Intrauterine and lactational exposure to fluoxetine enhances endothelial modulation of aortic contractile response in adult female rats.

The study aimed to evaluate if maternal exposure to fluoxetine (FLX) during pregnancy and lactation would result in altered aortic reactivity in adult offspring. We also sought to understand the role of endothelium derived relaxing factors in aortic response. Wistar rats (75­80 days old), whose progenitors had received FLX (5 mg/kg, FLX offspring) or tap water (control offspring) during pregnancy and lactation were anesthetized, after which the aorta was removed and cut into two rings, one with (Endo+) and the other without (Endo-) endothelium. Concentration-effect curves for acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine (Phe) were performed. The vasodilation to ACh and SNP was similar between control and FLX groups in both male and female offspring. In male rats, the response to Phe was similar between the FLX and control groups on Endo+ and Endo- rings. The response to Phe was reduced on Endo+ rings from female FLX when compared with the control group. The endothelium removal, as well as L-NAME, indomethacin, and tranylcypromine incubation corrected the reduced Phe-induced contraction in the aorta from the female FLX group. On the other hand, catalase, NS-398, and L-NIL did not interfere with the vasoconstriction. The aortic level of nitric oxide (NO) was higher in the female FLX than the control group. Although endothelial NO synthase isoform and cyclooxygenase (COX)-1 expressions were similar between the groups, there was a notable increment in neuronal NO synthase expression in the aorta of FLX-exposed female rats, suggesting an important role of this enzyme in the higher levels of NO. Our results show that developmental exposure to FLX causes sex-specific alteration in aortic function through a mechanism involving endothelial factors, probably NO and COX-1 products.

Role of nitric oxide and endothelin in endothelial maintenance of vasoconstrictor responses in aortas of diabetic female rats.

BACKGROUND: Diabetes differentially affects the vascular system in males and females. Although various results have been reported, very few studies have focused on responses in females. In the present study, we investigated contractile responses to norepinephrine in aortas of alloxan-diabetic female rats and evaluated endothelial modulation of these responses. METHODS: Concentration-response curves were constructed to norepinephrine in the absence or presence of N(G) -nitro-l-arginine methyl ester (l-NAME), indomethacin, losartan, tezosentan, and calphostin C; pre-pro-endothelin mRNA expression was evaluated; and norepinephrine-stimulated expression of phosphorylated (p-) Akt Ser(473) , p-endothelial nitric oxide synthase (eNOS) Ser(1177) , and p-eNOS Ser(633) was determined in endothelial cells incubated in the presence of low (5 mmol/L) or high (25 mmol/L) glucose concentrations. RESULTS: Similar maximal responses (Rmax ) to norepinephrine were seen in control and diabetic endothelium-intact aortas; however, Rmax was reduced in diabetic endothelium-denuded aortas. Incubation of endothelium-intact aortas with 100 µmol/L l-NAME increased Rmax in the control group only. Inhibition of cyclo-oxygenase (10 µmol/L indomethacin) and blockade of angiotensin II receptors (10 µmol/L losartan) reduced Rmax in endothelium-intact aortas in both the control and diabetic groups. Blockade of endothelin receptors (0.1 µmol/L tezosentan) and inhibition of protein kinase C (PKC; 0.1 µmol/L calphostin C) reduced Rmax only in endothelium-intact aortas from diabetic rats. Pre-pro-endothelin mRNA expression was increased in aortas from diabetic female rats. Finally, p-Akt Ser(473) , p-eNOS Ser(1177) , and p-eNOS Ser(633) levels were enhanced after norepinephrine stimulation only in low glucose-treated endothelial cells. CONCLUSIONS: In aortas of diabetic female rats, reductions in smooth muscle contractile responses to norepinephrine are counterbalanced by the endothelium via reduced eNOS activation and increased endothelin release and PKC activation.

Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(2)O(2)-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(2)O(2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(2+) ([Ca(2+)](i)), and document that activity of the L-type Ca(2+) channel is necessary for the H(2)O(2)-promoted increase in sarcomere shortening and of [Ca(2+)](i). Using the chemical NO sensor Cu(2)(FL2E), we discovered that the H(2)O(2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(2+) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(2)O(2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(2+)](i) as well as the activation of protein kinase C (PKC). We also found that H(2)O(2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(2+) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(2)O(2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(2)O(2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(2+) channel, but is independent of PKC. These studies establish key roles for Ca(2+)- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(2)O(2).

Angiotensin-II and MARCKS: a hydrogen peroxide- and RAC1-dependent signaling pathway in vascular endothelium.

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.

Anti-inflammatory and antinociceptive activities a of eugenol essential oil in experimental animal models / Atividades antiinflamatória e antinociceptiva do eugenol em modelos experimentais em animais

Eugenia caryophyllata, popularmente conhecida como "cravo-da-índia", cresce naturalmente na Indonésia e é cultivada em várias partes do mundo, incluindo o Brasil. O cravo-da-índia é utilizado em culinária, em farmácia, perfumaria e cosméticos. O óleo essencial extraído do cravo-da-índia cujo principal componente é o eugenol tem sido utilizado em odontologia como anti-séptico e analgésico. O objetivo deste estudo foi avaliar as atividades antiinflamatória e antinociceptiva do eugenol de uso odontológico, administrado oralmente, em modelos experimentais in vivo. A atividade antiinflamatória do eugenol foi avaliada através do volume de exsudato e migração leucocitária no teste de pleurisia e do edema de pata de rato induzido pela carragenina. A atividade antinociceptiva foi avaliada através dos testes de contorções induzidas pelo ácido acético e da placa quente. O eugenol (200 e 400 mg/kg) reduziu o volume de exsudato pleural sem interferir na contagem de leucócitos totais presentes na pleura. Na dose de 200 mg/kg, o eugenol inibiu significativamente o edema de pata, 2-4 h após a injeção do agente flogístico. No teste da placa quente, a administração do eugenol (100 mg/kg) mostrou atividade significativa à reação de desconforto-tempo dependente, avaliada como a latência da resposta, inibida pela meperidina. Eugenol na doses de 50, 75 e 100 mg/kg apresentou efeito antinociceptivo significativo no teste de contorções abdominais induzidas pelo ácido acético em comparação com o grupo controle. Os dados obtidos indicam que o eugenol apresenta atividade antiinflamatória e antinociceptiva periférica.

Eugenia caryophyllata, popular name "clove", is grown naturally in Indonesia and cultivated in many parts of the world, including Brazil. Clove is used in cooking, food processing, pharmacy; perfumery, cosmetics and the clove oil (eugenol) have been used in folk medicine for manifold conditions include use in dental care, as an antiseptic and analgesic. The objective of this study was evaluated the anti-inflammatory and antinociceptive activity of eugenol used for dentistry purposes following oral administration in animal models in vivo. The anti-inflammatory activity of eugenol was evaluated by inflammatory exudates volume and leukocytes migration in carrageenan-induced pleurisy and carrageenan-induced paw edema tests in rats. The antinociceptive activity was evaluated using the acetic acid-induced writhing and hot-plate tests in mice. Eugenol (200 and 400 mg/kg) reduced the volume of pleural exudates without changing the total blood leukocyte counts. At dose of 200 mg/kg, eugenol significantly inhibited carrageenan-induced edema, 2-4 h after injection of the flogistic agent. In the hot-plate test, eugenol administration (100 mg/kg) showed unremarkable activity against the time-to-discomfort reaction, recorded as response latency, which is blocked by meperidine. Eugenol at doses of 50, 75 and 100 mg/kg had a significant antinociceptive effect in the test of acetic-acid-induced abdominal writhing, compared to the control animals. The data suggest that eugenol possesses anti-inflammatory and peripheral antinociceptive activities.