Amanda C. Pereira

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way.

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC50). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E max=240s) is similar to measured for SNP (210s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1μM). In a similar way, 1μM ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ.

Hypotensive and vasorelaxing effects of the new NO-donor [Ru(terpy)(bdq)NO(+)](3+) in spontaneously hypertensive rats.

Drugs that release nitric oxide (NO) usually have limitations due to their harmful effects. Sodium nitroprusside (SNP) induces a rapid hypotension that leads to reflex tachycardia, which could be an undesirable effect in patients with heart disease, a common feature of hypertension. The nitrosyl ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) is a NO donor that is less potent than SNP in denuded aortic rings. This study evaluated the hypotension and vasorelaxation induced by this NO donor in Wistar (W) and spontaneously hypertensive rats (SHR) and compared to the results obtained with SNP. Differently from the hypotension induced by SNP, the action of TERPY was slow, long lasting and it did not lead to reflex tachycardia in both groups. The hypotension induced by the NO-donors was more potent in SHR than in W. TERPY induced relaxation with similar efficacy to SNP, although its potency is lower in both strains. The relaxation induced by TERPY is similar in W and SHR, but SNP is more potent and efficient in SHR. The relaxation induced by TERPY is partially dependent on guanylate cyclase in SHR aorta. The NO released from the NO donors measured with DAF-2 DA by confocal microscopy shows that TERPY releases similar amounts of NO in W and SHR, while SNP releases more NO in SHR aortic rings.

Long-lasting hypotensive effect in renal hypertensive rats induced by nitric oxide released from a ruthenium complex.

Abstract: In this study, we investigated the effect of the ruthenium complex [Ru(terpy)(bdq)NO+]3+ (TERPY) on the arterial pressure from renal hypertensive 2 kidney-1 clip (2K-1C) rats, which was compared with sodium nitroprusside (SNP). The most interesting finding was that the intravenous bolus injection of TERPY (2.5, 5.0, 7 mg/kg) had a dose-dependent hypotensive effect only in 2K-1C rats. On the other hand, SNP (35 and 70 &mgr;g/kg) presented a similar hypotensive effect in both normotensive (2K) and 2K-1C although the effect of 70 &mgr;g/kg was >35 &mgr;g/kg. The injection of the nonselective NO-synthase inhibitor N&ohgr;-nitro-L-arginine methyl ester (L-NAME) increased the arterial pressure in 2K and 2K-1C rats with a similar magnitude. After infusion of L-NAME, the hypotensive effect induced by TERPY and SNP was potentiated in both 2K and in 2K-1C rats. The administration of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl increased the hypotensive effect induced by TERPY or SNP in both 2K and 2K-1C rats. The hypotensive effect induced by TERPY was longer than that produced by SNP. Taken together, our results show that the TERPY has a long-lasting hypotensive effect, which has a dose dependence and higher magnitude in 2K-1C compared with in 2K rats. In comparison with SNP, TERPY is less potent in inducing arterial pressure fall, but it presents a much longer hypotensive effect.

The new NO donor Terpy induces similar relaxation in mesenteric resistance arteries of renal hypertensive and normotensive rats.

The present work aimed to investigate the cellular mechanisms involved on the vasorelaxation induced by the new nitric oxide donor [Ru(terpy)(bdq)NO](3+) (Terpy) in isolated mesenteric resistance artery and to compare the vascular responses in isolated vessels from 2K and 2K-1C hypertensive rats. We have used this artery because it is important to the control of vascular resistance and consequently to the blood pressure control. The NO donor Terpy induced relaxation in a concentration-dependent way in mesenteric resistance arteries. There were no differences between renal hypertensive (2K-1C) and normotensive (2K) in Terpy-induced relaxation neither in NO released. The relaxation induced by Terpy was inhibited by the soluble guanylyl-cyclase (sGC) inhibitor ODQ both in 2K and in 2K-1C with similar amplitude. In agreement with these data, the protein expression of the subunits α1 and β1 of the enzyme sGC was not different between 2K-1C and 2K mesenteric bed. The relaxation induced by Terpy was inhibited by the cGMP-dependent protein kinase (G kinase) inhibitor or by the non-selective K(+) channel blocker tetraethylamonium (TEA), but with no difference between 2K-1C and 2K arteries. The relaxation induced by Terpy was also inhibited by the SERCA inhibitor thapsigargin in both groups. Taken together, these results show that the vascular relaxation induced by the NO donor [Ru(terpy)(bdq)NO](3+) involves the activation of NO/sGC/cGMP/GK pathway, activation of K(+) channels sensitive to TEA and SERCA in normotensive and renal hypertensive rat mesenteric resistance arteries. Surprisingly, Terpy-induced vasorelaxation is similar in mesenteric resistance arteries of renal hypertensive and normotensive rats.

