Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca2+ influx. To this end, the effect of PA on phenylephrine- and KCI-induced increases in cytosolic calcium concentration ([Ca2+](c)) measured by the variation in the ratio of fluorescence intensities (R340/ 380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 mu mol L-1) and KCI (30 or 90 mmol L-1) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (110, 100, 200 mu mol L-) attenuated the contraction induced by CaCl2 (0.5 nmol L(-)1 or 2.5 mmol L-1) in denuded rat aorta exposed to Ca2+- free medium containing phenylephrine (0.1 mu mol L-1) or KCI (30 mmol L-1). Interestingly, the inhibitory effect displayed by PA on CaCl2-induced contraction was more pronounced when KCI was used as the stimulant. Phenylephrine- and KCI-induced increases in (Ca2+,](c) were inhibited by PA. Similarly, verapamil, a Ca2+-channel blocker, also inhibited the increase in [Ca2+](c) induced by either phenylephrine or KCI. Finally, bolus injection of PA (1-15 mg kg(-1)) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca2+ influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca2+ sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca2+ released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca2+ entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca2+-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca2+ release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca2+ entry through L-type Ca2+.

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M) or KCl (45 mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10(-5) M), L-NAME (10(-4) M), apamin (10(-6) M), and glibenclamide (10(-5) M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, K(ATP) and SK(Ca), and maybe other K(+) channels in isolated rat thoracic aorta. (C) 2011 Elsevier B.V. All rights reserved.

Structural characterization of exopolysaccharides from biofilm of a cariogenic streptococci

Soluble (EPS-SOL), as well as insoluble extracellular polysaccharide (EPS-INSOL), extracted from biofilm of Streptococcus mutans, were analyzed by nuclear magnetic resonance spectroscopy, methylation analysis, and a controlled Smith degradation. EPS-SOL was a branched alpha-glucan containing a (1 -> 6)-and (1 -> 3)-linkages. EPS-INSOL was a branched alpha-glucan with similar linkages, but with a (1 -> 3)-linked main-chain partially substituted at O-6 with Glcp-(1 -> 6)-Glcp-side chains. Biofilm EPS had a distinct chemical structure compared with those synthesized by plankton cells or by purified enzymes from S. mutans, which could indicate different mechanisms for its degradation. (C) 2011 Published by Elsevier Ltd.

Impaired calcium influx despite hyper-reactivity in contralateral carotid following balloon injury: eNOS involvement

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore. Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids. (C) 2010 Elsevier B.V. All rights reserved.

An Apparent Paradox: Attenuation of Phenylephrine-mediated Calcium Mobilization and Hyperreactivity to Phenylephrine in Contralateral Carotid After Balloon Injury

Balloon catheter injury promotes hyperreactivity to phenylephrine (Phe) in the contralateral carotid. Phe-induced contraction involves calcium mobilization, a process that may be sensitive to reactive oxygen species. In this study, we investigated whether increased reactivity to Phe in the contralateral carotid is due to alterations in calcium mobilization by Phe and reactive oxygen species signaling. Concentration-response curves to Phe were obtained in control and contralateral arteries 4 days after balloon injury. Tiron did not modify E(max) to Phe in control arteries but reduced this parameter in the contralateral carotid to control levels. Moreover, immunofluorescence to dihydroethydine showed increased basal oxidative stress in the contralateral artery compared with control artery. Intracellular calcium mobilization by Phe in the contralateral artery was not different from control, but Phe-induced extracellular calcium mobilization was reduced in the contralateral artery compared with that in the control. These data were confirmed by confocal microscopy using Fluo 3-AM. Tiron and SC-236 increased Phe-induced calcium influx in the contralateral artery, which was similar to controls in the same conditions. However, catalase did not modify this response. Taken together, our results suggest that superoxide anions and prostanoids from cyclooxygenase-2 alter pathways downstream of alpha(1)-adrenoceptor activation in the contralateral carotid in response to injury. This results in reduced Phe-induced calcium influx, despite hyperreactivity to Phe.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3 beta-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3 beta-ol (PA-3 beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3 beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3 beta-ol (10, 50 and 100 mu mol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3 beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). PA-3 beta-ol (1-300 mu mol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3 beta-ol (100 mu mol/l) and verapamil (1 mu mol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 mu mol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the PA-3 beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 mu mol/l), 1400 W (1 mu mol/l), indomethacin (10 mu mol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3 beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3 beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway. (C) 2009 Elsevier B.V. All rights reserved.

Functional characterization of the Aspergillus fumigatus PHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (Pi) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of Pi at normal or high external Pi concentrations) and a high-affinity (activated in response to Pi starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PH080 homologue, PhoB(PHO80). We show that the Delta phoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and Delta phoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the Delta phoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1 mM P-i. Epifluorescence microscopy revealed accumulation of poly-phosphate in Delta phoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, Delta phoB(PHO80) and Delta phoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis. (C) 2008 Elsevier Inc. All rights reserved.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women

Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the 6-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to nonpregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.

Mercury speciation in seafood samples by LC-ICP-MS with a rapid ultrasound-assisted extraction procedure: Application to the determination of mercury in Brazilian seafood samples

This paper describes a simple method for mercury speciation in seafood samples by LC-ICP-MS with a fast sample preparation procedure. Prior to analysis, mercury species were extracted from food samples with a solution containing mercaptoethanol, L-cysteine and HCl and sonication for 15 min. Separation of mercury species was accomplished in less than 5 min on a C8 reverse phase column with a mobile phase containing 0.05%-v/v mercaptoethanol, 0.4% m/v L-cysteine and 0.06 mol L(-1) ammonium acetate. The method detection limits were found to be 0.25, 0.20 and 0.1 ng g(-1) for inorganic mercury, ethylmercury and methylmercury, respectively. Method accuracy is traceable to Certified Reference Materials (DOLT-3 and DORM-3) from the National Research Council Canada (NRCC). With the proposed method there is a considerable reduction of the time of sample preparation. Finally, the method was applied for the speciation of mercury in seafood samples purchased from the Brazilian market. (C) 2010 Elsevier Ltd. All rights reserved.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+) -independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X7 in macrophages. (C) 2008 Elsevier Inc. All rights reserved.

The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.