Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes.

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

Relaxant effect of a metal-based drug in human corpora cavernosa and its mechanism of action.

We studied the mechanisms involved in the human corpora cavernosa (HCC) relaxation induced by a new metal-based nitric oxide (NO) donor, the ruthenium complex cis-[Ru(bpy)2Imn(NO)](+3) (FOR0811). FOR0811 produced relaxation in phenylephrine (PE)-precontracted HCC with a maximal response that achieved 112.9 ± 10.6%. There was no difference between the maximal relaxation induced by FOR0811 when compared with sodium nitroprusside (SNP) (106.8 ± 7.3%), BAY41-2272 (107.6 ± 4.1%) or vardenafil (103.4 ± 3.8%), however, FOR0811 was less potent than SNP and vardenafil. L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, had no effect in the concentration-response curve elicited by FOR0811. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a heme-site inhibitor of soluble guanylyl cyclase (sGC) was able to either block or reverse the relaxation induced by FOR0811. On the other hand, the relaxation induced by FOR0811 was not affected by glibenclamide, a blocker of ATP-sensitive potassium channels. FOR0811 (10 µM) was able to increase cyclic guanosine monophosphate (cGMP) levels in corpora cavernosa strips. FOR0811 completely relaxes HCC by a sGC-cGMP-dependent mechanism and can be a lead compound in the development of new stable NO donors.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones.

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

Uroguanylin induces electroencephalographic spikes in rats.

Uroguanylin (UGN) is an endogenous peptide that acts on membrane-bound guanylate cyclase receptors of intestinal and renal cells increasing cGMP production and regulating electrolyte and water epithelial transport. Recent research works demonstrate the expression of this peptide and its receptor in the central nervous system. The current work was undertaken in order to evaluate modifications of electroencephalographic spectra (EEG) in anesthetized Wistar rats, submitted to intracisternal infusion of uroguanylin (0.0125 nmoles/min or 0.04 nmoles/min). The current observations demonstrate that 0.0125 nmoles/min and 0.04 nmoles/min intracisternal infusion of UGN significantly enhances amplitude and frequency of sharp waves and evoked spikes (p = 0.03). No statistical significance was observed on absolute alpha and theta spectra amplitude. The present data suggest that UGN acts on bioelectrogenesis of cortical cells by inducing hypersynchronic firing of neurons. This effect is blocked by nedocromil, suggesting that UGN acts by increasing the activity of chloride channels.

Chronic treatment with D-chiro-inositol prevents autonomic and somatic neuropathy in STZ-induced diabetic mice.

AIM: D-chiro-inositol (DCI) has been shown to prevent and reverse endothelial dysfunction in diabetic rats and rabbits. The present study evaluates the preventive effect of DCI on experimental diabetic neuropathy (DN). METHODS: Streptozotocin-induced (STZ) diabetic mice were treated by oral gavage for 60 days with DCI (20 mg/kg/12 h) or saline (NaCl 0.9%; 0.1 ml/10 g/12 h; Diab) and compared with euglycaemic groups treated with saline (0.1 ml/10 g/12 h; Eugly). We compared the response of the isolated sciatic nerve, corpora cavernosa or vas deferens to electrical stimulation. RESULTS: The electrically evoked compound action potential of the sciatic nerve was greatly blunted by diabetes. The peak-to-peak amplitude (PPA) was decreased from 3.24 ± 0.7 to 0.9 ± 0.2 mV (p < 0.05), the conduction velocity (CV) of the first component was reduced from 46.78 ± 4.5 to 26.69 ± 3.8 ms (p < 0.05) and chronaxy was increased from 60.43 ± 1.9 to 69.67 ± 1.4 ms (p < 0.05). These parameters were improved in nerves from DCI-treated mice (p < 0.05). PPA in the DCI group was 5.79 ± 0.8 mV (vs. 0.9 ± 0.2 mV-Diab; p < 0.05) and CV was 45.91 ± 3.6 ms (vs. 26.69 ± 3.8 ms-Diab; p < 0.05). Maximal relaxation of the corpus cavernosum evoked by electrical stimulation (2-64 Hz) in the Diab group was 36.4 ± 3.8% compared to 65.4 ± 2.8% in Eugly and 59.3 ± 5.5% in the DCI group (p < 0.05). Maximal contraction obtained in the vas deferens was 38.0 ± 9.2% in Eugly and 11.5 ± 2.6% in Diab (decrease of 69.7%; p < 0.05), compared to 25.2 ± 2.3% in the DCI group (p < 0.05 vs. diabetic). Electron microscopy of the sciatic nerves showed prevention of neuronal damage. CONCLUSIONS: DCI has a neuroprotective action in both autonomic and somatic nerves in STZ-induced DN.

