Free Extracellular Medium Research Articles

Short-term mechanisms activated by the nerve growth factor NGF to induce pulmonary arterial hyperreactivity

We have previously shown a pathophysiological role for NGF in pulmonary hypertension, particularly in pulmonary arterial (PA) hyperreactivity. Our experiments have suggested different mechanisms activated by NGF depending on a short–or a long-term PA treatment. The aim of the present study was to determine the mechanisms activated after a NGF short–term PA treatment (1 h) leading to PA hyperreactivity. Basal and agonist-induced calcium responses were evaluated in vitro in primary rat PA smooth muscle cells (rPASMC) using calcium imaging (Fura-2, Fluo-4), in the absence or presence of NGF (100 ng/ml, 1 h). Calcium sources involved in NGF-increased calcium responses were determined using endoplasmic reticulum calcium stores depleters (thapsigargin, 1 μM or cyclopiazonic acid, 10 μM), or a calcium–free extracellular medium. NGF–induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was evaluated in vitro by Western blotting in primary human PA smooth muscle cells (hPASMC). Contractions of rat PA were induced ex vivo by endothelin-1 (ET-1, 10 -12 –10 -6 M) or phenylephrine (PHE, 10 -10 –10 -6 M) in the absence or presence of NGF (100 ng/ml, 1 h), with or without K252a (TrkA kinase inhibitor, 300 nM) or Y–27632 (Rho-associated protein kinase -ROCK-inhibitor, 10 μM). In vitro, NGF short-term treatment increases both basal and agonist-induced intracellular calcium responses in rPASMC, with endoplasmic reticulum being the main calcium source contributing to these effects. In addition, short–term treatment with NGF induced MYPT1 phosphorylation in hPASMC, involving a TrkA/ROCK-dependent pathway. Ex vivo, short-term treatment with NGF increased rat PA reactivity to PHE and ET–1. This effect was totally abolished after treatment with K252a or Y-27632. In conclusion, our results show that NGF can induce PA hyperreactivity through activation of its TrkA receptor, leading to both modulation of calcium intracellular responses and activation of ROCK-dependent calcium sensitization mechanisms. Such mechanisms may therefore contribute to NGF-dependent PA hyperreactivity in pulmonary hypertension.

Probenecid affects sarcoplasmic reticulum Ca2+ release and depresses contractile activation in mouse skeletal muscle

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation-contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

Neural depolarization triggers Mg2+ influx in rat hippocampal neurons

Homeostasis of magnesium ion (Mg2+) plays key roles in healthy neuronal functions, and deficiency of Mg2+ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg2+ concentration ([Mg2+]i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg2+]i. The aim of this study is to explore the direct relationship between neural activity and [Mg2+]i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg2+]i and [Ca2+]i dynamics were simultaneously visualized with a highly selective fluorescent Mg2+ probe, KMG-104, and a fluorescent Ca2+ probe, Fura Red, respectively. [Mg2+]i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg2+]i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K+ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca2+ increase. Furthermore, the [Mg2+]i increase was abolished in Mg2+-free extracellular medium, indicating [Mg2+]i increase is due to Mg2+ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na+ channel opener, induced [Mg2+]i increase, and this [Mg2+]i increase was suppressed by the pretreatment of a non-specific Mg2+ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg2+]i increase results from Mg2+ influx through 2-APB-sensitive channels in rat hippocampal neurons.

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the Human Umbilical Artery: An <i>in Vitro</i> Study

The vasoactive effects of oxidative stress induced by hydrogen peroxide (H2O2) on human umbilical artery strips as well as the possible mechanisms involved are studied. Contraction responses to cumulative H2O2 (10–7 M-3 × 10–2 M) in endothelium intact and denuded umbilical arteries and responses to cumulative H2O2 after incubation with L-NAME (10–4 M) (n = 8), indomethacin (10–5 M) (n = 8) and verapamil (10–6) (n = 8) were recorded. Responses elicited with cumulative H2O2 in Ca2+ free extracellular medium and the responses to cumulative Ca2+ (10–4 M-2 × 10–3 M) after H2O2 (10–3 M) induced contraction were also studied. The Emax for each experiment was calculated. p 2O2 elicited contraction was greater in endothelium denuded artery strips compared to endothelium intact strips (p 2O2 (p 2+ free extracellular medium caused decreases in cumulative H2O2 elicited contractions and cumulative Ca2+ caused concentration dependent increases in the contraction caused by a single bolus of H2O2 (p 2O2 causes concentration-dependent constriction in human umbilical arteries. The presence of the endothelium and NOS enzyme activation influences the H2O2 responses. Removal of the endothelium increases the H2O2 elicited contractions more than incubation with L-NAME suggesting beside NO, other endothelial vasodilators are also involved in vascular tonus of the umbilical arteries. Both intracellular and extracellular Ca2+ ions and constrictor cyclooxygenase metabolites play a role in the contractile responses elicited by H2O2 in human umbilical arteries.