A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle.

Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K(+) channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca(2+) sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca(2+) concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (E(max)) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPY-induced relaxation was reduced by tetraethylammonium (K(+) channels blocker) and by pre-contraction with 75mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca(2+) influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca(2+) concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca(2+) and K(+) across the membrane, it is more effective in reducing cytosolic Ca(2+) concentration and inducing relaxation in the rat trachea than the standard drug, SNP.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization.

The cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K(+) channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation. NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K(+) channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO(0) and SNP released both NO(-) and NO(0). The concentration-effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K(+) channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO(0) specie generated by RuBPY induces vasorelaxation due to activation of sGC, K(+) channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO(0) generated from RuBPY can also directly activate the K(+) channels in an independent way of sGC.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation.

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.

Osteossarcoma e condrossarcoma: diferenciação radiográfica por meio da tomografia computadorizada

A proposta deste trabalho consiste no estudo associativo e comparativo entre os aspectos radiograficos das tomografias computadorizadas de osteossarcomas e de condrossarcomas, com os exames histopatologicos destas lesoes. Foram analisadas as tomografias computadorizadas (TC) de 5 pacientes com osteossarcoma e 5 pacientes com condrossarcoma, na regiao de cabeca e pescoco. A sensibilidade da TC foi calculada a partir da confirmacao do exame histopatologico. Concluimos que o padrao radiografico dos osteossarcomas e condrossarcomas, observado na TC, pode auxiliar e facilitar o diagnostico final, por meio de caracteristicas individuais destas lesoes que, atraves do estudo qualitativo, puderam ser identificadas e analisadas.The purpose of this paper was to associate and compare the radiographic patterns of osteosarcoma and those of chondrosarcoma by means of computed tomography (CT). Computed tomographs of five cases of osteosarcoma and five cases of chondrosarcoma of the head and neck region were analyzed. The sensitivity of CT was calculated based on the results of histopathological examinations. We concluded that CT can facilitate the establishment of the final diagnosis since it is possible to distinguish the individual characteristics of osteosarcomas and chondrosarcomas in that exam.

Análise de carcinomas epidermóides por meio de radiografia panorâmica e tomografia computadorizada

The purpose of this work was to compare radiographic findings, such as localization and extension of tumors toward the bone and soft tissues, in panoramic radiography and computed tomography (CT). Four radiologists assessed the radiographic findings of 48 patients with the histopathological diagnosis of squamous cell carcinoma in different sites of the maxillofacial region. Panoramic radiographs and computed tomographs were obtained at the University of Iowa Hospitals and Clinics, at FUNDECTO - USP and at the hospital of the University of Sao Paulo (USP). We observed a considerable limitation of the panoramic radiography in determining the localization and extension of tumors, since it revealed unclear delimitations. Regarding CT, better results were obtained: it was possible to observe the invasion of the tumor toward adjacent soft tissues, as well as the extension of bone destruction and the depth of the lesion, which were confirmed by surgical findings. We concluded that computed tomography demonstrated to be a sensitive radiographic technique for the detection of the involvement of bone and soft tissues, contributing for a more precise diagnosis, surgical planning and intervention. On the other hand, panoramic radiography was considered less sensitive and less efficient than CT, since it shows only unclear borders of the lesions and is not able to assess the involvement of soft tissues.The purpose of this work was to compare radiographic findings, such as localization and extension of tumors toward the bone and soft tissues, in panoramic radiography and computed tomography (CT). Four radiologists assessed the radiographic findings of 48 patients with the histopathological diagnosis of squamous cell carcinoma in different sites of the maxillofacial region. Panoramic radiographs and computed tomographs were obtained at the University of Iowa Hospitals and Clinics, at FUNDECTO-USP and at the hospital of the University of São Paulo (USP). We observed a considerable limitation of the panoramic radiography in determining the localization and extension of tumors, since it revealed unclear delimitations. Regarding CT, better results were obtained: it was possible to observe the invasion of the tumor toward adjacent soft tissues, as well as the extension of bone destruction and the depth of the lesion, which were confirmed by surgical findings. We concluded that computed tomography demonstrated to be a sensitive radiographic technique for the detection of the involvement of bone and soft tissues, contributing for a more precise diagnosis, surgical planning and intervention. On the other hand, panoramic radiography was considered less sensitive and less efficient than CT, since it shows only unclear borders of the lesions and is not able to assess the involvement of soft tissues.