in normal animals, had to be increased about 20 fold to be active in diabetic animals. Increased osmolarity of extracellular fluid caused a significant and equivalent increase in latency of the vasoconstrictor response of microvessels to noradrenaline in normal and diabetic animals. Concentration-effect curves, constructed from the response of isolated aortae to noradrenaline, were similar in normal and diabetic animals, provided the endothelium was removed. Diabetes only affected preparations in which the endothelium was left intact. In these, the median effective concentrations of noradrenaline were greatly increased in comparison with normal values. Precontracted aortae from normal and diabetic animals were equally relaxed by acetylcholine and histamine, provided the endothelium was left intact. Loss of the relaxant response of the preparations in all groups of animals was observed following removal of endothelial cells. It is suggested that different mechanisms may be involved in the effects of vasodilator agents on large arteries in vitro or small vessels in situ. Histamine and bradykinin which are potent permeability-increasing factors, may antagonize the vasoconstrictor response of microvessels to noradrenaline through an action on endothelial cells with increased vascular permeability and temporary changes in composition of extracellular fluid. The reactive process of endothelial cells to permeability factors was affected by diabetes mellitus. However, the response of microvessels to acetylcholine and papaverine which are devoid of permeability-increasing properties, was not influenced by diabetes.

Aminoguanidine and the prevention of leukocyte dysfunction in diabetes mellitus: a direct vital microscopic study.

1. Defective leukocyte-endothelial interactions are observed in experimental diabetes mellitus. The present study investigated the effect of aminoguanidine, an inhibitor of advanced glycation end products formation, on leukocyte-endothelial interactions in alloxan-induced diabetic rats. 2. In rats anaesthetized with sodium pentobarbitone, the internal spermatic fascia was exteriorized and the microcirculation was observed by a closed-circuit TV coupled to a microscope. The number of leukocytes rolling along the venular endothelium and sticking to the vascular wall was determined after topical application of zymosan-activated plasma (1 mg ml(-1)), as well as the number of adherent and migrated cells after an irritative stimulus (carrageenan 100 microg). 3. The diabetic state decreased the number of rolling, sticking and migrated leukocytes. Pretreatment of diabetic animals with aminoguanidine (250 mg kg(-1) day(-1), for 18 days) normalized these values. To be effective, aminoguanidine had to be administered chronically, starting treatment before induction of the diabetic state. 4. The preventive effect was unrelated to the number of circulating leukocytes, or to the hyperglycaemia or to the hyperosmolality secondary to hyperglycaemia. 5. A non-dialyzed (>12,000-Mr) material in plasma from diabetic, but not normal animals, decreased the number of rolling, sticking and migrated leukocytes in recipient rats. This effect was completely abolished by chronic treatment of diabetic plasma donors with aminoguanidine. 6. The results suggest that a protein modified by glycosylation (>12 kDa) is associated with leukocyte dysfunction in diabetes mellitus and that the ability of aminoguanidine to prevent such dysfunction is related to an inhibitory effect on advanced glycation end products formation.

Vascular reactivity in diabetes mellitus: possible role of insulin on the endothelial cell.

The response to vasoactive agents of microvessels of the rat was tested in vivo by direct microscopic observation of the exteriorized mesentery and assessment of cutaneous vascular permeability changes with Evans blue. The constrictor response to a standard amount of noradrenaline in mesenteric microvessels was fully antagonized by acetylcholine in normal, diabetic, adrenalectomized and diabetic-adrenalectomized rats. In contrast, the minimum doses of histamine or bradykinin, effective in normal or adrenalectomized animals, had to be increased about 20 fold to be active in diabetic or diabetic-adrenalectomized animals. Topical application of insulin to mesenteric microvessels of diabetic animals, in amounts not causing any increase in serum insulin levels, improved or restored the capacity of the animals to respond to histamine or bradykinin, acting as antagonists of the vasoconstrictor response to noradrenaline. Topical insulin, however, was ineffective in normal animals given 2-deoxyglucose, the acute effects of which result from cellular glucopaenia unrelated to insulin deficiency. Vascular permeability responses to intracutaneous histamine or bradykinin were decreased in animals pretreated with 2-deoxyglucose as much as in diabetic animals. Pretreatment of normal animals with indomethacin produced no effect on the responses of these animals to histamine or bradykinin, tested as antagonists of noradrenaline on mesenteric microvessels, or as vascular permeability-increasing factors in the skin. Pretreatment of normal animals with chloroquine, mepacrine or dexamethasone had no effect on the reactivity of mesenteric microvessels to histamine and bradykinin, acting as antagonists to noradrenaline. 7 It is suggested that vasoactive substances, endowed with permeability-increasing properties, evoke relaxation of microvessels through an insulin-dependent action on endothelial cells, unrelated to the release of arachidonic acid metabolites. This action would lead to increased vascular permeability, with opening of interendothelial junctions, and temporary changes in composition of extravascular fluid, which in turn, would provide the basis for vasodilatation. Diabetes mellitus apparently impairs such responses as a result of the accompanying cellular glucopaenia. Adrenal corticosteroids are not involved in the impaired responses.

