Regina Scivoletto

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

1 The response to vasoactive agents of microvessels in situ and large arteries in vitro was compared in normal and alloxan‐diabetic rats. 2 Noradrenaline was equally effective in evoking a constrictor response of mesenteric microvessels in normal and diabetic animals. 3 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. 4 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. 5 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. 6 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. 7 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.

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(-).

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.

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

1 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. 2 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. 3 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. 4 Vascular permeability responses to intracutaneous histamine or bradykinin were decreased in animals pretreated with 2‐deoxyglucose as much as in diabetic animals. 5 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. 6 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.

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.

Metabolic consequences of food restriction in rats

Abstract Some aspects of carbohydrate metabolism were studied in rats subjected to food restriction (single daily meal offered during two hours, either diurnal or nocturnal) for a week. Nocturnal preference for the nocturnal meal was patent in spite of food restriction, inasmuch as the rats fed from 8:00 to 10:00 p.m. ingested significantly more than those fed from 8:00 to 10:00 a.m. The amount of food ingested by rats of both groups was lower than that ingested by the animals with free access to food (as evaluated in 24 hours); nevertheless, both food-restricted groups did not lose weight for the duration of the experiments. The more prominent carbohydrate metabolic adaptations to food-restriction were: (1) high hepatic glycogen concentration during the intermeal periods; (2) hyperglycemia in the 12, 14 and 16 hour intermeal periods; (3) insulinemia was lower than in rats having free access to food all day long; (4) gastric emptying was delayed.

Cardionatrin causes vasodilation in vitro which is not dependent on the presence of endothelial cells

Vasodilation, in vitro, evoked by atrial extracts (cardionatrin) was compared with the relaxation due to acetylcholine and papaverine in rat precontracted aortae with and without endothelium. Ventricular extracts were tested as controls. Atrial extracts and papaverine caused a similar concentration-dependent relaxation of both preparations whereas acetylcholine failed to relax preparations without endothelium. Ventricular extracts were ineffective on both preparations. It is concluded that the response of the preparations to atrial extracts (cardionatrin) is not dependent on the presence of the endothelial cells. Some pathophysiological implications are discussed.

Gender differences in vascular reactivity to endothelin-1 in deoxycorticosterone-salt hypertensive rats.

In experimental models of hypertension, blood pressure reaches a higher level in male than in female rats. Because endothelin-1 (ET-1) seems to play a role in blood pressure elevation in deoxycorticosterone acetate (DOCA)-salt hypertension, we hypothesized that male DOCA-salt rats would display a greater vascular responsiveness to ET-1 than female DOCA-salt rats. Male and female Wistar rats were uninephrectomized, received DOCA injections (50 mg/kg/week) and water plus 1.0% NaCl/0.2% KCl. Control rats received vehicle and tap water. Responses to ET-1, norepinephrine (NE), serotonin (5-HT), IRL-1620, a selective endothelin-B- (ET(B)) receptor agonist, and acetylcholine (ACh) were evaluated in isolated aortic rings and also in vivo in the mesenteric microcirculation. Endothelium-intact aortas from male and female DOCA rats displayed increased sensitivity (p < 0.05) to NE and 5-HT, but decreased relaxation to ACh in comparison to aortas from respective control male and female rats. Endothelium-denuded, but not endothelium-intact, arteries from male DOCA rats displayed increased sensitivity (-log EC20) to ET-1, but no changes in ET-1 sensitivity were observed in female DOCA aortas. IRL-1620 induced contraction in male DOCA aortas, but not in female DOCA or control endothelium-denuded aortas. In the microcirculation, IRL-1620 induced vasodilation in male and female control rats, but marked vasoconstriction in male DOCA and minimal changes in vessels diameter in female DOCA rats. Bosentan, an ET(A)/ET(B)-receptor antagonist, induced a greater decrease in mean arterial blood pressure in male than in female DOCA-salt hypertensive rats. These data support the hypothesis that DOCA-salt rats exhibit gender differences in ET-1 vascular reactivity, which probably result from functional changes in ET(B)-receptors. The increased ET(B) responses in male DOCA-salt hypertensive rats may play a role in their higher blood pressure levels.

Thyroid hormones and vascular reactivity: role of the endothelial cell

Decreased maximal responses to noradrenaline, adrenaline and phenylephrine were observed in aortas isolated from rats pretreated with triiodothyronine and thyroxine for 8 days. In addition, a potent relaxant effect occurred when supramaximal concentrations of noradrenaline and adrenaline, but not of phenylephrine, were used. The relaxant effect was not observed in preparations in which the endothelium had been removed, and it was antagonized by propranolol added to the bathing fluid. Practolol was ineffective. In vivo, adrenaline and noradrenaline evoked pressor effects which were of shorter duration and smaller magnitude in hormone-treated animals than in the controls. Furthermore, a subsequent fall in blood pressure to below normal was observed, not only with adrenaline but also with noradrenaline, in animals receiving thyroid hormones. We conclude that when large amounts of thyroid hormones circulate in the body, large arteries are more prone to distention in the presence of increased quantities of released catecholamines, and resistance vessels are less responsive to their pressor effects due to a superimposing dilator effect. These changes might represent adaptative mechanisms in hyperthyroid states, and might depend on a greater sensitivity of endothelial beta 2-receptors to noradrenaline and adrenaline.