Effects of NO/cGMP inhibitors in a rat model of anaphylactoid shock.

Anaphylactic shock can be defined as an acute syndrome, and it is the most severe clinical manifestation of allergic diseases. Anaphylactoid reactions are similar to anaphylactic events but differ in the pathophysiological mechanism. Nitric oxide (NO) inhibitors during anaphylaxis suggest that NO might decrease the signs and symptoms of anaphylaxis but exacerbate associated vasodilation. Therefore, blocking the effects of NO on vascular smooth muscle by inhibiting the guanylate cyclase (GC) would be a reasonable strategy. This study aimed to investigate the effects of NO/cGMP pathway inhibitors methylene blue (MB), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and indigo carmine (IC) in shock induced by compound 48/80 (C48/80) in rats. The effect was assessed by invasive blood pressure measurement. Shock was initiated by C48/80 intravenous bolus injection 5 min before (prophylactic) or after (treatment) the administration of the inhibitors MB (3 mg/kg), L-NAME (1 mg/kg), and IC (3 mg/kg). Of the groups that received drugs as prophylaxis for shock, only the IC group did not present the final systolic blood pressure (SBP) better than the C48/80 group. Regarding shock treatment with the drugs tested, all groups had the final SBP similar to the C48/80group. Altogether, our results suggested that inhibition of GC and NO synthase in NO production pathway was not sufficient to revert hypotension or significantly improve survival.

Effects of NO/cGMP inhibitors in a rat model of anaphylactoid shock

Anaphylactic shock can be defined as an acute syndrome, and it is the most severe clinical manifestation of allergic diseases. Anaphylactoid reactions are similar to anaphylactic events but differ in the pathophysiological mechanism. Nitric oxide (NO) inhibitors during anaphylaxis suggest that NO might decrease the signs and symptoms of anaphylaxis but exacerbate associated vasodilation. Therefore, blocking the effects of NO on vascular smooth muscle by inhibiting the guanylate cyclase (GC) would be a reasonable strategy. This study aimed to investigate the effects of NO/cGMP pathway inhibitors methylene blue (MB), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and indigo carmine (IC) in shock induced by compound 48/80 (C48/80) in rats. The effect was assessed by invasive blood pressure measurement. Shock was initiated by C48/80 intravenous bolus injection 5 min before (prophylactic) or after (treatment) the administration of the inhibitors MB (3 mg/kg), L-NAME (1 mg/kg), and IC (3 mg/kg). Of the groups that received drugs as prophylaxis for shock, only the IC group did not present the final systolic blood pressure (SBP) better than the C48/80 group. Regarding shock treatment with the drugs tested, all groups had the final SBP similar to the C48/80group. Altogether, our results suggested that inhibition of GC and NO synthase in NO production pathway was not sufficient to revert hypotension or significantly improve survival.

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect.

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (ΔBP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 µM). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control.

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (ΔBP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 μM). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control.

Morphological, immunohistochemical and biochemical effects of non-pulsatile ex vivo perfusion with crescent pressures in human saphenous veins.

AIM: On the average, 15% to 25% of peripheral grafts and 10% to 30% of coronary grafts fail within 5 years. Changes in mechanical forces to which the vein is subjected could be an explanation for this phenomenon. We submitted human saphenous vein segments to non-pulsatile ex vivo perfusion with crescent pressures and evaluated morphology, nitric oxide synthase immunohistochemical expression; tissue levels of nitrite/nitrate and oxidative stress products. METHODS: Intact segments of human saphenous veins were obtained from 30 patients submitted to elective coronary artery bypass graft surgery. Ex vivo perfusion was performed during 3 hours, using oxygenated Krebs solution, flow of 100 mL/min and pressures of 0, 50, 100, 200 and 300 mmHg, defining five groups. RESULTS: Optical microscopy showed that veins of groups perfused with 200 and 300 mmHg presented increased luminal area and endothelial denuding. Electron microscopy transmission showed alterations in veins perfused with 200 and 300 mmHg. Immunohistochemical expression of the three nitric oxide synthase isoforms was observed in all vein layers, without significant difference among groups. Tissue levels of nitrite/nitrate were not significantly different among distinctive perfusion. Nitrotyrosine was not immunohistochemically expressed in all veins and malondialdehyde tissue levels were not different among groups. CONCLUSION: Non-pulsatile ex vivo perfusion during 3h caused morphological alterations in human saphenous veins (HSVs), which were not accompanied by immunohistochemical and biochemical alterations. Even with mechanical lesions, HSVs maintained the ability of express nitric oxide synthase (NOS) and release nitric oxide.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta.

AIM: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. METHODS: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-l-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2×10(-5) M) and methyl-ß-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 µM), in the presence and absence of DMB (2×10(-5) M). RESULTS: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+) exchanger (NCX), and partially blunted by the caveolae disassembly. CONCLUSIONS: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.

Effects of partial liver ischemia followed by global liver reperfusion on the remote tissue expression of nitric oxide synthase: lungs and kidneys.