Involvement of Tetrodotoxin-Resistant Na+ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100 nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

Acetylcholine increases Ca 2+ influx by activation of CaMKII in mouse oocytes

IP 3-induced Ca 2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca 2+ oscillation. However, other mechanisms remain to explain intracellular Ca 2+ elevation. We here report that ACh induces Ca 2+ influx via T-type Ca 2+ channel by activation of Ca 2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca 2+ influx facilitates the generation of Ca 2+ oscillation in the mouse ovulated oocytes (oocytes MII). ACh increased Ca 2+ current by 50 ± 21%, and produced Ca 2+ oscillation. However, the currents and Ca 2+ peaks were reduced in Ca 2+-free extracellular medium. ACh failed to activate Ca 2+ current and to produce Ca 2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca 2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.

A constitutively active nonselective cation conductance underlies resting Ca 2+ influx and secretion in bovine adrenal chromaffin cells

We have combined fluorimetric measurements of the intracellular free Ca 2+ concentration ([Ca 2+] i) with the patch clamp technique, to investigate resting Ca 2+ entry in bovine adrenal chromaffin cells. Perfusion with nominally Ca 2+-free medium resulted in a rapid, reversible decrease in [Ca 2+] i, indicating a resting Ca 2+ permeability across the plasma membrane. Simultaneous whole-cell voltage-clamp showed a resting inward current that increased when extracellular Ca 2+ (Ca 2+ o) was lowered. This current had a reversal potential of around 0 mV and was carried by monovalent or divalent cations. In Na +-free extracellular medium there was a reduction in current amplitude upon removal of Ca 2+ o, indicating the current can carry Ca 2+. The current was constitutively active and not enhanced by agents that promote Ca 2+-store depletion such as thapsigargin. Extracellular La 3+ abolished the resting current, reduced resting [Ca 2+] i and inhibited basal secretion. Abolishment of resting Ca 2+ influx depleted the inositol 1,4,5-trisphosphate-sensitive Ca 2+ store without affecting the caffeine-sensitive Ca 2+ store. The results indicate the presence of a constitutively active nonselective cation conductance, permeable to both monovalent and divalent cations, that can regulate [Ca 2+] i, the repletion state of the intracellular Ca 2+ store and the secretory response in resting cells.

Caged Vanilloid Ligands for Activation of TRPV1 Receptors by 1- and 2-Photon Excitation

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: Involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca 2+] i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca 2+] i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y 2 receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca 2+-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y 2 receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.

Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.

Capacitative calcium entry in testosterone-induced intracellular calcium oscillations in myotubes

Ca2+ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca2+ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca2+ entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations in a majority of myotubes. Spectral analysis of the Ca2+ oscillations revealed a periodicity of 20.3 +/- 1.8 s (frequency of 49.3 +/- 4.4 mHz). In Ca(2+)-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. This intracellular Ca2+ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP3). Intracellular Ca2+ store depletion in Ca(2+)-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca2+, gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca(2+)-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La3+, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by either La3+ or cytochalasin D. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE.

Calcium channels activated by endothelin-1 in human trophoblast.