Purification and biological effects of a C-type lectin isolated from bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15 percent. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules.

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 muM and 10 muM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 muM concentrations. Comparing control and REN concentration of 1 muM, JHCO(3)(-), nmol cm(-2) s(-1)-1,76+/-0,11(control)x1,29+/-0,08(REN 10 muM); P<0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 muM (JHCO(3)(-), nmol cm(-2) s(-)1-0.80+/-0.07(control)x0.60+/-0.06(REN 1 muM); P<0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na(+)/H(+)exchanger and H(+)-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.

Antivenom action on renal effects induced by Thalassophryne nattereri venom

Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 miug/mL), the antivenom alone (1 miug/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat Kidney, T. nattereri venom (1 miug/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (% TNa more control equal 81.1 more or less 0.86; % TNa more 60 equal 78.04 more or less 1.18; % TNa more 90 equal -5.16 more or less 3.34; %TNa more 120 equal 79.49 more or less 0.87) and potassium (%TKcontrol equal 72.29 more or less 1.12; %TK more 60 equal 75.41 more or less 0.65; % TK more 90 equal 71.23 more or less 2.55; % TK more 120 equal 76.62 more or less 1.04) tubular transporto. The administration of the antivenom (1 miug/mL) incubated with venom (1 miug/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space.(...)

Sodium fluoride and chlorhexidine effect in the inhibition of mutans streptococci in children with dental caries: a randomized, double-blind clinical trial.

OBJECTIVE: We aimed to compare the effect of sodium fluoride and chlorhexidine on salivary levels of mutans streptococci (MS), in a double-blind, randomized clinical trial. METHODS: Thirty-five healthy volunteers, aged 4-8 years, with at least one active carious lesion and no previous history of allergies were selected to participate in the study. A gel formulation containing either 1.23% sodium fluoride or 1% chlorhexidine was topically administered to the dentition every 24 h for 6 consecutive days. Salivary MS levels were measured at baseline (D1) and on the 6th (D6), 15th (D15), and 30th (D30) days. For microbiological analysis, Mitis Salivarius-Bacitracin agar medium was used. RESULTS: Difference between treatments was only verified on D6. On the last day of treatment 1% chlorhexidine gel was significantly more effective than fluoride (P = 0.0000). The use of sodium fluoride did not cause a statistically significant variation in salivary MS levels throughout the duration of the study. Following treatment, a subsequent increase in MS counts between D6 and D15 (P = 0.0001) was observed with chlorhexidine. CONCLUSION: A 6-day treatment with a 1% chlorhexidine gel was effective in reducing salivary MS; there was a significant MS increase once treatment was suspended. The use of 1.23% sodium fluoride under the same regimen was not able to reduce salivary MS levels. Our results suggest repeated treatment with 1% chlorhexidine as a means for maintaining low salivary MS levels in children with dental caries.

BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects.

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 microM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 microM; guanylin - 0.2 microM) it promoted increases in urine flow (DeltaUF of 0.25 +/- 0.09 mL.g(-1)/min, P < 0.05) and Na+ excretion (% Delta ENa+ of 18.20 +/- 2.17, P < 0.05). BTCI (1.0 microM) also increased %ENa+ (from 22.8 +/- 1.30 to 34.4 +/- 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 microM) induced increases in glomerular filtration rate (GFR; from 0.96 +/- 0.02 to 1.28 0.02 mL.g(-1)/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 µM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 µM; guanylin - 0.2 µM) it promoted increases in urine flow (deltaUF of 0.25 ± 0.09 mL.g-1/min, P < 0.05) and Na+ excretion ( percent delta ENa+ of 18.20 ± 2.17, P < 0.05). BTCI (1.0 µM) also increased percentENa+ (from 22.8 ± 1.30 to 34.4 ± 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 µM) induced increases in glomerular filtration rate (GFR; from 0.96 ± 0.02 to 1.28 0.02 mL.g-1/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

