Rutin ameliorates nitrergic and endothelial dysfunction on vessels and corpora cavernosa of diabetic animals.

Endothelial dysfunction is an early complication of diabetes and it is related to both micro- and macroangiopathies. In addition, >70% of diabetic patients develop autonomic neuropathies. Increased oxidative stress has a major role in the development of both nitrergic and endothelial dysfunction. The aim of this work is to evaluate whether rutin, a potent antioxidant, could ameliorate nitrergic and/or endothelial dysfunction in diabetic animals. Primary and secondary treatment protocols with rutin were investigated on rat aortic rings and the mesenteric arteriolar bed, and on rabbit aortic rings and corpora cavernosa (RbCC) from both euglycemic and alloxan-diabetic animals. Acetylcholine endothelium-dependent and sodium nitroprusside endothelium-independent relaxations were compared in tissues from euglycemic or diabetic animals. Electrical field stimulation (EFS)-induced relaxation was performed only in the RbCC. Endothelial-dependent relaxations were blunted by 40% in vessels and neuronal relaxation was blunted by 50% in RbCC taken from diabetic animals when compared to euglycemic animals. Pre-treatment with rutin restored both neuronal and endothelial dependent relaxations in diabetic animals towards the values achieved in control euglycemic tissues. Rutin was able to ameliorate both endothelial dysfunction and nitrergic neuropathy in animal experimental models. Rutin could be a lead compound in the primary or secondary preventive ancillary treatment of endothelial and nitrergic dysfunction in the course of diabetes.

Nitroprusside─Expanding the Potential Use of an Old Drug Using Nanoparticles.

For more than 70 years, sodium nitroprusside (SNP) has been used to treat severe hypertension in hospital emergency settings. During this time, a few other clinical uses have also emerged such as in the treatment of acute heart failure as well as improving mitral incompetence and in the intra- and perioperative management during heart surgery. This drug functions by releasing nitric oxide (NO), which modulates several biological processes with many potential therapeutic applications. However, this small molecule has a short lifetime, and it has been administered through the use of NO donor molecules such as SNP. On the other hand, SNP also has some setbacks such as the release of cyanide ions, high water solubility, and very fast NO release kinetics. Currently, there are many drug delivery strategies that can be applied to overcome many of these limitations, providing novel opportunities for the use of old drugs, including SNP. This Perspective describes some nitroprusside properties and highlights new potential therapeutic uses arising from the use of drug delivery systems, mainly silica-based nanoparticles. There is a series of great opportunities to further explore SNP in many medical issues as reviewed, which deserves a closer look by the scientific community.

Diterpenoids from Leaves of Cultivated Plectranthus ornatus.

Two new diterpenoid derivatives 7α,12ß,17-triacetoxy-6ß,19-dihydroxy-13ß,16-spirocicloabiet-8-ene-11,14-dione ( 1: ) and 6ß-acetoxy-3ß,7α,12α-trihydroxy-13ß,16-spirocicloabiet-8-ene-11,14-dione ( 2: ) along with 11 ( 3: - 13: ) miscellaneous compounds were isolated from the leaves of Plectranthus ornatus Codd. Their structures were elucidated by spectroscopic analysis and gauge independent atomic orbitals 13C NMR calculations. The isolated compounds were screened for their effects on intestinal motility using guinea-pig ileum and duodenum and by their cytotoxicity against 4 human cancer cell lines (HCT-116, SF-295, PC-3, and HL-60). Compounds 6: and 9: were moderately cytotoxic against HL-60, whereas 6: and 13: were more active on SF-295 and HCT-116.

Pentacyanoferrate(II) complex of pyridine-4- and pyrazine-2-hydroxamic acid as source of HNO: investigation of anti-tubercular and vasodilation activities.

A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO.)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na3[FeII(CN)5] moiety. The corresponding pentacyanoferrate(II) complex Na4[FeII(CN)5(PyzCONHO-)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with H2O2 was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H2O2- and N-coordinated pyrazine FeII species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.

A divergent mode of activation of a nitrosyl iron complex with unusual antiangiogenic activity.