Ca2+ transfer across the syncytiotrophoblast (ST) of the human placenta is essential for normal fetal development. However, the nature of Ca2+ conductance in the ST and the mechanisms by which it is regulated are poorly understood. With the major signal transduction pathway of endothelin-1 (ET1) acting via phospholipase C (PLC) and Ca2+, we used ET1 to analyse the nature of Ca2+ channels on cultured trophoblastic cells by means of cytofluorimetric analysis using the ratiometric Ca2+ indicator Indo-1. Results indicate that ET1 (10(-7) M) stimulates a biphasic (transient and sustained) increase in [Ca2+]i in trophoblastic cells. This response is mediated by the endothelin receptor B (ETB) coupled to PLC, since treatment with BQ788 (10(-6) M) or U73122 (2 microM) totally abolished the response. Persistence of the rapid transient rise in [Ca2+]i in Ca2+-free extracellular medium confirms the release of Ca2+ from intracellular stores in response to ET1 stimulation. Furthermore, abolition of the sustained increase in [Ca2+]i in Ca2+-free extracellular medium argues in favour of the entry of Ca2+ during the plateau phase. Abolition of this plateau phase by Ni2+ (1 mM) in the presence of extracellular Ca2+ confirmed the existence of an ET1-induced Ca2+ entry. No evidence for the presence of voltage-operated channels was demonstrated during ET1 action since nifedipine (10(-6) M) did not reduce the Ca2+ response and depolarization with a hyper-potassium solution had no effect. Pharmacological studies using the imidazole derivatives SK&F96365 (30 microM) and LOE 908 (10 microM) partially inhibited the ET1-evoked Ca2+ response, thus providing evidence for the presence of both store-operated Ca2+ channels and non-selective cationic channels in the human ST.

Na +/Mg 2+ transporter acts as a Mg 2+ buffering mechanism in PC12 cells

Mg 2+ buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na +/Mg 2+ transporter by using a newly developed Mg 2+ indicator, KMG-20, and also a Na + indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg 2+ concentration ([Mg 2+] i). The rate of decrease of [Mg 2+] i was slower in a Na +-free extracellular medium, suggesting the coupling of Na + influx and Mg 2+ efflux. Na + influxes were different for normal and imipramine- (a putative inhibitor of the Na +/Mg 2+ transporter) containing solutions. FCCP induced a rapid increase in [Na +] i in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg 2+] i in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg 2+ depends on the Na +/Mg 2+ transporter in PC12 cells.

Low-concentration lidocaine rapidly inhibits axonal transport in cultured mouse dorsal root ganglion neurons.

Axonal transport plays a critical role in supplying materials for a variety of neuronal functions such as morphogenetic plasticity, synaptic transmission, and cell survival. In the current study, the authors investigated the effects of the analgesic agent lidocaine on axonal transport in neurites of cultured mouse dorsal root ganglion neurons. In relation to their effects, the effects of lidocaine on the growth rate of the neurite were also examined. Isolated mouse dorsal root ganglion cells were cultured for 48 h until full growth of neurites. Video-enhanced microscopy was used to observe particles transported within neurites and to measure the neurite growth during control conditions and in the presence of lidocaine. Application of 30 microM lidocaine immediately reduced the number of particles transported in anterograde and retrograde axonal directions. These effects were persistently observed during the application (26 min) and were reversed by lidocaine washout. The inhibitory effect was dose-dependent at concentrations from 0.1 to 1,000 microM (IC50 = 10 microM). In Ca2+-free extracellular medium, lidocaine failed to inhibit axonal transport. Calcium ionophore A23187 (0.1 microM) reduced axonal transport in both directions. The inhibitory effects of lidocaine and A23187 were abrogated by 10 microM KN-62, a Ca2+-calmodulin-dependent protein kinase II inhibitor. Application of such low-concentration lidocaine (30 microM) for 30 min reduced the growth rate of neurites, and this effect was also blocked by KN-62. Low-concentration lidocaine rapidly inhibits axonal transport and neurite growth via activation of calmodulin-dependent protein kinase II.

Inhibition of Neutrophil Spreading during Adhesion to Fibronectin Reveals Formation of Long Tubulovesicular Cell Extensions (Cytonemes)

Human neutrophils developed long thin tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated substrata, when spreading was blocked. We observed extension formation when neutrophils were plated to fibronectin-coated substrata in Na+-free extracellular medium or in the presence of drugs capable of inhibiting spreading: 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na+ ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and scanning electron microscopy revealed two types of extensions: (1) highly dynamic, flexible tubulovesicular extensions with unattached tips 0.2–0.4 μm in diameter, which can achieve 70–80 μm in length during 20 min, and (2) thinner straight extensions with flattened tips, which were formed in the presence of phorbol 12-myristate 13-acetate and connected cells to substratum or to the neighboring cells several cell diameters away. The latter may have derived from the former through tension after attachment of the tips. Spreading and extension formation may represent two states of the cell adhesive and communicative mechanism.