Guanilina e uroguanilina são peptídeos pequenos, ricos em cisteína, envolvidos na regulação da homeostase de fluidos e eletrólitos através da ligação e ativação da guanilato ciclase expressa no intestino e nos rins. A guanilina é menos potente do que a uroguanilina como agente natriurético e é degradada in vitro pela quimiotripsina devido a características estruturais únicas no domínio bioativo do peptídeo. Portanto o objetivo deste trabalho foi verificar se a guanilina é degradada por proteases tipo quimiotripsina, presentes na membrana da borda em escova dos rins. Para esta investigação, foi usado o modelo do rim isolado de rato perfundido. A Guanilina (0,2 µM) não induziu mudanças na função renal. Entretanto, quando pré-tratada com inibidor de tripsina e de quimiotripsina de black-eyed pea (BTCI - 1,0 µM; guanilina - 0,2 µM) promoveu um aumento no fluxo urinário (deltaUF de 0,25 ± 0,09 mL.g-1/min, P < 0,05) e na excreção de Na+ ( por centoDENa+ de 18,20 ± 2,17, P < 0,05). BTCI (1,0 µM) também aumenta por centoENa+ (de 22,8 ± 1,30 a 34,4 ± 3,48, P < 0,0590 minutos). Além disto, BTCI (3,0 µM) induziu um aumento da taxa de filtração glomerular (GFR; de 0,96 ± 0,02 para 1,28 ± 0,02 mL.g-1/min, P < 0,05, 60 minutos). O presente trabalho sugere fortemente que proteases semelhantes à quimiotripsina desempenham um papel no metabolismo renal de guanilinas e descreve, pela primeira vez, os efeitos renais induzidos por um membro da família de inibidores de proteases do tipo Bowman-Birk.

The role of indomethacin and tezosentan on renal effects induced by Bothrops moojeni Lys49 myotoxin I.

Renal changes determined by Lys49 myotoxin I (BmTx I), isolated from Bothrops moojeni are well known. The scope of the present study was to investigate the possible mechanisms involved in the production of these effects by using indomethacin (10 microg/mL), a non-selective inhibitor of cyclooxygenase, and tezosentan (10 microg/mL), an endothelin antagonist. By means of the method of mesenteric vascular bed, it has been observed that B. moojeni myotoxin (5 microg/mL) affects neither basal perfusion pressure nor phenylephrine-preconstricted vessels. This fact suggests that the increase in renal perfusion pressure and in renal vascular resistance did not occur by a direct effect on renal vasculature. Isolated kidneys from Wistar rats, weighing 240-280 g, were perfused with Krebs-Henseleit solution. The infusion of BmTx-I increased perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. Sodium, potassium and chloride tubular transport was reduced after addition of BmTx-I. Indomethacin blocked the effects induced by BmTx-I on perfusion pressure and renal vascular resistance, however, it did not revert the effect on urinary flow and sodium, potassium and chloride tubular transport. The alterations of glomerular filtration rate were inhibited only at 90 min of perfusion. The partial blockade exerted by indomethacin treatment showed that prostaglandins could have been important mediators of BmTx-I renal effects, but the participation of other substances cannot be excluded. The blockage of all renal alterations observed after tezosentan treatment support the hypothesis that endothelin is the major substance involved in the renal pathophysiologic alterations promoted by the Lys49 PLA(2) myotoxin I, isolated from B. moojeni. In conclusion, the rather intense renal effects promoted by B. moojeni myotoxin-I were probably caused by the release of renal endothelin, interfering with the renal parameters studied.

Inositols prevent and reverse endothelial dysfunction in diabetic rat and rabbit vasculature metabolically and by scavenging superoxide.

