Scielo RSS <![CDATA[Biological Research]]> https://scielo.conicyt.cl/rss.php?pid=0716-976020060003&lang=es vol. 39 num. 3 lang. es <![CDATA[SciELO Logo]]> https://scielo.conicyt.cl/img/en/fbpelogp.gif https://scielo.conicyt.cl <![CDATA[<strong>Editorial</strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300001&lng=es&nrm=iso&tlng=es <![CDATA[MI HOMENAJE<b> <strong>GUAYO ROJAS, COMPAÑERO DE RUTA</strong></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300002&lng=es&nrm=iso&tlng=es <![CDATA[Large conductance Ca<sup>2+</sup>-activated K<sup>+</sup> (BK) channel: Activation by Ca<sup>2+</sup> and voltage]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300003&lng=es&nrm=iso&tlng=es Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism. <![CDATA[<strong>Pituitary cell type-specific electrical activity, calcium signaling and secretion </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300004&lng=es&nrm=iso&tlng=es All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells. <![CDATA[<strong>The action potential</strong>: <strong>From voltage-gated conductances to molecular structures </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300005&lng=es&nrm=iso&tlng=es All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells. <![CDATA[<strong>Apical Maxi-chloride channel from human placenta</strong>: <strong>12 years after the first electrophysiological recordings </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300006&lng=es&nrm=iso&tlng=es The Maxi-chloride channel was the first ion channel described by electrophysiological methods in placenta. Because it is difficult to access a complex epithelium such as the placenta for electrophysiological procedures, the studies of ion channels from placental membranes have been performed only very recently. It was only in 1993 that a direct demonstration of a high-conductance chloride channel in apical membranes of intact trophoblastic epithelium was mentioned, and two years later, the description of this channel was reported from purified placental apical membranes reconstituted into artificial lipid membranes suitable for patch-clamp recordings. This brief review comments on the work done with regard to the electrophysiological characterization and regulation of the large-conductance or "Maxi" chloride channel and its contribution to the development of a cellular model for syncytiotrophoblast ion transport. <![CDATA[<strong>Histidines 13 and 14 in the A</strong><strong>β</strong><strong> sequence<sub> </sub>are targets </strong><b> <strong>for inhibition of Alzheimer's<sub> </sub>disease A</strong></b><strong>β</strong><strong> ion channel </strong><b> <strong>and cytotoxicity </strong></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300007&lng=es&nrm=iso&tlng=es The fact that Alzheimer's beta amyloid (Aβ) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Aβ ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Aβ channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Aβ channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Aβ cytotoxicity. These data thus contribute to the definition of the region of the Aβ sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Aβ. These data also emphasize the potential value in using inhibition of Aβ ion channel activity as an end point for Alzheimer's disease drug discovery. <![CDATA[Testing of a new prototype surgical stapler that automates the rollover sleeve technique for venous anastomoses]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300008&lng=es&nrm=iso&tlng=es The creation of successful vascular anastomoses is of primary importance in many surgical fields. Numerous attempts to automate this process have been made. These techniques have slowly gained acceptance, but their use is still limited. This report details feasibility testing of a new prototype stapler that automates the rollover sleeve technique for venous vascular anastomoses. Male and female mongrel dogs (n=7) (25-32 kg) were used. A segment of the right (n=5) or left (n=2) iliac vein was harvested for interposition grafts after the contra lateral side was transected. In each dog, two end-to-end venous anastomoses at the interposition grafts were performed. The standard anastomosis employed continuous mattress sutures. The experimental anastomosis was performed with a new prototype surgical stapler. The stapled anastomosis was proximal and the sutured was distal. In all experiments, it was possible to perform the experimental anastomosis with the stapler. Complications included two small leaks, one due to misfiring of a single pin in one experimental site. These leaks required suture reinforcement. One dog died of hemorrhage due to a slipped suture at the vein harvest site. One vein had thrombus seen at the sutured site although no technical abnormalities at either of the anastomoses could be found. After two weeks, grafts were inspected grossly and histologically. Healing appeared normal. There was a trend for less inflammatory cells infiltrating stapled sites; however, this was not statistically significant. The experiments demonstrate that this device can automate the rollover sleeve technique for venous anastomoses. <![CDATA[<strong>Altered calcium currents in cultured sensory neurons of normal and trisomy 16 mouse fetuses, an animal model for human trisomy 21 (Down Syndrome)</strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300009&lng=es&nrm=iso&tlng=es Down syndrome is determined by the presence of an extra copy of autosome 21 and is expressed by multiple abnormalities, with mental retardation being the most striking feature. The condition results in altered electrical membrane properties of fetal dorsal root ganglia (DRG) neurons, as in the trisomy 16 fetal mouse, an animal model of the human condition. Cultured trisomic DRG neurons from human and mouse fetuses present faster rates of depolarization and repolarization in the action potential compared to normal controls and a shorter spike duration. Also, trisomy 16 