Fresh and nonfibrillar amyloid beta protein(1-40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AbetaP-channel-mediated cellular toxicity.

Alzheimer's disease (AD) is primarily nonfamilial or sporadic (SAD) in origin, although several genetic linkages are reported. Tissues from AD patients contain fibrillar plaques made of 39 to 43 amino acid-long amyloid beta peptide (AbetaP), although the mechanisms of AbetaP toxicity are poorly understood. AbetaP(1-40) is the most prevalent AbetaP present in the neuronal and non-neuronal tissues from SAD patients. AbetaP(1-40) toxicity has been examined mainly after prolonged incubation and correlates with the age and fibrillar morphology of AbetaP(1-40). Globular and nonfibrillar AbetaPs are released continually during normal cellular metabolism; they elevate cellular Ca(2+) and form cation-permeable channels. However, their role in cellular toxicity is poorly understood. We have used an integrated atomic force and light fluorescence microscopy (AFM-LFM), laser confocal microscopy, and calcium imaging to examine real-time and acute effect of fresh and globular AbetaP(1-40) on cultured, aged human, AD-free fibroblasts. AFM images show that freshly prepared AbetaP(1-40) in phosphate-buffered saline (PBS) are globular and do not form fiber for an extended time period. AbetaP(1-40) induced rapid structural modifications, including cytoskeletal reorganization, retraction of cellular processes, and loss of cell-cell contacts, within minutes of incubation. This led to eventual cellular degeneration. AbetaP(1-40)-induced degeneration was prevented by anti-AbetaP antibody, zinc, and Tris, but not by tachykinin neuropeptides. In Ca(2+)-free extracellular medium, AbetaP(1-40) did not induce cellular degeneration. In the presence of extracellular Ca(2+), AbetaP(1-40) induced a sustained increase in the cellular Ca(2+). Thus, short-term and acute AbetaP(1-40) toxicity is mediated by Ca(2+) uptake, most likely via calcium-permeable AbetaP pores. Such rapid degeneration does not require fibrillar plaques, suggesting that the plaques may not have any causative role.

Lysophosphatidic acid-mediated Ca2+ mobilization in human SH-SY5Y neuroblastoma cells is independent of phosphoinositide signalling, but dependent on sphingosine kinase activation

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0. 1 and 1 microM, at which point [Ca(2+)](i) was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca(2+) from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca(2+) uptake, and by the persistence of their action in nominally Ca(2+)-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca(2+) entry. MCh produced a marked elevation in cellular Ins(1,4,5)P(3) and stimulated [(3)H]InsP accumulation in the presence of Li(+). In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P(3) receptor (InsP(3)R) proteins with MCh did not affect Ca(2+) responses to LPA. In addition, heparin, a competitive antagonist of InsP(3)Rs, blocked Ca(2+)-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P(3), but failed to inhibit Ca(2+)-release induced by LPA. Elevation of [Ca(2+)](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca(2+)](i) responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca(2+), N,N-dimethylsphingosine (DMS, 30 microM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca(2+) responses by almost 90%. In addition, MCh-induced Ca(2+) responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca(2+)-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P(3). Furthermore, the Ca(2+)-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.

17β-Oestradiol increases intracellular Ca2+ concentration in rat enterocytes

The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17β-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 µM) suppressed the stimulatory effect of 0.1 nM 17β-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17β-oestradiol effect at 5 min. 17β-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17β-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17β-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 µM La3+, whereas nitrendipine (1 µM) and verapamil (10 µM) separately were without effect. The permeability of the 17β-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17β-oestradiol regulation of enterocyte extracellular Ca2+ influx.

17β-Oestradiol increases intracellular Ca2+ concentration in rat enterocytes

The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17beta-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 microM) suppressed the stimulatory effect of 0.1 nM 17beta-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17beta-oestradiol effect at 5 min. 17beta-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17beta-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17beta-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 microM La3+, whereas nitrendipine (1 microM) and verapamil (10 microM) separately were without effect. The permeability of the 17beta-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17beta-oestradiol regulation of enterocyte extracellular Ca2+ influx.

Extracellular potassium rapidly inhibits axonal transport of particles in cultured mouse dorsal root ganglion neurites.

Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.