Bruno Escalante

Antiinflammatory activity of extracts from Aloe vera gel

We studied the effects of aqueous, chloroform, and ethanol extracts of Aloe vera gel on carrageenan-induced edema in the rat paw, and neutrophil migration into the peritoneal cavity stimulated by carrageenan. We also studied the capacity of the aqueous extract to inhibit cyclooxygenase activity. The aqueous and chloroform extracts decreased the edema induced in the hind-paw and the number of neutrophils migrating into the peritoneal cavity, whereas the ethanol extract only decreased the number of neutrophils. The antiinflammatory agents indomethacin and dexamethasone also decreased carrageenan-induced edema and neutrophil migration. The aqueous extract inhibited prostaglandin E2 production from [14C]arachidonic acid. The chemical tests performed in the aqueous extract for anthraglycosides, reductor sugars and cardiotonic glycosides were positive. In the ethanol extract, the chemical tests performed for saponins, carbohydrates naftoquinones, sterols, triterpenoids and anthraquinones were also positive. In the chloroform extract, the chemical tests performed for sterols type delta 5, and anthraquinones were positive. These results demonstrated that the extracts of Aloe vera gel have antiinflammatory activity and suggested its inhibitory action on the arachidonic acid pathway via cyclooxygenase.

Age-dependent changes in nitric oxide synthase activity and protein expression in striata of mice transgenic for the Huntington’s disease mutation

Huntingtons disease (HD) is an autosomal hereditary neurodegenerative disorder caused by an abnormal expansion of the CAG repeats that code for a polyglutamine tract in a novel protein called huntingtin (htt). Both patients and experimental animals exhibit oxidative damage in specific areas of the brain, particularly the striatum. Nitric oxide (NO) is involved in many different physiological processes, and under pathological conditions it may promote oxidative damage through the formation of the highly reactive metabolite peroxynitrite; however, it may also play a role protecting cells from oxidative damage. We previously showed a correlation between the progression of the neurological phenotype and striatal oxidative damage in a line of transgenic mice, R6/1, which expresses a human mutated htt exon 1 with 116 CAG repeats. The purpose of the present work was to explore the participation of NO in the progressive oxidative damage that occurs in the striata of R6/1 mice. We analyzed the role of NO by measuring the activity of nitric oxide synthase (NOS) in the striata of transgenic and control mice at different ages. There was no difference in NOS activity between transgenic and wild-type mice at 11 weeks of age. In contrast, 19-week-old transgenic mice showed a significant increase in NOS activity, compared with same age controls. By 35 weeks of age, there was a decrease in NOS activity in transgenic mice when compared with wild-type controls. NOS protein expression was also determined in 11-, 19- and 35-week-old transgenic mice and wild-type littermates. Our results show increased neuronal NOS expression in 19-week-old transgenic mice, followed by a decreased level in 35-week-old mice, compared with controls, a phenomenon that parallels the changes in NOS enzyme activity. The present results suggest that NO is involved in the process leading to striatal oxidative damage and that it is associated with the onset of the progressive neurological phenotype in mice transgenic for the HD mutation.

Nitric oxide and nitric oxide synthases in the fetal cerebral cortex of rats following transient uteroplacental ischemia.

The effect of transient uteroplacental ischemia on nitric oxide (NO) levels, enzymatic activity, and expression of NO synthase (NOS) isoforms was studied in fetal rat brains. Fetuses were subjected to ischemia by clamping the uterine arteries for 5 min on gestational day 17 (GD17). At different times after ischemia, fetuses were delivered by Cesarean section under anesthesia to obtain the brains. Transient uteroplacental ischemia produced a time dependent increase in nitrite levels in the brain, reaching a maximum value (300 +/- 25% of baseline) 24 h after uterine artery occlusion and remaining elevated as long as 48 h. Significantly increased nitrite levels were found in the cerebral cortex but not in the mesencephalon and cerebellum. The ischemia-induced increment in nitrite levels was totally blocked by either L-NAME (10 mg/kg) or AMT (0.65 mg/kg) administered i.p. 1 h before uterine artery occlusion. Both Ca(2+)-dependent and Ca(2+)-independent NOS activities in the cerebral cortex remained significantly increased with respect to controls after 24 h following the ischemia. Reverse transcriptase-polymerase chain reaction showed augmented levels of mRNAs for both nNOS and iNOS when compared with controls at 8 h after ischemia. At 36 h, nNOS mRNA returned to basal levels whereas eNOS mRNA levels increased and iNOS mRNA remained elevated. Our results show that the three NOS isoforms participate in increasing NO levels after transient ischemia and suggest a biphasic and differential regulation of the expression of constitutive NOS isoforms in the rat cerebral cortex.

