A Novel Prototype Biosensor Array Electrode System for Detecting the Bacterial Pathogen Salmonella typhimurium.

Salmonellosis caused by Salmonella sp. has long been reported all over the world. Despite the availability of various diagnostic methods, easy and effective detection systems are still required. This report describes a dialysis membrane electrode interface disc with immobilized specific antibodies to capture antigenic Salmonella cells. The interaction of a specific Salmonella antigen with a mouse anti-Salmonella monoclonal antibody complexed to rabbit anti-mouse secondary antibody conjugated with HRP and the substrate o-aminophenol resulted in a response signal output current measured using two electrode systems (cadmium reference electrode and glassy carbon working electrode) and an agilent HP34401A 6.5 digital multimeter without a potentiostat or applied potential input. A maximum response signal output current was recorded for various concentrations of Salmonella viz., 3, 30, 300, 3000, 30,000 and 300,000 cells. The biosensor has a detection limit of three cells, which is very sensitive when compared with other detection sensors. Little non-specific response was observed using Streptococcus, Vibrio, and Pseudomonas sp. The maximum response signal output current for a dialysis membrane electrode interface disc was greater than that for gelatin, collagen, and agarose. The device and technique have a range of biological applications. This novel detection system has great potential for future development and application in surveillance for microbial pathogens.

Bio-Fabrication of Human Amniotic Membrane Zinc Oxide Nanoparticles and the Wet/Dry HAM Dressing Membrane for Wound Healing.

The preparation of unique wet and dry wound dressing products derived from unprocessed human amniotic membrane (UP-HAM) is described. The UP-HAM was decellularized, and the constituent proteins were cross-linked and stabilized before being trimmed and packed in sterile Nucril-coated laminated aluminium foil pouches with isopropyl alcohol to manufacture processed wet human amniotic membrane (PW-HAM). The dry type of PD-HAM was prepared by decellularizing the membrane, UV irradiating it, lyophilizing/freeze-drying it, sterilizing it, and storing it at room temperature. The UP-HAM consists of a translucent yellowish mass of flexible membranes with an average thickness of 42 µm. PW-HAM wound dressings that had been processed, decellularized, and dehydrated had a thinner average thickness of 30 µm and lacked nuclear-cellular structures. Following successful decellularization, discrete bundle of fibrous components in the stromal spongy layers, microvilli and reticular ridges were still evident on the surface of the processed HAM, possibly representing the location of the cells that had been removed by the decellularization process. Both wet and dry HAM wound dressings are durable, portable, have a shelf life of 3-5 years, and are available all year. A slice of HAM dressing costs 1.0 US$/cm2. Automation and large-scale HAM membrane preparation, as well as storage and transportation of the dressings, can all help to establish advanced technologies, improve the efficiency of membrane production, and reduce costs. Successful treatment of wounds to the cornea of the eye was achieved with the application of the HAM wound dressings. The HAM protein analysis revealed 360 µg proteins per gram of tissue, divided into three main fractions with MWs of 100 kDa, 70 kDa, and 14 kDa, as well as seven minor proteins, with the 14 kDa protein displaying antibacterial properties against human pathogenic bacteria. A wide range of antibacterial activity was observed after treatment with 75 µg/ml zinc oxide nanoparticles derived from human amniotic membrane proteins (HAMP-ZnO NP), including dose-dependent biofilm inhibition and inhibition of Gram-positive (S. aureus, S. mutans, E. faecalis, and L. fusiformis) and Gram-negative bacteria (S. sonnei, P. aeruginosa, P. vulgaris, and C. freundii).

When day meets night: Subsiding calcium signalling translates daylight into new neurones.

Day/night cycle controls neurogenesis; melatonin released from pineal gland in darkness stimulates intracellular Ca2+ dynamics thus decreasing proliferation of neural stem cells. In the daylight intracellular Ca2+ activity subsides, which stimulates neural stem cells division and increases generation of newborn neurones.

Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs.

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

Vasopressin and oxytocin in sensory neurones: expression, exocytotic release and regulation by lactation.

The neurohormones arginine-vasopressin (AVP) and oxytocin (OT) synthesised in supraoptic and paraventricular nuclei of neurohypophysis regulate lactation, systemic water homeostasis and nociception. Using transgenic rats expressing AVP and OT tagged with fluorescent proteins we demonstrate that both neurohormones are expressed in sensory neurones both in vitro, in primary cultures, and in situ, in the intact ganglia; this expression was further confirmed with immunocytochemistry. Both neurohormones were expressed in nociceptive neurones immunopositive to transient receptor potential vannilloid 1 (TRPV1) channel antibodies. The AVP and OT-expressing DRG neurones responded to AVP, OT, 50 mM K+ and capsaicin with [Ca2+]i transients; responses to AVP and OT were specifically blocked by the antagonists of V1 AVP and OT receptors. Probing the extracellular incubation saline with ELISA revealed AVP and OT secretion from isolated DRGs; this secretion was inhibited by tetanus toxin (TeNT) indicating the role for vesicular release. Expression of OT, but not AVP in DRG neurones significantly increased during lactation. Together, the results indicate novel physiological roles (possibly related to nociception and mood regulation) of AVP and OT in the sensory neurones.

Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells.

Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca(2+) signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca(2+) channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca(2+) stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca(2+)]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca(2+)]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca(2+)]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from -40 to -50mV in cells in basal to -80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K(+)-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium formulation is associated with significant changes in their Ca(2+) signaling and membrane properties.

Sodium-calcium exchanger and R-type Ca(2+) channels mediate spontaneous [Ca(2+)]i oscillations in magnocellular neurones of the rat supraoptic nucleus.

Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes.

Physiology of spontaneous [Ca(2+)]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus.

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca(2+) signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca(2+)]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca(2+)]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca(2+)]i dynamics. In rats dehydrated for 3 or 5days almost 90% of neurones displayed spontaneous [Ca(2+)]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca(2+) signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

Physiology of Ca(2+) signalling in stem cells of different origins and differentiation stages.

Stem cells (SCs) of different origins have brought hope as potential tools for the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Calcium signalling plays a key role in SC differentiation and proliferation, and dysregulation of Ca(2+) homeostasis may instigate pathological scenarios. Currently, the role of ion channels and receptors in SCs is not fully understood. In the recent years, we found that (i) the pre-differentiation of human embryonic SCs (hESCs) led to the activation of Ca(2+) signalling cascades and enhanced the functional activities of these cells, (ii) the Ca(2+) homeostasis and the physiological properties of hESC-derived neural precursors (NPs) changed during long term propagation in vitro, (iii) differentiation of NPs derived from human induced pluripotent SCs affects the expression of ion channels and receptors, (iv) these neuronal precursors exhibited spontaneous activity, indicating that their electrophysiological and Ca(2+) handling properties are similar to those of mature neurones, and (v) in mesenchymal SCs isolated from the adipose tissue and bone marrow of rats the expression profile of ion channels and receptors depends not only on the differentiation conditions but also on the source from which the cells were isolated, indicating that the fate and functional properties of the differentiated cells are driven by intrinsic mechanisms. Together, identification and assignment of a unique ion channel and a Ca(2+) handling footprint for each cell type would be necessary to qualify them as physiologically suitable for medical research, drug screening, and cell therapy.

Human Multipotent Mesenchymal Stromal Cells in the Treatment of Postoperative Temporal Bone Defect: An Animal Model.

Canal wall down mastoidectomy is one of the most effective treatments for cholesteatoma. However, it results in anatomical changes in the external and middle ear with a negative impact on the patient's quality of life. To provide complete closure of the mastoid cavity and normalize the anatomy of the middle and external ear, we used human multipotent mesenchymal stromal cells (hMSCs), GMP grade, in a guinea pig model. A method for preparing a biomaterial composed of hMSCs, hydroxyapatite, and tissue glue was developed. Animals from the treated group were implanted with biomaterial composed of hydroxyapatite and hMSCs, while animals in the control group received hydroxyapatite alone. When compared to controls, the group implanted with hMSCs showed a significantly higher ratio of new bone formation (p = 0.00174), as well as a significantly higher volume percentage of new immature bone (p = 0.00166). Our results proved a beneficial effect of hMSCs on temporal bone formation and provided a promising tool to improve the quality of life of patients after canal wall down mastoidectomy by hMSC implantation.

Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.

Primary cilia act as physical-chemical sensors and their functions include the perception of the extracellular milieu, regulation of organogenesis, and cell polarity. In general, these cells are monociliated and the single cilium possesses diverse receptors and channels which are involved in morphogenesis and growth signaling, and are, therefore, important for cell proliferation and differentiation. In this study, we used an in vitro model of C2C12 myoblasts to evaluate the effect of DNA damage induced by gamma ionizing radiation on primary cilia incidence. A significantly higher number of ciliated cells were observed after 1 day post-irradiation with 2-20 Gy when compared with non-irradiated cells. After 3 days post-irradiation, the cilia incidence in cells had decreased slightly when treated with 2, 6, and 10 Gy, although an increase in incidence rate was observed in cells treated with 20 Gy. Multi-ciliated cells were also detected in myoblasts irradiated with 10 and 20 Gy but not in non-irradiated cells or after low irradiation (2-6 Gy). Irradiation also caused a dose-dependent decrease in cell viability and proliferation and corresponding cell cycle arrest. Furthermore, an activation of caspases 3/7, 8, and 9 was observed after higher radiation (10 and 20 Gy) with increased apoptosis. Together, our results show that irradiation by gamma rays promotes myoblast ciliogenesis, with pronounced effects observed after 3 days post-irradiation. We conclude that irradiation doses of 10 and 20 Gy are sufficient to induce cell death and are responsible for the formation of multiple cilia originating from multiple basal bodies.

Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nucleus.

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 µM) and the TRPV1 antagonists, capsazepine (10 µM) and BCTC (10 µM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

Getting it right before transplantation: example of a stem cell model with regenerative potential for the CNS.

