Osteocalcin improves insulin resistance and inflammation in obese mice: Participation of white adipose tissue and bone.

AIMS/HYPOTHESIS: The discovery of osteocalcin, a protein synthetized by osteoblasts, as a hormone that has positive effects on insulin resistance, contributed to support the concept of bone as an endocrine organ. However, very little is known about the molecular pathways involved in osteocalcin improved-insulin resistance. The present study aimed to investigate the mechanisms of action of osteocalcin on insulin resistance and inflammation in obese mice and 3T3-L1 adipocytes. METHODS AND RESULTS: Lean control, saline-treated obese and uncarboxylated osteocalcin (uOC)-treated obese mice were subjected to insulin tolerance test in vivo. Blood was collect for biochemical/metabolic profile analysis; and, skeletal muscle, white adipose tissue (WAT) and bone were collected for protein (Western blotting) and mRNA (RT-qPCR) analysis. uOC effects on insulin resistance and inflammation were also investigated in 3T3-L1 adipocytes challenged with tumor necrosis factor. Osteocalcin treatment improved in vivo insulin resistance in obese mice. In WAT, osteocalcin had positive effects such as (1) WAT weight reduction; (2) upregulation of glucose transporter (GLUT) 4 protein and its mRNA (Slc2a4); (3) improved insulin-induced AKT phosphorylation; (4) downregulation of several genes involved in inflammation and inflammassome transcriptional machinery, and (5) reduction of the density of macrophage in crown-like structures (histomorphometrical analysis). Notably, in 3T3-L1 adipocytes, osteocalcin restored Slc2a4/GLUT4 content and reduced the expression of inflammatory genes after TNF-a challenge; moreover, osteocalcin treatment increased AKT phosphorylation induced by insulin. Finally, it was observed that in bone, osteocalcin improves insulin resistance by increasing insulin-induced AKT phosphorylation and reducing the expression of genes involved in bone insulin resistance, resulting in increased secretion of uncarboxylated osteocalcin in circulation. CONCLUSION: We provided some mechanisms of action for osteocalcin in the amelioration of insulin resistance in obesity: in WAT, osteocalcin improves insulin resistance by decreasing inflammation, and increasing insulin signaling and the expression of Slc2a4/GLUT4; and, in bone, osteocalcin increases the secretion of uncarboxylated osteocalcin by improving insulin resistance.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice.

INTRODUCTION: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. METHODS: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. RESULTS: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90-110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. CONCLUSIONS: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling.

Stat3 and Socs3 expression patterns during murine placenta development.

Signal transducers and activators of transcription 3 (Stat3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of Stat3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (Socs3) expression. Stat3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, Stat3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for Stat3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, Socs3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of Stat3, its serine activation and Socs3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice. 

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa.

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis.

Maternal diabetes affects cell proliferation in developing rat placenta.

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

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.

Characterization and distribution of hyaluronan and the proteoglycans decorin, biglycan and perlecan in the developing embryonic mouse gonad.

The morphogenesis of tissues and organs requires dynamic changes in cells and in extracellular matrix components. It is known that various extracellular matrix molecules are of fundamental importance for gonad differentiation and growth. In the adult testis, the extracellular matrix represents an important component of the interstitium, participating in the transport of biologically active substances needed for the communication between different cellular components, as well as for the regulation of spermatogenesis and hormone production. The present study was designed in order to identify the proteoglycans biglycan, decorin and perlecan, as well as the glycosaminoglycan hyaluronan, during testis development in mouse embryos. Our data profile the chronology of testis differentiation, as well as the distribution of these extracellular matrix components during testis development in mice. We show that these extracellular matrix molecules are present early in the development of the gonads, suggesting that they play a role in gonad development. In addition, we found no decorin in the testicular cords. Furthermore, of the proteoglycans analysed, only biglycan was seen surrounding immature Sertoli cells and Leydig cell precursors in the testicular cords. This indicates that specific sets of extracellular matrix molecules are required in the various compartments of the developing gonad.

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.

Perlecan and syndecan-4 in uterine tissues during the early pregnancy in mice.

PROBLEM: During early pregnancy in mice, there is recruitment of specific immune cells, remodeling of the endometrium, cell differentiation and synthesis of new molecules. METHOD OF STUDY: Immunohistochemistry was used to determine the distribution of perlecan and syndecan-4 in the uteri before and after embryo implantation. RESULTS: During pre-implantation, perlecan was identified in basement membranes and extracellular spaces of the endometrial stroma. In contrast, expression of syndecan-4 was quite weak. In the peri-implantation period, perlecan remained in the basement membranes, and it was no longer observed in the stroma and it was identified in the embryonic cells. On day 4 of pregnancy, syndecan-4 increased in the fibroblasts of the subepithelial stroma. After implantation, syndecan-4 was pronounced in pre-decidual and mature decidual cells. CONCLUSIONS: The coordinate balance between the pre- and post-implantation periods suggests a role of these two molecules in the adaptive modification of the uterine microenvironment to receive and implant the embryo.

