Características clínicas, manejo y riesgo de complicaciones a un año en pacientes con insuficiencia cardíaca con y sin diabetes tipo 2 en España / Clinical characteristics, management, and one-year risk of complications among patients with heart failure with and without type 2 diabetes in Spain

Objetivos: Describir las características clínicas y el manejo terapéutico y determinar los eventos cardiovasculares tras un año de seguimiento en una población contemporánea con insuficiencia cardíaca (IC) con y sin diabetes tipo 2 en España. También se analizó en la población DAPA-HF (pacientes que cumplieron la mayoría de los criterios de inclusión del estudio DAPA-HF) y en los pacientes tratados basalmente con inhibidores SGLT2.Métodos: Estudio observacional, retrospectivo, poblacional, empleando la base de datos BIG-PAC. La fecha índice fue 1 de enero de 2019. Se seleccionaron sujetos≥18 años que recibieron tratamiento por IC en 2019. Se analizaron los eventos durante 2019.Resultados: Se identificaron 21.851 pacientes con IC (78±11,3 años; 53% varones; 50,9% IC con fracción de eyección reducida; 44,5% en clase funcional NYHA II). La prevalencia de IC fue del 1,88% y la incidencia 2,83 por 1.000 pacientes-año. El 66,1% tomaba inhibidores del sistema renina-angiotensina, el 69,4% betabloqueantes, el 31,2% antialdosterónicos y el 7,5% sacubitrilo/valsartán. Durante el año de seguimiento, el 29,8% fue hospitalizado por descompensación de la IC (tiempo medio primer evento 120,9±72,5 días), un 12,3% murieron, un 8,1% murieron durante la hospitalización. Los eventos fueron más frecuentes en los pacientes con diabetes tipo 2. Las hospitalizaciones por IC fueron más comunes en la población similar a DAPA-HF.Conclusiones: En España, la población con IC es anciana y tiene muchas comorbilidades. Aproximadamente la mitad de los pacientes tienen IC con fracción de eyección reducida. Existe margen de mejora en el manejo de la IC, en particular mediante el empleo de aquellos fármacos que reducen tanto la hospitalización por IC como la mortalidad, para disminuir la carga de IC (AU)

Objective: This work aims to describe the clinical characteristics and therapeutic management and to determine cardiovascular outcomes after one year of follow-up in a contemporaneous population with heart failure (HF) with and without type 2 diabetes in Spain. These factors were also analyzed in the DAPA-HF-like population (patients who met most inclusion criteria of the DAPA-HF trial) and in patients treated with SGLT2 inhibitors at baseline.Methods: This work is an observational, retrospective, population-based study using the BIG-PAC database. The index date was January 1, 2019. People aged≥18 years who received care for HF in 2019 were selected. Events that occurred in 2019 were analyzed.Results: We identified 21,851 patients with HF (age 78.0±11.3 years, 53.0% men, 50.9% with HF with reduced left ventricular ejection fraction, 44.5% in NYHA functional class II). HF prevalence was 1.88% and incidence was 2.83 per 1,000 person-years. Regarding HF treatments, 66.1% were taking renin-angiotensin system inhibitors, 69.4% beta blockers, 31.2% aldosterone antagonists, and 7.5% sacubitril/valsartan. During the year of follow-up, 29.8% had HF decompensation which led to hospitalization (mean time to first event of 120.9±72.5 days), 12.3% died, and 8.1% died during hospitalization. Events were more common among patients with type 2 diabetes. Hospitalizations for HF were more common in the DAPA-HF-like population.Conclusions: In Spain, the population with HF is elderly and has many comorbidities. Approximately half of patients have HF with reduced left ventricular ejection fraction. There is room for improvement in HF management, particularly through the use of drugs that reduce both HF hospitalization and mortality, in order to reduce the burden of HF (AU)

Clinical characteristics, management, and one-year risk of complications among patients with heart failure with and without type 2 diabetes in Spain.