The burden of neurodegenerative disorders in an aging population has become a challenge for the modern world. While the biomarkers available and the methods of diagnosis have improved to detect the onset of these diseases at early stages, the question of adapted and efficient therapies is still a major issue. The prospect of replacing the loss of functional neural cells remains an attractive but still audacious approach. A huge progress has been made in the generation of neurons derived from human stem cell lines and transplantation assays are tested in animals for a wide range of pathologies of the central nervous system. Here we take one step back and examine neuronal differentiation and the characterization of neural progenitors derived from human embryonic stem cells. We gather results from our previous studies and present a cell model that was successfully used in functional analyses and engraftment experiments. These neuronal precursors exhibit spontaneous and evoked activity, indicating that their electrophysiological and calcium handling properties are similar to those of matured neurons. Hence this summarized information will serve as a basis to design better stem cell-based therapies to improve neural regeneration.

The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice.

Bone marrow-derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP(+)) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP(+) lin(-) Sca-1(+) cells into non-haematopoietic tissues. The transplantation of BM cells or GFP(+) lin(-) Sca-1(+) cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP(+) cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP(+) lin(-) Sca-1(+) cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.

INTRODUCTION: The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues. METHODS: Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity. RESULTS: We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca(2+) channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity. CONCLUSIONS: We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]i) evoked by high K(+), adenosine-5'-triphosphate (ATP), glutamate, γ-aminobutyric acid (GABA), and caffeine in correlation with the expression of various neuronal markers in different passages (P6 through P10) during the course of hESC differentiation. We found that only differentiated NPs from P7 exhibited significant and specific [Ca(2+)]i responses to various stimuli. About 31% of neuronal-like P7 NPs exhibited spontaneous [Ca(2+)]i oscillations. Pharmacological and immunocytochemical assays revealed that P7 NPs express L- and P/Q-type Ca(2+) channels, P2X2, P2X3, P2X7, and P2Y purinoreceptors, glutamate receptors, and ryanodine (RyR1 and RyR3) receptors. The ATP- and glutamate-induced [Ca(2+)]i responses were concentration-dependent. Higher glutamate concentrations (over 100 µM) caused cell death. Responses to ATP were observed in the presence or in the absence of extracellular Ca(2+). These results emphasize the notion that with time in culture, these cells attain a transient period of operative Ca(2+) signaling that is predictive of their ability to act as stem elements.

Vasopressin-induced intracellular Ca²âº concentration responses in non-neuronal cells of the rat dorsal root ganglion.

Arginine-vasopressin (AVP) is a nonapeptide of hypothalamic origin that has been shown to exert many important cognitive and physiological functions in neurons and terminals of both the central and peripheral nervous system (CNS and PNS). Here we report for the first time that AVP induced an increase in intracellular Ca²âº concentration ([Ca²âº](i)) in non-neuronal cells isolated from the rat dorsal root ganglion (DRG) and cultured in vitro. The ratiometric [Ca²âº](i) measurements showed that AVP evoked [Ca²âº](i) responses in the non-neuronal cells and these concentration-dependent (100 pM to 1 µM) responses increased with days in vitro in culture, reaching a maximum amplitude after 4-5 day. Immunostaining by anti-S-100 antibody revealed that more than 70% of S-100 positive cells were AVP-responsive, indicating that glial cells responded to AVP and increased their [Ca²âº](i). The responses were inhibited by depletion of the intracellular Ca²âº stores or in the presence of inhibitors of phospholipase C, indicating a metabotropic response involving inositol trisphosphate, and were mediated by the V1 subclass of AVP receptors, as evidenced by the use of the specific blockers for V1 and OT receptors, (d(CH2)5¹,Tyr(Me)²,Arg8)-Vasopressin and (d(CH2)5¹,Tyr(Me)²,Thr4,Orn8,des-Gly-NH29)-Vasotocin, respectively. V(1a) but not V(1b) receptor mRNA was expressed sustainably through the culture period in cultured DRG cells. These results suggest that AVP modulates the activity of DRG glial cells via activation of V(1a) receptor.

Neuroendocrine signalling: natural variations on a Ca2+ theme.

This special issue on Ca(2+) signalling in neuroendocrine cells is an opportunity to assess, through a range of first-class review articles, the complex world of endocrine signalling, a complexity that is probably best captured by calling it "diversity in unity". The unity comes from the fact that all the endocrine cells are excitable cells, able to generate action potentials and are using Ca(2+) as an essential informational molecule, coupling cell stimulation with the activation of secretion, through the exocytotic process. The 'diversity' element, illustrated by almost all the reviews, stems from the modalities employed to achieve the increase in cytosolic Ca(2+) signal, the balance between the participation of Ca(2+) entry through the plasma membrane voltage-operated Ca(2+) channels and the release of Ca(2+) from intracellular Ca(2+) stores, and the cross-talk between the Ca(2+) and cyclic AMP signalling pathways.