The small proteoglycan biglycan is associated with thick collagen fibrils in the mouse decidua.

In mice, embryo implantation induces profound changes in the endometrium. These changes include redifferentiation of endometrial fibroblasts and extensive remodeling of extracellular matrix components. We have previously shown that, during this process, there is an impressive increase in the thickness of collagen fibrils present in decidualised areas that surround the implantation site, while collagen fibrils in non-decidualised areas and in the interimplantation site remain thin. In vitro and in vivo experiments have identified small leucine rich proteoglycans (SLRPs) as regulators of collagen fibrillogenesis. In a previous study, we demonstrated a difference between the pre-implantation and the post-implantation expression and distribution of four SLRPs types in uterine tissues. The present study, utilising immunocytochemical electron microscopy, shows that biglycan is associated with the presence of thick collagen fibrils in decidualised regions of the endometrium and that decorin is associated exclusively with thin collagen fibrils in non-decidualised endometrial areas. These results strongly indicate that biglycan plays a role in collagen fibrillogenesis and probably participates in the determination of collagen fibril thickness in the mouse decidua.

Distribution of versican and hyaluronan in the mouse uterus during decidualization

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-æm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation

Distribution of biglycan and decorin in rat dental tissue

Biglycan and decorin are small leucine-rich proteoglycans that play several biological and structural roles in different tissues and organs. Several reports have indicated that biglycan participates in odontoblast and ameloblast differentiation and in the calcification process. In the present study we show that the expression of biglycan changes from within the ameloblasts and odontoblasts to the extracellular space according to the stage of animal development. In predentin and in the pulp space, however, biglycan was continually expressed throughout the period of investigation. In contrast, decorin was absent in odontoblasts and in ameloblasts and was exclusively expressed in predentin throughout the period of observation. In young rats, however, decorin was expressed in the extracellular spaces of the pulp, where it was concentrated mainly in the peripheral pulp

Distribution of biglycan and decorin in rat dental tissue.

Biglycan and decorin are small leucine-rich proteoglycans that play several biological and structural roles in different tissues and organs. Several reports have indicated that biglycan participates in odontoblast and ameloblast differentiation and in the calcification process. In the present study we show that the expression of biglycan changes from within the ameloblasts and odontoblasts to the extracellular space according to the stage of animal development. In predentin and in the pulp space, however, biglycan was continually expressed throughout the period of investigation. In contrast, decorin was absent in odontoblasts and in ameloblasts and was exclusively expressed in predentin throughout the period of observation. In young rats, however, decorin was expressed in the extracellular spaces of the pulp, where it was concentrated mainly in the peripheral pulp.

Distribution of versican and hyaluronan in the mouse uterus during decidualization.

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-microm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

Increase of interstitial collagen in the mouse endometrium during decidualization.

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [alpha1(V)](3), was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.

Small leucine-rich proteoglycans (SLRPs) in uterine tissues during pregnancy in mice.

Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.

Exercise training causes skeletal muscle venular growth and alters hemodynamic responses in spontaneously hypertensive rats.

OBJECTIVE: To investigate whether training changes skeletal muscle venular profile and hemodynamic responses to exercise we studied spontanesouly hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats submitted to training programme (T = 50-60% of VO2max). DESIGN: Training (T) was performed on a treadmill over a period of 13 weeks. Age-matched control groups were kept sedentary (S). T and S rats were chronically instrumented for hindlimb flow (HLF) and arterial pressure (AP) measurements at rest, during dynamic exercise and recovery in two different situations: control and after extensive intravenous blockade (hexamethonium + losartan + Nomega-nitro-L-arginine methyl ester + hydralazine). For morphometric analysis, skeletal muscle samples (gracilis) were obtained after transcardiac perfusion with fixative. RESULTS: T caused a significant reduction of resting mean arterial pressure (MAP) (-11%) only in the SHR group without changing basal HLF. In the sedentary SHR (SHRs), basal relative hindlimb resistance was increased by 45%, but was significantly reduced after T (P < 0.05). During dynamic exercise, MAP increased similarly (10-20 mmHg) in all groups. HLF increases were similar for the four groups up to 0.8 km/h; at higher workloads, HLF was higher in trained SHR (SHRT) versus trained WKY (WKYT) (3.9- versus 2.9-fold increase over basal HLF, respectively). After blockade (and pressure correction with IV phenylephrine infusion), steady-state exercise was performed with similar hindlimb vasodilation in all groups and was accompanied by MAP reduction (-17 +/- 8 mmHg) only in SHRT group. Skeletal muscle venular profile (density, diameter and lumen cross-sectional area) was similar in WKY(T), WKY(S) and SHR(S), but significantly increased in SHR(T). In this group the two-fold increase in venule density was correlated with both the reduction in baseline MAP and the increase in HLF during dynamic exercise. CONCLUSIONS: The results suggest that increased venule density is a specific adaptation of SHR skeletal muscle to training. Venular growth may contribute to both the pressure-lowering effect and the large HLF at high exercise intensities observed in the trained SHR.

