Glutamate‐induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium‐dependent pathways and the microtubule cytoskeleton

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium‐dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate‐induced changes in the pattern of dendrite outgrowth. We used Fura‐2 AM and inhibitors of the calcium‐dependent proteins, calmodulin and calpain, to identify the role of specific calcium‐dependent pathways in glutamate‐regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate‐induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca²⁺ ]_i) changes in a biphasic manner over a 12‐h period in the presence of glutamate. A transient increase in [Ca²⁺ ]_i over the first hour of glutamate exposure correlated with a calmodulin‐associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca²⁺ ]_i was correlated with calpain‐associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin‐associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain‐associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity‐dependent regulation of dendrite morphology during development and after pathology. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 159–172, 2000     

Modulation of Ca2+ Influx in Leech Retzius Neurons II. Effect of Extracellular Ca2+

We investigated the cytosolic free calcium concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular Ca²⁺ concentration via the bathing solution ([Ca²⁺]_B). Changing [Ca²⁺]_B had only an effect on [Ca²⁺]_i if the cells were depolarized by raising the extracellular K⁺ concentration. Surprisingly, raising [Ca²⁺]_B from 2 to 10 mm caused a decrease in [Ca²⁺]_i, and an increase was evoked by reducing [Ca²⁺]_B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca²⁺]_B moderate membrane depolarizations were sufficient to evoke a [Ca²⁺]_i increase, while progressively larger depolarizations were necessary at higher [Ca²⁺]_B. The changes in the relationship between [Ca²⁺]_i and membrane potential upon varying [Ca²⁺]_B could be reversed by changing extracellular pH. We conclude that [Ca²⁺]_B affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels via the electrochemical Ca²⁺ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca²⁺ concentration enhances the electrochemical Ca²⁺ gradient and hence Ca²⁺ influx, but it attenuates Ca²⁺ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa. Received: 23 January 2001/Revised: 23 June 2001     

Potentiation of Carbachol-Induced Ca2+ Release by Peroxynitrite in Human Neuroblastoma SH-SY5Y Cells

We have investigated the effect of 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, on carbachol-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human neuroblastoma SH-SY5Y cells by means of single cell imaging of [Ca²⁺]_i. SIN-1 potentiated carbachol-induced [Ca²⁺]_i rise regardless of external Ca²⁺, and the potentiation was completely inhibited by superoxide dismutase, indicating that peroxynitrite may enhance Ca²⁺ release from intracellular stores. On the other hand, SIN-1 reduced carbachol-induced inositol 1,4,5-trisphosphate (IP₃) formation. Genistein, a tyrosine kinase inhibitor, potentiated carbachol-induced rise of [Ca²⁺]_i regardless of external Ca²⁺. These results suggest that peroxynitrite may potentiate the release of Ca²⁺ from intracellular stores through the perturbation of regulation in tyrosine phosphorylation-dephosphorylation system.     

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca²⁺]_i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca²⁺]_i change with initial transient phase followed by a plateau phase with highly synchronized [Ca²⁺]_i oscillations. Here, we aimed to correlate the plateau [Ca²⁺]_i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca²⁺]_i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca²⁺]_i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca²⁺]_i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca²⁺]_i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca²⁺]_i. However, the mechanism of silica- induced cytotoxicity relative to [Ca²⁺]_i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca²⁺]_i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca²⁺]_i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca²⁺]_o markedly inhibited the [Ca²⁺]_i rise (≈90%), suggesting that Ca²⁺ influx from extracellular source is a major mechanism for silica-induced [Ca²⁺]_i rise. When used at low doses but sufficient to cause a transient [Ca²⁺]_i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca²⁺]_o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca²⁺ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca²⁺ influx is inhibited by the Ca²⁺ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca²⁺ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca²⁺]_i rise was not due to the impairment of Ca²⁺-pumps, which restricts Ca²⁺ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca²⁺]_i rise. Taken together, these results suggest that the elevation of [Ca²⁺]_i caused by silica is due mainly to Ca²⁺ influx through plasma membrane Ca²⁺ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca²⁺ leakage during phagocytosis was attributed to the silica-induced [Ca²⁺]_i rise. © 1993 Wiley-Liss, Inc.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

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.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Modulation of Ca2+ Influx in Leech Retzius Neurons. I. Effect of Extracellular pH

We investigated the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca²⁺]_i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K⁺ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca²⁺]_i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca²⁺]_i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca²⁺]_i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca²⁺]_i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Appearance of Depolarization- and Maitotoxin-Induced [Ca2+]i Elevation in Single LAN-1 Human Neuroblastoma Cells on Exposure to Retinoic Acid

Abstract: LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca²⁺]_i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca²⁺]_i elevation elicited by 55 mM K⁺. Maitotoxin, a putative activator of voltage-sensitive Ca²⁺ channels, did not evoke an elevation of [Ca²⁺]_i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca²⁺]_i when superfused in retinoic acid-treated cells. Both high K⁺- and maitotoxin-induced [Ca²⁺]_i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd³⁺, an inorganic Ca²⁺-entry blocker, and by the snail toxin ω-conotoxin GVIA, which interacts with the N sub-type of voltage-sensitive Ca²⁺ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel sub-type, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca²⁺]_i due to Ca²⁺ influx elicited by membrane depolarization. K⁺-induced [Ca²⁺]_i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K⁺-induced increase of [Ca²⁺]_i was still present. In conclusion, the results of the present study demonstrated that retinoic acid-induced differentiation of LAN-1 cells, which lack a high K⁺-evoked [Ca²⁺]_i increase in the undifferentiated state, induces the functional expression of an ω-conotoxin GVIA-sensitive, dihydropyridine-insensitive N-type voltage-sensitive Ca²⁺ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.     

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.     

Caffeine-Induced Ca2+ Transients and Exocytosis in Paramecium Cells. A Correlated Ca2+ Imaging and Quenched-Flow/Freeze-Fracture Analysis

Caffeine causes a [Ca²⁺] _i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca²⁺]^rest _i ∼50 to 80 nm, [Ca²⁺]^act _i rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca²⁺ spread during the subsequent ≥2 sec. Chelation of Ca²⁺ _o considerably attenuated [Ca²⁺] _i increase. Therefore, caffeine may primarily mobilize cortical Ca²⁺ pools, superimposed by Ca²⁺ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca²⁺-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca²⁺] _i signals were reduced by [Ca²⁺] _o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca²⁺] _i signal was generated by caffeine stimulation (with Ca²⁺ _i and Ca²⁺ _o available). We conclude the following. (i) Caffeine can mobilize Ca²⁺ from cortical stores independent of the presence of Ca²⁺ _o . (ii) To yield adequate signals for normal exocytosis, Ca²⁺ release and Ca²⁺ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca²⁺] _i increase entails caffeine-mediated access of Ca²⁺ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca²⁺] _i -values for membrane fusion and contents discharge. Received: 23 May 1997/Revised: 18 August 1997     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.