Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Mandin-Darby canine kidney cells

The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration [Ca2+]i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture. Changes in [Ca2+]i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm). MDCK cells exhibited a mean baseline [Ca2+]i of 98 +/- 10 nM. The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times basal levels. In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in [Ca2+]i. The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in [Ca2+]i which never exceeded the basal level in the absence of ANP. The data indicate that ANG II increases cell [Ca2+]i, as expected, and provide the new observation that ANP reduces [Ca2+]i in these cells. Furthermore, ANP reduces the increase in [Ca2+]i elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i.     

Role of intracellular calcium in fast and slow desensitization of P2-receptors in PC12 cells

1. Combined whole-cell patch clamp recording and confocal laser scanning microscopy of [Ca2+]i transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+]i. ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate. 2. Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+]i increases. Long applications (2 s) of ATP (5 mM) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P2-receptors. The associated [Ca2+]i increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization. 3. Both membrane currents and [Ca2+]i transients were linearly dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+]i elevation. This view was supported by experiments carried out in Ca(2+)-free solution. 4. Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+]i rises indicating a role for [Ca2+]i in controlling slow desensitization of P2-receptors. 5. This notion was confirmed in experiments with various [Ca2+]i chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization. 6. These results suggest a role for [Ca2+]i in slow rather than fast desensitization of P2-receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP.     

Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in eicosanoid and cytokine production between injury/hemorrhage and bacteremic shock in the pig

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from the anterior pituitary gland of 6- to 8-day-old rats. GnRH-induced [Ca2+]i oscillations were recorded indirectly by monitoring the activity of apamin-sensitive Ca(2+)-activated K+ channels using the perforated patch-clamp technique and fast microperfusion system. Melatonin (1 nM) inhibited the initiation or attenuated the amplitude of oscillatory current responses induced by 10 nM GnRH in 72% of GnRH-sensitive cells. Analysis of the melatonin dose-inhibition relationship showed that melatonin inhibited the initiation of [Ca2+]i oscillations with IC50 = 0.35 nM. In partially inhibited cells, melatonin reduced the GnRH-induced current amplitude by 55% on the average, prolonged the delay in onset of response to GnRH and decreased the frequency of oscillations. Once initiated by GnRH, the amplitude and frequency of oscillatory currents was inhibited by melatonin after a latency of 10-30 s. These effects of melatonin were fully reversible. After pretreatment of neonatal gonadotropes with pertussis toxin, no inhibition by melatonin was observed. The inhibitory effect of melatonin on initiation, amplitude and frequency of GnRH-induced oscillatory current persisted in the absence of external Ca2+. Melatonin alone did not induce any transmembrane current or membrane potential changes. These observations suggest that melatonin reduces GnRH-induced calcium mobilization from intracellular stores.     

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.     

Spontaneous and repetitive calcium transients in C2C12 mouse myotubes during in vitro myogenesis

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i) in the mouse muscle cell line C2Cl2 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2Cl2 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2+]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.     

Oscillations of receptor-operated cationic current and internal calcium in single guinea-pig ileal smooth muscle cells

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at -40 mV or -50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[gamma S] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CCh-evoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachol's action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.     

Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+]i in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphasic increase in free [Ca2+]i, an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+]i. Thapsigargin did not affect either response to H2O2, but activated capacitive calcium entry, an action abolished by 10 microM La3+. Simultaneous recordings of membrane potential and [Ca2+]i demonstrated the same biphasic [Ca2+]i response to H2O2 and showed that the late increase in [Ca2+]i coincided temporally with cell membrane potential collapse. Buffering Ca2+i to low nanomolar levels prevented both phases of increased [Ca2+]i and the H2O2-induced depolarization. The H2O2-induced late rise in [Ca2+]i was prevented by extracellular application of 100 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+]i, activates NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.     

Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats

We have characterized two different types of Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch-clamp and Fura-2 microfluorescence technique to measure simultaneously ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. Exposure of CPAE cells to 28% hypotonic solution induces cell swelling without a change in membrane capacitance and [Ca2+]i, and concomitantly activates a current. This current, I(Cl, vol), is closely correlated with the changes in cell volume and shows a modest outward rectification. It slowly inactivates at potentials more positive than +60 mV but is time- and voltage-independent at other potentials. Increase in [Ca2+]i by different maneuvers, such as application of vasoactive agonists (ATP), ionomycin, or loading of the cells directly with Ca2+ also activates a Cl- current, I(Cl, Ca). This current slowly activates at positive potentials, inactivates quickly at negative potentials and shows strong outward rectification. A time-independent component of the current activated by elevation of [Ca2+]i alone can be inhibited by cell shrinking by exposing the cells to hypertonic solution, indicating that an increase in [Ca2+]i also co-activates I(Cl, vol). Forskolin or cAMP never activated a current in CPAE cells, which indicates the lack of cAMP-activated channels in these cells. There is also no evidence for the existence of voltage-gated Cl- channels in resting, nonstimulated cells. Challenging a cell with elevated [Ca2+]i and hypotonic solutions activated I(Cl, vol) on top of I(Cl, Ca), suggesting that I(Cl, Ca) and I(Cl, vol) are different channels. We conclude that CPAE cells do not express voltage-gated (ClC-type) or cAMP-gated (CFTR-type) Cl- channels, but activate large Cl- currents after volume (mechanical?) or chemical (Ca2+) stimulation.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca2+ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonist-induced Ca2+ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 microM ATP or by 100 microM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2-100 microM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20-30% decrease in the magnitude of [Ca2+]i elevation induced by a low concentration of ATP (<1 microM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20-40% increase of ATP-induced elevation of [Ca2+]i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, Km, value in ATP-treated cells and of the maximal [Ca2+]i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2+ and/or ion channels.     