Granulated decidual cells in the mouse deciduoma: a putative source of decidual prolactin in mice.

Decidual cells are endometrial fibroblasts that redifferentiate during pregnancy in several species of mammals. In this work, we describe a subpopulation of resident decidual cells in the mouse endometrium that are joined by intercellular junctions and have cytoplasmic granules. Decidualization was induced in pseudopregnant mice on the 4th day of pseudopregnancy by injection of 30 microl of arachis oil into the uterine lumen. The uteri were collected on day 8 of pseudopregnancy (at 4 p.m., 8 p.m. and 11 p.m.) and on day 9 (at 8 a.m.). The tissues were fixed for light and electron microscopy. During day 8 of pseudopregnancy, granulated cells were present at the antimesometrial pole of the endometrium; they were concentrated at the periphery of the antimesometrial decidua and disappeared on day 9 of pseudopregnancy. The cytoplasm of the granulated decidual cells had acidophilic granules that stained also with periodic acid-Schiff (PAS). These granules stained with anti-rat prolactin antibody in both light and electron microscope immunocytochemical preparations. Vacuoles of various sizes were always present in the granulated cells. A PAS-positive and prolactin-stained material was often deposited at the periphery of the vacuoles. Our results indicate that the granulated decidual cells are the source of decidual prolactin which accumulates in cytoplasmic granules. These granulated cells therefore form a transient gland in the mouse antimesometrial endometrium (granulated decidual gland).

Exercise training normalizes wall-to-lumen ratio of the gracilis muscle arterioles and reduces pressure in spontaneously hypertensive rats.

OBJECTIVE: To investigate mechanisms underlying the training-induced blood pressure-lowering effect we analyzed the hemodynamic responses and morphometric changes of the skeletal muscle microcirculation of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats during an exercise training program. DESIGN TRAINING: (50-60% VO2 max) was performed on a treadmill for 13 weeks and control groups were kept sedentary over the same period of time. Trained and sedentary rats were chronically instrumented for hindlimb flow and arterial pressure (AP) recordings under conscious unrestrained conditions. Gracilis and myocardial muscle samples were obtained for morphometric analysis after transcardiac perfusion of fixative. RESULTS: SHR, when compared to WKY presented an elevated blood pressure, an increased relative hindlimb vascular resistance, capillary rarefaction in both gracilis and myocardium and an increased wall-to-lumen ratio of gracilis arterioles. Training increased significantly both capillary density and capillary/fiber ratio in the gracilis and myocardium of WKY and SHR groups, causing a complete reversal of capillary rarefaction in trained SHR. In SHR, training also reduced resting blood pressure and caused normalization of both relative hindlimb vascular resistance and gracilis arterioles wall-to-lumen ratio. Regression analysis revealed strong positive correlation between hindlimb vascular resistance and mean AP (MAP) and between arterioles wall-to-lumen ratio and MAP. CONCLUSIONS: The results suggest that low-intensity training can significantly reduce pressure in SHR while normalizing both the arteriole morphology and the resistance of the skeletal muscle microcirculation.

Influence of enalapril on the endothelial function of DOCA-salt hypertensive rats.

In the present study, we investigated whether the correction of endothelial dysfunction can be independent of the normalization of high blood pressure levels by enalapril in deoxycorticosterone (DOCA-salt) hypertensive rats. Aorta morphology and the response of aortas with (E+) and without (E-) endothelium to noradrenaline, acetylcholine, and sodium nitroprusside were studied. DOCA-salt hypertensive and normotensive (control) rats were or were not treated with enalapril (5 mg/day/rat in the drinking fluid) for 1, 7, or 15 days. Blood pressure was measured before and after 1, 3, 7, and 15 days of enalapril treatment. Enalapril normalized the high blood pressure levels in 50% (responders) of the hypertensive rats after 3 to as many as 15 days but not after 1 day of treatment. Initial blood pressure levels were not different between responders and nonresponders. Blood pressure levels of normotensive control rats were not altered by enalapril treatment. The tunica media of aortas of DOCA-salt hypertensive rats treated or not treated with enalapril for 15 days was thicker than aortas from normotensive rats. Enalapril corrected the reduced response to acetylcholine observed in aorta from hypertensive rats from the first day of treatment. This treatment rendered aortas from normotensive control rats more sensitive (lower EC(50)) to acetylcholine without a change in the maximal responses. The responses to sodium nitroprusside, a nitric oxide donor, were unaltered in aorta E+ or E- from control and hypertensive rats before and after enalapril treatment. Enalapril did not correct the increased responses to noradrenaline observed in aorta E+ of hypertensive rats. These results suggest that the high blood pressure in DOCA-salt hypertension is not correlated with the altered response to endothelium-dependent agents (either dilator or constrictors). The endothelium-dependent vasodilation by antihypertensive agents can be corrected independently of normalization of blood pressure levels or the vascular morphology.