G-protein tonic inhibition of calcium channels in pancreatic ß-cells.

Voltage-gated calcium channels (CaV) conduct Ca2+ influx promoting neurotransmitters and hormone release. CaV are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic ß-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca2+ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca2+ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca2+ channels in pancreatic ß-cells. Experiments were conducted in pancreatic ß-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca2+ channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP2). Furthermore, carbachol or intramembrane lipids, such as PIP2, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca2+ channels in pancreatic ß-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca2+ channels. The novelty of these results strengthens the critical role of Gßγ's in Ca2+ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes.

The FDA's approval of peptide drugs such as Ziconotide or Exendin for pain relief and diabetes treatment, respectively, enhanced the interest to explore novel conotoxins from Conus species venom. In general, conotoxins can be used in pathologies where voltage-gated channels, membrane receptors, or ligands alter normal physiological functions, as in metabolic diseases such as Type 2 diabetes. In this study, the synthetic cal14.2b (s-cal14.2b) from the unusual Californiconus californicus demonstrated bioactivity on NIT-1 insulinoma cell lines stimulating insulin secretion detecting by high performance liquid chromatography (HPLC). Accordingly, s-cal14.2b increased the CaV1.2/1.3 channel-current by 35 ± 4% with a recovery τ of 10.3 ± 4 s in primary cell culture of rat pancreatic ß-cells. The in vivo results indicated a similar effect of insulin secretion on mice in the glucose tolerance curve model by reducing the glucose from 500 mg/dL to 106 mg/dL in 60 min, compared to the negative control of 325 mg/dL at the same time. The PET-SCAN with radiolabeling 99mTc-s-cal14.2b demonstrated biodistribution and accumulation in rat pancreas with complete depuration in 24 h. These findings show the potential therapeutic use of s-cal14.2b in endocrinal pathologies such as early stages of Type 2 Diabetes where the pancreas's capability to produce insulin is still effective.

Motor learning impairment in rats under a high sucrose diet.

Motor learning skills are reliable indicators of behavioral acquisition and cognitive disorders. The ease with which learning skills are measured disparities the complexity of the interpretation concerning neural plasticity. Conversely, a wealth of information regarding metabolic derangements has long been reported with direct connection to high sucrose diets. However, the impact of excessive sucrose consumption on undergoing cognitive processes has been only scarcely addressed up to now. Therefore, the goal of this work was to describe the associative relationship between high sucrose consumption and changes in motor learning skills acquisition. Motor learning impairments conditioned by central alterations are hypothesized. Rotarod, elevated plus-maze and open field trials, along with metabolic and pro-inflammatory biomarkers tests in Wistar rats under a high sucrose treatment, were performed. Motor learning impairment in high sucrose diet-treated rats was found while spontaneous locomotor activity remained unchanged. Even though, no anxiety-like behavior under high sucrose diet-treatment was observed. Consistently, the worst outcome in the glucose tolerance test was developed, the worst motor learning performance was observed. Furthermore, insulin resistance correlated positively with a pro-inflammatory state and a decreased latency to fall in the rotarod test. Indeed, C-reactive protein and tumor necrosis factor-α serum levels, along with the homeostasis model assessment of insulin resistance (HOMA-IR), significantly increased in motor learning impairment. Together, these results support behavioral, metabolic and pro-inflammatory changes associated with deleterious changes in central nervous system likely involving crucial motor learning structures. Underlying pro-inflammatory-triggered processes may explain cognitive disorders in advanced states of metabolic derangements.

Dynamical phase transition in spike neuronal firing patterns of hippocampal cells.

There is increasing evidence that the brain resides in a state of criticality. The purpose of the present work is to characterize the dynamics of individual hippocampal CA1 pyramidal cells and to investigate how it is influenced by changes in Kv7.2/7.3 (M-channel) ion channel modulation, which is known to be key in determining the neuronal excitability. We show that the resting activity of CA1 neurons exhibit random dynamics with low information content, while changes in M-channel modulation move the neuronal activity near a phase transition to richer non-trivial dynamics. We interpret these results as the basis upon which the state of self-organized criticality is built.

