Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store.

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts.

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.

Electrophysiological characterization of ion channels in osteoclasts isolated from human deciduous teeth.

Ion channels contribute to several important processes in osteoclasts, including proton transport and volume regulation. Although ion channels have been described in osteoclasts from several species, little is known about their properties in human osteoclasts. We devised a method for isolation of authentic human osteoclasts from deciduous teeth undergoing root resorption, and characterized currents in these cells using patch-clamp techniques. Three types of K(+) current were identified. Hyperpolarization elicited an inwardly rectifying K(+) current in most osteoclasts, which was inhibited by Ba(2+) in a voltage- and time-dependent manner. Depolarization elicited an outwardly rectifying and tetraethylammonium-sensitive current, consistent with a large-conductance Ca(2+)-dependent K(+) channel. In addition to these basal currents, extracellular adenosine 5'-triphosphate (ATP) elicited a linear current that was identified as a Ca(2+)-dependent K(+) current, based on its reversal potential close to that predicted for K(+), its blockade by quinine, and its activation by Ca(2+) ionophore. Last, an outwardly rectifying current was observed to activate spontaneously or in response to ATP, with properties of a swelling-activated Cl(-) current. This current reversed direction close to the Cl(-) equilibrium potential and was blocked by the anion channel blocker, niflumic acid, identifying it as a Cl(-) current. In summary, we have developed a novel method for isolation of authentic human osteoclasts and have characterized K(+) and Cl(-) currents. Cl(-) current mediates charge compensation during electrogenic H(+) transport, so activation of Cl(-) current may contribute to the stimulatory effects of extracellular ATP on bone resorption.

Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms.

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated 45Ca2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC50 of 19 microM. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 microM) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.

Mapping of IP(3)-mediated Ca(2+) signals in single human neuroblastoma SH-SY5Y cells: cell volume shaping the Ca(2+) signal.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP(3)-mediated Ca(2+) release signals in human SH-SY5Y neuroblastoma cells. [Ca(2+)](i) increases were not affected by ryanodine (30 microgM) or caffeine (10 mM) and largely insensitive to removal of external Ca(2+), indicating predominance of IP(3)-induced Ca(2+) release. Ca(2+) signals evoked by high concentration (10 microM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca(2+) changes in most cells displayed striking spatiotemporal heterogeneity. The Ca(2+) response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca(2+) changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca(2+) release signals.

P2X(4) purinoceptors mediate an ATP-activated, non-selective cation current in rabbit osteoclasts.

Extracellular nucleotides act as signaling molecules in numerous tissues. In bone, nucleotides stimulate osteoclast formation and activity; however, the receptors and signaling mechanisms underlying these effects have yet to be identified. To identify specific P2X purinoceptor subtypes in osteoclasts, degenerate oligonucleotide primers were used to PCR-amplify DNA fragments from a rabbit osteoclast cDNA library. A 372-base-pair fragment was obtained that encoded an amino acid sequence with 88% identity to the rat P2X(4) purinoceptor. The presence of P2X(4) mRNA in purified osteoclasts was confirmed by reverse transcription-PCR. Endogenous purinoceptors were functionally characterized in isolated rabbit osteoclasts by patch-clamp recording in whole-cell configuration. At negative membrane potentials, application of ATP or ADP rapidly activated an inward current followed by an outward current. In contrast, UTP or ADPbetaS elicited only an outward current, due to activation of a Ca(2+)-dependent K(+) conductance. The initial inward current was non-selective for cations and inactivated during agonist application. Furthermore, the inward current was insensitive to suramin and Cibacron blue, and was potentiated by Zn(2+). These characteristics are consistent with properties of P2X(4) purinoceptors. Activation of P2X(4) purinoceptors leads to cation influx and depolarization. Nucleotides, released at sites of trauma or inflammation, may act through these receptors on osteoclasts to stimulate bone resorption.

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.

Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca(2+) pumps.

Xestospongins, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are potent blockers of the inositol 1,4,5-trisphosphate (IP(3))-induced Ca2+ release in bi-directional Ca2+-flux conditions. We have now studied the effects of xestospongin C on the (45)Ca2+ uptake and the uni-directional (45)Ca2+ efflux in permeabilized A7r5 smooth-muscle cells. Xestospongin C not only inhibits the IP(3)-induced Ca2+ release, but is also an equally potent blocker of the endoplasmic-reticulum Ca2+ pump, while it has no effect on the passive Ca2+ leak. The inhibition of the IP(3) receptor did not depend on the IP(3), Ca2+ or ATP concentration. Xestospongin C can, therefore, not be considered as a selective blocker of IP(3) receptors.

