Pharmacologic evidence for involvement of phospholipase C in pemphigus IgG-induced inositol 1,4,5-trisphosphate generation, intracellular calcium increase, and plasminogen activator secretion in DJM-1 cells, a squamous cell carcinoma line

The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (IP3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3, PA secretion, and cell-cell detachment in DJM-1 cells. [Ca+2]i and IP3 contents were determined with or without 30-min pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxymethylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell-cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++]i and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 microM), but not with U73343 (10 microM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 caused no effects on cell viability and IgG binding to the cell surface. These results suggest that phospholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.     

Effects of a time-varying strong magnetic field on transient increase in cytosolic free Ca2+ induced by bradykinin in cultured bovine adrenal chromaffin cells

We tested the effects of exposure to a time-varying magnetic field changing between 0.07 and 1.7 T at an interval of 3 s on transient increase in intracellular Ca2+ stimulated by bradykinin in bovine adrenal chromaffin cells. Addition of bradykinin induced the increase in intracellular Ca2+ within a few minutes. The exposure to the magnetic field perfectly suppressed the increase in intracellular Ca2+ in Ca2+-free medium. The inhibition occurred for 15 min when the maximum magnetic flux density was more than 1.4 T. These results suggest that the exposure inhibits Ca2+ release from intracellular Ca2+ stores.     

Effects of selegiline hydrochloride on intracellular Ca2+ contents in cultured neuronal cells

The effects of Selegiline hydrochloride (Selegiline HCl) on the intracellular Ca2+ contents of primarily cultured rat striatal, mesencephalic neuronal cells and PC-12 cells were examined by the use of a Ca2+ imaging analyzer. In the former two cell types, Selegiline HCl (10(-5)-10(-6) M) induced a transient inflow of extracellular Ca2+ through the voltage-dependent N-type Ca2+ channel. In addition, all cells indicating an increase in the intracellular Ca2+ content were found to be catecholaminergic neurons which showed a positive reaction with anti-tyrosine hydroxylase antibodies. Furthermore, a transient intracellular influx of Ca2+ was observed in the NGF-pretreated PC-12 cells. From these results, it is suggested that Selegiline HCl elicits various functions, including antioxidation, activation of neurotrophic factor biosynthesis and neuronal protection probably via an unidentified specific proteins of tyrosine hydroxylase-positive neurons.     

Estimation of fiber diameters in the spinal dorsal columns from clinical data

In the rat cerebellar slice preparation in vitro, excessive DL-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-receptor activation elicits a characteristic type of excitotoxicity of Purkinje cells (PCs) known as dark cell degeneration (DCD). DCD models neurotoxicity of PCs and hippocampal pyramidal neurons in vivo following hyperexcitable states. The intent of this study was to: a) determine whether AMPA-induced neurotoxicity of PCs is correlated with temporally and spatially restricted rises in intracellular Ca2+ and b) whether GYKI 52466 and nominal external Ca2+, conditions that reduced expression of AMPA-elicited DCD, altered the induced Ca2+ patterns. Employing the Ca2+-sensitive dye Fluo-3 and a confocal laser scanning microscope, we evaluated changes in intracellular Ca2+ within PCs in a cerebellar slice preparation. AMPA application alone (30 microM for 30 min) caused a significant initial rise in perinuclear and cytoplasmic Ca2+ that returned to control levels during the latter part of the AMPA exposure period. Following removal of AMPA (expression period), perinuclear and cytoplasmic Ca2+ displayed a significant delayed rise peaking transiently 60 min after AMPA removal. The efficacy of GYKI 52466 and nominal external Ca2+ conditions to attenuate AMPA-induced DCD was correlated to reductions in AMPA-induced transient elevations in perinuclear and cytoplasmic Ca2+ levels during the expression phase and to a lesser extent during the exposure period. The present data suggest that during the expression phase, the delayed perinuclear and cytoplasmic Ca2+ transient may be the harbinger of impending loss of Ca2+ homeostasis and cell damage.     

Histamine-induced calcium released from cultured human mucosal microvascular endothelial cells from nasal inferior turbinate

Changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human mucosal microvascular endothelial cells (HMMECs) from nasal inferior turbinate were measured using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy. Histamine caused a transient increase in intracellular free Ca2+ in cell populations and in individual cells, followed by a decrease to a sustained elevation. Histamine (100 microM) elevated [Ca2+]i in HMMECs up to 563 +/- 20 nM from a resting level of 60 +/- 45 nM (means +/- SD, n = 31). Promethazine (a histamine H1 receptor antagonist) inhibited [Ca2+]i increase during histamine stimulation, whereas cimetidine (a H2 receptor antagonist) and thioperamide (a H3 receptor antagonist) showed no inhibition. These results suggest that the histamine increase [Ca2+]i in HMMECs induces both a Ca2+ release from stores and a Ca2+ influx through activation of the H1 receptor.     

Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

Recognition of acute cardiac allograft rejection from serial integrated backscatter analyses in human orthotopic heart transplant recipients. Comparison with conventional echocardiography

The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively. Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells. These elevations were insensitive to compounds known to release Ca2+ from intracellular stores. Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.     

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis

We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.     

Hemolytic properties of Ca(2+)-treated human erythrocytes under hydrostatic pressure

The effect of intracellular Ca2+ on high pressure-induced hemolysis of human erythrocytes was examined. Red cells were incubated with Ca2+ (0.01-1 mM) in the presence of ionophore A23187. The Ca(2+)-loaded cells were subjected to a pressure of 200 mPa. Treatment with 0.1 mM Ca2+ had the greatest suppressive effect on the hemolysis. On removal of intracellular Ca2+, red cells showed a morphological change from echinocytes to normal discocytes but the hemolysis remained unaltered. Measurement of intracellular K+ and viscosity demonstrated that the suppressive effect of Ca2+ on the hemolysis is irreversible and is largely associated with the increase of intracellular viscosity induced by K+ efflux.     