Endothelial dysfunction (ED) is an early feature of cardiovascular risk and diabetes. Hyperglycemia and hyperlipidemia are causative factors. Excessive endothelial mitochondrial superoxide (ROS) production with hyperglycemia and hyperlipidemia is a key mechanism. Inositol components of an insulin inositol glycan mediator, d-chiro-inositol (DCI) and 3-O-methyl DCI (pinitol), decrease hyperglycemia and hyperlipidemia. We tested whether these, myoinositol and dibutyryl DCI (db-DCI), would prevent or reverse ED in diabetic rats and rabbits. Oral inositols reduced hyperglycemia and hypertriglyceridemia with different potencies and prevented ED in rat aortic rings and mesenteric beds. Inositols added in vitro to five diabetic tissues reversed ED. Relaxation by Ach, NO, and electrical field stimulation was potentiated by inositols in vitro in rabbit penile corpus cavernosa. Inositols in vitro restored impaired contraction by the eNOS inhibitor l-NAME and increased NO effectiveness. DCI and db-DCI decreased elevated ROS in endothelial cells in high glucose and db-DCI reduced PKC activation, hexosamine pathway activity, and advanced glycation end products to basal levels. Xanthine/xanthine oxidase generated superoxide was reduced by superoxide dismutase or inositols, with db-DCI efficacious in a mechanism requiring chelated Fe(3+). Histochemical examination of rat aortic rings for protein SNO demonstrated a decrease in diabetic rings with restoration by inositols. In summary, inositols prevented and reversed ED in rat and rabbit vessels, reduced elevated ROS in endothelial cells, potentiated nitrergic or vasculo-myogenic relaxations, and preserved NO signaling. These effects are related to their metabolic actions, direct superoxide scavenging, and enhancing and protecting NO signaling. Of the inositols tested, db-DCI was most effective.

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom.

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.

Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; P<0.05; n = 8) and ODQ (100 microM) (62.7+/-5.1 vs 26.8+/-3.9%; P<0.05; n = 8). Charybdotoxin and apamin (K(Ca)-channel blockers) did not affect the phentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; P<0.05; n = 8) of the phentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; P<0.05; n = 8). The results suggest that phentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0 percent), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2 percent), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0 percent), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom.

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 micro g/ml) in the absence and presence of thalidomide (15 micro M) or pentoxifylline (500 micro M) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 +/- 1.3; venom = 137.1 +/- 1.5; Thalid = 121.0 +/- 2.5; Ptx = 121.4 +/- 4.0 mmHg), renal vascular resistance (control = 4.5 +/- 0.2; venom = 7.3 +/- 0.6; Thalid = 4.5 +/- 0.9; Ptx = 4.8 +/- 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 +/- 0.001; venom = 0.44 +/- 0.01; Thalid = 0.22 +/- 0.007; Ptx = 0.21 +/- 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 +/- 0.06; venom = 1.91 +/- 0.11; Thalid = 0.75 +/- 0.04; Ptx = 0.77 +/- 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 +/- 0.9; venom = 73.9 +/- 0.66; Thalid = 76.6 +/- 1.1; Ptx = 81.8 +/- 2.0%), percent tubular chloride transport (control = 77.1 +/- 1.2; venom = 71.4 +/- 1.1; Thalid = 77.6 +/- 1.7; Ptx = 76.8 +/- 1.2%), and percent tubular potassium transport (control = 72.7 +/- 1.1; venom = 63.0 +/- 1.1; Thalid = 72.6 +/- 1.0; Ptx = 74.8 +/- 1.0%), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom.

Effects of Thalassophryne nattereri fish venom in isolated perfused rat kidney.

Thalassophryne nattereri, popularly known as Niquim, is a venomous fish responsible for many accidents in fishermen in the Northeast of Brazil. The effects of T. nattereri venom on renal physiology has not been tested. Isolated kidneys from Wistar rats of 240-280 g weight were perfused with Krebs-Henseleit solution containing 6g% of previously dialyzed bovine serum albumin. The effects of Niquim venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). The venom of T. nattereri (0.3, 1.0, and 3.0 microg/ml) was always added to the system 30 minutes after the beginning of each experiment (n=6). All experiments were preceded by 30 minutes internal control period and an external control group, where kidneys were perfused with only Krebs-Henseleit solution. All three doses tested promoted increases in PP and RVR. The first two doses also increased GFR and UF. The higher dose promoted decreases in GFR, UF, %TNa(+), %TK(+), %TCl(-). In the treated groups we observed hyalin casts inside all tubules and proteinaceous material in the urinary space. We conclude that the effects resulted from niquim venom agents that promoted a direct effect in kidney cells causing the release of vasoactive factors.