Expression of bone-related proteins in vascular calcification and its serum correlations with coronary artery calcification score.

Since vascular calcification is considered a process regulated similar to that of bone tissue mineralization, we investigated the participation of bone formation proteins. We analyzed the correlation of serum circulating bone markers, osteoprotegerin (OPG) and receptor activator of nuclear factor ĸB ligand (RANKL) in chronic kidney disease (CKD) patients, to coronary artery calcification score. We also considered the effect of inorganic phosphate on pro- and anti-calcifying tissue factors. We confirmed that circulating OPG is an independent calcium score predictor with its high serum concentration favoring high coronary artery calcification. In tissue samples of non-diseased human renal arteries, the expression of OPG and receptor activator of nuclear factor ĸB (RANK) was positive, while expression of RANKL was absent. In atherosclerotic specimens and arteries with medial calcification, the most upregulated was expression of bone morphogenetic proteins, BMP-2 and BMP-7, as well as expression of RANK and RANKL. In the diseased arteries, OPG expression was present only in areas where bone structures were formed. In atherosclerotic and medial calcification arteries, loss of alpha-smooth muscle actin (α-SMA) expression was observed. These data suggest a possible regulatory role of the examined proteins, especially OPG and RANKL, in vascular calcification, as well as their possible clinical significance as circulating predictors of vascular calcification.

SFPQ associates to LSD1 and regulates the migration of newborn pyramidal neurons in the developing cerebral cortex.

The development of the cerebral cortex requires the coordination of multiple processes ranging from the proliferation of progenitors to the migration and establishment of connectivity of the newborn neurons. Epigenetic regulation carried out by the COREST/LSD1 complex has been identified as a mechanism that regulates the development of pyramidal neurons of the cerebral cortex. We now identify the association of the multifunctional RNA-binding protein SFPQ to LSD1 during the development of the cerebral cortex. In vivo reduction of SFPQ dosage by in utero electroporation of a shRNA results in impaired radial migration of newborn pyramidal neurons, in a similar way to that observed when COREST or LSD1 expressions are decreased. Diminished SFPQ expression also associates to decreased proliferation of progenitor cells, while it does not affect the acquisition of neuronal fate. These results are compatible with the idea that SFPQ, plays an important role regulating proliferation and migration during the development of the cerebral cortex.

BDNF and schizophrenia: from neurodevelopment to neuronal plasticity, learning, and memory.

Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that has been related not only to neurodevelopment and neuroprotection, but also to synapse regulation, learning, and memory. Research focused on the neurobiology of schizophrenia has emphasized the relevance of neurodevelopmental and neurotoxicity-related elements in the pathogenesis of this disease. Research focused on the clinical features of schizophrenia in the past decades has emphasized the relevance of cognitive deficits of this illness, considered a core manifestation and an important predictor for functional outcome. Variations in neurotrophins such as BDNF may have a role as part of the molecular mechanisms underlying these processes, from the neurodevelopmental alterations to the molecular mechanisms of cognitive dysfunction in schizophrenia patients.

Calcium mediates dorsoventral patterning of mesoderm in Xenopus.

Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel.

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

Modulation of the kinetics of inositol 1,4,5-trisphosphate-induced [Ca2+]i oscillations by calcium entry in pituitary gonadotrophs.

