Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Relation of the disaccharidases in the small intestine of the rat to the degree of experimentally induced iron-deficiency anemia

Hypolactasia associated with severe iron-deficiency anemia has been reported in several studies. The objective of the present study was to determine whether hypolactasia is associated with the degree and duration of iron-deficiency anemia. Newly weaned male Wistar rats were divided into a control group receiving a diet supplemented with iron (C) and an experimental group (E) receiving a diet not supplemented with iron (iron-deficiency diet). The animals were studied on the 3rd, 5th, 7th, 14th, 21st, 28th and 35th days of the experiment, when overall and iron nutritional status and disaccharidase activity in the small intestine were determined by the Dahlqvist method. A reduction in weight occurred in the anemic animals starting on the 5th day of the study. Anemia was present in the experimental animals, with a progressive worsening up to the 14th day (hemoglobin: C = 13.27 and E = 5.37) and stabilizing thereafter. Saccharase and maltase activities did not differ significantly between groups, whereas lactase showed a significant reduction in total (TA) and specific activity (SA) in the anemic animals starting on the 21st day of the study. Median lactase TA for the C and E groups was 2.27 and 1.25 U on the 21st day, 2.87 and 1.88 U on the 28th day, and 4.20 and 1.59 U on the 35th day, respectively. Median lactase SA was 0.31 and 0.20 U/g wet weight on the 21st day, 0.39 and 0.24 U/g wet weight on the 28th day, and 0.42 and 0.23 U/g wet weight on the 35th day, respectively. These findings suggest a relationship between the enzymatic alterations observed and both the degree and duration of the anemic process. Analysis of other studies on intestinal disaccharidases in anemia suggests that the mechanism of these changes may be functional, i.e., that the enterocytes may suffer a reduction in their ability to synthesize these enzymes.

21-Hydroxylase deficiency in Brazil

We determined the frequency of large rearrangements and point mutations in 130 Brazilian patients with 21-hydroxylase deficiency and correlated genotype with phenotype. The frequency of CYP21 deletions was lower (4.4%) than in most of the previous series described, whereas the frequency of large gene conversions was similar to the frequency reported in the literature (6.6%). The most frequent point mutations were I2 splice (41.8% in salt wasting - SW), I172N (32.6% in simple virilizing - SV) and V281L (40.2% in the late onset form - LO). The frequency of the nine most common point mutations was similar to that reported for other countries. The 93 fully genotyped patients were classified into 3 mutation groups based on the degree of enzymatic activity (A<2%, B @ 2%, C>20%). In group A, 62% of cases presented the SW form; in group B, 96% the SV form, and in group C, 88% the LO form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. To diagnose these remaining alleles we sequenced the CYP21 gene of one patient with the SV form and identified a heterozygous G->A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region and was also found in a compound heterozygous state in 4 other patients. The mutation G424S presented a linkage disequilibrium with CYP21P and C4A gene deletions and HLA DR17, suggesting a probable founder effect. Search for the G424S mutation in other populations will reveal if it is restricted to the Brazilian patients or if it has a wider ethnic distribution.

Nitric oxide synthase blockade and body fluid volumes

The influence of chronic nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on body fluid distribution was studied in male Wistar rats weighing 260-340 g. Extracellular, interstitial and intracellular spaces, as well as plasma volume were measured after a three-week treatment with L-NAME (~70 mg/kg per 24 h in drinking water). An increase in extracellular space (16.1 ± 1.1 vs 13.7 ± 0.6 ml/100 g in control group, N = 12, P<0.01), interstitial space (14.0 ± 0.9 vs 9.7 ± 0.6 ml/100 g in control group, P<0.001) and total water (68.7 ± 3.9 vs 59.0 ± 2.9 ml/100 g, P<0.001) was observed in the L-NAME group (N = 8). Plasma volume was lower in L-NAME-treated rats (2.8 ± 0.2 ml/100 g) than in the control group (3.6 ± 0.1 ml/100 g, P<0.001). Blood volume was also lower in L-NAME-treated rats (5.2 ± 0.3 ml/100 g) than in the control group (7.2 ± 0.3 ml/100 g, P<0.001). The increase in total ratio of kidney wet weight to body weight in the L-NAME group (903 ± 31 vs 773 ± 45 mg/100 g in control group, P<0.01) but not in total kidney water suggests that this experimental hypertension occurs with an increase in renal mass. The fact that the heart weight to body weight ratio and the total heart water remained constant indicates that, despite the presence of high blood pressure, no modification in cardiac mass occurred. These data show that L-NAME-induced hypertension causes alterations in body fluid distribution and in renal mass.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS) and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS). Addition of L-NAME (500 µM) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.