Extracellular nucleotides activate non-selective cation and Ca(2+)-dependent K+ channels in rat osteoclasts.

1. Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 microM) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3. The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 +/- 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to -21 +/- 3 mV, indicating that ATP activates a non-selective cation current, consistent with involvement of P2X receptors. 4. The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I-V relationship, and reversed at -71 +/- 4 mV. The reversal potential shifted 51 mV per 10-fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5. In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca(2+)-free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br-A23187) to elevate [Ca2+]i, IK activated when [Ca2+]i exceeded approximately 400 nM, with half-maximal activation at 580 +/- 50 nM. 6. In cell-attached patches, ATP activated a channel with a conductance of 48 +/- 6 pS, that reversed director, near EK. Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7. These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non-selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca(2+)-dependent K+ channel.

Tetrodotoxin-sensitive fast Na+ current in embryonic chicken osteoclasts.

A voltage-dependent, fast, transient inward current was characterized in embryonic chicken osteoclasts using the permeabilized patch configuration of the patch-clamp technique. The current was activated by depolarizations to higher than -28 +/- 4 mV from a holding potential of -80 mV. It peaked within 1-1.5 ms, and inactivated within 3.3-6.9 ms. The 50% inactivation voltage was -59 +/- 6 mV with a steepness factor of 0.11 +/- 0.06. The current disappeared with the removal of extracellular Na+ and was reversibly blocked by tetrodotoxin (K0.5 < 15 nM) but not by verapamil (< or = 100 microM). We conclude that this new current in embryonic chicken osteoclasts is a sodium current known from excitable cells.

Permeabilization of cells of hemopoietic origin by extracellular ATP4-: elimination of osteoclasts, macrophages, and their precursors from isolated bone cell populations and fetal bone rudiments.

Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5' triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.

Differential depolarization-activated calcium responses in fetal and neonatal rat osteoblast-like cells.

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

A Ca(2+)-dependent K(+)-channel in freshly isolated and cultured chick osteoclasts.

Calcium-activated potassium channels were found in embryonic chick osteoclasts using the patch-clamp technique. The activity of the channel was increased by both membrane depolarisation and an increase in intracellular Ca2+ concentration in the range 10(-5) to 10(-3) M. In the cell-attached-patch configuration the channel was only active at extreme depolarising potentials. Ca2+ addition to the cytoplasm via ionomycin increased channel activity at the resting membrane potential of the osteoclast. The channel had a single-channel conductance of 150 pS in the inside-out patch under symmetrical K+ conditions (150 mM) and was selective for potassium ions. During sustained application of increased [Ca2+] at the cytoplasmic side of inside-out patches, channel activity sometimes decreased again after the initial increases (desensitization). The results established the properties of the single channels underlying an outward rectifying K+ conductance in chick osteoclasts described previously by us.

Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch-activated channels in OCL locomotion.

No evidence for effects of mild microwave irradiation on electrophysiological and morphological properties of cultured embryonic rat dorsal root ganglion cells.

Effects of mild microwave treatment (1 hr, 37 degrees C) on the in vitro development of rat mechanically dissociated dorsal root ganglion (DRG) neurons were investigated to establish whether microwave irradiation effects exist on nervous tissue other than heat induced tissue fixation. Phase contrast microscopy and immunocytochemical neurofilament stainings did not reveal significant differences between irradiated (2 hr after isolation) and control cultures, maintained up till 21 days. The electrophysiological properties of microwave exposed and non-exposed DRG neurons were compared using the whole-cell patch-clamp technique. Control neurons, in culture for 0-12 days, were excitable. In cultured cells (1-12 days), microwaved 2 hr after isolation, the action potentials were similar to or slightly different from those of the control cells. No acute microwave effects were found on neurons irradiated after 1 day of culture. These results suggest that mild microwave irradiation has neither significant acute nor strong long-term effects on DRG culture development and DRG neuron membrane properties, consistent with the notion that microwave effects essentially are temperature effects.