Hepatic ischemia followed by reperfusion (IR) results in mild to severe remote organ injury. Oxidative stress and nitric oxide (NO) seem to be involved in the IR injury. Our aim was to investigate the effects of liver I/R on hepatic function and lipid peroxidation, leukocyte infiltration and NO synthase (NOS) immunostaining in the lung and the kidney. We randomized 24 male Wistar rats into 3 groups: 1) control; 2) 60 minutes of partial (70%) liver I and 2 hours of global liver R; and 3) 60 minutes of partial (70%) liver I and 6 hours of global liver R. Groups 2 and 3 showed significant increases in plasma alanine and aspartate aminotransferase levels and in tissue malondialdehyde and myeloperoxidase contents. In the kidney, positive endothelial NOS (eNOS) staining was significantly decreased in group 3 compared with group 1. However, staining for inducible NOS (iNOS) and neuronal NOS (nNOS) did not differ among the groups. In the lung, the staining for eNOS and iNOS did not show significant differences among the groups; no positive nNOS staining was observed in any group. These results suggested that partial liver I followed by global liver R induced liver, kidney, and lung injuries characterized by neutrophil sequestration and increased oxidative stress. In addition, we supposed that the reduced NO formation via eNOS may be implicated in the moderate impairment of renal function, observed by others at 24 hours after liver I/R.

[Hormone replacement therapy after total thyroidectomy. Can the combined treatment be considered effective to get metabolic adequacy? Preliminary results]. / Terapia ormonale sostitutiva dopo tiroidectomia totale. Il trattamento ormonale combinato può essere considerato efficace strumento di adeguatezza metabolica? Risultati preliminari.

INTRODUCTION: Even if T4 is standard treatment for hypothyroidism after thyroidectomy, a treatment with T4 plus T3 has been proposed as an alternative reproducing carefully the physiology of the thyroid. We performed an observational study about the effects of the combined replacement therapy with T4 and T3 in patients who underwent total thyroidectomy. PATIENTS AND METHODS: Fifty thyroidectomized patients (not for cancer), in T4 replacement therapy, were included in the study. Such a therapy has been changed by administering T3 and T4 in proportion 1/16. Adverse effects, clinical parameters and general health perceptions (SF36 Questionnaire) have been considered at the time of enrollment (T0), after 30 days (T1) and after 60 days (T2). RESULTS: No differences in weight, cardiac frequency and blood pression have been found between T0 and T2. A not statistically significant reduction has been found in total cholesterol (3 mg/dL) and triglycerides (3.29 mg/dL) levels. A reduction of complaints referred by the patients at T0 has been revealed in T2: anxiety from 21 to 13 patients; headache from 22 to 13; tiredness from 17 to 8 (p<0.05); sleepiness from 25 to 15 (p<0.05). About the questionnaire, in the General Health Perception sub-score has been found a not significant increase of the parameter. DISCUSSION AND CONCLUSIONS: T4 replacement therapy is of proved efficacy, notwithstanding some complaints afflict a share of patients. To improve the quality of life of these patients, we consider relevant the reduction of complaints and the improvement of well-being and cognitive function obtained by the combined T4 and T3 treatment. Moreover, even if the production of T3 from T4 usually ensures euthyroidism in all tissues, contemporary pathologies and alterations determined by the oldness can disturb the enzymatic activity, which is the essential requirement of T4 therapy. These preliminary findings in a small group of patients encourage further studies on a larger patient population.

Effects of acid-base imbalance on vascular reactivity.

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

Effects of acid-base imbalance on vascular reactivity

Acid-base homeostasis maintains systemic arterial pH within a narrow range. Whereas the normal range of pH for clinical laboratories is 7.35-7.45, in vivo pH is maintained within a much narrower range. In clinical and experimental settings, blood pH can vary in response to respiratory or renal impairment. This altered pH promotes changes in vascular smooth muscle tone with impact on circulation and blood pressure control. Changes in pH can be divided into those occurring in the extracellular space (pHo) and those occurring within the intracellular space (pHi), although, extracellular and intracellular compartments influence each other. Consistent with the multiple events involved in the changes in tone produced by altered pHo, including type of vascular bed, several factors and mechanisms, in addition to hydrogen ion concentration, have been suggested to be involved. The scientific literature has many reports concerning acid-base balance and endothelium function, but these concepts are not clear about acid-base disorders and their relations with the three known mechanisms of endothelium-dependent vascular reactivity: nitric oxide (NO/cGMP-dependent), prostacyclin (PGI2/cAMP-dependent) and hyperpolarization. During the last decades, many studies have been published and have given rise to confronting data on acid-base disorder and endothelial function. Therefore, the main proposal of this review is to provide a critical analysis of the state of art and incentivate researchers to develop more studies about these issues.

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated.

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.

[Diagnostic and therapeutic strategies in post-infarct]. / Strategie diagnostiche e terapeutiche nel post-infarto.

It is of primary importance for the clinical cardiologist to keep in mind the parameters allowing an adequate prognostic stratification in post-infarct patients in view of making the best diagnostic and therapeutic choices. A diagnostic strategy, based on a pathophysiologic approach, should evaluate four aspects: spontaneous and stress-induced ischemia, myocardial viability, and ventricular arrhythmias. Spontaneous ischemia has an undefined prognostic value, especially in the thrombolytic era; therefore it seems reasonable to perform invasive procedures in patients who are not stabilized by an adequate medical therapy or with large jeopardized areas. In asymptomatic patients, a provocative stress test allows a more articulated decisional iter. It is preferably to perform the test by the most physiological exercise EKG, together with the echocardiographic imaging, after the acute phase. It has a high negative predictive value, but a low positive predictive value. The detection of myocardial viability is frequently performed, mainly in patients with large post-ischemic myocardial dysfunction. Among all the proposed methods, the echo-dobutamine test mainly allows to estimate patients in whom revascularization may result in more benefit. The role of ventricular arrhythmias as an independent prognostic factor is debated and has to be always considered in relationship to other parameters, particularly left ventricular function. Regarding the therapeutic strategy, the indications from recent trials, related to antithrombotic drugs, beta-blockers, ACE-inhibitors, nitrates, Ca-blockers and antiarrhythmic drugs, are emphasized.