OBJECTIVE: This work aims to describe the clinical characteristics and therapeutic management and to determine cardiovascular outcomes after one year of follow-up in a contemporaneous population with heart failure (HF) with and without type 2 diabetes in Spain. These factors were also analyzed in the DAPA-HF-like population (patients who met most inclusion criteria of the DAPA-HF trial) and in patients treated with SGLT2 inhibitors at baseline. METHODS: This work is an observational, retrospective, population-based study using the BIG-PAC database. The index date was January 1, 2019. People aged ≥ 18 years who received care for HF in 2019 were selected. Events that occurred in 2019 were analyzed. RESULTS: We identified 21,851 patients with HF (age 78.0 ± 11.3 years, 53.0% men, 50.9% with HF with reduced left ventricular ejection fraction, 44.5% in NYHA functional class II). HF prevalence was 1.88% and incidence was 2.83 per 1,000 person-years. Regarding HF treatments, 66.1% were taking renin-angiotensin system inhibitors, 69.4% beta blockers, 31.2% aldosterone antagonists, and 7.5% sacubitril/valsartan. During the year of follow-up, 29.8% had HF decompensation which led to hospitalization (mean time to first event of 120.9 ± 72.5 days), 12.3% died, and 8.1% died during hospitalization. Events were more common among patients with type 2 diabetes. Hospitalizations for HF were more common in the DAPA-HF-like population. CONCLUSIONS: In Spain, the population with HF is elderly and has many comorbidities. Approximately half of patients have HF with reduced left ventricular ejection fraction. There is room for improvement in HF management, particularly through the use of drugs that reduce both HF hospitalization and mortality, in order to reduce the burden of HF.

FDG-PET parameters predicting mediastinal malignancy in lung cancer.

BACKGROUND: Staging of mediastinal lymph nodes in non-small cell lung cancer (NSCLC) is mandatory. The maximum Standard Uptake Value (SUVmax) obtained using F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) is the best non-invasive technique available for this evaluation, but its performance varies from center to center. The aim of the present study was to identify FDG-PET predictors of mediastinal malignancy that are able to minimize intercenter variability and improve the selection of subsequent staging procedures. METHOD: A multicenter study of NSCLC patients staged through FDG-PET and endobronchial ultrasonography with needle aspiration (EBUS-NA) was performed using therapeutic surgery with systematic nodal dissection as gold standard. Intercenter variability and predictive power for mediastinal malignancy of different FDG-PET measures were assessed, as well as the role of these measures for selecting additional staging procedures. RESULTS: One hundred and twenty-one NSCLC patients, of whom 94 (72%) had ≥1 hypermetabolic spots in the mediastinum, were included in the study. Mean SUVmax of the primary tumor was 12.3 (SD 6.3), and median SUVmax of the highest hypermetabolic spots in the mediastinum was 3.9 (IQR 2.4-7). Variability of FDG-PET measures between hospitals was statistically significant (p = 0.016 and p < 0.001 respectively), but lost significance when SUVmax in the mediastinum was expressed as a ratio or a subtraction from the primary tumor (SUVmax mediastinum/tumor, p = 0.083; and SUVmax mediastinum - tumor, p = 0.428 respectively). SUVmax mediastinum/tumor showed higher accuracy in the ROC analysis (AUC 0.77 CI 0.68-0.85, p < 0.001), and showed predictive power for mediastinal malignancy when using a 0.4 cutoff (OR 6.62, 95%CI 2.98-14.69). Sensitivities and negative predictive values of clinical staging through EBUS-NA attained values ranging between 57% and 92% after FDG-PET, which improved with additional techniques when the tumor had a diameter >3 cm and/or a SUVmax mediastinum/tumor ratio >0.4. CONCLUSION: The SUVmax mediastinum/tumor ratio is a good predictor of regional tumor extension in NSCLC. This measure is not influenced by intercenter variability and has an accuracy of over 70% for the identification of malignancy when using a 0.4 cutoff.

Prevalence of metabolic syndrome in Galicia (NW Spain) on four alternative definitions and association with insulin resistance.