Possible Involvement of Endothelium-Derived Hyperpolarizing Factor in Vascular Responses of Abdominal Aorta From Pregnant Rats

Increased relaxant response to acetylcholine during pregnancy is proposed to be due to an estrogen-mediated increase in nitric oxide release. We studied acetylcholine-induced pathways of relaxation in the thoracic and abdominal aortic rings from pregnant and nonpregnant Wistar-Kyoto rats and measured basal and stimulated release of nitrites in these vessels. Endothelium-dependent relaxation was significantly greater in pregnant than in nonpregnant rats. Acetylcholine provoked a concentration-dependent relaxation on thoracic and abdominal aortic rings from nonpregnant and pregnant rats. After N118-nitro-L-arginine methyl ester pretreatment, the relaxation was significantly inhibited in the two preparations of nonpregnant and pregnant rodents. The relaxation was not inhibited by indomethacin in any of the aortic segments from pregnant and nonpregnant rats. After cytochrome P450 arachidonic acid metabolism inhibitor clotrimazole, a nonsignificant decrease in the Emax to acetylcholine-induced relaxation was observed in the thoracic segments of pregnant and nonpregnant rats. On the other hand, in abdominal aorta, clotrimazole decreased maximal relaxation in rings from pregnant rats (P<.05) but did not change the acetylcholine-induced relaxation from nonpregnant rats. Our results show an increase in the acetylcholine-stimulated release of nitrites in thoracic aortic rings from pregnant rats compared with rings from nonpregnant rats, which cannot be evidenced in abdominal aortic rings. These results suggest that acetylcholine-induced vasodilation in the abdominal segment from pregnant rats is mediated only in part by nitric oxide, the remainder apparently due to an endothelium-derived vasodilator, cytochrome P450-dependent, which may be endothelium-derived hyperpolarizing factor/epoxyeicosatrienoic acid.

Effect of treatment with losartan on salt sensitivity and SGLT2 expression in hypertensive diabetic rats

Sodium-glucose cotransporters (SGLTs) in the kidney, may be involved in hypertension, diabetes and salt sensitivity. We evaluate the effect of losartan on blood pressure (BP) and SGLT2 expression in diabetic rats with high or normal salt diet. Losartan prevented an increase in BP and SGLT2 expression in diabetic rats.

Arachidonic acid metabolism modulates vasopressin-induced renal vasoconstriction

Previous studies have shown that cytochrome P450-Arachidonic Acid (P450-AA) metabolites modify the vascular tone of several vessels and that vasopressin (AVP) stimulates P450-AA metabolism. Thus, in the present study, we decided to investigate if the vasoconstrictor effect of AVP is related to activation of P450-AA metabolism. We used the isolated perfused kidney of a rat, to test this hypothesis. Bolus injection of AVP (5.5, 11, 22 and 45 ng) increased the perfusion pressure of the isolated kidney of a rat by 66 +/- 2, 87 +/- 4, 110 +/- 2 and 130 +/- 3 mmHg respectively. This AVP-induced vasoconstriction was significantly reduced by inhibition of AA metabolism with ETYA, or 7 ethoxyresorsorufin (7ER). Furthermore, in vivo induction of P450 system with dexamethasone, enhanced the AVP-induced vasoconstrictor effect. Conversely, depletion of P450 system with SnCl2 diminished the vasoconstrictor response to AVP. Measurement of P450-14cAA metabolites in the renal effluent, showed the presence of 3 radioactive peaks. The % of the recovered radioactivity was 0.12 +/- 0.05%, 0.11 +/- 0.03% and 1.13 +/- 0.5% and corresponded to Dihydroxyeicosatrienoic acids (DHTs), Hydroxyeicosatetraenoic acids (HETEs) and Epoxyeicosatrienoic acids (EETs) respectively, when kidneys were stimulated by AVP (300 ng) the % recovered were 0.34 +/- 0.01%, 0.38 +/- 0.01% and 3.11 +/- 0.7% for the DHTs, HETEs and EETs respectively. Treatment with dexamethasone or SnCl2 potentiated or inhibited the AVP-dependent release of the P450-AA metabolites. In conclusion, we suggest that AVP stimulates AA metabolism via P450 pathway in the kidney and that these AA metabolites participate in the vasoconstrictor effect of AVP in the renal circulation.

Acute lead exposure induces renal haeme oxygenase-1 and decreases urinary Na excretion

The effects of acute lead exposure on renal function, lipid peroxidation and the expression of haeme oxygenase (HO) in rat kidney were determined. A single injection of lead acetate (50 mg Pb/kg) was given to rats. Changes in renal function, characterized by a significant reduction in the Na excretion was observed six hours after Pb exposure; this effect persisted for 24 hours. TBARS levels increased in kidney cortex 24 hours after Pb administration. In kidney cortex, Pb exposure affected the expression of HO-1, a renal protein associated with oxidative stress. HO-1 mRNA increased 2.3-fold, three hours after Pb administration and remained increased for six, 12 and 24 hours. HO enzymatic activity and HO-1 protein increased six and three hours after Pb administration, respectively, and remained increased at 24 hours. HO inhibition by tin-protoporphyrin, potentiated Pb-induced increase in TBARS and prevented the Pb-induced reduction in Na excretion. Our data suggest that Pb may be acting through the generation of oxidant products and induction of HO.

