Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

The prestalk/prespore differentiation and polarized cell movement inDictyostelium discoideum slugs. A possible involvement of the intracellular Ca2+-concentration

Summary Intracellular free calcium ion concentrations ([Ca²⁺]_i) in the anterior prestalk and posterior prespore cells of theDictyostelium discoideum slug were determined, using the highly selective Ca²⁺ indicators, quin-2/AM and fura-2/AM. Temporal changes in [Ca²⁺]_i in response to chemotactic stimulation with cAMP were also monitored at the single-cell level and compared between the two types of cells. The results obtained showed that resting [Ca²⁺]_i in the prestalk cells is considerably higher than that in the prespore cells. Moreover, transient increase in [Ca²⁺]_i upon stimulation with a low concentration of cAMP (20 nM) was noticed only in the prestalk cells, but not in the prespore cells. These facts are discussed in relation to the polarized movement and cellular differentiation in the migrating slug.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - DIF differentiation-inducing factors - IP₃ inositol 1,4,5-triphosphate     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Photolysis of caged Ca2+ induces trichocyst discharge inParamecium caudatum

Summary Trichocyst discharge, ciliary reversal, and cell body contraction inParamecium spp. have all been claimed to be regulated by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the cortical region of the cell. We injected caged Ca²⁺ intoP. caudatum cells and applied ultraviolet (UV) light to the cell for 125 ms. This did not induce trichocyst discharge but did induce both ciliary reversal and cell body contraction. A re-application of UV for 125 ms triggered trichocyst discharge. These results demonstrate that (1) trichocyst discharge and ciliary reversal and cell body contraction are controlled by [Ca²⁺]_i and (2) the threshold of [Ca²⁺]i for trichocyst discharge is higher than those for the other two functions.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ICL infraciliary lattice - [Ca²⁺]_i intracellular Ca²⁺ concentration - NP-EG o-nitrophenyl EGTA - PMT photomultiplier tube - UV ultraviolet     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line

The effects of the -galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca²⁺]_i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca²⁺]_i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by -galactosides. The observed Ca²⁺-response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca²⁺]_i compared to wild type lectin.Abbreviation [Ca²⁺]_i concentration of intracytoplasmic free calcium - CM carboxamidomethylation - CRD earbohydrate recognition domain - C2S mutant lectin protein in which Cys2 was replaced by serine - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N,N - N-tetraacetic acid HEPES,N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPL14 human -galactoside-binding placental lectin - Rec H recombinant human 14 kDa lectin - W68Y mutant lectin protein in which Trp68 was substituted to tyrosine     

Mitochondrial membrane potential (DeltaPsi) and Ca<Superscript>2+</Superscript>-induced differentiation in HaCaT keratinocytes

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca²⁺]₀, induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca²⁺]₀ results in acute and sustained increases in intracellular calcium concentration, [Ca²⁺]_i. We find that the Ca²⁺-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca²⁺]_i led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K⁺ channels, which is known to inhibit cell proliferation, and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a blocker of plasma membrane Cl^– channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

Determination of cytoplasmic calcium concentration inDryopteris spores

Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca²⁺ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca²⁺]_i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm^–1, half-life (time constant) 230 s). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca²⁺ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca²⁺] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N-tetraacetic acid (EGTA)) by 1 mM CaCl₂ caused a fast increase of [Ca²⁺]_i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl₂ by EGTA decreased [Ca²⁺]_i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]_i according to the Ca²⁺ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca²⁺-free medium, [Ca²⁺]_i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca²⁺-containing medium and analysed in EGTA, [Ca²⁺]_i was significantly higher ( 500 nM). In red-light-irradiated spores, [Ca²⁺]_i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.Dedicated to Professor H. Mohr on the occasion of his 60th birthday     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Apoptotic granulosa cells have moderately increased intracellular free calcium during phosphatidylserine exposure and a normal resting calcium level during DNA fragmentation

Alterations in intracellular free calciumconcentration ([Ca²⁺]_i) areinstrumental in apoptosis. We have previously shown that a[Ca²⁺]_i increase above 1000 nM isrelated to the appearance of apoptosis in serum-free cultures ofgranulosa cell sheets. In the present study we examined how the[Ca²⁺]_i increase relates toindicators of distinct phases of the apoptotic cascade. We used adouble staining technique whereby loading with theCa²⁺ indicator fura-2 and capture of a[Ca²⁺]_i image, was followed bystaining with annexin-V, as an early apoptotic marker or withacridine orange, marking the late degradation phase. Calcium imagingshowed a large heterogeneity of cellular[Ca²⁺]_i levels. [Ca²⁺]_i was moderately increased to230 nM in annexin positive cells but was at resting levelin cells with nuclear manifestations of apoptosis as evidenced byacridine orange. Our results suggest that a moderate[Ca²⁺]_i increase is related tophosphatidylserine translocation and that[Ca²⁺]_i has already recovered inapoptotic cells displaying chromatin condensation and/or nuclearfragmentation. Granulosa cells with[Ca²⁺]_i above 1000 nM were neverobserved to stain positive for the apoptotic markers used; therefore,large [Ca²⁺]_i increases areprobably related to the apoptosis initiation phase occurring upstreamof phosphatidylserine exposure.     

Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration

Summary Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pH_i or [Ca²⁺]_i in crayfish septate axons uncoupled by acidification. For measuring [Ca²⁺]_i a new neutral carrier sensor sensitive to picomolar [Ca²⁺] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pH_i or [H⁺]_i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pH_i and junctional resistance curves caused significant hysteresis in the pH_i versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca²⁺]_i, increased by approximately one order of magnitude from basal values of 0.1–0.3 m. The curves describing the time course of changes in [Ca²⁺]_i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca²⁺]_i recovered following a single exponential decay with a time constant of 2 min. Different rates of acidification caused increases in [Ca²⁺]_i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca²⁺]_i than to [H⁺]_i.     

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N−'tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca²⁺]_i, we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 μM BAPTA/AM reduced the number of cells showing deregulation of [Ca²⁺]_i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCI were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3–30 μM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 μM BAPTA/AM, the time constant of decline of [Ca²⁺]_i was increased by a factor of 4.1 ± 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 μM BAPTA/AM, the K⁺-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca²⁺]_i-induced cell damage, however, at the expense of a disturbed calcium signalling.     

Cytosolic free calcium concentrations in synaptosomes during histotoxic hypoxia

Altered cytosolic free calcium concentrations ([Ca²⁺]_i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca²⁺]_i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca²⁺]_i. However, with external CaCl₂ concentrations as small as 13 M, KCl depolarization increased [Ca²⁺]_i instantaneously while hypoxia gradually raised [Ca²⁺]_i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K⁺ and KCN on [Ca²⁺]_i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca²⁺]_i due to hypoxia, but enhanced the KCl response. The changes in ATP following K⁺ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca²⁺]_i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca²⁺]_i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca²⁺]_i and the effects were additive with K⁺ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca²]_i are due to an inability of the synaptosome to buffer entering calcium.