The present study evaluated the prevalence of the metabolic syndrome (MS) in a representative sample (no.=2860) of adults from the Spanish region of Galicia using the definitions of a) the World Health Organization; b) the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults; c) the European Group for Study of Insulin Resistance; and d) the International Diabetes Federation. In addition, we assess concordance among the different definitions, and the relationships of MS with insulin resistance (IR) as assessed by the homeostatic model assessment (HOMA-IR) index. Our results indicate a high prevalence of MS under all 4 definitions. MS prevalence was higher in men than women on all 4 definitions, and increased significantly with body mass index and age. IR was high among subjects with MS, and the HOMA index was a good discriminator of MS and non-MS on all 4 definitions, suggesting that HOMA index may be a useful predictive tool in clinical practice.

[Tracheal intubation guided by fibrobronchoscopy in patients with difficult airway. Predictive factors of the outcome]. / Intubación traqueal guiada por fibrobroncoscopio en pacientes con vía aérea difícil. Factores predictores del resultado.

INTRODUCTION AND OBJECTIVE: Tracheal intubation (TI) guided by fibrobronchoscopy (FB) is one of the essential techniques in the approach to the difficult airway (DAW). Few works have been published on the possible causes of TI failure with this procedure. This study aims to discover which factors could predict TI failure with FB. MATERIAL AND METHODS: An observational and retrospective study in which the last 122 consecutive TIs guided by FB (between January 2000 and April 2008) performed by our group were included. A multivariate analysis of the factors that could influence in the outcome was conducted: cause of the DAW, TI pathway, type of endotracheal tube, elective or urgent indication of the procedure, sedation level and experience of the bronchoscopist. RESULTS: Tracheal intubation in individuals who are going to undergo surgical interventions accounts for 92.6% of the total. The most frequent indications of TI by FB were: limitation of neck movement (60 cases), airway stenosis (24), increase of soft tissues (13), narrow oral aperture (9), airway compression (6), and vocal cord paralysis (6). In 10 (8.2%) cases, TI by FB was not possible. The variables that best predicted IT failure in the multivariate analysis were profound sedation/ general anesthesia (OR = 12.2; 95% CI, 1.8-84; p = 0.01) and limited experience of the bronchoscopist (OR = 25.3; 95% CI, 3.5-181.8; p = 0.001). CONCLUSIONS: TI guided by FB performed by bronchoscopist is successful in more than 90% of the cases with DAW. The skill and experience of the bronchoscopist is one of the primary determining factors of success of the procedure. Profound sedation may condition TI guided by FB failure.

Intubación traqueal guiada por fibrobroncoscopio en pacientes con vía aérea difícil. Factores predictores del resultado / Tracheal intubation guided by fibrobronchoscopy in patients with difficult airway. Predictive factors of the outcome

Introducción y objetivo. La intubación traqueal (IT) guiada por fibrobroncoscopio (FB) es una de las técnicas esenciales en el abordaje de la vía aérea difícil (VAD). Hay pocos trabajos publicados sobre las causas del fallo de la IT con este procedimiento. El objetivo del presente estudio es analizar cuáles son los factores que podrían predecir el fallo de la IT con FB. Material y métodos. Estudio observacional retrospectivo en el que se incluyeron las últimas 122 IT consecutivas guiadas por FB (enero de 2000 a abril de 2008) realizadas por nuestro grupo. Se llevó a cabo un análisis multivariable de los factores que podrían influir en el resultado: causa de VAD, vía de IT, tipo de tubo endotraqueal, indicación programada o urgente del procedimiento, grado de sedación y experiencia del broncoscopista. Resultados. Las IT en individuos que se sometieron a intervenciones quirúrgicas representaron el 92,6% del total. Las indicaciones más frecuentes de la IT por FB fueron: limitación del movimiento del cuello (60 casos), estenosis de la vía aérea (24), aumento de tejidos blandos (13), apertura oral limitada (9), compresión de la vía aérea (6) y parálisis de cuerdas vocales (6). En 10 (8,2%) casos la IT por FB no fue posible. Las variables que mejor predijeron el fracaso de la IT en el análisis multivariable fueron la sedación profunda/anestesia general (odds ratio [OR] = 12,2; intervalo de confianza [IC] del 95%, 1,8-84; p = 0,01) y la escasa experiencia del broncoscopista (OR = 25,3; IC del 95%, 3,5-181,8; p = 0,001). Conclusiones. En más del 90% de los casos con VAD, la IT con FB realizada por broncoscopistas es exitosa. La habilidad y la experiencia del broncoscopista son uno de los principales determinantes del éxito del procedimiento. La sedación profunda puede condicionar el fallo de la IT por FB (AU)