Influence of diabetes on the reactivity of mesenteric microvessels to histamine, bradykinin and acetylcholine.

1 Noradrenaline (NA) evoked a vasoconstrictor response in rat mesenteric microvessels in situ, the latency and nature of which was analogous in normal and alloxan-diabetic animals.2 Histamine and bradykinin (Bk) were capable of antagonizing the response to NA in normal but not in diabetic animals. In contrast, acetylcholine (ACh) was equally effective as an antagonist to NA in both groups of animals.3 The altered responses to histamine and Bk were not associated with hyperglycaemia since fasting rendered the diabetic animals normoglycaemic and yet did not restore the reactivity of microvessels. Previous administration of insulin to diabetic animals corrected the impaired responses to histamine and Bk.4 A similar condition of impaired responses to histamine and Bk was produced in normal animals by the intravenous injection of 2-deoxyglucose although ACh remained fully active.5 Apparently, the functional changes observed in the response to histamine or Bk, as antagonists of the vasoconstrictor reaction to NA, were not associated with a defective response of all smooth muscle. First, because ACh remained active in diabetic animals, and, second, because extravascular smooth muscles obtained from either normal or diabetic rats were equally relaxed by histamine or Bk in vitro.6 It is suggested that histamine and Bk antagonized the vasoconstrictor response of microvessels to NA through an action on lining endothelial cells resulting in increased vascular permeability and hyperosmolarity of extracellular fluids.7 The process depended on the availability of insulin, and, therefore, might be affected by intracellular glucopaenia as occurring in diabetes.8 Intracellular glucopaenia markedly affected other structures. Reduced atria rates were observed in diabetes, despite the fact that the isolated preparation responded normally to NA, ACh or tyramine. Partial substitution of glucose in the bathing fluid by 2-deoxyglucose or addition of NaF to the organ bath evoked similar changes in atria from normal animals.9 ACh which has little effect on vascular permeability must exert its vasodilator effects through mechanisms which are different from those influenced by the biochemical changes occurring in diabetes.

Deoxycorticosterone acetate-salt hypertensive rats display gender-related differences in ET(B) receptor-mediated vascular responses.

1. Male DOCA-salt rats exhibit vasoconstriction upon ET(B) activation. Because hypertension is less severe in female than male DOCA rats, we hypothesized that female DOCA rats would display attenuated ET(B) vasoconstrictor responses. 2. Uninephrectomized Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Systolic blood pressure was higher in male vs female DOCA rats. Responses to endothelin-1 (ET-1), IRL-1620, an ET(B) agonist, and acetylcholine were evaluated in isolated aortas and in vivo in the mesenteric microcirculation. 3. Endothelium-denuded aortas from male, but not female, DOCA rats displayed increased sensitivity to ET-1. IRL-1620 contracted aortas from male DOCA rats, but not control or female DOCA aortas. Noradrenaline-constricted and endothelium-intact aortas from male, but not female, DOCA rats displayed increased relaxation to IRL-1620 compared to control aortas. 4. In vivo, increased vasoconstriction to ET-1 was observed in male and female DOCA rats. IRL-1620 induced vasodilation in control rats, but vasoconstriction in male DOCA rats. There were minimal changes in diameter in vessels from female DOCA rats. 5. The initial fall in blood pressure induced by ET-1 and IRL-1620 was attenuated in male DOCA rats. Bosentan, a mixed ET(A)/ET(B) receptor antagonist, lowered blood pressure in male and female DOCA rats, but a greater and marked decrease occurred in the male DOCA group. 6. The gender-related differences in ET-1/ET(B)-mediated effects both in the vasculature and blood pressure suggest that sex-related functional up-regulation of ET(B) receptors may play a role in the more severe hypertension in male DOCA hypertensive rats.