Effects of SCN substitution for Cl- on tension, [Ca2+]i, and ionic currents in vascular smooth muscle

Substitution of thiocyanate ions (SCN-) for chloride ions (Cl-) in the extracellular medium of aortic rings and strips causes a biphasic contractile response; initial relaxation followed by sustained contraction. Alterations in these responses are sex-specific, and may elucidate fundamental differences in vascular function between males and females. In order to investigate the role of changes in intracellular Ca2+ ([Ca2+]i) in these changes in tension, we investigated effects of SCN- on [Ca2+]i and ionic currents in vascular smooth muscle cells (VSMC). Extracellular substitution of SCN- for Cl- caused a biphasic change in [Ca2+]i. Initially, [Ca2+]i decreased, reaching a minimum within 1-2 min, subsequently returned to original levels within 4-5 min, and then increased to a higher plateau over the next 10 minutes. This pattern of change in [Ca2+]i is identical to the pattern of tension changes in aortic rings, but it occurs somewhat faster. Partial substitution of SCN- for Cl- elicited increased, but no preceding decrease in [Ca2+]i. In the absence of external Ca2+, anion substitution elicited the decrease in [Ca2+]i but not the subsequent increase. Verapamil (1 microM) blocked the increased [Ca2+]i phase but not the decreased [Ca2+]i phase; whereas, R+ verapamil (up to 5 microM for 20 min), an inactive enantiomer, caused no change. Ionic current measurements obtained using whole cell patch and current clamp techniques revealed two responses to anion substitution: (a) a rapid, transient outward shift in holding current, and (b) a sustained increase in peak current and a hyperpolarizing shift in voltage sensitivity of Ca2+ channels. The calcium channel blocker PN200-110 blocked SCN(-)-enhanced current but had no effect on the changes in holding current. S- verapamil, but not R+ verapamil, reduced SCN(-)-enhanced current. In current clamp mode, SCN- caused an initial hyperpolarization followed by a slow depolarization punctuated by spikes. Thus, SCN- causes changes in vascular smooth muscle [Ca2+]i that could underlie both phases of its effects on tension in isolated aortas and may be explained by the following model: an initial outward shift in current causes hyperpolarization with a consequent decrease in cell excitability, and the somewhat slower increase in Ca2+ channel excitability eventually leads to enhanced calcium influx and tension. These data shed light on possible mechanisms underlying gender-related differences in VSMC physiology.     

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.     

Fluid flow modulates vascular endothelial cytosolic calcium responses to adenine nucleotides

OBJECTIVE: To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. METHODS: Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca(2+)-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+]i) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. RESULTS: Continuous perfusion with ATP (0.3-3.0 microM) or ADP (0.1-1.0 microM) evoked repetitive oscillations in [Ca2+]i in individual BAEC. The frequency of the [Ca2+]i oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]i oscillations. In the presence of nonhydrolyzable nucleotide analog, ATP gamma S or ADP beta S, application of flow resulted in similar shear-dependent [Ca2+]i oscillations, suggesting that flow modulation of the [Ca2+]i response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. CONCLUSIONS: These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.     

Patch-clamp analysis of the mechanism of PACAP-induced excitation in rat supraoptic neurones

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP) causes an increase in [Ca2+]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca2+]i revealed that the membrane depolarization correlated well with increases in [Ca2+]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of -70 or -80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was -33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAP receptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca2+]i and vasopressin (VP) release observed in SON neurones.     

Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase

Oxidative stress can cause changes in intracellular free calcium concentration ([Ca2+]i) that resemble those occurring under normal cell signaling. In the alveolar macrophage, hydroperoxide-induced elevation of [Ca2+]i modulates the respiratory burst and other important physiologic functions. The source of Ca2+ released by hydroperoxide is intracellular but separate from the endoplasmic reticulum pool released by receptor-mediated stimuli (Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171). Previous studies in other cells have suggested that mitochondria are a potential source of oxidant-induced [Ca2+]i elevation. In this study we have identified another potential source of hydroperoxide-releasable intracellular calcium, that bound to annexin VI on the inner surface of the plasma membrane. Translocation of annexin VI from the membrane during exposure to t-butyl hydroperoxide matched elevation of [Ca2+]i as a function of time and t-butyl hydroperoxide concentration. The translocation was possibly due to a combination of ATP depletion and oxidative modification of membrane lipids and proteins. A sustained increase in [Ca2+]i occurring > 50 pmol/10(6) cells (50 microM under these conditions) appeared to be a consequence of membrane Ca2+-ATPase dysfunction. These results suggest that exposure to oxidative stress results in early alterations to the plasma membrane and concomitant release of Ca2+ into the cytosol. In addition it suggests a mechanism for participation of annexin VI translocation that may underlie the alterations in macrophage function by oxidative stress.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.