Introduction and objective. Tracheal intubation (TI) guided by fibrobronchoscopy (FB) is one of the essential techniques in the approach to the difficult airway (DAW). Few works have been published on the possible causes of TI failure with this procedure. This study aims to discover which factors could predict TI failure with FB. Material and methods. An observational and retrospective study in which the last 122 consecutive TIs guided by FB (between January 2000 and April 2008) performed by our group were included. A multivariate analysis of the factors that could influence in the outcome was conducted: cause of the DAW, TI pathway, type of endotracheal tube, elective or urgent indication of the procedure, sedation level and experience of the bronchoscopist. Results. Tracheal intubation in individuals who are going to undergo surgical interventions accounts for 92.6% of the total. The most frequent indications of TI by FB were: limitation of neck movement (60 cases), airway stenosis (24), increase of soft tissues (13), narrow oral aperture (9), airway compression (6), and vocal cord paralysis (6). In 10 (8.2%) cases, TI by FB was not possible. The variables that best predicted IT failure in the multivariate analysis were profound sedation/ general anesthesia (OR = 12.2; 95% CI, 1.8-84; p = 0.01) and limited experience of the bronchoscopist (OR = 25.3; 95% CI, 3.5-181.8; p = 0.001). Conclusions. TI guided by FB performed by bronchoscopist is successful in more than 90% of the cases with DAW. The skill and experience of the bronchoscopist is one of the primary determining factors of success of the procedure. Profound sedation may condition TI guided by FB failure (AU)

Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain.

OBJECTIVE: Toxic thyroid adenoma (TA) is a common cause of hyperthyroidism. Mutations in the TSH receptor (TSHR) gene, and less frequently in the adenylate cyclase-stimulating G alpha protein (GNAS) gene, are well established causes of TA in Europe. However, genetic causes of TA remain unknown in a small percentage of cases. We report the first study to investigate mutations in TSHR, GNAS, protein kinase, cAMP-dependent, regulatory, type I alpha (PRKAR1A) and RAS genes, in a large series of TA from Galicia, an iodine-deficient region in NW Spain. DESIGN AND METHODS: Eighty-five TA samples were obtained surgically from 77 hyperthyroid patients, operated on for treatment of non-autoimmune toxic nodular goitre. After DNA extraction, all coding exons of TSHR, GNAS and PRKAR1A genes, and exons 2 and 3 of HRAS, KRAS and NRAS were amplified by PCR and sequenced. Previously unreported mutants were cloned in expression vectors and their basal constitutive activities were determined by quantification of cAMP response element (CRE)-luciferase activity in CO7 cells transfected with wild-type and mutant plasmids. RESULTS: TSHR gene mutations were found in 52 (61.2%) samples, GNAS gene mutations in 4 (4.71%) samples and no PRKAR1A or RAS mutations were found. Only three previously unreported mutations were found, two affecting the TSHR, A623F and I635V, and one affecting the G-protein alpha-subunit (Gsalpha), L203P. All mutant proteins showed higher CRE-luciferase activity than their wild-type counterparts. CONCLUSIONS: TA in a hyperthyroid population living in Galicia, a Spanish iodine-deficient region, harbours elevated frequencies of TSHR and GNAS mutations activating the cAMP pathway. However, the genetic cause of TA was undetermined in 34% of the TA samples.

Prevalence, awareness, treatment and control of hypertension in Galicia (Spain) and association with related diseases.

