Cardiovascular effects of the essential oil of Alpinia zerumbet leaves and its main constituent,Terpinen-4-ol,in rats: role of the autonomic nervous system

Cardiovascular effects of intravenous ( i. v.) treatment with the essential oil of Alpinia zerumbet (EOAZ) were investigated in rats. Additionally this study examined (I) whether the autonomic nervous system is involved in the mediation of EOAZ-induced changes in mean aortic pressure (MAP) and heart rate (HR), and (II) whether these changes could be, at least in part, attributed to the actions of terpinen-4-ol (Trp-4-ol), the major constituent of EOAZ. In both pentobarbitone-anaesthetised and conscious rats, i. v. bolus injections of EOAZ (1 to 20 mg/kg) elicited immediate and dose-dependent decreases in MAP. In anaesthetised rats, EOAZ decreased HR only at higher doses (10 and 20 mg/kg), while changes of this parameter were not uniform in conscious rats. Hypotensive responses to EOAZ were of the same order of magnitude or duration, irrespective of whether the animal was under general anaesthesia. Pretreatment of anaesthetised rats with bilateral vagotomy did not modify significantly the hypotensive and bradycardic responses to EOAZ. In conscious rats, i. v. injections of bolus doses (1 to 10 mg/kg) of Trp-4-ol also elicited immediate and dose-dependent decreases in MAP. However, these hypotensive effects were significantly greater than those evoked by the same doses of EOAZ (1 to 10 mg/kg). Intravenous pretreatment of conscious rats with either methylatropine (1 mg/kg) or hexamethonium (30 mg/kg) had no significant effects on the EOAZ-induced hypotension. These data show that i. v. treatment with the EOAZ in either anaesthetised or conscious rats induced an immediate and significant hypotension, an effect that could be partially attributed to the actions of Trp-4-ol. The hypotension appears independent of the presence of an operational sympathetic nervous system, suggesting that the EOAZ may be a direct vasorelaxant agent.     

Effects of Crotalus durissus collilineatus venom in the isolated rat kidney.

Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A2. C. d. collilineatus (Cdc; 30 microg mL(-1)), crotoxin (CTX; 10 microg mL(-1)) and phospholipase A2 (PLA2; 10 microg mL(-1)) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control120 = 110.3 +/- 3.69 mmHg; Cdc120 = 96.7+/-8.1 mmHg), renal vascular resistance (RVR; control120 = 6.42+/-0.78 mmHg mL g(-1) min(-1); Cdc120 = 4.8+/-0.56 mmHg/mL g(-1) min(-1)), urinary flow (UF; control120 = 0.19+/-0.03 mL g(-1) min(-1); Cdc120 = 0.12 +/- 0.01 mL g(-1) min(-1)), and glomerular filtration rate (GFR; control120 = 0.79 +/- 0.07 mL g(-1) min(-1); Cdc120 = 0.53 +/- 0.09 mL g(-1) min(-1)), but had no effect on the percent of sodium tubular transport (%TNa+), percent of chloride tubular transport (%TK+) and percent of potassium tubular transport (%TCl-). CTX and PLA2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK+ (control120 = 69.94 +/- 6.49; CTX120 = 33.28 +/- 4.78) and %TCl- (control120 = 79.53 +/- 2.67; CTX120 = 64.62 +/- 6.93). PLA2 reduced the %TK+, but exerted no effect on the %TNa+ or on that of TCl-. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.     

Terpinen-4-ol: mechanisms of relaxation on rabbit duodenum

The effect of terpinen-4-ol was studied on rabbit duodenum in-vitro. Terpinen-4-ol induced relaxation of the basal tonus (IC50 170.2 (95% confidence interval, 175-204) microM) with a maximal relaxant response of 180.4+/-3.9% (n=6) of the contraction induced by 60 mM [K(+)]. The maximal relaxation induced in control conditions was not affected (P>0.05) by pretreatment of the tissues with phentolamine (50 microM) or propranolol (10 microM), N(G) nitro-L-arginine methyl ester (L-NAME; 1 mM), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM), hexamethonium (1 mM), tetrodotoxin (1 microM), the mixture charybdotoxin-apamin (1 microM), glibenclamide (10 microM), 4-aminopyridine (10 microM) or tetraethyl-ammonium (100 microM). In addition, terpinen-4-ol completely relaxed tissues precontracted with 60 mM [K(+)] solutions (IC50 325.9 (245.1-433.1) microM) and also blocked (IC50 154.7 (117.7-191.7) microM) the phasic component of this contraction. At a concentration of 195 and 650 muM it reduced by 41.3+/-3.4% and 75.4+/-3.1%, respectively the maximal contractile response to Ca(2+) in depolarized duodenum. Terpinen-4-ol completely blocked the component of carbachol-induced contraction, which was resistant to nifedipine (100 microM) pretreatment or to a Ca(2+)-free solution. These data show that terpinen-4-ol relaxes intestinal smooth muscle and suggest that this effect is myogenic in nature and depends on calcium antagonism.     

Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys.

Envenomation by Crotalus durissus terrificus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure (ARF). The most serious systemic change and primary cause of death is ARF. In this work, we used RP-HPLC to isolate crotoxin, convulxin and gyroxin from venom of the related subspecies Crotalus durissus cascavella and investigated the effects of these toxins on renal function in the isolated rat kidneys perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). Crotoxin (5 microg/ml) increased the PP, RVR, GFR, UF and decreased %TNa(+), %TK(+) and %TCl(-), with gyroxin (5 micro g/ml) the GFR remained stable during the 120 min of perfusion, whereas PP and RVR increased significantly and the %TNa(+), %TK(+) and %TCl(-) decreased significantly. Convulxin (5 micro g/ml) had no effect on renal function. Crotoxin caused alterations in all renal parameters. Gyroxin produced a minor effect compared to crotoxin. These results indicated that crotoxin is the main componenet responsible for acute nephrotoxicity caused by C. d. cascavella venom.     

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6kDa to 13kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2_Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action.     

Renal effects of supernatant from macrophages activated by Crotalus durissus cascavella venom: the role of phospholipase A2 and cyclooxygenase

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 microg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 microg/ml) was not statistically different from the control values. The most intense effects were seen at 10 microg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 microg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 microM) and quinacrine (10 microM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 microM) and nordiidroguaretic acid (1 microM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.     

Renal effects and vascular reactivity induced by Tityus serrulatus venom.

Tityus serrulatus, popularly known as yellow scorpion, is one of the most studied scorpion species in South America and its venom has supplied some highly active molecules. The effects of T. serrulatus venom upon the renal physiology in human showed increased renal parameters, urea and creatinine. However, in perfused rat kidney the effects were not tested until now. Isolated kidneys from Wistar rats, weighing 240-280 g, were perfused with Krebs-Henseleit solution containing 6% (g weight) of previously dialysed bovine serum albumin. The effects of T. serrulatus venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-). Tityus serrulatus venom (TsV; 10 microg/mL) was added to the system 30 min after the beginning of each experiment (n=6). This 30 min period was used as an internal control. The mesenteric bed was perfused with Krebs solution kept warm at 37 degrees C by a constant flow (4 mL/min), while the variable perfusion pressure was measured by means of a pressure transducer. The direct vascular effects of TsV (10 microg/mL/min; n=6), infused at a constant rate (0.1 mL/min), were examined and compared to the infusion of the vehicle alone at the same rate. TsV increased PP (PP30'=127.8+/-0.69 vs PP60'=154.2+/-14 mmHg*, *p<0.05) and RVR (RVR30'=6.29+/-0.25 vs RVR60'=8.03+/-0.82 mmHg/mLg(-1)min(-1)*, *p<0.05), decreased GFR (GFR30'=0.58+/-0.02 vs GFR60'=0.46+/-0.01mLg(-1)min(-1)*, *p<0.05) and UF (UF30'=0.135+/-0.001 vs UF60'=0.114+/-0.003mLg(-1)min(-1)*, *p<0.05). Tubular transport was not affected during the whole experimental period (120 min). On the other hand, the infusion of TsV (10 microg/mL/min) increased the basal perfusion pressure of isolated arteriolar mesenteric bed (basal pressure: 74.17+/-3.42 vs TsV 151.8+/-17.82 mmHg*, *p<0.05). TsV affects renal haemodynamics probably by a direct vasoconstrictor action leading to decreased renal flow.     

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom.

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.     

Comparative proteomic analysis of human whole saliva of children with protein-energy undernutrition

ObjectiveThe aim of the present study was to investigate the protein profile of children with different levels of protein–energy undernutrition (PEU) through a proteomic approach of human whole saliva.MethodsInitially, saliva samples of children with mild, moderate, and severe PEU were collected and lyophilized. Saliva samples of healthy children were used as controls. Samples were analyzed for total protein using the Bradford method. Saliva samples were analyzed by two-dimensional electrophoresis according to their isoelectric point (pI) and their molecular weights (MWs).ResultsComparisons of protein bands among the healthy and mildly, moderately, and severely undernourished children showed significant differences in the MWs (P = 0.001) and pI values (P = 0.03). In total 159 spots were identified in the healthy children; 156, 168, and 221 spots were observed in mildly, moderately, and severely undernourished children, respectively. Mildly undernourished children presented with the spot with the highest MW of 293 kDa (pI = 7.77) and the lowest MW of 5 kDa (pI = 4.83). Moderately undernourished children were the only ones who did not present with a protein band with an MW of 30 kDa. The presence of a protein band with an MW of 123 kDa (pI = 516), possibly a cyclin-dependent protein kinase, was also observed only in this group.ConclusionThe protein profile in saliva varies according to the presence or absence of PEU, and these variations are specifically expressed in different grades of undernutrition. Thus, saliva may be an important diagnostic tool for the assessment of PEU.