Dietary cellulose has no effect on the regeneration of hemoglobin in growing rats with iron deficiency anemia

The objective of the present study was to determine the effect of cellulose on intestinal iron absorption in rats during recovery from iron deficiency anemia. Twenty-one-day-old male Wistar-EPM rats were fed an iron-free ration for two weeks to induce anemia. At 5 weeks of age, the rats were divided into two groups (both groups receiving 35 mg of elemental iron per kg diet): cellulose group (N = 12), receiving a diet containing 100 g of cellulose/kg and control (N = 12), receiving a diet containing no cellulose. The fresh weight of the feces collected over a 3-day period between the 15th and 18th day of dietary treatment was 10.7 ± 3.5 g in the group receiving cellulose and 1.9 ± 1.2 g in the control group (P<0.001). Total food intake was higher in the cellulose group (343.4 ± 22.0 g) than in the control (322.1 ± 13.1 g, P = 0.009) during the 3 weeks of dietary treatment. No significant difference was observed in weight gain (cellulose group = 132.8 ± 19.2, control = 128.0 ± 16.3 g), hemoglobin increment (cellulose group = 8.0 ± 0.8, control = 8.0 ± 1.0 g/dl), hemoglobin level (cellulose group = 12.3 ± 1.2, control = 12.1 ± 1.3 g/dl) or in hepatic iron levels (cellulose group = 333.6 ± 112.4, control = 398.4 ± 168.0 µg/g dry tissue). We conclude that cellulose does not adversely affect the regeneration of hemoglobin, hepatic iron level or the growth of rats during recovery from iron deficiency anemia.

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Impact of congenital calcitonin deficiency due to dysgenetic hypothyroidism on bone mineral density

The objective of the present study was to determine the effect of chronic calcitonin deficiency on bone mass development. The results of 11 patients with thyroid dysgenesis (TD) were compared to those of 17 normal individuals (C) and of 9 patients with other forms of hypothyroidism (OH): 4 with hypothyroidism due to inborn errors of thyroid hormone synthesis and 5 with Hashimoto's thyroiditis. The subjects received an intravenous calcium stimulus and blood was collected for the determination of ionized calcium (Ca2+), calcitonin, and intact parathyroid hormone. Bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry. After calcium administration the levels of Ca2+ in the two groups of hypothyroidism were significantly higher than in the normal control group (10 min after starting calcium infusion: C = 1.29 ± 0.08 vs TD = 1.34 ± 0.03 vs OH = 1.34 ± 0.02 mmol/l; P < 0.05), and only the TD group showed no calcitonin response (5 min after starting calcium infusion: C = 27.9 ± 5.8 vs TD = 6.6 ± 0.3 vs OH = 43.0 ± 13.4 ng/l). BMD values did not differ significantly between groups (L2-L4: C = 1.116 ± 0.02 vs TD = 1.109 ± 0.03 vs OH = 1.050 ± 0.04 g/cm²). These results indicate that early deficiency of calcitonin secretion has no detrimental effect on bone mass development. Furthermore, the increased calcitonin secretion observed in patients with inborn errors of thyroid hormone biosynthesis does not confer any advantage in terms of BMD.

Newborn screening for biotinidase deficiency in Brazil: biochemical and molecular characterizations

Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10% of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30% of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09%). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H³]-arginine to L-[H³]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Morphological and functional alterations of the intestine of rats with iron-deficiency anemia

The present study was designed to assess the intestinal absorption of D-xylose and jejunal morphometry in rats with iron-deficiency anemia. Male Wistar rats were randomly divided into a control group (diet containing 50 mg Fe/kg, N = 12) and an anemic group (diet containing <5 mg Fe/kg, N = 12). The animals were housed in individual metabolic cages and deionized water and diet were provided ad libitum for 6 weeks. Hemoglobin and hematocrit were determined at 0, 2, 4, and 6 weeks. At the end of the study the rats were submitted to a D-xylose absorption test (50 mg/100 g body weight) and sacrificed and a jejunal specimen was obtained for morphometric study. At the end of the study the hemoglobin and hematocrit of the anemic rats (8.7 ± 0.9 g/dl and 34.1 ± 2.9%, respectively) were significantly (P < 0.05) lower than those of the controls (13.9 ± 1.4 g/dl and 47.1 ± 1.5%, respectively). There was no statistical difference in D-xylose absorption between the anemic (46.5 ± 7.4%) and control (43.4 ± 9.0%) groups. The anemic animals presented statistically greater villus height (445.3 ± 36.8 µm), mucosal thickness (614.3 ± 56.3 µm) and epithelial surface (5063.0 ± 658.6 µm) than control (371.8 ± 34.3, 526.7 ± 62.3 and 4401.2 ± 704.4 µm, respectively; P < 0.05). The increase in jejunum villus height, mucosal thickness and epithelial surface in rats with iron-deficiency anemia suggests a compensatory intestinal mechanism to increase intestinal iron absorption.

Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.