Valor diagnóstico de la excreción fraccional de minerales para evaluar el metabolismo del calcio, magnesio y fósforo en el perro

The aim of this work was to establish reference values of the fractional excretion (FE) of calcium (Ca), phosphorus (P) and magnesium (Mg) in dogs, proposing a methodology for their obtention. The dogs were divided into groups SS: standard diet and CS: standard diet plus calcium supplement for 10 days. We observed increased plasma concentration of P, both fasting and postprandial, in CS respect to SS. More FECa in CS respect to SS in fasting and postprandial. SS showed a lower FEP and FEMg in fasting than postprandial, parameters that were stabilized in CS. (p<0.01). Thanks to the proposed methodology and the associated analysis of minerals we can see the lack of calcium in SS group despite having EFCa values that are at the bibliography.Nuestro objetivo fue establecer valores de referencia de la excreción fraccional (EF) de calcio (Ca), fósforo (P) y magnesio (Mg) en perros, proponiendo una metodología para su obtención. Los perros se dividen en grupos SS: con dieta estándar y CS: con dieta estándar más suplemento de calcio durante 10 días. Se observa en CS mayor concentración plasmática de P tanto en ayuno como postprandial respecto a SS. Mayor EFCa en CS respecto a SS en ayuno y postprandial. Menor EF de P y Mg postprandial en SS respecto al ayuno, parámetros que se estabilizan en CS. (p<0.01). Gracias a la metodología propuesta y al análisis asociado de los minerales se pudo determinar la carencia de calcio en el grupo SS a pesar de tener valores dentro de los bibliográficos de EFCa

Antibodies against the cardiac sodium/bicarbonate co-transporter (NBCe1) as pharmacological tools

Background and purpose: Na⁺/HCO3⁻ co-transport (NBC) regulates intracellular pH (pHi) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. Experimental approach: We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pHi was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H⁺ flux, JH, in mmol·L-1 min-1 at pHi 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpHi) respectively. Key results: The potassium pulse produced a pHi increase of 0.18 ± 0.006 (n= 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased JH by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pHi increase than control,(0.25 ± 0.018) whereas the recovery of pHi from acidosis was faster (JH was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. Conclusions and implications: Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Effect of supragingival plaque control on subgingival microflora and periodontal tissues

