Pharmacological and functional characterization of excitatory amino acid mediated cytotoxicity in cerebral cortical neurons

The cytotoxic action of the excitatory amino acids (EAAs) glutamate, N-methyl- D-aspartate (NMDA), quisqualate (QA), kainate (KA) and (RS)-2-amino-3(3-hydoxy-5-methylisoxazol-4-yl) propionate (AMPA) was studied in cerebral cortical neurons in culture. The pharmacological profile of these actions was characterized using the NMDA selective antagonist D-(-)-2-amino-5- phosphonopentanoate (APV) and the non-NMDA selective antagonists 6.7- dinitroquinoxaline-2,3-dione (DNQX), 2-amino-3[3-(carboxymethoxy)-5- methylisoxazol-4-yl]-propionate (AMOA) and 2-amino-3-[2-(3-hydroxy-5- methylisoxazol-4-yl)methyl-3-methyl-3-oxoisoxazolin-4-yl] propionate (AMNH). The role of intracellular Ca⁺⁺ homeostasis and cGMP production for development of EAA mediated cytotoxicity was assessed by measurements of changes in [Ca⁺⁺]i using the flourescent Ca⁺⁺ chelator Fluo-3 and in cGMP concentrations using a conventional radioimmune assay. It was found that glutamate toxicity involves both NMDA and non-NMDA receptor activation and that aberrations in Ca⁺⁺ homeostasis brought about by Ca⁺⁺ influx and/or liberation of Ca⁺⁺ from internal stores aare important for development of toxicity. The drug dantrolene which prevents release of Ca⁺⁺ from such stores can prevent toxicity induced by glutamate, NMDA and QA completely but has no effect on KA and AMPA toxicity. Changes in cGMP levels appear to play a role for development of glutamate, NMDA and KA toxicity but does not seem to be involved in that triggered by QA and AMPA.Abbreviations AMNH: (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate) - AMOA: (2-amino-3[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propinate) - AMPA: ( (RS) —2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinate) - APV: (D-(-)-2-amino-5-phosphonopentanoate) - DNQX: (6,7-dinitroquinoxaline-2,3-dione) - KA (kinate) - QA (quisqualate)     

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.     

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca²⁺]_i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca²⁺ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca²⁺]_i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca²⁺]_i after expoxure to 50 mM K⁺ which was of the same magnitude as the increase in [Ca²⁺]_i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca²⁺ channels verapamil had no effect on the hyposmolarity induced increase in [Ca²⁺]_i. On the contrary, this increase in [Ca²⁺]_i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca²⁺ channels Mg²⁺ and La³⁺. Dantrolene which prevents release of Ca²⁺ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca²⁺ which represents an influx of Ca²⁺ rather than a release of Ca²⁺ from internal, dantrolene sensitive stores.     

NMDA Receptor-Mediated Calcium Influx in Cerebral Cortical Synaptosomes of the Hypoxic Guinea Pig Fetus

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca²⁺]_i observed under hypoxic conditions is the result of an NMDA-mediated Ca²⁺ influx. Changes of [Ca²⁺]_i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca²⁺]_i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca²⁺]_i was not observed in a Ca²⁺ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca²⁺]_i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca²⁺ via the altered NMDA receptor with subsequent release of Ca²⁺ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.     

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.     

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.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

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.     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

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     

Mapping of a carboxyl-terminal active site of parathyroid hormone by calciummimaging

We recently showed that the C-terminal fragment PTH (52–84) effectively increases intracellular free calcium ([Ca²⁺]_i in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1–34). Here we characterize the active site on C-terminal PTH (52–84) with respect to calcium (Ca²⁺)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73–76 to be the core region for increasing [Ca²⁺]_i. Ryanodine (1 μM), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2–5 μMI), agents that interfere with intracellular Ca²⁺ release, all failed to block PTH (52–84) induced [Ca²⁺]_i increases. Depletion of extracellular calcium ([Ca²⁺]_o) blocked PTH (52–84) induced [Ca²⁺]; increases, indicating a transmembrane Ca²⁺ influx. In contrast to voltage-gated and Ca²⁺ release activated Ca²⁺ influx, PTH (52–84) evoked Ca²⁺ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73–76 are essential for activation of a nickel-insensitive Ca²⁺ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca²⁺]_i. Carbachol evoked an increase in the [Ca²⁺]_i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca²⁺]_i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca²⁺]_i in myocytes. The absence of the slow component in the [Ca²⁺]_i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca²⁺ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca²⁺]_i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca²⁺]_i induced by both CCh and ATP; therefore, the release of Ca²⁺ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca²⁺ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.     

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.     

Intracellular Ca2+ homeostasis in rat round spermatids

Intracellular calcium, [Ca²⁺]_i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca²⁺]_i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca²⁺ and immunodetection of plasma membrane (PM) Ca²⁺-ATPases, we report that: a) rat round spermatids maintain [Ca²⁺]_i levels of 60 ± 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca²⁺]_i by actively extruding it using a PM Ca²⁺-ATPase; c) rat spermatids also actively transport Ca²⁺ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca²⁺_i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca²⁺ entry mechanisms by the release of Ca²⁺ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca²⁺ signals upon activation of Ca²⁺ channels or Ca²⁺ release from intracellular stores.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

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.     

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.     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Galactocerebroside mediated transmembrane Ca2+ signalling in U-87 MG cells: Possible involvement of phosphoinositides

Antibody to galactocerebroside (anti- GalC) has been shown to evoke a Ca²⁺ response in cultured glioma U- 87 MG cells. The rise in [Ca²⁺]_i was due to release of Ca²⁺ from the intracellular stores and influx through the plasma membrane. The rise in [Ca²⁺]_i was markedly inhibited by neomycin sulphate and phorbol dibutyrate suggesting the involvement of phosphoinositides in Ca²⁺ mobilization. The Ca²⁺ response induced by anti- GalC was rapidly desensitized and repeated addition of anti- GalC did not elevate the [Ca²⁺]_i. Heterologous desensitization was observed with bradykinin and adenosine triphosphate. The intracellular Ca²⁺ store mobilized by anti- GalC appears to be the IPin3 sensitive pool of endoplasmic reticulum. The influx of Ca²⁺ is mediated by a channel. The Ca²⁺ influx was also prevented by pretreatment of cells with neomycin sulphate or phorbol dibutyrate. We propose that galactocerebroside may be associated with phospholipase C or other proteins linked to the phosphoinositide pathway of transmembrane signalling and anti- GalC activates the breakdown of phosphoinositides and thus mobilizes Ca²⁺ in U-87 MG cells.     

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.