Functional and cellular interactions between nitric oxide and prostacyclin.

Nitric oxide (NO) and prostacyclin (PGI(2)) can be released by vascular agents to synergize their effects on vascular relaxation. In the present study we assess whether NO could affect PGI(2) production. We evaluated the effect of NO on PGI(2)-mediated arachidonic acid (AA)-induced relaxation in the perfused heart. We used cultured endothelial cells to characterize the mechanism involved in the NO effect on PGI(2) synthesis. AA-induced PGI(2) synthesis was enhanced when NO synthesis was inhibited. NO inhibited AA-induced relaxation and PGI(2) release in the coronary circulation. S-Nitroso-acetyl-DL-penicillamine (SNAP) decreased PGI(2) production in cultured endothelial cells. The SNAP effect was blunted by the inhibitor of soluble guanylate cyclase (LY-83,583) and the blocker of cGMP-dependent protein kinases (H-9). Specific cyclooxygenase-1 (COX-1) immunoprecipitation was associated to co-precipitation of four proteins. COX-1 showed neither serine nor threonine phosphorylation. One of the proteins that co-precipitated with COX-1 presented increased serine phosphorylation in the presence of SNAP. This effect was inhibited by the H-9. We suggest that NO, through cGMP-dependent protein kinases, produces the phosphorylation of a 104-kDa protein that is associated with inhibition in the activity of the COX-1, decreasing PGI(2) synthesis and thereby decreasing coronary PGI(2)-mediated vasodilatation.

The effect of ovariectomy on depressed contractions to phenylephrine and KCl and increased relaxation to acetylcholine in isolated aortic rings of female compared to male rabbits

1 Differences in vascular responses to phenylephrine, acetylcholine (ACh) and potassium chloride (KC1) were studied in rabbit aorta from female and male rabbits, in the absence and presence of an inhibitor of nitric oxide (NO) production, NG‐nitro‐l‐arginine methyl ester (L‐NAME, 100 μm). 2 Phenylephrine and KC1‐induced contractions, were significantly reduced in amplitude (P < 0.01) in the rings from female rabbits compared to those from male rabbits. 3 ACh‐induced relaxation was greater (P < 0.01) in aortic rings from females than from males. 4 Incubation of the rings with L‐NAME abolished the phenylephrine‐induced contraction differences between rings from male and female rabbits. 5 Ovariectomy eliminated the differences in vascular responses to phenylephrine, KC1 and ACh of aortic rings from the female rabbits. 6 Both basal and ACh‐stimulated release of nitrites from aortic rings was greater (P < 0.01) in vascular tissue from female than male rabbits. 7 These results indicate that differences in vascular reactivity in aortic rings from male and female rabbits may be associated with a higher release of NO, resulting in an increased vasodilator response in the female rabbits.

Prevention of Renal Injury and Endothelial Dysfunction by Chronic l-Arginine and Antioxidant Treatment

We evaluated the effects of vitamins with antioxidant properties (a combination of vitamins C and E) and l-arginine treatment on renal failure in mice by measuring survival rate. The molecular changes were elucidated by determining endothelial tetrahydrobiopterin (BH4) levels and nitric oxide synthase (eNOS) mRNA expression in mice with renal ablation. Previous studies have shown that endothelial dysfunction in 5/6 nephrectomized mice is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. WTC57 mice were divided into three groups: Group 1 was the sham-operated group (C); Group 2 was the 5/6 nephrectomized group (Nfx); and Group 3 was a group of 5/6 nephrectomized mice, treated with l-arginine and vitamins with antioxidant properties (NfxTx; 200 mg/kg l-arginine, 83 mg/kg vitamin C, and 46.6 mg/kg vitamin E). After 20 weeks of treatment, urinary protein excretion, blood pressure, BH4 and dihydrobiopterin (BH2) levels, eNOS mRNA, oxidative stress, and survival rate were determined. An increase in urinary protein excretion, blood pressure, and oxidative stress was prevented in the NfxTx group, but not in the Nfx group. BH4 and eNOS mRNA expression was increased by 32% and 78%, respectively, in the NfxTx group. Furthermore, the treatment increased the survival rate by 33%. Our results indicate that under normal conditions, NO appears to protect renal function. However, this NO-dependent protection is lost during kidney failure, probably due to increased reactive oxygen species synthesis. The treatment restores the viability of NO and prevents the BH4 oxidation. Therefore, this treatment may represent a therapeutic approach for the management of kidney disease.