The role of thromboxane A2 in the altered microvascular reactivity in two-kidney, one-clip hypertension.

To investigate the nature of the arachidonic acid metabolite involved in the altered reactivity of microvessels of two-kidney, one-clip hypertensive rats and the possible contribution of this product to the elevated blood pressure levels found in two-kidney, one-clip hypertension, mesenteric arterioles either perfused in vitro or studied in vivo were used along with blood pressure determinations. The decreased response to acetylcholine observed was normalized by ridogrel, a thromboxane A2 receptor antagonist, and dazoxiben, a thromboxane A2 synthase inhibitor. The smooth muscle response to nitric oxide, tested with sodium nitroprusside, was unaltered in two-kidney, one-clip hypertensive microvessels. Neither ridogrel nor dazoxiben modified the response to this vasodilator. In contrast, the potentiated response to noradrenaline was corrected by ridogrel and dazoxiben in vitro but not in vivo. Noradrenaline and acetylcholine increased the release of thromboxane A2 from the mesenteric microvessels of two-kidney, one-clip hypertensive rats. Ridogrel and dazoxiben decreased but did not normalize the elevated blood pressure of hypertensive rats. Based on these results, we concluded that: 1) the decreased responsiveness of smooth muscle to acetylcholine resulted from an increase in thromboxane A2 formation rather than a decrease in sensitivity to nitric oxide; 2) thromboxane A2 contributes to the increased noradrenaline response in mesenteric microvessels perfused in vitro while in in vivo other blood borne vasoactive agents may also be involved since the potentiated noradrenaline response was not corrected by inhibiting thromboxane A2 synthesis or receptors; 3) in addition to thromboxane A2, another as yet unidentified factor, may contribute to the elevated blood pressure in two-kidney, one-clip hypertension.

Sex-related differences in the response of spontaneously hypertensive rats to angiotensin-converting enzyme inhibitor.

We have compared the endothelium-dependent responses of thoracic aortic rings obtained from age-matched male and female SHR in order to explore gender differences in the effectiveness of antihypertensive drug therapy in correcting the endothelial dysfunction found in these animals. For this, concentration-effect curves to acetylcholine and sodium nitroprusside were obtained using aortic rings with and without endothelium isolated from male and female rats which had or had not been pre-treated with enalapril for 72 h (acute) or 15 d (chronic). The maximal responses achieved and the EC50s were determined. The blood pressure of male and female spontaneously hypertensive rats (SHR) decreased to normal levels within 72 h of initiating treatment with enalapril and remained normal during the remainder of the treatment period (15 d). However, enalapril was not effective in restoring a normal blood pressure in all of the male and female SHR. Female SHR were more responsive to enalapril after both acute and chronic treatment (70% of the females and 45% of the males became normotensive). Enalapril corrected the decreased response to acetylcholine in male but not in female SHR. An increased sensitivity to sodium nitroprusside, an endothelium-independent vasodilator, was observed after acute or chronic treatment with enalapril in aortic rings with endothelium from male SHR. Indomethacin restored the decreased response to acetylcholine in aortic rings from enalapril-treated females and potentiated the response to acetylcholine in aortic rings from treated male SHR. We conclude that: a) there are significant differences in the responses of male and female SHR to enalapril, b) the imbalance in endothelium-dependent relaxing and contracting factors in SHR is corrected by enalapril in male but not in female SHR, c) correction of the endothelial dysfunction probably occurs independently of the normalization of blood pressure levels and appears to be gender-dependent.