Inositol 1,4,5-trisphosphate (InsP3) binds to its receptor channels and causes liberation of Ca2+ from intracellular stores, frequently in an oscillatory manner. In addition to InsP3, the activation and inactivation properties of these intracellular channels are controlled by Ca2+. We studied the influence of Ca2+ entry on the kinetics of InsP3-triggered oscillations in cytosolic calcium ([Ca2+]i) in gonadotrophs stimulated with gonadotropin-releasing hormone, an agonist that activates InsP3 production. The natural expression of voltage-gated Ca2+ channels (VGCC) in these cells was employed to manipulate Ca2+ entry by voltage clamping the cells at different membrane potentials (Vm). Under physiological conditions, the frequency of the GnRH-induced oscillations increased with time, while the amplitude decreased, until both reached stable values. However, in cells with Vm held at -50 mV or lower, both parameters progressively decreased until the signal was abolished. These effects were reverted by a depolarization of the membrane positive to -45 mV in both agonist- and InsP3-stimulated gonadotrophs. Depolarization also led to an increase in the fraction of time during which the [Ca2+]i remained elevated; this effect originated from both an increase in the mean duration of spikes and a decrease in the interval between spikes. The frequency and amplitude of spiking depended on the activity of VGCC, but displayed different temporal courses and voltage relationships. The depolarization-driven recovery of the frequency was instantaneous, whereas the recovery of the amplitude of spiking was more gradual. The midpoints of the Vm sensitivity curve for amplitude and duration of spiking (-15 mV) were close to the value observed for L-type Ca2+ current and for depolarization-induced increase in [Ca2+]i, whereas this parameter was much lower (-35 mV) for interval between spikes and frequency of oscillations. These observations are compatible with at least two distinct effects of Ca2+ entry on the sustained [Ca2+]i oscillations. Calcium influx facilitates its liberation from intracellular stores by a direct and instantaneous action on the release mechanism. It also magnifies the Ca2+ signal and decreases the frequency because of its gradual effect on the reloading of intracellular stores.

Molecular determinants of ion conduction and inactivation in K+ channels.

K+ channel-forming proteins can be grouped into three families that differ by the number of potential membrane-spanning segments. The largest of these families is composed of tetrameric channels with subunits containing six putative membrane-spanning segments (S1-S6). Inward rectifiers comprise a second family of K+ channels with subunits having two transmembrane domains (M1, M2). Monomers in the third family are proteins containing only one membrane-spanning segment, and they give origin to minK+ channels. Joining together segments S5 and S6 in the case of voltage-gated K+ channels and M1 and M2 in inward rectifiers, there is a highly conserved region with a hairpin shape called the H5 or P region. The P region, the loop connecting the S4 and S5 domains and the S6 transmembrane segment in Shaker-type K+ channels and the COOH-terminal in inward rectifiers, appears to play crucial roles in ion conduction. In Shaker K+ channels the NH2-terminal has been identified as responsible for fast inactivation (N-type inactivation). If the fast-inactivation gate is removed, a slower inactivation process persists, and its rate can be altered by mutations of amino acid residues forming part of the region in the neighborhood of the COOH-terminal (C-type inactivation). In this review we discuss the strategies followed to identify the different structures of K+ channels involved in ion conduction and inactivation processes and how they interplay.

Control of calcium spiking frequency in pituitary gonadotrophs by a single-pool cytoplasmic oscillator.

The mechanisms by which the generation and frequency of cytoplasmic Ca2+ oscillations are controlled were investigated in pituitary gonadotrophs. In these cells, two Ca(2+)-mobilizing receptors, the gonadotropin-releasing hormone and endothelin receptors, induce frequency-modulated Ca2+ spiking at the rate of up to 30 min-1. The cytoplasmic oscillator is also activated by discharge of luminal Ca2+ (initiated by ionomycin, thapsigargin, or thimerosal) but not by increased voltage-sensitive Ca2+ influx or treatment with caffeine. The basic difference between these two types of Ca2+ oscillations is related to their requirement for inositol-1,4,5-triphosphate (InsP3). Thapsigargin-, thimerosal-, and ionomycin-induced spiking occurs without the rise in InsP3 production that is essential for the generation of receptor-controlled oscillatory responses. The differential requirement for InsP3 in the two types of Ca2+ spiking is indicated by two lines of evidence. First, agonist-induced Ca2+ spiking of frequency similar to that of non-receptor-mediated oscillations was accompanied by a significant increase in InsP3, whereas none of the non-receptor-mediated oscillations was associated with measurable changes in inositol phosphate production. Second, agonist-induced InsP3 formation and Ca2+ spiking were abolished by treatment with the phospholipase C inhibitors U73122 and neomycin sulfate, whereas non-receptor-mediated Ca2+ spiking was not affected by these agents. When the oscillator was activated by agents that do not increase InsP3 formation, it operated only at the basal rate of approximately 5 min-1 and spiking frequency did not rise with increasing drug concentrations, in contrast to the situation in agonist-stimulated gonadotrophs. However, both types of oscillations were affected by depletion of luminal Ca2+ and by changes in the intracellular Ca2+ concentration ([Ca2+]i) but were not inhibited by ryanodine. These findings are consistent with the operation of a single-pool Ca2+ oscillator that is responsible for generation of both types of Ca2+ oscillations. The oscillator is controlled by the coagonist actions of InsP3 and Ca2+ on the InsP3 receptor channels and by the activation of Ca(2+)-ATPase by rising [Ca2+]i. It can be induced to operate at low frequency without an increase in InsP3 production by agents that reduce intraluminal [Ca2+]i, and it exhibits a dose-dependent increase in spiking frequency during agonist stimulation.