O objetivo deste trabalho foi estudar, clínica e microbiologicamente, 44 sítios em 11 pacientes com periodontite crônica generalizada. IP, IG, SS, PS e NI foram registrados. Amostras de placa subgengival foram colhidas nos mesmos sítios para cultivo de bactérias anaeróbias e determinação dos morfotipos microbianos por MCE. Os registros clínicos e estudos microbiológicos foram tomados no "baseline" e 4 semanas após a incorporação em um programa de controle de placa e cálculo supragengival. A análise microbiológica categorizou o grau de desenvolvimento em: 0 - não detectado, 1 - escasso, 2 - moderado e 3 - abundante. Os registros clínicos no "baseline" e dia 28 foram: IP - 1,73 ± 0,10 e 0,30 ± 0,08, IG - 1,73 ± 0,08 e 1,41 ± 0,08, SS - 0,91 ± 0,04 e 0,59 ± 0,07, PS - 6,43 ± 0,20 e 5,77 ± 0,25, NI - 6,86 ± 0,32 e 6,52 ± 0,34, respectivamente. A redução do IP, IG, SS e PS foi significativa. Não foram registradas diferenças significativas no NI. As proporções relativas dos morfotipos bacterianos observados por MCE no "baseline" e dia 28 foram: células cocóideas - 21,16 ± 3,77 e 36,00 ± 4,66, bacilos móveis - 44,86 ± 2,65 e 39,50 ± 2,64, treponemas totais - 24,66 ± 3,08 e 19,25 ± 2,75. No "baseline" e no dia 28 foi observado: Pi/n - 1,36 ± 0,18 e 0,43 ± 0,11, Pg - 0,48 ± 0,16 e 0,32 ± 0,13, Aa - 0,23 ± 0,09 e 0,23 ± 0,10, Fusobacterium nucleatum - 0,32 ± 0,14 e 0,41 ± 0,13 e peptostreptococos - 0,82 ± 0,19 e 0,54 ± 0,16, respectivamente. Houve um aumento significativo das células cocóideas, diminuição de treponemas e de Pi/n.The aim of this study was to investigate, clinically and microbiologically, forty-four sites in 11 patients presenting with generalized chronic periodontitis. Plaque Index (PI), Gingival Index (GI), Probing Bleeding (PB), Probing Depth (PD) and Insertion Level (IL) were registered. Samples of subgingival plaque were collected in the same sites for cultivation of anaerobic bacteria and determination of microbiological morphotypes using dark field microscopy. Clinical and microbiological data were recorded on the baseline and 4 weeks after the adoption of a program to control supragingival plaque and calculus. The microbiological analysis categorized the degree of development as follows: 0 - not detected, 1 - scarce, 2 - moderate and 3 - abundant. The clinical results at the baseline and on the 28th day were, respectively: PI - 1.73 ± 0.10 and 0.30 ± 0.08; GI - 1.73 ± 0.08 and 1.41 ± 0.08; PB - 0.91 ± 0.04 and 0.59 ± 0.07; PD - 6.43 ± 0.20 and 5.77 ± 0.25; and IL - 6.86 ± 0.32 and 6.52 ± 0.34. There was significant decrease in PI, GI, PB and PD. However, the difference in IL was not significant. The relative proportions of the microbial morphotypes observed under dark field microscopy at the baseline and on the 28th daywere, respectively: coccoid cells - 21.16 ± 3.77 and 36.00 ± 4.66; mobile bacillus - 44.86 ± 2.65 and 39.50 ± 2.64; and total treponemes - 24.66 ± 3.08 and 19.25 ± 2.75 . The cultures presented, at the baseline and on the 28th day, respectively: Prevotella intermedia/nigrescens (Pi/n) - 1.36 ± 0.18 and 0.43 ± 0.11; Porphyromonas gingivalis - 0.48 ± 0.16 and 0.32 ± 0.13; Actinobacillus actinomycetemcomitans - 0.23 ± 0.09 and 0.23 ± 0.10; Fusobacterium nucleatum - 0.32 ± 0.14 and 0.41 ± 0.13; and peptostreptococci - 0.82 ± 0.19 and 0.54 ± 0.16. There was a significant increase in the number of coccoid cells and a decrease in the number of treponemes and Pi/n

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation, reactive oxygen species and NHE-1

Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1 nmol l⁻¹ Ang II or 5 nmol l⁻¹ ET1. Ang II increased ~45 % cell surface area (CSA) and ~37 % [³H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT₁ and ETA receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2- p90RSK kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187±9 %, 149±8 % and 163.7±6 % of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT₁, ETA and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90RSK and NHE-1 in adult cardiomyocytes.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Aldosterone stimulates the cardiac Na +/H + exchanger via transactivation of the epidermal growth factor receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na/H exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90 RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.Facultad de Ciencias Médica

Early Hypertrophic Signals After Myocardial Stretch : Role of Reactive Oxygen Species and the Sodium/Hydrogen Exchanger

In this chapter the enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy.We propose that the chain of events triggered by stretch begins with the release of small amounts of angiotensin II which in turn induce the release/formation of endothelin. The actions of these hormones trigger the production of mitochondrial reactive oxygen species that enhances NHE-1 activity, causing an increment in the intracellular Na+ concentration which promotes the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger. This [Ca2+]i increase would trigger cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.Centro de Investigaciones Cardiovasculare

Estrés oxidativo: nexo entre aldosterona y el daño del miocardio mediado por el NHE1

En los estudios clínicos RALES, EPHESUS y EMPHASIS se demostraron los efectos beneficiosos del antagonismo de la aldosterona (Aldo) en la insuficiencia cardíaca (IC), aunque no se conocen los mecanismos implicados. Por otro lado, el estrés oxidativo, aumentado en la IC, resulta deletéreo por diferentes mecanismos, siendo uno de ellos la activación de quinasas redox-sensibles que fosforilan y estimulan al NHE1. Además, la inhibición del NHE1 es beneficiosa en modelos experimentales de hipertrofia e IC.Facultad de Ciencias Médica

Early signals after stretch leading to cardiac hypertrophy : Key role of NHE-1

The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCXrev), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.Centro de Investigaciones Cardiovasculare