Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.     

Effect of carbachol and prostaglandin E2 on chloride secretion and signal transduction in the exocrine glands of frog skin (Rana esculenta)

Carbachol (CCH) increased the short-circuit current across frog skin glands in a biphasic manner, which coincided with an increase in the transepithelial Cl- net flux. CCH also induced a biphasic increase in [Ca2+]i. Both these responses were mediated via muscarinic receptors. The plateau phase of the CCH-induced Cl- secretion was modestly inhibited by indomethacin and unaffected by tetrodotoxin or tetrodotoxin plus indomethacin, indicating that CCH can increase Cl- secretion directly via receptors on the secretory cells. Prostaglandin E2 (PGE2) increased secretion and cAMP production, indicating expression of EP2 and/or EP4 receptors. PGE2 failed to increase [Ca2+]i ruling out involvement of EP1 receptors. The secretory response to CCH was potentiated by prestimulation with PGE2, and it was investigated whether this potentiation is caused by interaction at the level of the messengers involved. Stimulation by CCH plus PGE2 failed to stimulate cAMP production further than PGE2 alone. Addition of PGE2 during the CCH-elevated [Ca2+]i plateau phase in most cases reduced the level of [Ca2+]i. These data show that the synergy between CCH and PGE2 is not based on interactions at the intracellular messenger level.     

Tumor-promoting phorbol esters suppress receptor-stimulated inositol phospholipid degradation and Ca2+ mobilization in mouse lymphocytes

Anti-immunoglobulin antibodies (anti-Ig) provoke the rapid breakdown of phosphatidylinositol bisphosphate (PIP2), elevation of cytoplasmic Ca2+ levels ([Ca2+]i) and activation of protein kinase C (PKC) in B lymphocytes. Tumor-promoting phorbol esters, like phorbol myristate acetate, also activate PKC, but inhibit anti-Ig-induced B cell proliferation. To investigate the basis of the latter effect, we studied the influence of phorbol esters on PIP2 degradation and [Ca2+]i in murine B cells. The results show that PKC-activating phorbol esters cause marked inhibition of anti-Ig-stimulated PIP2 breakdown and Ca2+ mobilization. In addition, these agents inhibit concanavalin A-provoked Ca2+ influx, lower resting cytoplasmic Ca2+ levels and reduce ionophore-induced Ca2+ influx in B cells. Apparently, PKC stimulation causes feedback inhibition of receptor signalling, not only by suppressing PIP2 degradation, but also by exerting additional complex effects on the control of [Ca2+]i in B cells. It is, however, not clear how these findings relate to the anti-proliferative effects of phorbol esters on B cells.     

Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias

Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dog-fish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 micromol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl- secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl-K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na(+)-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl- secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.     

Effect of low temperatures on compound 48/80-induced intracellular Ca2+ changes and exocytosis of rat peritoneal mast cells

It has been well documented that compound 48/80-induced exocytosis of mast cells is accompanied by changes in intracellular Ca2+ concentration ([Ca2+]i) showing a biphasic pattern: an initial phase which constitutes an abrupt increase, followed by a plateau phase. The former is caused by Ca2+ release from intracellular Ca2+ stores, and the latter is the result of secondary Ca2+ influx. Low temperatures lead to the inhibition of exocytosis, but the precise mechanism remains unclear. The present study aims to reveal whether [Ca2+]i changes are affected by the environmental temperature. To this end, we developed a novel imaging method to record [Ca2+]i changes and exocytotic processes simultaneously. Rat peritoneal mast cells were loaded by Indo-1/AM or Fluo-3/AM for measuring [Ca2+]i, and the exocytosed granule matrices were stained by sulforhodamine-B. Cells were stimulated by compound 48/80, and [Ca2+]i changes and exocytosis were recorded by means of a real-time confocal microscope. At 37 degrees C, [Ca2+]i changes in stimulated mast cells showed a sustained plateau phase. Granule discharge was observed at the cell surface, and, in addition, most of the intracellular granule matrices were involved in compound exocytosis. The granule discharge and compound exocytosis proceeded over a period of a few minutes. At 4 degrees C, the plateau phase of [Ca2+]i changes declined rapidly, although the initial phase was not suppressed. Granule discharge occurred at the cell surface, but compound exocytosis ceased within a few minutes. These findings indicate that a low temperature inhibits compound exocytosis which can be caused by Ca2+ influx. The present imaging method represents a powerful tool for investigating the stimulus-secretion coupling of mast cells.     