Fast Inactivation of CaV2.2 Channels Is Prevented by the Gß1 Subunit in Rat Sympathetic Neurons.

Voltage-dependent regulation of CaV2.2 channels by G-proteins is performed by the ß (Gß) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of CaV2.2 channels in superior cervical ganglion neurons that overexpressed Gß subunits. CaV2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gß1 subunit reduced inactivation, while Gß5 subunit did not alter at all inactivation kinetics compared to control recordings. CaV2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gß1-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gß1-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gß1 subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of CaV2.2 channels resting in the inactivated state. These results support that the Gß1 subunit inhibits the fast inactivation of CaV2.2 channels in SCG neurons. They explain the long-observed sustained Ca2+ current under G-protein modulation.

Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

A Conus regularis conotoxin with a novel eight-cysteine framework inhibits CaV2.2 channels and displays an anti-nociceptive activity.

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits CaV2.2 channel current in a dose-dependent manner with an EC50 of 2.8 µM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for CaV2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.

PIP2 hydrolysis is responsible for voltage independent inhibition of CaV2.2 channels in sympathetic neurons.

GPCRs regulate Ca(V)2.2 channels through both voltage dependent and independent inhibition pathways. The aim of the present work was to assess the phosphatidylinositol-4,5-bisphosphate (PIP2) as the molecule underlying the voltage independent inhibition of Ca(V)2.2 channels in SCG neurons. We used a double pulse protocol to study the voltage independent inhibition and changed the PIP(2) concentration by means of blocking the enzyme PLC, filling the cell with a PIP(2) analogue and preventing the PIP(2) resynthesis with wortmannin. We found that voltage independent inhibition requires the activation of PLC and can be hampered by internal dialysis of exogenous PIP(2). In addition, the recovery from voltage independent inhibition is blocked by inhibition of the enzymes involved in the resynthesis of PIP(2). These results support that the hydrolysis of PIP(2) is responsible for the voltage independent inhibition of Ca(V)2.2 channels.

Gating charges per channel of Ca(V)2.2 channels are modified by G protein activation in rat sympathetic neurons.

It has been suggested that voltage-dependent G protein modulation of Ca(V)2.2 channels is carried out at closed states of the channel. Our purpose was to estimate the number of gating charges of Ca(V)2.2 channel in control and G protein-modulated conditions. By using a Cole-Moore protocol we observed a significant delay in Ca(V)2.2 channel activation according to a transit of the channel through a series of closed states before channel opening. If G protein voltage-dependent modulation were carried out at these closed states, then we would have expected a greater Cole-Moore lag in the presence of a neurotransmitter. This prediction was confirmed for noradrenaline, while no change was observed in the presence of angiotensin II, a voltage-insensitive G protein modulator. We used the limiting slope method for calculation of the gating charge per channel. Effective charge z was 6.32+/-0.65 for Ca(V)2.2 channels in unregulated conditions, while GTPgammaS reduced elementary charge by approximately 4 e(0). Accordingly, increased concentration of noradrenaline induced a gradual decrease on z, indicating that this decrement was due to a G protein voltage-sensitive modulation. This paper shows for the first time a significant and reversible decrease in charge transfer of Ca(V)2.2 channels under G protein modulation, which might depend on the activated G protein inhibitory pathway.

Construcción y validación clínica de un prototipo para el registro continuo de la presión arterial de forma no invasiva y ambulatoria / Construction and clinical validation of an automated measuring device for continuous non-invasive ambulatory blood pressure measurement