Nitric oxide (NO) and nitroxyl (HNO) have gained broad attention due to their roles in several physiological and pathophysiological processes. Remarkably, these sibling species can exhibit opposing effects including the promotion of angiogenic activity by NO compared to HNO, which blocks neovascularization. While many NO donors have been developed over the years, interest in HNO has led to the recent emergence of new donors. However, in both cases there is an expressive lack of iron-based compounds. Herein, we explored the novel chemical reactivity and stability of the trans-[Fe(cyclam)(NO)Cl]Cl2 (cyclam = 1,4,8,11-tetraazacyclotetradecane) complex. Interestingly, the half-life (t1/2) for NO release was 1.8 min upon light irradiation, vs 5.4 h upon thermal activation at 37 °C. Importantly, spectroscopic evidence supported the generation of HNO rather than NO induced by glutathione. Moreover, we observed significant inhibition of NO donor- or hypoxia-induced HIF-1α (hypoxia-inducible factor 1α) accumulation in breast cancer cells, as well as reduced vascular tube formation by endothelial cells pretreated with the trans-[Fe(cyclam)(NO)Cl]Cl2 complex. Together, these studies provide the first example of an iron-nitrosyl complex with anti-angiogenic activity as well as the potential dual activity of this compound as a NO/HNO releasing agent, which warrants further pharmacological investigation.

Renal protective effect of pinitol in experimental diabetes.

Pinitol is a natural antidiabetic agent shown to prevent or ameliorate metabolic and overall vascular and neural function. In the present study we have evaluated the potential benefits of pinitol on renal function of streptozotocin (STZ)-induced diabetic rats. Both euglycemic or 8-week or 16-week diabetic rats were treated with either saline (1 ml/kg/12h; p.o) or pinitol (20 mg/kg/12h; p.o). The renal function was evaluated by using metabolic cages, renal hemodynamic and tubular parameters measurements. Histological examination and evaluation of the protein expression of renal markers such as nephrin, TGFß and pERK were also performed. Pinitol decreased by 50% the increased urinary albumin/creatinine ratio in both 8-week and 16 week diabetic rats. In addition, the glomerular volume of 16-week rats increased by 55% and this increase was blunted by pinitol. Remarkably, pressure-natriuresis was completely blunted in both 8 and 16-week diabetic rats but this impairment was prevented by pinitol in both treatment regimens. Pinitol ameliorated renal lesions and also prevented the decrease in nephrin expression and the increase of pERK and TGFß expression in both diabetic groups. Natriuresis due to high renal perfusion pressure increased 7-fold in control animals but was blocked in 16-week diabetic rats and remarkably pinitol partially restored pressure natriuresis (3-fold increase in sodium excretion during pressure natriuresis). Pinitol prevents and ameliorates albuminuria, glomerular expansion, impairment of pressure-natriuresis, renal structural alterations and changes of renal markers and has the potential to be tested for the prevention of diabetic kidney disease.

Antihypertensive potential of cis-[Ru(bpy)2(ImN)(NO)]3+, a ruthenium-based nitric oxide donor.

The aim of this study was to investigate the antihypertensive properties of cis-[Ru(bpy)2ImN(NO)]3+ (FOR0811) in normotensive and in Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Vasorelaxant effects were analyzed by performing concentration response curve to FOR0811 in rat aortic rings in the absence or presence of 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ), L-cysteine or hydroxocobalamin. Normotensive and L-NAME-hypertensive rats were treated with FOR0811 and the effects in blood pressure and heart rate variability in the frequency domain (HRV) were followed. FOR0811 induced relaxation in rat aortic rings. Neither endothelium removal nor L-cysteine altered the FOR0811 effects. However, the incubation with ODQ and hydroxocobalamin completely blunted FOR0811 effects. FOR0811 administered intravenously by bolus infusion (0.01-1 mg/bolus) or chronically by using subcutaneous implanted osmotic pumps significantly reduced the mean arterial blood pressure. The effect was long lasting and did not induce reflex tachycardia. FOR0811 prevented both LF and VLF increases in L-NAME hypertensive rats and has antihypertensive properties. This new ruthenium complex compound might be a promising nitric oxide donor to treat cardiovascular diseases.

