AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.     

Estradiol Inhibits Depolarization-Evoked Exocytosis in PC12 Cells via N-Type Voltage-Gated Calcium Channels

Fast neuromodulatory effects of 17-β-estradiol (E2) on cytosolic calcium concentration ([Ca²⁺] _i ) have been reported in many cell types, but little is known about its direct effects on vesicular neurotransmitter secretion (exocytosis). We examined the effects of E2 on depolarization-evoked [Ca²⁺] _i in PC12 cells using fluorescence measurements. Imaging of [Ca²⁺] _i with FURA-2 revealed that depolarization-evoked calcium entry is inhibited after exposure to 10 nM and 10 μM E2. Calcium entry after exposure to 50 μM E2 decreases slightly, but insignificantly. To relate E2-induced changes in [Ca²⁺] _i to functional effects, we measured exocytosis using amperometry. It was observed that E2 in some cells elicits exocytosis upon exposure. In addition, E2 inhibits depolarization-evoked exocytosis with a complex concentration dependence, with inhibition at both physiological and pharmacological concentrations. This rapid inhibition amounts to 45% at a near physiological level (10 nM E2), and 50% at a possible pharmacological concentration of 50 μM. A small percentage (22%) of cells show exocytosis during E2 exposure (“Estrogen stimulated”), thus vesicle depletion could possibly account (at least partly) for the E2-induced inhibition of depolarization-evoked exocytosis. In cells that do not exhibit E2-stimulated release (“Estrogen quiet”), the E2-induced inhibition of exocytosis is abolished by a treatment that eliminates the contribution of N-type voltage-gated calcium channels (VGCCs) to exocytosis. Overall, the data suggest that E2 can act on N-type VGCCs to affect secretion of neurotransmitters. This provides an additional mechanism for the modulation of neuronal communication and plasticity by steroids.     

Multiple Cation Channels Mediate Increases in Intracellular Calcium Induced by the Volatile Irritant, Trans-2-Pentenal in Rat Trigeminal Neurons

Trans-2-Pentenal (pentenal), an α,β-unsaturated aldehyde, induces increases in [Ca²⁺]_i in cultured neonatal rat trigeminal ganglion (TG) neurons. Since all pentenal-sensitive neurons responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC) and neurons from TRPA1 knockouts failed to respond to pentenal, TRPA1 appears to be sole initial transduction site for pentenal-evoked trigeminal response, as reported for the structurally related irritant, acrolein. Furthermore, because the neuronal sensitivity to pentenal is strictly dependent upon the presence of extracellular Na⁺/Ca²⁺, as we showed previously, we investigated which types of voltage-gated sodium/calcium channels (VGSCs/VGCCs) are involved in pentenal-induced [Ca²⁺]_i increases as a downstream mechanisms. The application of tetrodotoxin (TTX) significantly suppressed the pentenal-induced increase in [Ca²⁺]_i in a portion of TG neurons, suggesting that TTX-sensitive (TTXs) VGSCs contribute to the pentenal response in those neurons. Diltiazem and ω-agatoxin IVA, antagonists of L- and P/Q-type VGCCs, respectively, both caused significant reductions of the pentenal-induced responses. ω-Conotoxin GVIA, on the other hand, caused only a small decrease in the size of pentenal-induced [Ca²⁺]_i rise. These indicate that both L- and P/Q-type VGCCs are involved in the increase in [Ca²⁺]_i produced by pentenal, while N-type calcium channels play only a minor role. This study demonstrates that TTXs VGSCs, L- and P/Q-type VGCCs play a significant role in the pentenal-induced trigeminal neuronal responses as downstream mechanisms following TRPA1 activation.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Differential Effects of Methionine and Cysteine Oxidation on [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Cultured Hippocampal Neurons

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca²⁺]_i in hippocampal neurons. We investigated the effects of H₂O₂ and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H₂O₂, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca²⁺]_i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H₂O₂ and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca²⁺]_i, but only partially reduced the effects of H₂O₂ and Ch-T on [Ca²⁺]_i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca²⁺]_i induced by H₂O₂ and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca²⁺]_i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.     

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca²⁺)-activated K⁺ (K_Ca) channels regulate membrane excitability and are activated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), leading to membrane hyperpolarization. Most patch clamp experiments that measure K_Ca currents use steady-state [Ca²⁺] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca²⁺]_i changes dynamically, for example during [Ca²⁺]_i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca²⁺]_i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K_Ca subtype in isolation were used to simultaneously measure agonist-evoked [Ca²⁺]_i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca²⁺]_i oscillations induced a corresponding K_Ca current that faithfully followed the [Ca²⁺]_i in 13–50% of cells, suggesting a good synchronization. However, [Ca²⁺]_i and K_Ca current was much less synchronized in 50–76% of cells that exhibited Ca²⁺-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca²⁺]_i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca²⁺]_i and K_Ca current was least synchronized, 36% of total [Ca²⁺]_i spikes occurred without activating a corresponding K_Ca current spike, suggesting that BK_Ca channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca²⁺]_i and K_Ca current suggests that this relationship is more complex than could be predicted from steady-state [Ca²⁺]_i and K_Ca current. These phenomena may be important for encoding stimulus–response coupling in various cell types.     

