The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Regulation of Na(+)/H(+) Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca(2+)/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA(1K3R/4E) and SB(1K3R/4E). Wild type and mutants were transfected into PS120 cells and their activity was examined by H(+) flux (J(H+)). The basal J(H+) of wild type was 4.71 +/- 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10(-12) and 10(-9) M) increased the J(H+) in wild type and SB. Ang II (10(-6) M) increased this parameter only in SA. Ang II (10(-9) M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10(-6) M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10(-7) M), a calcium chelator, suppressed the effect of Ang II (10(-9) M) in wild type. With Ang II (10(-6) M), Bapta failed to affect wild type or SA, but it increased the J(H+) in SB. W13 or calmidazolium chloride (10(-5) M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10(-9) M) in wild type or SB. With Ang II (10(-6) M), W13 or calmidazolium chloride decreased the J(H+) in wild type or SA and increased it in SB. Thus, with Ang II (10(-12) and 10(-9) M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10(-6) M), site B is important to maintain its basal activity. Copyright (C) 2010 S. Karger AG, Basel

Actions of angiotensin II and dopamine in the medial preoptic area on prolactin secretion

Dopamine (DA) is known as a primary regulator of prolactin secretion (PRL) and angiotensin II (Ang II) has been recognized as one brain inhibitory factor of this secretion. In this work, estrogen-primed or unprimed ovariectornized rats were submitted to the microinjection of saline or Ang II after previous microinjection of saline or of DA antagonist (haloperidol, sulpiride or SCH) both in the medial preoptic area (MPOA). Our study of these interactions has shown that 1) estrogen-induced PRL secretion is mediated by Ang II and DA actions in the MPOA, i.e. very high plasma PRL would be prevented by inhibitory action of Ang II, while very low levels would be prevented in part by stimulatory action of DA through D-2 receptors, 2) the inhibitory action of Ang II depends on estrogen and is mediated in part by inhibitory action of DA through D, receptors and in other part by inhibition of stimulatory action of DA through D2 receptors.

Interstitial expression of angiotensin II and AT1 receptor are increased in patients with progressive glomerulopathies

In animal models, interstitial angiotensin II (ang II) and AT1 receptor (AT1R) are key mediators of renal inflammation and fibrosis in progressive chronic nephropathies. We hypothesized that these molecules were overexpressed in patients with progressive glomerulopathies. In this observational retrospective study, we described the expression of ang II and AT1R by immunohistochemistry in kidney biopsies of 7 patients with minimal change disease (MCD) and in 25 patients with progressive glomerulopathies (PGPs). Proteinuria, serum albumin, and serum creatinine were not statistically different between MCD and PGP patients. Total expression of ang II and AT1R was not statistically different between MCD (108.7 +/- 11.5 and 73.2 +/- 13.6 cells/mm(2), respectively) and PGN patients (100.7 +/- 9.0 and 157.7 +/- 13.8 cells/mm(2), respectively; p>0.05). Yet, interstitial expression of ang II and AT1R (91.6 +/- 16.0 and 45.6 +/- 5.4 cells/mm(2), respectively) was higher in patients with PGN than in those with MCD (22.0 +/- 4.1 and 17.9 +/- 2.9 cells/mm(2), respectively, p<0.05), as was the proportion of interstitial fibrosis (11.0 +/- 0.7% versus 6.1 +/- 1.2%, p<005). In patients with MCD, ang II and AT1R expressions predominate in the tubular compartment (52% and 36% of the positive cells, respectively). In those with PGP, the interstitial expression of ang II and AT1R predominates (58% and 45%, respectively). In conclusion, interstitial expression of ang II and AT1R is increased in patients with progressive glomerulopathies. The relationship of these results and interstitial fibrosis and disease progression in humans warrants further investigations.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)). (C) 2008 Elsevier B.V. All rights reserved.

Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Effect of some antioxidants on oxidative DNA damage induced by autoxidation of microquantities of sulfite in the presence of Ni(II)/Gly-Gly-L-His

Ni(II)GGH (GGH, glycylglycyl-L-histidine) reacts rapidly with S(IV), in air-saturated solution, to produce Ni(III)GGH. A mechanism is proposed where Ni(III) oxidizes SO(3)(2-) to SO(3)(center dot-), which reacts with dissolved oxygen to produce SO(5)(center dot-), initiating radical chain reactions. DNA strand breaks and 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodGuo) formation were observed in air-saturated solutions containing micromolar concentrations of nickel(II) and S(IV). The efficacies of melatonin, (-)-epigallocatechin-gallate (from green tea), resveratrol, tannic, and ascorbic acids in terms of their inhibitory activities of DNA strand breaks and 8-oxodGuo formation were evaluated.

Effect of propolis gel on the in vitro reduction of dentin permeability

OBJECTIVE: The aim of this study was to evaluate the capacity of potassium oxalate, fluoride gel and two kinds of propolis gel to reduce the hydraulic conductance of dentin, in vitro. MATERIAL AND METHODS: The methodology used for the measurement of hydraulic conductance of dentin in the present study was based on a model proposed in literature. Thirty-six 1-mm-thick dentin discs, obtained from extracted human third molars were divided into 4 groups (n=9). The groups corresponded to the following experimental materials: GI-10% propolis gel, pH 4.1; GII-30% propolis gel; GIII-3% potassium oxalate gel, pH 4,1; and GIV-1.23% fluoride gel, pH 4.1, applied to the dentin under the following surface conditions: after 37% phosphoric acid and before 6% citric acid application. The occluding capacity of the dentin tubules was evaluated using scanning electron microscopy (SEM) at ×500, ×1,000 and ×2,000 magnifications. Data were analyzed statistically by two-way ANOVA and Tukey's test at 5% significance level. RESULTS: Groups I, II, III, IV did not differ significantly from the others in any conditions by reducing in hydraulic conductance. The active agents reduced dentin permeability; however they produced the smallest reduction in hydraulic conductance when compared to the presence of smear layer (P<0.05). The effectiveness in reducing dentin permeability did not differ significantly from 10% or 30% propolis gels. SEM micrographs revealed that dentin tubules were partially occluded after treatment with propolis. CONCLUSIONS: Under the conditions of this study, the application of 10% and 30% propolis gels did not seem to reduce the hydraulic conductance of dentin in vitro, but it showed capacity of partially obliterating the dentin tubules. Propolis is used in the treatment of different oral problems without causing significant great collateral effects, and can be a good option in the treatment of patients with dentin sensitivity.