In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. There are many complications presented by the current treatment, as high toxicity, high cost and parasite resistance, making the development of new therapeutic agents indispensable. The present study aims to evaluate the leishmanicidal potential of ruthenium nitrosyl complex cis-[Ru(bpy)2(SO3)(NO)](PF6) against Leishmania (Viannia) braziliensis. The effect of this metal complex on parasite-host interaction was evaluated by in vitro efficacy test in dermal fibrobast cells in the presence of different concentrations (1, 10, 50 and 100 µM) and by in vivo efficacy tests performed in the presence of two different concentrations of complex (100 µg/kg/day or 300 µg/kg/day) evaluating its effect on the size of the lesion and the number of parasites present in the draining lymph nodes in hamsters. Even at the lowest concentration of 1 µM of ruthenium complex, it was observed a significant decrease of the infected cells, after 24 h exposure in vitro, with total reduction at 50 µM of the ruthenium complex. In the in vivo cutaneous infection model, administration of daily doses of 300 µg/kg/day of complex reduced significantly lesion size by 51% (p < 0.05), with a 99.9% elimination of the parasites found in the lymph nodes (p < 0.001). The results suggest a promising leishmanicidal effect by that ruthenium nitrosyl complex against L. (V.) braziliensis.

Early consumption of high-fat diet worsens renal damage in spontaneously hypertensive rats in adulthood.

The association between hypertension and obesity has been shown to be an important cause of kidney disease. We aimed to investigate the impact of a high-fat diet (HFD) administered in spontaneously hypertensive rats (SHR) after weaning in renal morphology and functional parameters. Male post-weaned SHR were divided into two groups: standard control diet (CD) (3% lipids; n = 8) or HFD (30% lipids; n = 8) during 8 weeks. The group HFD showed an increase in serum triglycerides (HFD: 96 ± 7 vs. CD: 33 ± 2 mg/dL) and glucose intolerance (HFD: 185 ± 7 vs. CD: 149 ± 4 mg/dL/min). Moreover, the HFD also showed an increase in almost 90% of the periepididymal and retroperitoneal adiposity. There was no difference in arterial blood pressure between groups. Renal morphofunctional parameters were decreased in HFD group for glomerular tuft area and diameter (4733 ± 65 µm2 and 82 ± 1 µm, respectively) when compared with CD group (5289 ± 171 µm2 and 88 ± 2 µm, respectively). HFD also showed a decrease of 50% of the renal function, which was associated with higher renal extracellular matrix and lipid deposition. Therefore, our data suggest that HFD since early period of life may contribute to renal damage in adults with hypertension, and this impairment can be associated with increased renal lipid accumulation.

Intra-Renal Angiotensin Levels Are Increased in High-Fructose Fed Rats in the Extracorporeal Renal Perfusion Model.

Overconsumption of fructose leads to metabolic syndrome as a result of hypertension, insulin resistance, and hyperlipidemia. In this study, the renal function of animals submitted to high fructose intake was analyzed from weaning to adulthood using in vivo and ex vivo methods, being compared with a normal control group. We investigated in ex vivo model of the role of the renin Angiotensin system (RAS) in the kidney. The use of perfused kidney from animals submitted to 8-week fructose treatment showed that high fructose intake caused metabolic and cardiovascular alterations that were consistent with other studies. Moreover, the isolated perfused kidneys obtained from rats under high fructose diet showed a 33% increase in renal perfusion pressure throughout the experimental period due to increased renal vascular resistance and a progressive fall in the glomerular filtration rate, which reached a maximum of 64% decrease. Analysis of RAS peptides in the high fructose group showed a threefold increase in the renal concentrations of angiotensin I (Ang I) and a twofold increase in angiotensin II (Ang II) levels, whereas no change in angiotensin 1-7 (Ang 1-7) was observed when compared with the control animals. We did not detect changes in angiotensin converting enzyme (ACE) activity in renal tissues, but there is a tendency to decrease. These observations suggest that there are alternative ways of producing Ang II in this model. Chymase the enzyme responsible for Ang II formation direct from Ang I was increased in renal tissues in the fructose group, confirming the alternative pathway for the formation of this peptide. Neprilysin (NEP) the Ang 1-7 forming showed a significant decrease in activity in the fructose vs. control group, and a tendency of reduction in ACE2 activity. Thus, these results suggest that the Ang 1-7 vasodilator peptide formation is impaired in this model contributing with the increase of blood pressure. In summary, rats fed high fructose affect renal RAS, which may contribute to several deleterious effects of fructose on the kidneys and consequently an increase in blood pressure.