The present study evaluated the prevalence of hypertension (HT) and its possible relationships with various risk factors in a representative sample (n=2884) of the adult population (>18 years old) of Galicia, a region of Spain. Subjects were selected by a two-step cluster sampling procedure from the Galician public health service database, which covers more than 95% of the population (2.7 million inhabitants). The overall prevalence of HT, defined as BP >140/90 mm Hg and/or current treatment with antihypertensive medication, was 25.5%, higher in men (31.1%) than in women (20.7%). Of the hypertensive subjects 50.6% were aware of the HT; of these, 72.0% were receiving treatment and 36.4% were treated and controlled. The prevalence of HT increased with age and was higher in subjects from urban areas than rural areas and higher in subjects with low educational level. Surprisingly, people with low educational level more frequently showed awareness of HT than people with high education level. Increased body mass index was related to increased prevalence of HT and close associations were observed between HT and cardiovascular diseases. Our data also show a linear upward trend in blood pressure from normal glucose metabolism to diabetes mellitus. Surprisingly, the prevalence of HT among people with known diabetes was higher than among people with undetected diabetes, which may indicate poor control of HT in diabetic subjects.

Iodine nutrition in the adult population of Galicia (Spain).

OBJECTIVE: To identify the current state of iodine nutrition in the adult population of Galicia (Spain), which is considered iodine sufficient based on results from studies carried out on schoolchildren. Urinary iodine concentration (UIC) and its relationship with different socio-demographic variables were assessed. DESIGN: A cross-sectional study was carried out on the population aged above 18 years in the Autonomous Community of Galicia (Spain) during 2004. The UICs were determined in an isolated urine sample using Dunn's colorimetric method. Iodine status was based on World Health Organization/International Council for the Control of Iodine Deficiency Disorders (WHO/ICCIDD) UIC. MAIN OUTCOME: A total of 2877 urine samples were taken. Median UIC for the total Galician population was 75.6 microg/L. About 30% of the population showed a UIC below 50 microg/L. Educational level, place of residence (coast vs. inland), and consumption of iodized salt were independent variables associated with the iodine nutrition of the adult population of Galicia. CONCLUSIONS: There is "mild" iodine deficiency (WHO) in the adult population of Galicia, which affects all the groups analyzed and which is particularly significant in the group of women of a fertile age. The data obtained on the state of iodine nutrition in school-age populations cannot be extended to the adult population.

Okadaic acid, a protein phosphatase inhibitor, stimulates the activity of Na+/H+ and Na+-independent Cl-/HCO3- exchangers in human lymphocytes.

The cytosolic pH (pHi) regulatory mechanisms of peripheral blood human lymphocytes and the effect of okadaic acid on the activity of these mechanisms were studied by means of fluorescence imaging microscopy of 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF)-loaded individual cells. Lymphocytes recover from a CO(2)-induced acid load in an extracellular Na+-dependent, intracellular Cl- -independent fashion. This pHi recovery is highly sensitive to the anion exchange inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and minimally sensitive to the Na+/H+ exchange inhibitor amiloride, suggesting that it is mostly due to the action of a Na+-dependent HCO3- transporter. Extracellular Cl- and Na+ removal experiments point to the existence of a DIDS-sensitive Na+-independent Cl-/HCO3- exchanger. Preincubation with okadaic acid stimulates the pHi recovery rate from a CO2-induced acid load in the presence of DIDS (0.002 pHu/min vs. 0.065 pHu/min), but not in the presence of amiloride. Okadaic acid also accelerates the pHi elevation induced by Cl- removal (0.039 pHu/min vs. 0.067 pHu/min). In summary, these results indicate that okadaic acid stimulates the activity of Na+/H+ and Na+-independent Cl-/HCO3- exchangers, but has no effect on the activity of the Na+-dependent HCO3- transporter of human lymphocytes.

Prolactin induces calcium influx and release from intracellular pools in human T lymphocytes by activation of tyrosine kinases.