Reduced inward rectifying and increased E-4031-sensitive K+ current density in arrhythmogenic subendocardial purkinje myocytes from the infarcted heart

Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Diversity of calcium signaling by metabotropic glutamate receptors

During prolonged application of glutamate (20 min), patterns of increase in intracellular Ca2+ concentration ([Ca2+]i) were studied in HEK-293 cells expressing metabotropic glutamate receptor, mGluR1alpha or mGluR5a. Stimulation of mGluR1alpha induced an increase in [Ca2+]i that consisted of an initial transient peak with a subsequent steady plateau or an oscillatory increase in [Ca2+]i. The transient phase was largely attributed to Ca2+ mobilization from the intracellular Ca2+ stores, but the sustained phase was solely due to Ca2+ influx through the mGluR1alpha receptor-operated Ca2+ channel. Prolonged stimulation of mGluR5a continuously induced [Ca2+]i oscillations through mobilization of Ca2+ from the intracellular Ca2+ stores. Studies on mutant receptors of mGluR1alpha and mGluR5a revealed that the coupling mechanism in the sustained phase of Ca2+ response is determined by oscillatory/non-oscillatory patterns of the initial Ca2+ response but not by the receptor identity. In mGluR1alpha-expressing cells, activation of protein kinase C selectively desensitized the pathway for intracellular Ca2+ mobilization, but the mGluR1alpha-operated Ca2+ channel remained active. In mGluR5a-expressing cells, phosphorylation of mGluR5a by protein kinase C, which accounts for the mechanism of mGluR5a-controlled [Ca2+]i oscillations, might prevent desensitization and result in constant oscillatory mobilization of Ca2+ from intracellular Ca2+ stores. Our results provide a novel concept in which oscillatory/non-oscillatory mobilizations of Ca2+ induce different coupling mechanisms during prolonged stimulation of mGluRs.     

A simulation model including ovulation rate, potential embryonic viability, and uterine capacity to explain litter size in mice: II. Responses to alternative criteria of selection

We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). Ca2+-mobilizing agonists CCK-8 and ACh induced a transient acidification totally dependent on release of Ca2+ from internal stores. Employment of different physiological tools including ionomycin and thapsigargin to increase the cytosolic Ca2+ concentration and capacitative calcium influx also induced cellular acidification. Application of 1mM LaCl3 reduced the CCK-8-evoked acidification. These data indicate that the mobilization of intracellular Ca2+ stores by CCK-8 decreases cellular pH by Ca2+/H+ exchanger.     

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P < 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.     

Dendritic cell development: multiple pathways to nature''s adjuvants

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 ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-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 Ca(2+)-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 microM ATP or 10 microM 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 ETB 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.     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

Chemotactic factor-induced membrane potential changes in rabbit neutrophils monitored by the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide

Rabbit neutrophil leucocytes take up the cationic, fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)). Treatment with valinomycin and K+ then produces characteristic changes in suspension fluorescence that indicate that the dye enters the cells in a potential-dependent fashion and that the resting membrane potential lies between -66 and -86 mV. The peptide, N-fMet-Leu-Phe, a potent chemoattractant for neutrophils, added to stained cell suspensions, induces fluorescence intensity changes. These occur over an 8-10 min period. The time course of this response is profoundly affected by the omission of Ca2+ from the medium. When this ion is present (1.26 mM) a small, transient increase in intensity is observed, superimposed on a sustained decrease. On the other hand, in the absence of added Ca2+ a large, transient increase is observed. The ED50 for this is 1.1 x 10(-10) M. These changes are not elicited by N-fMet-Phe (10(-9) M) and are inhibited by the antagonist Boc-Leu-Phe-Leu-Phe. However, a component of zymosan-activated rabbit plasma, which is complement-derived, induces identical fluorescence changes that are not inhibited by the antagonist, confirming that neutrophil activation by complement operates through an independent receptor. The fluorescence responses to the chemotactic peptide and the activated-plasma component may be interpreted in terms of changes in neutrophil membrane potential brought about by alterations in cell ionic permeability at an early stage during activation. The transient increase corresponds to a depolarisation that may be associated with a change in Na+ permeability, while the sustained decrease corresponds to a membrane hyperpolarisation.     

The germinal vesicle of the mouse oocyte contains elements of the phosphoinositide cycle: what is their role at meiosis resumption?

The role of the nuclear phosphoinositide (PI) cycle during meiotic resumption in mouse oocytes was examined. First, using indirect immunofluorescence staining with specific monoclonal antibodies (mAbs) against elements of this cycle, the presence of inositol trisphosphate receptors (IP3Rs) (IP3R-1 or IP3R-3) or phosphoinositide-phospholipase (PLC) isoforms (PLC beta 1 or PLC gamma 1) was monitored in the germinal vesicle (GV). Using confocal laser scanning microscopy, we analysed the effects of the nuclear microinjection of these antibodies on both spontaneous nuclear calcium oscillations and meiosis resumption. Immunostainings showed that IP3R-1 and PLC beta 1 isoforms were both present in the GV, whereas IP3R-3 and PLC gamma 1 isoforms were not. The anti-IP3R-1 mAbs or the anti-PLC beta 1 mAbs microinjected into the GV, induced inhibition of both the nuclear Ca2+ oscillations and the meiotic process, whereas the anti-IP3R-3 mAbs and the anti-PLC gamma 1 mAbs did not. We concluded that a specific nuclear PI cycle is present in the mouse oocyte and meiosis resumption requires a specific nuclear phosphoinositide-dependent Ca2+ signal.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.