Effect of co-administration of two different phosphodiesterase inhibitors and a ß3-adrenoceptor agonist in an experimental model of detrusor overactivity.

The purpose of this study was to evaluate in vitro the effect of the combination of BRL 37344 (ß3-adrenoceptor agonist) with tadalafil (phosphodiesterase type 5 inhibitor) or rolipram (phosphodiesterase type 4 inhibitor) in an experimental model of detrusor overactivity. The experiments were carried out in two phases using bladder strips of mice. In the first phase, on the top of 40 mM potassium-induced contraction, strips isolated from control mice were exposed to increasing concentrations of each study drug. In another series of experiments, prior to contraction, strips were incubated with either tadalafil or rolipram, followed by the addition of increasing concentrations of BRL 37344. In the second phase, the same protocols were performed with animals previously treated with L-NAME for 30 days. Chronic L-NAME administration leads to detrusor overactivity due to nitric oxide synthase inhibition. In phase one, preincubation with tadalafil enhanced relaxation response to BRL 37344 at two concentrations. Pretreatment with rolipram had no effect on BRL 37344-induced relaxation. In L-NAME-treated mice, rolipram induced more relaxation than the other drugs, enhancing relaxation response to BRL 37344 at almost all concentrations, but no synergistic effect with tadalafil was observed. The relaxant effect of BRL 37344 was enhanced by rolipram but not by tadalafil, suggesting that PDE4 inhibition, especially when associated with ß3-adrenoceptor stimulation, could represent a potential treatment for overactive bladder.

Low-level laser therapy (904nm) can increase collagen and reduce oxidative and nitrosative stress in diabetic wounded mouse skin.

BACKGROUND AND OBJECTIVE: Over the last decade we have seen an increased interest in the use of Low-Level Laser Therapy (LLLT) in diseases that involve increased oxidative stress. It is well established that hyperglycemia in diabetes elicits a rise in reactive oxygen species (ROS) production but the effect of LLLT remains unclear. This study aimed to investigate whether LLLT was able to improve oxidative/nitrosative stress parameters in the wound healing process in diabetic mice. STUDY DESIGN/MATERIALS AND METHODS: Twenty male mice were divided into four groups: non-irradiated control (NIC), irradiated control (IC), non-irradiated and diabetic (NID), irradiated and diabetic (ID). Diabetes was induced by administration of streptozotocin. Wounds were created 120days after the induction of diabetes in groups IC and ID and these groups were irradiated daily for 5days (superpulsed 904nm laser, average power 40mW, 60s). All animals were sacrificed 1day after the last irradiation and histology, collagen amount, catalase activity, nitrite and thiobarbituric acid reactive substances (TBARS) were measured. RESULTS: Histology showed that collagen fibers were more organized in IC and ID when compared to NID group, and significant differences in collagen content were found in group ID versus NID. Catalase activity was higher in IC group compared to other groups (p<0.001). TBARS levels were higher in IC versus NIC, but were lower in ID versus NID (p<0.001). Nitrite was lower in both irradiated groups versus the respective non-irradiated groups (p<0.001). CONCLUSIONS: Delayed wound healing in diabetes is still a challenge in clinical practice with high social costs. The increased production of collagen and decreased oxidative and nitrosative stress suggests that LLLT may be a viable therapeutic alternative in diabetic wound healing.

The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.