The early events related to intracellular signals after prolactin (PRL) activation in T lymphocytes are not clearly established. The aim of this work was to study the effect of PRL in cytosolic calcium levels in human T lymphocytes. By using the dye FURA-2 AM, the variations in cytosolic Ca(2+) were studied in peripheral human T lymphocytes isolated from extracted blood from healthy donors. Fifty nanograms per milliliter PRL induces a small increase in cytosolic calcium. When the cells are preincubated overnight (16-20 h) in the presence of PRL, the increase in calcium is higher. This high increase is due to the release from intracellular pools and to the influx from the extracellular media. That is, after overnight incubation with PRL, calcium influx in T cells follows the capacitative model. Since PRL receptor (PRL-R) activation involves the tyrosine kinase pathway, we check calcium effect in the presence of genistein, a known inhibitor of tyrosine kinases. When cells are preincubated in the presence of 10 microM genistein, and PRL is immediately added, no increase in cytosolic calcium is observed. The presence of genistein also completely blocks the increase in cytosolic calcium stimulated by PRL after overnight incubation with PRL. In the presence of PRL and N,N-dimethyl-D-erythro-sphingosine (DMS), a stimulus that increases cytosolic calcium in T cells by tyrosine kinase stimulation, a high, even insignificant, calcium influx is induced. However, when the cells are incubated overnight in the presence of PRL, and then DMS is added, a significant increase in cytosolic calcium levels takes place. This increase is associated with an increase in calcium release from intracellular pools and an increase in calcium uptake. Genistein reduces the influx of external calcium induced by DMS after short incubation with PRL and significantly inhibits both, calcium pools empty, and calcium influx is induced by DMS after overnight incubation with PRL. In summary, PRL induces calcium influx in normal T lymphocytes. The influx is magnified after long PRL exposures, intracellular Ca(2+) pool-dependent, and activated through tyrosine kinases.

Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes: effect of 4-aminopyridin, ammoniun chloride and ionomycin.

Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca(2+), H(+) and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca(2+) were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bis-oxonol. In a bicarbonate-free medium, cell alkalinization with NH(4)Cl slightly decreased intracellular Ca(2+) concentration ([Ca(2+)](i)) due to efflux of Ca(2+) from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca(2+)](i), either in the presence or absence of external Ca(2+). The increase in Ca(2+)-free medium is likely to be due to Ca(2+) release from thapsigargin and caffeine-independent intracellular stores. Both 4-AP or NH(4)Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca(2+) levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca(2+).

Characterization of the Na+/Ca2+ exchanger on rat mast cells. Evidence for a functional role on the regulation of the cellular response.

The Na+/Ca2+ exchanger has not been characterized in rat mast cells, although its existence has been previously suggested. In this work, we determine that this exchanger exists on rat mast cells and that it has an important regulatory role on the cellular function. We have studied uptake and release of 45Ca in the presence of different external sodium concentrations and, under the same conditions, the simultaneous uptake of 22Na and 45Ca. The results show that uptake and release of 45Ca in these cells are related to the concentration of sodium in the extracellular medium and that there is also a perfect coupling between 22Na and 45Ca fluxes. In these conditions, we evaluated the intracellular calcium levels in fura-2 loaded cells. When the extracellular sodium concentration was lower than 60 mM, we observed an increase in intracellular calcium, reaching its maximum when the extracellular medium has no sodium. Then we investigated the effect on histamine release. The ionophore A23187 elicits histamine release in rat mast cells, depending on the extracellular calcium concentration. This drug releases more histamine (up to twofold) with sodium concentrations <60 mM. In the presence of 2,4-dichlorobenzamil hydrochloride, a Na+/Ca2+ exchanger inhibitor, the release of histamine induced by the ionophore was lower than in controls in media with low external sodium, and, on the contrary, at extracellular sodium concentrations >60 mM, the histamine release was higher than in controls. In the same conditions, but when the Na+-K+ ATPase was inhibited by ouabain, and as a consequence more sodium was inside the cells, a high increase in histamine release induced by A23187 in a sodium-free medium was observed. Under the same conditions, a high increase in intracellular calcium takes place. The overall data are preliminary evidence suggesting the existence of a Na+/Ca2+ exchanger in rat mast cells with a threshold to get reversed at 60 mM external sodium, lower concentrations of this ion increasing internal calcium and producing higher histamine release.

Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes.

