Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Lack of tolerance for the anti-dyskinetic effects of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in rats

7-Nitroindazole (7-NI) inhibits neuronal nitric oxide synthase in vivo and reduces l-DOPA-induced dyskinesias in a rat model of parkinsonism. The aim of the present study was to determine if the anti-dyskinetic effect of 7-NI was subject to tolerance after repeated treatment and if this drug could interfere with the priming effect of l-DOPA. Adult male Wistar rats (200-250 g) with unilateral depletion of dopamine in the substantia nigra compacta were treated with l-DOPA (30 mg/kg) for 34 days. On the 1st day, 6 rats received ip saline and 6 received ip 7-NI (30 mg/kg) before l-DOPA. From the 2nd to the 26th day, all rats received l-DOPA daily and, from the 27th to the 34th day, they also received 7-NI before l-DOPA. Animals were evaluated before the drug and 1 h after l-DOPA using an abnormal involuntary movement scale and a stepping test. All rats had a similar initial motor deficit. 7-NI decreased abnormal involuntary movement induced by l-DOPA and the effect was maintained during the experiment before 7-NI, median (interquartile interval), day 26: 16.75 (15.88-17.00); day 28: 0.00 (0.00-9.63); day 29: 13.75 (2.25-15.50); day 30: 0.5 (0.00-6.25); day 31: 4.00 (0.00-7.13), and day 34: 0.5 (0.00-14.63), Friedman followed by Wilcoxon test,vs day 26, P < 0.05;. The response to l-DOPA alone was not modified by the use of 7-NI before the first administration of the drug (l-DOPA vs time interaction, F1,10 = 1.5, NS). The data suggest that tolerance to the anti-dyskinetic effects of a neuronal nitric oxide synthase inhibitor does not develop over a short-term period of repeated administration. These observations open a possible new therapeutic approach to motor complications of chronic l-DOPA therapy in patients with Parkinson’s disease.

Morphometric evaluation of nitric oxide synthase isoforms and their cytokine regulators predict pulmonary dysfunction and survival in systemic sclerosis

Because histopathological changes in the lungs of patients with systemic sclerosis (SSc) are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO) and plasminogen activator inhibitor-1 (PAI-1) synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP). We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS) and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS) related significantly to staining of septal cells for interleukin (IL)-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal basic fibroblast growth factor (bFGF; P=0.02). Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.

Changes in cardiac aldosterone and its synthase in rats with chronic heart failure: an intervention study of long-term treatment with recombinant human brain natriuretic peptide

The physiological mechanisms involved in isoproterenol (ISO)-induced chronic heart failure (CHF) are not fully understood. In this study, we investigated local changes in cardiac aldosterone and its synthase in rats with ISO-induced CHF, and evaluated the effects of treatment with recombinant human brain natriuretic peptide (rhBNP). Sprague-Dawley rats were divided into 4 different groups. Fifty rats received subcutaneous ISO injections to induce CHF and the control group (n=10) received equal volumes of saline. After establishing the rat model, 9 CHF rats received no further treatment, rats in the low-dose group (n=8) received 22.5 μg/kg rhBNP and those in the high-dose group (n=8) received 45 μg/kg rhBNP daily for 1 month. Cardiac function was assessed by echocardiographic and hemodynamic analysis. Collagen volume fraction (CVF) was determined. Plasma and myocardial aldosterone concentrations were determined using radioimmunoassay. Myocardial aldosterone synthase (CYP11B2) was detected by quantitative real-time PCR. Cardiac function was significantly lower in the CHF group than in the control group (P<0.01), whereas CVF, plasma and myocardial aldosterone, and CYP11B2 transcription were significantly higher than in the control group (P<0.05). Low and high doses of rhBNP significantly improved hemodynamics (P<0.01) and cardiac function (P<0.05) and reduced CVF, plasma and myocardial aldosterone, and CYP11B2 transcription (P<0.05). There were no significant differences between the rhBNP dose groups (P>0.05). Elevated cardiac aldosterone and upregulation of aldosterone synthase expression were detected in rats with ISO-induced CHF. Administration of rhBNP improved hemodynamics and ventricular remodeling and reduced myocardial fibrosis, possibly by downregulating CYP11B2 transcription and reducing myocardial aldosterone synthesis.

Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

Caracterização imunoquímica da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em frutos climatéricos

Com o objetivo de caracterizar, por via imunoquímica, a enzima ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em frutos climatéricos, foram preparados anticorpos policlonais específicos para esta proteína. Utilizou-se, como antígeno, uma proteína recombinante, produzida em Escherichia coli K38/pGP1,2, contendo o vetor de expressão pT7-7A4 no qual foi inserido um clone de DNA da ACC oxidase. A especificidade dos anticorpos foi demonstrada pela técnica de "Western blot", a partir de extratos protéicos de maçãs e tomates em diferentes estágios de maturação. Verificou-se que o aumento da produção de etileno, quando os frutos passaram do estágio pré-climatérico para o climatérico, está diretamente correlacionada com o aumento da síntese da ACC oxidase. Em estágios de maturação mais avançados houve uma redução da produção de etileno e da atividade ACC oxidase, mas esta proteína continuava presente. Quando o "Western blot" foi realizado com tomates transgênicos, onde a produção de etileno e a síntese da ACC oxidase foram inibidos em mais de 95%, nenhuma reação imunoquímica foi detectada. O conjunto de resultados obtidos indica que os anticorpos detectam especificamente ACC oxidase.

Inibição da síntese da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em maçãs frigoconservadas em atmosfera controlada

Foi estudada a expressão da ACC oxidase em maçãs, cv. Jonagold, colhidas no estádio pré-climatérico e armazenadas sob refrigeração em atmosfera normal (0ºC, 95% UR - AN) e controlada (0ºC, 95% UR, 1,5% O2 e 2,5% CO2 - AC), durante 180 dias. Na instalação do experimento, aos 90 e aos 120 dias, foram coletadas amostras para a determinação da firmeza de polpa, da acidez total titulável, dos sólidos solúveis totais, da produção de etileno, da atividade ACC oxidase e para a detecção imunoquímica das isoformas desta enzima. A dosagem da atividade ACC oxidase foi realizada por cromatografia gasosa a partir de extrato protéico solúvel acrescido de 250µM de ACC, 10µL de sulfato ferroso e 30µL de ascorbato de sódio. Para a detecção imunoquímica utilizou-se a técnica "western blot", com anticorpos policlonais anti-ACC oxidase de maçã, após separação das proteínas em eletroforese e isoeletrofocalização. Não foi detectada ACC oxidase em maçãs pré-climatéricas. Porém, após 120 horas em condições ambientais, houve a síntese dessa enzima e um incremento na produção de etileno. A refrigeração não exerceu controle na síntese da ACC oxidase e produção de etileno, resultando em significativas perdas físico-químicas nas frutas armazenadas em AN. Já a utilização de AC permitiu controlar a via de biossíntese do etileno, pela inibição da síntese da ACC oxidase, mantendo o material com boa qualidade para o consumo in natura. A adição de ACC e dos cofatores aumentou a atividade ACC oxidase e alterou o pI da ACC oxidase.