As lectinas são descritas como (glico)proteínas que se ligam, especificamente e reversivelmente, a carboidratos. Lectinas de leguminosas isoladas da subtribo Diocleinae (Canavalia, Dioclea eCratylia) são estruturalmente homólogas em relação às suas estruturas primárias. Demonstrou-se que as lectinas de DiocleinaeCanavalia brasiliensis, Dioclea guianensis eCanavalia ensiformis alteram diferentemente parâmetros fisiológicos em rins isolados de ratos. Dessa maneira, o objetivo deste estudo foi investigar o papel da lectina de Cratylia floribunda (CFL) na hemodinâmica renal e no transporte de íons em ratos. Em rins isolados perfundidos, CFL (10 mg/mL, n=5) aumentou a pressão de perfusão renal, a resistência vascular renal e reduziu o percentual do transporte tubular de K+, mas não alterou o fluxo urinário, a taxa de filtração glomerular e o percentual de transporte tubular dos íons sódio e cloreto. No leito mesentérico isolado perfundido, CFL (3 e 10 mg/mL/min, n=4) não alterou o tônus basal ou a contração do tecido induzida por fenilefrina (1 mM/mL/min). Em conclusão, a lectina de sementes de Cratylia floribunda altera parâmetros hemodinâmicos renais, provavelmente de origem tubular, e não por alterações hemodinâmicas.

Effects of the essential oil of Croton zehntneri and its major components, anethole and estragole, on the rat corpora cavernosa.

AIMS: The effects of the essential oil of Croton zehntneri (EOCz) and its major components anethole, estragole and methyl eugenol were evaluated in phenylephrine precontracted rat corpora cavernosa (RCC). MAIN METHODS: RCC strips were mounted in 5 ml organ baths for isometric recordings of tension, precontracted with 10 µM phenylephrine and exposed to test drugs. KEY FINDINGS: All major compounds relaxed RCC. The order of potency was estragole>anethole>methyl eugenol. The maximal relaxation to EOCz and methyl eugenol was 62.67% (IC50 of 1.67 µM) and 45.8% (IC50 of 1.7 µM), respectively. Estragole relaxed RCC with an IC50 of 0.6 µM (maximal relaxation-76.6%). The maximal relaxation to estragole was significantly reduced by L-NAME (43.46%-IC50 of 1.4 µM), ODQ (53.11%-IC50 of 0.83 µM) and indomethacin (24.41%-IC50 of 1.3 µM). On the other hand, anethole relaxed RCC by 66.73% (IC50 of 0.96 µM) and this relaxation was blunted by indomethacin (35.65%-IC50 of 1.6 µM). Both estragole and anethole increased the relaxation achieved upon electrical stimulation. Both compounds increased the levels of cAMP (estragole by 3-fold and anethole by 2-fold when compared to controls). Estragole also increased the levels of cGMP (0.5-fold). SIGNIFICANCE: The higher potency of these compounds to relax corpora cavernosa smooth muscle may form the pharmacological basis for the use of such substances as leading compounds in the search of alternative treatments of erectile dysfunction.

Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound.

PURPOSE: The aim of this study was to evaluate the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitrosil-ruthenium, named trans-[Ru(NH3)4(caffeine)(NO)]C13 (Rut-Caf) and sodium nitroprusside (SNP). MATERIALS AND METHODS: The tissues, immersed in isolated bath systems, were pre-contracted with phenilephrine (PE) (1 µM) and then concentration-response curves (10 (-12) - 10(-4) M) were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM), oxyhemoglobin (10 µM), L-cysteine (100 µM), hydroxicobalamine (100 µM), glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. RESULTS: The substances provoked significant relaxation of the cavernous smooth muscle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50) and maximum effect (E(max)). The substances showed activity through activation of the soluble guanylyl cyclase (sGC), because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. L-cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09). There were no alterations of potency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP during the relaxation process. CONCLUSIONS: In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO in the intracellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels.

Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound

PURPOSE: The aim of this study was to evaluate the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitrosil-ruthenium, named trans-[Ru(NH3)4(caffeine)(NO)]C13 (Rut-Caf) and sodium nitroprusside (SNP). MATERIALS AND METHODS: The tissues, immersed in isolated bath systems, were pre-contracted with phenilephrine (PE) (1 µM) and then concentration-response curves (10-12 - 10-4 M) were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM), oxyhemoglobin (10 µM), L-cysteine (100 µM), hydroxicobalamine (100 µM), glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. RESULTS: The substances provoked significant relaxation of the cavernous smooth muscle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50) and maximum effect (Emax). The substances showed activity through activation of the soluble guanylyl cyclase (sGC), because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. L-cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09). There were no alterations of potency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP during the relaxation process. CONCLUSIONS: In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO in the intracellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels.