Membrane potential regulates inositol 1,4,5-trisphosphate-controlled cytoplasmic Ca2+ oscillations in pituitary gonadotrophs.

The influence of membrane potential (Vm) on cytoplasmic calcium ([Ca2+]i) oscillations during the sustained extracellular Ca(2+)-dependent phase of the Ca2+ signaling response to gonadotropin-releasing hormone (GnRH) was analyzed in cultured pituitary gonadotrophs. In agonist- and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-stimulated cells, sustained [Ca2+]i oscillations were extinguished by hyperpolarization after 3-15 min despite the availability of Ca2+ in the extracellular medium. Single depolarizing pulses transiently restored the amplitude of the sustained spiking in a dihydropyridine- and extracellular Ca(2+)-sensitive manner. The responses to depolarization showed a marked dependence on Vm that was correlated with the steady-state inward Ca2+ current. In addition, repetitive application of brief depolarizing pulses modulated the frequency of agonist- and Ins(1,4,5)P3-controlled spiking; depolarization pulses at frequencies lower than the intrinsic rate of episodic Ca2+ release triggered large transients between the autonomous spikes, whereas higher frequencies of depolarizing pulses overcame the original Ca2+ spiking frequency. These extrinsically driven and extracellular Ca(2+)-dependent oscillations were sensitive to the Ca(2+)-ATPase blocker, thapsigargin, but not to ryanodine. On the other hand, spontaneous firing and application of depolarizing pulses to nonstimulated cells failed to induce thapsigargin-sensitive oscillations. These findings demonstrate that the pattern of Ca2+ signaling in gonadotrophs does not depend exclusively on the Ins(1,4,5)P3 concentration, but also on the excitable status of the cell. Such modulation of the Ins(1,4,5)P3-controlled Ca2+ signaling system by changes in Vm could provide a mechanism for the integration of multiple inputs that utilize diverse signal transduction pathways.

Mechanism of agonist-induced [Ca2+]i oscillations in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH) activates oscillatory Ca2+ signaling in pituitary gonadotrophs at a frequency (up to 25 min-1) that is dose-dependent and is determined by the degree of receptor-mediated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation. Similar dose-dependent and frequency-modulated Ca2+ oscillations were elicited by intracellular administration of Ins(1,4,5)P3 and its nonhydrolyzable analogs, consistent with models in which Ins(1,4,5)P3 levels determine the frequency of Ca2+ oscillations but do not fluctuate in synchrony with [Ca2+]i. At constant agonist concentrations, Ca2+ spiking varied in amplitude, with a number of progressively larger transients before the onset of maximal oscillations, followed by a gradual decrease in spike amplitude that was accompanied by an increase in spiking frequency. The decline in the amplitude and increase in frequency of Ca2+ transients during stimulation by GnRH were not related to a decrease in the propagation of the Ca2+ signal within the cell but were associated with gradual depletion of the agonist-sensitive Ca2+ pool. Once initiated, the pattern of Ca2+ spiking was not altered by blockade of receptor occupancy, by inhibition of phospholipase C, or by reduction of extracellular [Ca2+]. Also, the endoplasmic reticulum (Ca2+)-ATPase blocker, thapsigargin, could substitute for Ins(1,4,5)P3 in initiating the oscillatory Ca2+ response. These findings indicate that although the Ins(1,4,5)P3 concentration determines the pattern of transients at the initiation of the oscillatory Ca2+ signal, maintenance of the signal does not require a sustained rise in Ins(1,4,5)P3. Since the frequency of Ca2+ oscillations is also influenced by depletion of luminal [Ca2+], it is possible that the Ins(1,4,5)P3-sensitive channels in the endoplasmic reticulum are tonically inhibited by high intraluminal Ca2+ levels and that Ins(1,4,5)P3 surmounts such inhibition by promoting Ca2+ discharge. When a critical level of Ca2+ discharge is attained, repetitive Ca2+ transients are generated by an autocatalytic mechanism in which a sustained rise in Ins(1,4,5)P3 is not an essential requirement.

Integration of cytoplasmic calcium and membrane potential oscillations maintains calcium signaling in pituitary gonadotrophs.

Pituitary gonadotrophs exhibit spontaneous low-amplitude fluctuations in cytoplasmic calcium concentration ([Ca2+]i) due to intermittent firing of nifedipine-sensitive action potentials. The hypothalamic neuropeptide, gonadotropin-releasing hormone, terminates such spontaneous [Ca2+]i transients and plasma-membrane electrical activity and initiates high-amplitude [Ca2+]i oscillations and concomitant oscillations in membrane potential (Vm). The onset of agonist-induced [Ca2+]i oscillations is not dependent on Vm or extracellular Ca2+ but is associated with plasma-membrane hyperpolarization interrupted by regular waves of depolarization with firing of action potentials at the peak of each wave. The Vm and Ca2+ oscillations are interdependent during continued gonadotropin-releasing hormone action (greater than 3-5 min), when sustained Ca2+ entry is necessary for the maintenance of [Ca2+]i spiking. The initial and sustained agonist-induced Ca2+ transients and Vm oscillations are abolished by blockade of endoplasmic reticulum Ca(2+)-ATPase, consistent with the role of Ca2+ re-uptake by internal stores in the oscillatory response during both phases. Such a pattern of synchronization of electrical activity and Ca2+ spiking in cells regulated by Ca(2+)-mobilizing receptors shows that the operation of the cytoplasmic oscillator can be integrated with a plasma-membrane oscillator to provide a long-lasting signal during sustained agonist stimulation.

Apamin-sensitive potassium channels mediate agonist-induced oscillations of membrane potential in pituitary gonadotrophs.

In cultured rat pituitary gonadotrophs, gonadotropin-releasing hormone (GnRH) induces rapid hyperpolarization of the cell membrane and causes cessation of the spontaneous electrical activity present in non-stimulated cells. This initial response to GnRH is followed by slow oscillations of membrane potential (Vm) which often exhibit brief bursts of action potentials (AP) fired from the peak of the oscillations. The hyperpolarization waves are synchronous with GnRH-induced elevations of cytoplasmic Ca2+ concentration ([Ca2+]i), such that Vm maxima alternate with the peak values of [Ca2+]i. The Vm oscillations result from repetitive activation of apamin-sensitive K+ channels by cytoplasmic Ca2+. Thus, GnRH activation of Ca2+ mobilization can generate a bursting pattern of membrane potential through the activation of K+ channels against a background of spontaneous electrical activity.

Charybdotoxin-sensitive K(Ca) channel is not involved in glucose-induced electrical activity in pancreatic beta-cells.

The effects of charybdotoxin (CTX) on single [Ca2+]-activated potassium channel (K(Ca)) activity and whole-cell K+ currents were examined in rat and mouse pancreatic beta-cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured beta-cells and beta-cells in intact islets were compared. K(Ca) activity was very infrequent at negative patch potentials (-70 less than Vm less than 0 mV), channel activity appearing at highly depolarized Vm. K(Ca) open probability at these depolarized Vm values was insensitive to glucose (10 and 20 mM) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K+ channels of smaller conductance. The venom from Leiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K(Ca) channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18 nM) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K+ currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20 nM). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet beta-cells in the presence of glucose were not affected by CTX. We conclude that the K(Ca) does not participate in the steady-state glucose-induced electrical activity in rodent pancreatic islets.

Characterization of potassium channels in pancreatic beta cells from ob/ob mice.

The patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic beta cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal beta cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent beta cells. Thus, potassium channels in ob/ob pancreatic beta cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.