Introducción: la monitorización ambulatoria de la presión arterial con equipos biomédicos, es un método útil y confiable para el diagnóstico de la hipertensión arterial. El propósito del estudio fue validar un nuevo equipo de monitorización ambulatoria de la presión arterial de 24 horas (MAPA-FCV) de bajo costo, producido en la Fundación Cardiovascular de Colombia. Métodos: el estudio se desarrolló de acuerdo con las recomendaciones de la Sociedad Americana para el Avance de la Instrumentación Médica (AAMI, por sus siglas en Inglés) y la Sociedad Británica de Hipertensión (BHS) para validación de equipos de monitorización de presión arterial. Se incluyeron 85 sujetos sanos, en quienes, una persona previamente entrenada, obtuvo tres medidas simultáneas de presión arterial (cada 10 minutos) y se compararon con las que se obtuvieron con el equipo MAPA-FCV. Resultados: los sujetos presentaron una presión arterial sistólica de 115 ± 15 mm Hg y diastólica de 71 ± 8 mm Hg. Se encontró una diferencia media de 0,63 ± 5,94 mm Hg en la presión arterial sistólica y de 0,17 ± 5,08 mm Hg en la diastólica entre el equipo y el observador entrenado. Así mismo, se observó que más de 93% de las mediciones simultáneas de presión arterial, presentaron una diferencia menor a 10 mm Hg. Conclusiones: en el estudio el monitor MAPA-FCV alcanzó altos grados de concordancia con los valores de presión arterial que obtuvo el personal capacitado; adicionalmente el equipo cumplió con los criterios de validación de la AAMI y BHS, lo que hace posible su recomendación para uso clínico en población adulta.

Introduction: ambulatory arterial pressure monitoring with biomedical devices is a useful and reliable method to diagnose hypertension. The aim of this study was to validate a new low cost Holter blood pressure monitor (MAPA-FCV) produced at the Fundación Cardiovascular de Colombia. Methods: the study was developed according to the guidelines for validation of automated blood pressure measuring devices of the Association for the Advancement of Medical Instrumentation (AAMI) and the British Hypertension Society (BHS). Three blood pressure measurements were taken in 85 healthy subjects in a 30 minutes period (every 10 minutes). Measurements were taken by one observer trained to measure blood pressure with a mercury column device, and were compared with those obtained with the automatic device. Results: mean systolic blood pressure obtained in the subjects was 115 ± 15 mmHg, and mean diastolic blood pressure was 71 ± 8 mmHg. The mean and standard deviation of the differences between the measurements obtained by the observer and those obtained with the automatic device were 0.63 ± 5.94 mmHg for systolic pressure and 0.17 ± 5.08 mmHg for diastolic pressure. In addition, about 93% of the differences between the ascultatory and MAPA-FCV were within 10 mmHg. Conclusions: in the present study a close agreement between systolic and diastolic pressure measurements obtained by the auscultatory method and the MAPA-FCV device was found. The data obtained show that the MAPA-FCV can be recommended for clinical use according to the guidelines of international entities.

La importancia de la rehabilitación cardiovascular: experiencia en la Fundación Cardiovascular de Colombia / Importance of cardiovascular rehabilitation: experience in the Colombian Cardiovascular Foundation

Introducción: los programas de rehabilitación cardiovascular constituyen una herramienta fundamental en el manejo integral de los pacientes con enfermedades cardiovasculares. Se presentan los datos históricos relevantes del programa de Rehabilitación de la Fundación Cardiovascular de Colombia y los resultados obtenidos durante su existencia, comparándolos con otros programas a nivel nacional e internacional.Resultados: se confirman los beneficios de los programas de rehabilitación cardiovascular a nivel de control de factores de riesgo, mejoría en la tolerancia al ejercicio, mejoría en la calidad de vida y reducción de la morbimortalidad. Conclusiones: los programas de rehabilitación cardiovascular apoyados por un equipo multidisciplinario, influyen de manera positiva en el mejoramiento de la calidad de vida de los pacientes que han presentado un evento cardiovascular y han sido manejados a través de medio médicos o quirúrgicos.

Introduction: the programs of cardiovascular rehabilitation constitute a fundamental tool in the integral treatment of patients with cardiovascular disease. Historical relevant data from the Colombian Cardiovascular Foundation Rehabilitation program, as well as the results obtained during its existence, are presented, comparing them to those of other national and international programs. Results: the benefits of the cardiovascular rehabilitation programs referring to the control of risk factors, improvement to exercise tolerance, improvement in the quality of life and morbid-mortality reduction, are confirmed. Conclusions: cardiovascular rehabilitation programs supported by a multidisciplinary team have a positive influence in the improvement of the quality of life of patients who have had a cardiovascular event and have been treated by medical or surgical means.