We studied the effects of external HCO3- on pHi regulation in human lymphocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pHi recovery was measured with the fluorescent dye BCECF. Cells recovering in HCO3--containing medium reached a higher final pHi, the H+ efflux rate was increased and shifted to alkaline pHi compared to that of cells recovering in HCO3--free solution. The resting pHi was higher in a HCO3--containing solution. Experiments carried out in the presence of amiloride, DIDS and in the absence of external Na+ suggest the existence of two major mechanisms acting in the pHi recovery of lymphocytes after an acid load: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-dependent HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitive fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H-89 showed protein-kinase-A-dependent downregulation of the amiloride-insensitive recovery of pHi in human lymphocytes. In summary, this paper provides functional evidence for the existence of a Na+/HCO3--dependent mechanism involved in pHi recovery in human lymphocytes following an acid load, that is electrogenic and downregulated by PKA.

Membrane potential changes associated with calcium signals in human lymphocytes and rat mast cells.

Human lymphocytes and rat mast cells, two non-excitable cellular models, were used to investigate membrane potential changes accompanying Ca2+ signals. Cells were stimulated with agents known to induce both Ca2+ release from internal stores and influx of extracellular Ca2+, namely thapsigargin, ionomycin and compound 48/80. Thapsigargin and ionomycin were used to activate lymphocytes, while compound 48/80 was used to stimulate mast cells. Membrane potential changes and Ca2+ concentration were monitored with the fluorescent dyes bis-oxonol and fura-2, respectively. In lymphocytes, thapsigargin induced a hyperpolarization temporally correlated with the increase in intracellular Ca2+ concentration. This hyperpolarization is due to activation of a K+ conductance which consists of two phases, a first phase independent on external Ca2+ and a second one blocked in a Ca2+-free medium. Ionomycin induced a Ca2+-dependent depolarization attributed to a massive influx of external Ca2+. On the other hand, stimulation of mast cells with compound 48/80 produced a fast hyperpolarization and an increase in intracellular Ca2+ levels. Besides different time-courses, this hyperpolarization differs from that induced by thapsigargin in lymphocytes in two aspects, it is mainly due to a Cl(-)-entry current and exit of K+ and it is completely inhibited in the absence of extracellular Ca2+. Compound 48/80-induced histamine release is not related to membrane potential changes.

Sodium, PMA and calcium play an important role on intracellular pH modulation in rat mast cells.

In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.

Expression of neurotrophins and their receptors in sciatic nerve of experimentally diabetic rats.

The levels of neurotrophins and their receptor mRNAs were measured in rat sciatic nerve, after 6 or 12 weeks of streptozotocin-induced diabetes. Expression of neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) was decreased by 50 and 29%, respectively, compared with age-matched controls after 12 weeks of diabetes. Expression of brain-derived neurotrophic factor (BDNF) was not detected. In addition, diabetes induced a reduction in the expression levels of the neurotrophin receptors: trkB mRNA decreased by 50% after 6 weeks of diabetes, but returned to control levels after 12 weeks; meanwhile the trkC and p75LNGFR transcripts were reduced by 20% of control at both times studied. trkA expression was below detection limits. Thus, these data suggest that a reduction in neurotrophin and neurotrophin receptors could contribute to the development and maintenance of diabetic neuropathy.

Functional characterization of the Na(+)-H+ exchanger in rat mast cells: crosstalks between different kinase pathways.

In our effort to understand the mechanisms by which rat mast cells regulate intracellular pH (pHi), we studied the effect of drugs acting on different transducting signals on the Na(+)-H+ antiport. We studied the activity of the antiporter in recovering pHi after an acid load with sodium propionate. The drugs used were okadaic acid, which inhibits the phosphatases 1 and 2A, pertussis toxin, which ADP-rybosylates the Gi-protein, cholera toxin, which ADP-rybosylates the Gs-protein, NaF which non-specifically activates G-proteins, and the phorbol esther 12-O-tetradecanoylphorbol 13-acetate (TPA) which specifically activates protein kinase C. The effect of TPA is a two-fold stimulation of the activity of the antiporter. A similar activation was observed with the combination okadaic acid plus cholera toxin. All the drugs alone did not modify the activity of the antiporter, and they all blocked the stimulatory activity of TPA. In a Ca(2+)-free medium, okadaic acid inhibits the activity of the antiporter. All the mechanisms affected by these drugs have some regulatory role on the Na(+)-H+ antiport. Our results indicate the great complexity of the crosstalks between the different signal transducing pathways.