Pulsed electromagnetic field enhances brain-derived neurotrophic factor expression through L-type voltage-gated calcium channel- and Erk-dependent signaling pathways in neonatal rat dorsal root ganglion neurons

Although pulsed electromagnetic field (PEMF) exposure has been reported to promote neuronal differentiation, the mechanism is still unclear. Here, we aimed to examine the effects of PEMF exposure on brain-derived neurotrophic factor (Bdnf) mRNA expression and the correlation between the intracellular free calcium concentration ([Ca²⁺]_i) and Bdnf mRNA expression in cultured dorsal root ganglion neurons (DRGNs). Exposure to 50 Hz and 1 mT PEMF for 2 h increased the level of [Ca²⁺]_i and Bdnf mRNA expression, which was found to be mediated by increased [Ca²⁺]_i from Ca²⁺ influx through L-type voltage-gated calcium channels (VGCCs). However, calcium mobilization was not involved in the increased [Ca²⁺]_i and BDNF expression, indicating that calcium influx was one of the key factors responding to PEMF exposure. Moreover, PD098059, an extracellular signal-regulated kinase (Erk) inhibitor, strongly inhibited PEMF-dependant Erk1/2 activation and BDNF expression, indicating that Erk activation is required for PEMF-induced upregulation of BDNF expression. These findings indicated that PEMF exposure increased BDNF expression in DRGNs by activating Ca²⁺- and Erk-dependent signaling pathways.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Background

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca²⁺] ([Ca²⁺]_o) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca²⁺]_o sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.

Methodology/Principal Findings

By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca²⁺]_o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.

Conclusions/Significance

Our experiments identify CaSR as the first component in the [Ca²⁺]_o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca²⁺]_o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca²⁺]_o following its entry via voltage-activated Ca²⁺ channels. CaSR may detects such falls in [Ca²⁺]_o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca²⁺]_o on release probability. CaSR is positioned to detect the dynamic changes of [Ca²⁺]_o and provide presynaptic feedback that will alter brain excitability.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca²⁺]_i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca²⁺]_i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca²⁺]_i transient in osteoblasts. Fluorescent dyes DiBAC₄(3) and fura-2 AM were respectively used to detect membrane potential and [Ca²⁺]_i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca²⁺]_i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca²⁺]_i transients. Replacing extracellular Na⁺ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca²⁺]_i transients. However, voltage-activated K⁺ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca²⁺ or blocking of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca²⁺]_i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca²⁺ mobilization wherein FSS activated SACs to promote Na⁺ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca²⁺ influx though L-VSCCs switched on [Ca²⁺]_i response in osteoblasts.     

Calcium signal induced by mechanical perturbation of osteoclasts

Multinucleated osteoclasts from rabbit long bone, 1–6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca²⁺]_i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca²⁺]_i responses decreases with decreasing bathing [Ca²⁺]. Following mechanical perturbation, a thapsigargin-induced [Ca²⁺]_i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca²⁺]_i is larger and longer than in control cells. Ni²⁺ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd³⁺ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca²⁺]_i signal via both a calcium influx and an internal calcium release. © 1995 Wiley-Liss, Inc.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Ca2+-Induced Increases in Steady-State Concentrations of Intracellular Calcium Are Not Required for Inhibition of Parathyroid Hormone Secretion

It has been well established that increases in extracellular calcium concentration ([Ca²⁺]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca²⁺] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca²⁺] results in an increase in steady-state levels of intracellular calcium ([Ca²⁺]_i). At present, it has not been fully resolved whether changes in [Ca²⁺]_i are related to changes in PTH secretion. In the current study, the effect of increased [Ca²⁺] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca²⁺]_i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca²⁺] (≥0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca²⁺]_i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca²⁺]_i varied among individual cells (∼40% to >300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca²⁺]_i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca²⁺]_i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca²⁺]_i were not observed when [Ca²⁺] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca²⁺] was increased by gradual or rapid addition.     

Oscillatory Cl current induced by angiotensin II in rat pulmonary arterial myocytes: Ca dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca²⁺]_i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca²⁺]_i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (E_rev) of these currents was close to the equilibrium potential for Cl⁻ ions (E_Cl = −2.1 mV) and was shifted towards more positive values in low-Cl⁻ solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca²⁺]_i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca²⁺]_i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca²⁺]_i Niflumic acid (25 μM) had no effect on agonist-induced [Ca²⁺]_i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca²⁺]_i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca²⁺]_i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca²⁺]_i and that both oscillatory phenomena are primarily IP₃ mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca²⁺ channels. The resulting Ca²⁺ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Activation of Phosphatidylinositol-linked Novel D<Subscript>1</Subscript> Dopamine Receptor Contributes to the Calcium Mobilization in Cultured Rat Prefrontal Cortical Astrocytes

Recent evidences indicate the existence of an atypical D₁ dopamine receptor other than traditional D₁ dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase C_β (PLC_β). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D₁ dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca²⁺]_i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca²⁺]_i. Application of D₁ receptor, but not D₂, α₁ adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca²⁺]_i rise, indicating that activation of the D₁ dopamine receptor was essential for this response. Increase in [Ca²⁺]_i was a two-step process characterized by an initial increase in [Ca²⁺]_i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca²⁺ entry. Furthermore, SKF83959-stimulated increase in [Ca²⁺]_i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D₁ receptor by SKF83959 mediates a dose-dependent mobilization of [Ca²⁺]_i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.