Calcium signalling in glial cells

Various electrical, mechanical, and chemical stimuli, including the influences of neurotrasmitters, neuromodulators, and hormones, trigger complex changes in [Ca²⁺] _i in all types of glial cells. Glial [Ca²⁺] _i responses are controlled by coordinated activity of several molecular cascades. The initiation of [Ca²⁺] _i signal in glial cells results from activation of either plasmalemmal, or intracellular Ca²⁺ permeable channels. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide the means for intracellular and intercellular information coding. Furthermore, glial [Ca²⁺] _i signals can travel with no decrement within glial networks. These intercellular Ca²⁺ waves can be regarded as a substrate for information exchange between the glial cells. Neuronal activity can trigger [Ca²⁺] _i signals in neighboring glial cells and, moreover, there is some evidence that glial [Ca²⁺] _i waves can activate neuronal electrical and/or [Ca²⁺] _i , responses. Glial Ca²⁺ signalling can be regarded as a form of glial excitability.     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

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-.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Relationship between Extra- and Intracellular Calcium in Distal Segments of the Renal Tubule. Role of the Ca2+ Receptor RaKCaR

The effect of extracellular calcium ([Ca²⁺] _e ) on cytosolic calcium ([Ca²⁺] _i ) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In cortical collecting ducts, basolateral but not apical changes in [Ca²⁺] _e were associated with parallel changes in [Ca²⁺] _i . Basal [Ca²⁺] _i was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La³⁺. Increasing peritubular [Ca²⁺] _e triggered Ca²⁺ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca²⁺-receptor RaKCaR, e.g., Ba²⁺, Mg²⁺, Gd³⁺, and neomycin, but was reproduced by Ni²⁺. Ni²⁺-induced mobilization of intracellular Ca²⁺ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca²⁺] _e . In the cortical thick ascending limb, removing basolateral Ca²⁺ hardly altered [Ca²⁺] _i but increasing [Ca²⁺] _e or adding Ba²⁺, Mg²⁺, Gd³⁺ and neomycin released intracellular calcium. These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions; (2) this influx occurs through nonvoltage gated channels, permeable to Ba²⁺, insensitive to verapamil and nifedipine, and blocked by La³⁺; (3) increasing [Ca²⁺] _e stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR; (4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni²⁺ but not to Ca²⁺ is present in the collecting duct. Received: 12 July 1996/Revised: 28 October 1996     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

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.     

Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002     

Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes

Abstract: Rilmenidine, a ligand for imidazoline and α₂-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca²⁺]_i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca²⁺]_i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca²⁺-free medium, indicating that rilmenidine evokes release of Ca²⁺ from intracellular stores. However, the sustained elevation of Ca²⁺ was completely dependent on extracellular Ca²⁺, consistent with rilmenidine activating Ca²⁺ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca²⁺ influx pathway. The α₂-adrenergic antagonist rauwolscine attenuated the increase in [Ca²⁺]_i induced by clonidine (a selective α₂ agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca²⁺ release from intracellular stores and Ca²⁺ influx by a mechanism independent of α₂-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca²⁺ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

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     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca²⁺]_i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca²⁺]_i) was monitored using Fura-2-based ([Ca²⁺]_i) microfluorimetry. The ET-triggered ([Ca²⁺]_i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-free extracellular solution and the ET-triggered [Ca²⁺]_i elevation was blocked by 500 nM thapsigargin indicating that the [Ca²⁺]_i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca²⁺]_i increase in Ca²⁺-free solution was shorter in duration. Restoration of normal extracellular [Ca²⁺] briefly after the ET application induced a second [Ca²⁺]_i increase indicating the presence of a secondary Ca²⁺ influx which prolongs the Ca²⁺ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca²⁺ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca²⁺ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ET_B receptors which induce the generation of intracellular [Ca²⁺]_i signals by activation of Ca²⁺ release from InsP₃-sensitive intracellular stores followed by a secondary Ca²⁺ influx.     

Osmotic Swelling-Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes

Abstract: Hyposmotic swelling-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca²⁺]_i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca²⁺ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca²⁺]_i elevation. Omission of external Ca²⁺ or addition of Cd²⁺, Mn²⁺, or Gd³⁺ did not reduce RVD, although it was decreased by La³⁺ (0.1–1 mM). Verapamil did not affect either the swelling-evoked [Ca²⁺]_i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca²⁺ stores, such as treatment (in Ca²⁺-free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5-di-tert-butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca²⁺]_i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca²⁺ content or cytosolic Ca²⁺ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca²⁺]_i or RVD. BAPTA-acetoxymethyl ester (20 µM) abolished [Ca²⁺]_i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca²⁺]_i rise occurs as a consequence of increased Ca²⁺ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.     

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     

Fluid Flow Modulates Calcium Entry and Activates Membrane Currents in Cultured Human Aortic Endothelial Cells

Human aortic endothelial cells (HAEC) respond to flow with Ca²⁺ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca²⁺] _i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca²⁺, fluid flow was unable to evoke the [Ca²⁺] _i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca²⁺. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999     

A Prototypic Intracellular Calcium Antagonist, TMB-8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium-Dependent Component of Glutamate Neurotoxicity

Abstract: The effect(s) of a prototypic intracellular Ca²⁺ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca²⁺ levels ([Ca²⁺]_i) that was dependent on the extracellular concentration of Ca²⁺ ([Ca²⁺]_o). In addition, this increase in [Ca²⁺]_i correlated with a decrease in cell viability that was also dependent on [Ca²⁺]_o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na⁺/Cl^?-dependent component observed within minutes of glutamate exposure, and a “delayed” Ca²⁺-dependent component (ED₅₀～50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB-8 (at >100 µM) protects granule cells from the Ca²⁺-dependent, but not the Na⁺/Cl^?-dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca²⁺]_i. These findings suggest that Ca²⁺ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca²⁺]_i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca²⁺ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca²⁺]_i and glutamate-induced neurotoxicity.     

Structural and physiological properties of leech giant glial cells as studied by confocal microscopy

We have studied the architecture of giant neuropile glial cells of the medicinal leech Hirudo medicinalis L. using confocal laser scanning microscopy. We also measured changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by activation of glutamate receptors or voltage-gated Ca²⁺ channels in different glial cell compartments. Glial cells of isolated segmental ganglia were filled iontophoretically with the Ca²⁺ indicator dye Fluo-3. The three-dimensional structure, calculated from serial sections, showed that numerous fine glial branches extend within the whole neuropile, where most of the synapses between neurones are established. Activation of glial glutamate receptors by glutamate or kainate, or depolarizing the cell membrane by elevating the external K⁺ concentration resulted in a transient increase in [Ca²⁺]_i, as measured by Fluo-3 fluorescence. The comparison of [Ca²⁺]_i changes in glial cell branches with changes in the cell body demonstrated that transients in the branches were 2–3 times larger than those in the cell body. The results suggest that glutamate receptors and voltage-gated Ca²⁺ channels are located in the membrane not only of the glial cell body but also of the cellular branches, which may extend close to synaptic domains.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.