brain and spinal cord tissue exhibit reduced acetylcholine secretion. Therefore, we decided to study Ca2+ currents in cultured DRG neurons from trisomy 16 and age-matched control mice, using the whole-cell patch-clamp technique. Trisomic neurons exhibited a 62% reduction in Ca2+ current amplitude and reduced voltage dependence of current activation at -30 and -20 mV levels. Also, trisomic neurons showed slower activation kinetics for Ca2+ currents, with up to 80% increase in time constant values. Kinetics of the inactivation phase were similar in both conditions. The results indicate that murine trisomy 16 alter Ca2+ currents, which may contribute to impaired cell function, including neurotransmitter release. These abnormalities also may alter neural development. <![CDATA[<strong>Developmental regulation of the expression of sodium currents in Xenopus primary neurons </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300010&lng=es&nrm=iso&tlng=es The electrophysiological properties of neurons are determined by the expression of defined complements of ion channels. Nonetheless, the regulation mechanisms of the expression of neuronal ion channels are poorly understood, due in part to the diversity of neuron subtypes. We explored the expression of voltage-gated currents of Xenopus primary spinal neurons unequivocally identified by means of single-cell RT-PCR. We found that identified spinal neurons exhibit heterogeneity in the temporal appearance of voltage-gated currents. Nevertheless, all neurons progress to similar functional phenotypes. A physiological feature is the onset and increase of the expression of sodium currents. To understand the mechanisms underlying this process, we studied the effect of a dominant negative form of the transcriptional silencer REST/NRSF and found that it associates to an increase in the density of sodium currents. This observation is compatible with a role of this factor in the regulation of gene expression in neurons. These experiments constitute a proof of principle for the feasibility of analyzing molecular mechanisms of the regulation of ion channel genes during early neuronal development and provide direct evidence of the role of REST/NRSF in the control of neuronal sodium channel expression. <![CDATA[<strong>Fast kinetics of calcium dissociation from calsequestrin </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300011&lng=es&nrm=iso&tlng=es We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25ºC calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s-1) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s-1) than the slower component (k = 6.9 s-1), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions. <![CDATA[<strong>Properties of voltage-gated Ca<sup>2+</sup> currents measured from mouse pancreatic </strong><strong>β</strong><strong>-cells in situ </strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300012&lng=es&nrm=iso&tlng=es We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic β-cells within intact mouse islets of Langerhans at 37C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I Cav), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of β-cell electrical activity. The temperature dependence of I Cav was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on β-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I Cav contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping β-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion. <![CDATA[Effect of the zinc chelator N,N,N',N'-tetrakis<b> <strong>(2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on </strong> <strong>synaptic transmission </strong></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300013&lng=es&nrm=iso&tlng=es An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butiric acid (GABA A) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 μM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 μM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP. <![CDATA[Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300014&lng=es&nrm=iso&tlng=es Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50%. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion. <![CDATA[<strong>Single-channel recording of inositol trisphosphate receptor in the isolated nucleus of a muscle cell line</strong>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300015&lng=es&nrm=iso&tlng=es Nuclear calcium appears to have an important role in the regulation of gene expression in many cells, but the mechanisms involved in controlling nuclear Ca2+ signaling are controversial and still poorly understood. We have described the presence of inositol 1,4,5 trisphosphate (IP3) receptors in the nuclei of skeletal muscle cells. Now, we have characterized the properties of the IP3 receptors channels present in the nuclei of the 1B5 cell line, which do not express any isoforms of the ryanodine receptor. Immunocytochemistry of isolated nuclei confirmed the presence of IP3R in the nuclear envelope and fluorescence measurements in nuclei suspensions allowed us to document ATP-dependent calcium loading by the nucleus and its release upon IP3 addition. Patch clamp of nuclear membranes was performed, and single-channel activity recorded was dependent on the presence of IP3 in the pipette; single-channel conductance was in the range reported in the literature for these channels, and the open probability was shown to be dependent on IP3 concentration. <![CDATA[<strong>Increased expression of SNARE proteins and</strong><b> <strong>synaptotagmin IV in islets from pregnant rats and</strong> <strong><i>in vitro</i> prolactin-treated neonatal islets </strong></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300016&lng=es&nrm=iso&tlng=es During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic β-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion. <![CDATA[Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000300017&lng=es&nrm=iso&tlng=es The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally.