Dihydropyridine-sensitive Ca2 Research Articles

Expression of Calcium Channel Subunit Variants in Small Mesenteric Arteries of WKY and SHR.

Enhanced function of dihydropyridine-sensitive Ca2+ channels (CaV) in hypertensive arterial myocytes (HAM) is well accepted. Increased protein expression of pore forming α1-subunits contributes to this effect, but cannot explain all of the differences in CaV properties in HAM. We hypothesized that differences in expression of CaV subunits and/or their splice variants also contribute. RNA, protein, and myocytes were isolated from small mesenteric arteries (SMA) of 20-week-old male WKY and SHR and analyzed by polymerase chain reaction (PCR), sequencing, immunoblotting, and patch clamp methods. Cav1.2 α1, β2c, and α2δ1d were the dominant subunits expressed in both WKY and SHR with a smaller amount of β3a. Real-time PCR indicated that the mRNA abundance of β3a and α2δ1 but not total Cav1.2 α1 or β2c were significantly larger in SHR. Analysis of alternative splicing of Cav1.2 α1 showed no differences in abundance of mutually exclusive exons1b, 8, 21 and 32 or alternative exons33 and 45. However, inclusion of exon9* was higher and a 73 nucleotide (nt) deletion in exon15 (exon15Δ73) was lower in SHR. Immunoblot analysis showed higher protein levels of Cav1.2 α1 (1.61±0.05), β3 (1.80±0.32), and α2δ1 (1.80±0.24) but not β2 in SHR. The lower abundance of exon15Δ73 transcripts in SHR results in a larger fraction of total Cav1.2 mRNA coding for full-length CaV protein, and the higher abundance of exon9* transcripts and CaVβ3a protein likely contribute to differences in gating and kinetics of CaV currents in SHR. Functional studies of Ca2+ currents in native SMA myocytes and HEK cells transiently transfected with CaV subunits support these conclusions.

Noradrenaline upregulates T-type calcium channels in rat pinealocytes.

The mammalian pineal gland is a neuroendocrine organ that responds to circadian and seasonal rhythms. Its major function is to secrete melatonin as a hormonal night signal in response to nocturnal delivery of noradrenaline from sympathetic neurons. Culturing rat pinealocytes in noradrenaline for 24 h induced a low-voltage activated transient Ca(2+) current whose pharmacology and kinetics corresponded to a CaV3.1 T-type channel. The upregulation of the T-type Ca(2+) current is initiated by β-adrenergic receptors, cyclic AMP and cyclic AMP-dependent protein kinase. Messenger RNA for CaV3.1 T-type channels is significantly elevated by noradrenaline at 8 h and 24 h. The noradrenaline-induced T-type channel mediated an increased Ca(2+) entry and supported modest transient electrical responses to depolarizing stimuli, revealing the potential for circadian regulation of pinealocyte electrical excitability and Ca(2+) signalling. Our basic hypothesis is that mammalian pinealocytes have cycling electrical excitability and Ca(2+) signalling that may contribute to the circadian rhythm of pineal melatonin secretion. This study asked whether the functional expression of voltage-gated Ca(2+) channels (CaV channels) in rat pinealocytes is changed by culturing them in noradrenaline (NA) as a surrogate for the night signal. Channel activity was assayed as ionic currents under patch clamp and as optical signals from a Ca(2+)-sensitive dye. Channel mRNAs were assayed by quantitative polymerase chain reaction. Cultured without NA, pinealocytes showed only non-inactivating L-type dihydropyridine-sensitive Ca(2+) current. After 24 h in NA, additional low-voltage activated transient Ca(2+) current developed whose pharmacology and kinetics corresponded to a T-type CaV3.1 channel. This change was initiated by β-adrenergic receptors, cyclic AMP and protein kinase A as revealed by pharmacological experiments. mRNA for CaV3.1 T-type channels became significantly elevated, but mRNA for another T-type channel and for the major L-type channel did not change. After only 8 h of NA treatment, the CaV3.1 mRNA was already elevated, but the transient Ca(2+) current was not. Even a 16 h wait without NA following the 8 h NA treatment induced little additional transient current. However, these cells were somehow primed to make transient current as a second NA exposure for only 60 min sufficed to induce large T-type currents. The NA-induced T-type channel mediated an increased Ca(2+) entry during short depolarizations and supported modest transient electrical responses to depolarizing stimuli. Such experiments reveal the potential for circadian regulation of excitability.

Detection of dihydropyridine- and voltage-sensitive intracellular Ca2+ signals in normal human parathyroid cells

We recently showed dihydropyridine- and voltage-sensitive Ca(2+) entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca(2+) entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca(2+) concentration ([Ca(2+)]I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca(2+) transient) following a 10-s exposure to a 150mM K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was reduced by dihydropyridine antagonists or 0.5mM Cd(2+), but enhanced by FPL-64176, an L-type Ca(2+)-channel agonist. Ca(2+) transients induced by the 10-s exposure to 3.0mM extracellular Ca(2+) ([Ca(2+)]o) were also inhibited by dihydropyridine antagonists or 0.5mM Cd(2+). These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca(2+) entry system that may be involved in the [Ca(2+)]o-induced change in [Ca(2+)]I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes

Cardiac transient receptor potential canonical (TRPC) channels are crucial upstream components of Ca(2+)/calcineurin/nuclear factor of activated T cells (NFAT) signaling, thereby controlling cardiac transcriptional programs. The linkage between TRPC-mediated Ca(2+) signals and NFAT activity is still incompletely understood. TRPC conductances may govern calcineurin activity and NFAT translocation by supplying Ca(2+) either directly through the TRPC pore into a regulatory microdomain or indirectly via promotion of voltage-dependent Ca(2+) entry. Here, we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca(2+) permeability but preserves monovalent permeation, abrogates agonist-induced NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to convert phospholipase C-linked stimuli into L-type (Ca(V)1.2) channel-mediated Ca(2+) entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca(2+) signal that is isolated from the NFAT pathway. Prevention of PKC-dependent modulation of TRPC3 by either inhibition of cellular kinase activity or mutation of a critical phosphorylation site in TRPC3 (T573A), which disrupts targeting of calcineurin into the channel complex, converts cardiac TRPC3-mediated Ca(2+) signaling into a transcriptionally silent mode. Thus, we demonstrate a dichotomy of TRPC-mediated Ca(2+) signaling in the heart constituting two distinct pathways that are differentially linked to gene transcription. Coupling of TRPC3 activity to NFAT translocation requires microdomain Ca(2+) signaling by PKC-modified TRPC3 complexes. Our results identify TRPC3 as a pivotal signaling gateway in Ca(2+)-dependent control of cardiac gene expression.

Flow regulation of endothelin‐1 production in the collecting duct

Collecting duct (CD) endothelin-1 (ET-1) inhibits Na reabsorption. Salt loading increases CD ET-1 production, however the responsible mechanisms are unknown. Tubule fluid flow increases in response to Na loading, hence we studied flow modulation of CD ET-1. A high salt diet in mice increased inner medullary CD (IMCD) ET-1 mRNA. Primary cultures of mouse IMCD detached in response to flow, consequently a CD cell line (mpkCCDcl4) was examined. Flow dose-dependently increased ET-1 mRNA between 2–10 dyne/cm2 and was first evident after 1 hr. Inhibition of calmodulin or dihydropyridine-sensitive Ca channels did not alter the flow response, however chelation of intracellular Ca prevented flow-stimulated ET-1 mRNA accumulation. Flow increased intracellular Ca concentration as assessed by Fluo-4 fluorescence. PKC inhibition markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 synthesis was partially dependent upon sodium delivery since hypertonic NaCl perfusate increased, while absence of perfusate Na (replacing NaCl with choline Cl) decreased, flow-stimulated ET-1 mRNA. Amiloride partially reduced the ET-1 response to flow. In summary, apical flow increases CD ET-1 biosynthesis through intracellular Ca and PKC, and partly through apical Na channel Na transport. These studies suggest a novel pathway for coupling extracellular fluid volume to CD Na reabsorption.

Flow regulation of collecting duct endothelin-1 production

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of CD Na(+) reabsorption. Salt loading is thought to increase CD ET-1 production; however, definitive evidence of this, as well as understanding of the mechanisms transducing this effect, is lacking. Tubule fluid flow increases in response to Na(+) loading; hence, we studied flow modulation of CD ET-1 production. Three days of a high-salt diet increased mouse and rat inner medullary CD (IMCD) ET-1 mRNA expression. Acute furosemide infusion increased urinary ET-1 excretion in anesthetized rats. Primary cultures of mouse or rat IMCD detached in response to flow using a closed perfusion chamber, consequently a CD cell line (mpkCCDcl4) was examined. Flow increased ET-1 mRNA at shear stress rates exceeding 1 dyne/cm(2), with the maximal effect seen between 2 and 10 dyne/cm(2). Induction of ET-1 mRNA was first evident after 1 h, and most apparent after 2 h, of flow. Inhibition of calmodulin or dihydropyridine-sensitive Ca(2+) channels did not alter the flow response; however, chelation of intracellular Ca(2+) or removal of extracellular Ca(2+) largely prevented flow-stimulated ET-1 mRNA accumulation. Downregulation of protein kinase C (PKC) using phorbol 12-myristate 13-acetate, or PKC inhibition with calphostin C, markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 mRNA accumulation was abolished by inhibition of phospholipase C (PLC). Taken together, these data indicate that flow increases CD ET-1 production and this is dependent on extracellular and intracellular Ca(2+), PKC, and PLC. These studies suggest a novel pathway for coupling alterations in extracellular fluid volume to CD ET-1 production and ultimately control of CD Na(+) reabsorption.

Modulation of Adrenergic Responses of Human Vas Deferens by K + Channel Inhibitors

The present study was designed to evaluate the role of K(+) channels in the adrenergic responses of human vas deferens as well as the intervention of dihydropyridine-sensitive Ca(2+) channels on modulation of adrenergic responses by K(+) channel inhibitors. Ring segments of the epididymal part of the vas deferens were taken from 32 elective vasectomies and mounted in organ baths for isometric recording of tension. We then studied the effects of K(+) channel blockers on neurogenic and norepinephrine-induced contractile responses. Addition of tetraethylammonium (TEA, 10(-3) M), a nonspecific K(+) channel blocker, or charybdotoxin (10(-7) M), a nonselective inhibitor of large- and intermediate-conductance Ca(2+)-activated K(+) channel, increased the contractile responses to norepinephrine and electrical field stimulation-induced contractions (P < .01), whereas iberiotoxin (10(-7) M), a selective blocker of large-conductance Ca(2+)-activated K(+) channels, apamin (10(-6) M), a blocker of small-conductance Ca(2+)-activated K(+) channels, or glibenclamide (10(-5) M), an inhibitor of ATP-sensitive K(+) channels, were without effect. TEA- and charybdotoxin-induced potentiation of contractions elicited by electrical field stimulation and norepinephrine was blocked by L-type Ca(2+) channel blocker nifedipine (10(-6) M). The results suggest that charybdotoxin-sensitive, but iberiotoxin-insensitive, K(+) channels are activated by stimulation with norepinephrine and electrical field stimulation to counteract the adrenergic-induced contractions of human vas deferens. Thus, inhibition of these channels increases significantly the contraction, an effect that appears to be mediated by an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels.

Interplay between Cytosolic Dopamine, Calcium, and α-Synuclein Causes Selective Death of Substantia Nigra Neurons

The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DA(cyt)) in cultured midbrain neurons, we confirm that elevated DA(cyt) and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DA(cyt) provide neuroprotection. L-DOPA increased DA(cyt) in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DA(cyt) was not altered by alpha-synuclein deletion, although dopaminergic neurons lacking alpha-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DA(cyt), and alpha-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+].

FGF-activated calcium channels control neural gene expression in Xenopus

In vertebrates, the formation of the nervous system starts at gastrulation with a process called neural induction. This process requires, at least in part, the inhibition of BMP signalling in the ectoderm by noggin, as well as FGF receptor activation and Ca 2+ signalling. Our studies with Xenopus embryos suggest that an increase in intracellular Ca 2+ concentration ([Ca 2+] i ), via dihydropyridine-sensitive Ca 2+ channels (DHP-sensitive Ca 2+ channels) is necessary and sufficient to direct the ectodermal cells toward a neural fate, and that Ca 2+ directly controls the expression of neural genes. The mechanism by which the DHP-sensitive Ca 2+ channels are activated during neural induction remains unknown. One possible mechanism is via the activation of FGF signalling. Using isolated ectoderm tissue, here we demonstrated that FGF-4 depolarises the membrane of ectodermal cells and induces an increase in [Ca 2+] i . This Ca 2+ increase can be blocked by SU5402, an FGF receptor inhibitor, and by DHP-sensitive Ca 2+ channel antagonists. These inhibitors also block the induction of neural genes. We discuss a possible gating mechanism for the activation of DHP-sensitive Ca 2+ channels via the FGF signalling pathway, which involves arachidonic acid and TRPC1 channel activation.

Dihydropyridine Receptor Blockade in the Treatment of Asthma

Asthma is a chronic airway disease resulting from inappropriate Th2-cell biased activation. Interleukin (IL)-4, IL-5 and IL-13 produced by Th2 cells contribute to the inflammatory process. Attempts for inhibiting interleukin-4 or IL-5 gave disappointing results. The simultaneous inhibition of several Th2-cytokines could be a more promising issue. Several arguments support the concept that Th2-cells express selective markers that could be targeted in asthma. Our group showed that Th2-cells selectively up-regulated dihydropyridine-sensitive Ca(2+) (DHP-Ca) channels essential for Ca(2+) signaling and type-2 cytokine production. Indeed, DHP-Ca antagonist effectively prevented or even reverted airway inflammation, airway remodeling and airway hyperresponsiveness in experimental models of asthma. Although it remains to be formally demonstrated that human lung infiltrating T-lymphocytes in asthmatic patients express DHP- Ca(2+) channels, we hypothesize that targeting DHP-Ca channels in T-lymphocytes could represent an efficient strategy in the treatment of asthma.This review article also discussed patents relevant to the field.

Indirect Activation of a Chloride Channel by a Trophic Ligand Is Required for Development of the Preimplantation Mouse Embryo.

Platelet activating factor (paf) is an autocrine survival factor that acts on the preimplantation embryo from the late zygote stage through to the end of the 2-cell stage. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient that recurs with a periodicity of approximately 60-90 min. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarization of the membrane potential in 2-cell embryos, accompanied by an increased outward ion current. Both the membrane hyperpolarization and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient. The aim of this study was to examine the role of the outward current in generating the transient hyperpolarization of the embryos plasma membrane, and to assess whether the current plays a role in normal mouse preimplantation development. We show that removal of extracellular anions prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient. This data suggests that the outward current resulted from activation of a Ca2+-activated Cl- channel. Niflumic acid (NFA, 100 μM), an inhibitor of Ca2+-activated Cl- channels, had no effect on the paf-induced [Ca2+]i, response but significantly reduced the amplitude of the hyperpolarization of membrane potential (p < 0.05) and the outward current (p < 0.01). In order to assess whether the outward Cl- current has a role in paf-induced embryo survival, the effect of NFA on embryo development was examined. The culture of embryos in medium containing 100 μM NFA from the 1-cell stage significantly reduced their development to the 4-cell stage (p < 0.05). By contrast, culture in medium containing NFA from the 8-cell stage had no effect on their development to the blastocyst stage. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. This channel is sensitive to NFA, and exposure of the zygote and 2-cell embryo to NFA blocked normal embryo development. Activation of a NFA-sensitive Cl- channel by a trophic ligand(s) during early development is required for the normal development of the mouse preimplantation embryo.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).

Calcium signalling during neural induction in Xenopus laevis embryos.

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.

257. Activation of a calcium-activated chloride channel by paf is required for normal preimplantation mouse embryo development in vitro

Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P &lt; 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development. (1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288. (2) Y. Li, M.L. Day &amp; C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.

Smooth muscle sparklet Cav channels defined: 1.2 is the number

Systemic blood pressure and tissue blood flow are regulated by the contractile status of smooth muscle cells located within the wall of small arteries and arterioles. One important signal that regulates arterial smooth muscle cell contractility is the intracellular calcium ion (Ca2+) concentration. Although Ca2+ was once considered to act only as a global intracellular signaling element, research performed over the past decade has revealed Ca2+ to act in a far more dynamic and versatile manner than previously thought possible.

Cav1.3 channels produce persistent calcium sparklets, but Cav1.2 channels are responsible for sparklets in mouse arterial smooth muscle

Ca(2+) sparklets are local elevations in intracellular Ca(2+) produced by the opening of a single or a cluster of L-type Ca(2+) channels. In arterial myocytes, Ca(2+) sparklets regulate local and global intracellular Ca(2+). At present, the molecular identity of the L-type Ca(2+) channels underlying Ca(2+) sparklets in these cells is undetermined. Here, we tested the hypotheses that voltage-gated calcium channel-alpha 1.3 subunit (Ca(v)1.3) can produce Ca(2+) sparklets and that Ca(v)1.2 and/or Ca(v)1.3 channels are responsible for Ca(2+) sparklets in mouse arterial myocytes. First, we investigated the functional properties of single Ca(v)1.3 channels in tsA201 cells. With 110 mM Ba(2+) as the charge carrier, Ca(v)1.3 channels had a conductance of 20 pS. This value is similar to that of Ca(v)1.2 and native L-type Ca(2+) channels. As previously shown for Ca(v)1.2 channels, Ca(v)1.3 channels can operate in two gating modes characterized by short and long open times. Expressed Ca(v)1.3 channels also produced Ca(2+) sparklets. Ca(v)1.3 sparklets had properties similar to those produced by Ca(v)1.2 and native L-type channels, including quantal amplitude, dihydropyridine sensitivity, bimodal gating, and dual-event duration times. However, the voltage dependencies of conductance and steady-state inactivation of the Ca(2+) current (I(Ca)) in arterial myocytes were similar to those recorded from cells expressing Ca(v)1.2 but not Ca(v)1.3 channels. Furthermore, nifedipine (10 microM) eliminated Ca(2+) sparklets in wild-type myocytes but not in myocytes expressing dihydropyridine-insensitive Ca(v)1.2 channels. Accordingly, Ca(v)1.3 transcript and protein were not detected in isolated arterial myocytes. We conclude that although Ca(v)1.3 channels can produce Ca(2+) sparklets, Ca(v)1.2 channels underlie I(Ca), Ca(2+) sparklets, and hence dihydropyridine-sensitive Ca(2+) influx in mouse arterial myocytes.

Characterization of Voltage-Gated Ionic Channels in Cholinergic Amacrine Cells in the Mouse Retina

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.