Effect of temporary cements on the shear bond strength of luting cements

OBJECTIVE: The purpose of this study was to evaluate, by shear bond strength (SBS) testing, the influence of different types of temporary cements on the final cementation using conventional and self-etching resin-based luting cements. Material and Methods: Forty human teeth divided in two halves were assigned to 8 groups (n=10): I and V (no temporary cementation); II and VI: Ca(OH)2-based cement; III and VII: zinc oxide (ZO)-based cement; IV and VIII: ZO-eugenol (ZOE)-based cement. Final cementation was done with RelyX ARC cement (groups I to IV) and RelyX Unicem cement (groups V to VIII). Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. RESULTS: Means were (MPa): I - 3.80 (±1.481); II - 5.24 (±2.297); III - 6.98 (±1.885); IV - 6.54 (±1.459); V - 5.22 (±2.465); VI - 4.48 (±1.705); VII - 6.29 (±2.280); VIII - 2.47 (±2.076). Comparison of the groups that had the same temporary cementation (Groups II and VI; III and VII; IV and VIII) showed statistically significant difference (p<0.001) only between Groups IV and VIII, in which ZOE-based cements were used. The use of either Ca(OH)2-based (Groups II and VI) or ZO-based (Groups III and VII) cements showed no statistically significant difference (p>0.05) for the different luting cements (RelyX TM ARC and RelyX TM Unicem). The groups that had no temporary cementation (Groups I and V) did not differ significantly from each other either (p>0.05). CONCLUSION: When temporary cementation was done with ZO- or ZOE-based cements and final cementation was done with RelyX ARC, there was an increase in the SBS compared to the control. In the groups cemented with RelyX Unicem, however, the use of a ZOE-based temporary cement affected negatively the SBS of the luting agent used for final cementation.

Effect of eugenol-based endodontic sealer on the adhesion of intraradicular posts cemented after different periods

OBJECTIVE: This study evaluated in vitro the influence of an eugenol-based sealer (EndoFill) on the retention of stainless steel prefabricated posts cemented with zinc phosphate and resin-based (Panavia F) cements after different periods of root canal obturation, using the pull-out test. MATERIAL AND METHODS: Sixty upper canines were decoronated and the roots were embedded in resin blocks. The specimens were distributed into 3 groups, according to the period elapsed between canal obturation and post cementation: Group I - immediately; Group II - 72 h and Group III - 4 months. The groups were subdivided according to the type of cement used for post cementation: A - zinc phosphate and B - Panavia F. Following the experimental periods, specimens were subjected to pullout test in an Instron machine with application of tensile force at a crosshead speed of 0.5 mm/min until post dislodgement. The maximum forces required for post removal were recorded (kN) and means were subjected to statistical analysis by 2-way ANOVA and Tukey-Kramer test (α=0.001) RESULTS: There were statistically significant differences (p<0.01) between the posts cemented with zinc phosphate cement (0.2112 kN) and Panavia F (0.0501 kN). However, no statistically significant differences (p>0.05) were found between the three post cementation periods, regardless of the cement. CONCLUSIONS: It was concluded that the eugenol-based sealer influenced the tensile strength of the posts cemented with the resin cement, but had no influence on the time waited between root canal obturation and post space preparation/post cementation.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Effect of Titanium Tetrafluoride and Amine Fluoride Treatment Combined with Carbon Dioxide Laser Irradiation on Enamel and Dentin Erosion

Objective: This in vitro study aimed to analyze the influence of carbon dioxide (CO(2)) laser irradiation on the efficacy of titanium tetrafluoride (TiF(4)) and amine fluoride (AmF) in protecting enamel and dentin against erosion. Methods: Bovine enamel and dentin samples were pretreated with carbon dioxide (CO(2)) laser irradiation only (group I), TiF(4) only (1% F, group II), CO(2) laser irradiation before (group III) or through (group IV) TiF(4) application, AmF only (1% F, group V), or CO(2) laser irradiation before (group VI) or through (group VII) AmF application. Controls remained untreated. Ten samples of each group were then subjected to an erosive demineralization and remineralization cycling for 5 days. Enamel and dentin loss were measured profilometrically after pretreatment, 4 cycles (1 day), and 20 cycles (5 days) and statistically analyzed using analysis of variance and Scheffe's post hoc tests. Scanning electron microscopy (SEM) analysis was performed in pretreated but not cycled samples (two samples each group). Results: After 20 cycles, there was significantly less enamel loss in groups V and IV and significantly less dentin loss in group V only. All other groups were not significantly different from the controls. Lased surfaces (group I) appeared unchanged in the SEM images, although SEM images of enamel but not of dentin showed that CO(2) laser irradiation affected the formation of fluoride precipitates. Conclusion: AmF decreased enamel and dentin erosion, but CO(2) laser irradiation did not improve its efficacy. TiF(4) showed only a limited capacity to prevent erosion, but CO(2) laser irradiation significantly enhanced its ability to reduce enamel erosion.