Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus . FSG and/or SAP-I induced elevations of [Ca²⁺]; and pH_i in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca²⁺]_i or pH_i of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca²⁺]_i and pH_i, At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca²⁺]_i, although SAP-I elevated the pH_i, its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K⁺-channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca²⁺]_i and pH_i by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca²⁺ and H⁺ transport systems.     

AMPA Receptor-Mediated Excitotoxicity in Human NT2-N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+

Abstract: Human NT2-N neurons express Ca²⁺-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca²⁺ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na⁺]_i, which induced a delayed [Ca²⁺]_i rise. Transfer of the neurons to a 5 m M Na⁺ medium after AMPA-GluR activation accelerated the delayed [Ca²⁺]_i rise and intensified excitotoxicity. Low-Na⁺ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca²⁺, suggesting that Ca²⁺ entry by reverse operation of Na⁺/Ca²⁺ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na⁺ loading. Our results serve to emphasize the central role of neuronal Na⁺ loading in AMPA-GluR-mediated excitotoxicity in human neurons.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Early Events in Free Radical-Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na⁺ ([Na⁺]_i) and intracellular Ca²⁺ ([Ca²⁺]_i) concentrations, and basal glutamate release of synaptosomes. Both H₂O₂ and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na⁺]_i over an incubation period of 15 min. A steady rise of the [Ca²⁺]_i due to peroxides was also observed that was external Ca²⁺ dependent and detected only at an inwardly directed Ca²⁺ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H₂O₂. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

Gene Transfer of Calbindin D28k cDNA via Herpes Simplex Virus Amplicon Vector Decreases Cytoplasmic Calcium Ion Response and Enhances Neuronal Survival Following Glutamatergic Challenge but Not Following Cyanide

Abstract: Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺])_i) and be followed by neuron death from hours to days later. If the rise in [Ca²⁺]_i is prevented, either by removing Ca²⁺ from the extracellular environment or by placing Ca²⁺ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D_28k responded to hypoglycemia with decreased [Ca²⁺]_i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca²⁺]_i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca²⁺]_i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

Tris stimulates ecdysteroid secretion via Ca2+ messenger system in the prothoracic glands of Locusta migratoria

Abstract.  Secretion of ecdysteroids by the prothoracic glands of the migratory locust, Locusta migratoria L., is enhanced in vitro by tris(hydroxymethyl)aminomethane (tris) in a dose-dependent manner. Glands from larvae on the second day of the penultimate stadium are most sensitive. This action of tris depends on the uptake of calcium; increased production of ecdysteroid does not occur in the presence of cadmium, verapamil or TMB-8, or in calcium-free media. The concentration of unbound Ca²⁺, [Ca²⁺]_i, in the cytoplasm is measured with the aid of FURA 2/AM. Tris causes a rise of [Ca²⁺]_i that is fully suppressed by lanthanum and partially by nitrendipine. Two antagonists of IP₃ receptors elicit mutually opposite effects: heparin blocks, whereas 2-APB accelerates, the rise in [Ca²⁺]_i. Ryanodine exerts only a slight effect. It is proposed that tris activates locust prothoracic glands in a Ca²⁺-dependent manner that exhibits many similarities to the transduction pathway of prothoracicotropic hormone.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.