Guanylin peptide family: history, interactions with ANP, and new pharmacological perspectives.

The guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. These hormones are synthesized in the intestine and released both luminally and into the circulation, and are also produced within the kidney. Stimulation of renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis by both cGMP-dependent and independent mechanisms. Uroguanylin may act as a hormone in a novel endocrine axis linking the digestive system and kidney as well as a paracrine system intrarenally to increase sodium excretion in the postprandial period. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess sodium in the urine. In addition, small concentrations of the atrial natriuretic peptide (ANP) can synergize with low concentrations of both guanylin or uroguanylin, which do not induce natriuresis per se, to promote significant natriuresis. Interestingly, the activation of the particulate guanylate cyclase receptors by natriuretic peptides can promote relaxation of animal and human penile erectile tissue and increase intracavernosal pressure to induce penile erection. These peptides can be prototypes for new drugs to treat erectile dysfunction, especially in patients with endothelial and nitrergic dysfunction, such as in diabetes.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2.

Bothrops marajoensis is found in the savannah of Marajó Island in the State of Pará and regions of Amapá State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A(2) (PLA(2)). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA(2) showed amino acid identity with other lysine K49 sPLA(2)s of snake venom. B. marajoensis venom (30 microg/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA(2) did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA(2) decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA(2) induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system.

1,8-Cineole induces relaxation in rat and guinea-pig airway smooth muscle.

OBJECTIVES: 1,8-Cineole is a monoterpene with anti-inflammatory, vascular and intestinal smooth muscle relaxant activity. We have evaluated the potential bronchodilatatory activity of this compound. METHODS: 1,8-Cineole was tested against carbachol, histamine, K+ 80 mM and ovalbumin-induced bronchial contractions in Wistar rat or guinea-pig tissues. Some of the guinea-pigs had been previously sensitized with an intramuscular injection of 5% (w/v) ovalbumin/saline solution. Control animals received 0.3 ml saline. In separate experimental groups the response to 1,8-cineole (1-30 mg/kg), phenoterol (0.05-5 mg/kg) or vehicle (0.3% Tween in saline) was studied. KEY FINDINGS: 1,8-Cineole decreased, in vivo, rat bronchial resistance with similar efficacy as phenoterol (66.7 +/- 3.2% vs 72.1 +/- 5.3%). On the other hand, the maximal relaxant response to 1,8-cineole in carbachol-precontracted rat tracheas was 85.5 +/- 5.7% (IC50 = 408.9 (328-5196) microg/ml) compared with 80.2 +/- 4.8% (IC50 = 5.1 (4.3-6.1) microg/ml) with phenoterol. The addition of 1,8-cineole to guinea-pig tracheal rings tonically contracted with K+ 80 mM induced a concentration-related relaxation. The maximal relaxation elicited by 1,8-cineole was 113.6 +/- 11.7% (IC50 127.0 (115.9-139.2) microg/ml) compared with 129.7 +/- 14.6% (IC50 0.13 (0.12-0.14) microg/ml) achieved after phenoterol administration. In addition, the incubation of tracheal rings with 1,8-cineole (100, 300 or 1000 microg/ml), 15 min before inducing phasic contractions with K+ 80 mM, decreased the maximal amplitude of the contraction by 31.6 +/- 4.6, 75.7 +/- 2.7 and 92.2 +/- 1.5%, respectively. In another set of experiments, neither the maximal response nor the IC50 for the 1,8-cineole-induced relaxation were different between normal and ovalbumin-sensitized tissues. Moreover, the relaxation of bronchial rings contracted after exposure to 1 microg/ml ovalbumin occurred at a faster rate in rings pre-incubated with 1,8-cineole when compared with rings pre-incubated with vehicle only (Tween 0.3%). Therefore, in the first minute after the antigen challenge, the tracheal tissue relaxed after the peak contraction by 6.5, 21.4 (P < 0.05 vs control) and 66.9% (P < 0.05 vs control) in the presence of 100, 300 or 1000 microg/ml 1,8-cineole, respectively. CONCLUSIONS: 1,8-Cineole relaxed rat and guinea-pig (nonsensitized and ovalbumin-sensitized) airway smooth muscle by a nonspecific mechanism.