Effects of long-term acidification of extracellular pH on ATP-induced calcium mobilization in rabbit lens epithelial cells

ATP-induced calcium (Ca2+) mobilization was investigated in rabbit lens epithelial cells that had been cultured in a medium with pH of 7.4 (group 1), 7.2 (group 2), or 7.0 (group 3) for 10 to 21 d. Intracellular free Ca2+ ([Ca2+]i and pH (pHi) were measured by using fluorescent dyes, fura-2 and BCECF, respectively. The long-term acidification decreased the pHi to 7.15 +/- 0.01, from 7.22 +/- 0.01, in group 2 and to 7.09 +/- 0.01 in group 3. The administration of 10 micromol/l ATP produced an initial peak followed by a sustained increase in [Ca2+]i in the lens cells of group 1. Both the initial peak and the sustained increase in [Ca2+]i were enhanced in groups 2 and 3. The initial peak was abolished by pretreatment with 1 micromol/l thapsigargin, an ER Ca2+ pump inhibitor, but was not affected by the removal of extracellular Ca2+. On the other hand, the sustained increase was suppressed either by the thapsigargin treatment or by the Ca2+ removal. Treatment with only thapsigargin caused a sustained increase in [Ca2+]i that was greater in group 3 than in group 1. These results suggest that (1) the ATP-induced initial peak in [Ca2+]i is due to Ca2+ release from the intracellular stores, (2) the sustained increase in [Ca2+]i is mediated through either Ca2+ influx from the extracellular space or Ca2+ release from the store triggered by the Ca2+ influx, and (3) long-term, moderate acidification enhances both the initial peak and the sustained increase in [Ca2+)]i in rabbit lens epithelial cells. One possible mechanism of the ATP-induced Ca2+ influx seems to be a capacitative Ca2+ entry pathway.     

Modulation of a pacemaker current through Ca(2+)-induced stimulation of cAMP production.

Brief increases in [Ca2+]i can result in prolonged changes in neuronal properties. A Ca(2+)-dependent modulation of the hyperpolarization-activated cation current (Ih) controls the slow recurrence of synchronized thalamocortical activity. Here we show that the persistent activation of Ih is initiated by rapidly increased [Ca2+]i and subsequent production of cAMP. The modulation is maintained via a facilitated interaction of cAMP with open (voltage-gated) h-channels, inducing prolonged activation of Ih that may outlast the presence of increased free [Ca2+]i and [cAMP]i. This persistent Ih activation may control the presence and periodicity of both normal and abnormal synchronized thalamocortical rhythms.     

The effect of calcium mobilization on LPS-induced IL-1 beta production depends on the differentiation stage of the monocytes/macrophages.

The role of elevated intracellular calcium concentration [Ca2+]i in the LPS-induced activation of interleukin-1 beta (IL-1 beta) production was examined in cells representing different stages of myeloid differentiation (undifferentiated monocytic leukaemia cell line THP-1, THP-1 cells induced to adherent, macrophage-like cells by phorbol ester treatment and normal peripheral blood-derived adherent monocytes). LPS did not elevate the [Ca2+]i as measured by the Fura-2 fluorescence technique. When these cells were stimulated with LPS in the presence of the calcium ionophore A23187, a clear increase in the IL-1 beta protein production was observed in the undifferentiated THP-1 cells but not in the more differentiated cell types. This ionophore-induced increase was also seen in the IL-1 beta mRNA levels. Thus these data confirm the previous findings demonstrating that elevation of [Ca2+]i is not involved in the LPS-dependent signal transmission. However, the LPS-induced signals are greatly potentiated by the elevated [Ca2+]i, but only in undifferentiated monocytic cells.     

Uptake of extracellular Ca2+ and not recruitment from internal stores is essential for T lymphocyte proliferation

Changes in free cytosolic calcium concentrations ([Ca2+]i) are thought to be important initiating events in the activation of T lymphocytes. Mitogen-induced increases in [Ca2+]i may result from net influx across the plasma membrane and/or release of Ca2+ from intracellular stores. In human T lymphocytes loaded with the fluorescent indicator indo-1, addition of phytohemagglutinin (PHA) or the anti-CD3 antibody UCHT-1 elicits a biphasic [Ca2+]i response. A major component of the initial transient peak was due to release from internal stores whereas the lower plateau phase was sustained by Ca2+ influx. Previous work suggested that Ca2+ influx is essential for interleukin 2 (IL 2) secretion and cell proliferation. To determine the relative effects of the initial and sustained phases of [Ca2+]i change, IL 2 secretion and cell proliferation, we introduced into the cell 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a high affinity intracellular Ca2+ chelator which neither contributes to nor interferes with the fluorescence determinations of [Ca2+]i. In cells preloaded with BAPTA, both PHA and UCHT-1 antibody failed to elicit the transient [Ca2+]i overshoot. Only the plateau phase could be observed in the presence of extracellular Ca2+. In contrast, BAPTA-loaded cells were found to be fully functional when assessed for IL 2 receptor expression, IL 2 secretion and cell proliferation. Thus, the mitogen-induced, maximal but transient increase in [Ca2+]i, contributed to mainly by release of Ca2+ from internal stores, does not appear to be essential for these T cell responses.     

Somatostatin inhibits prolactin secretion by multiple mechanisms involving a site of action distal to increased cyclic adenosine 3',5'-monophosphate and elevated cytosolic Ca2+ in rat lactotrophs

The release of prolactin (PRL) from a clonal cell-line of anterior pituitary cells (GH4C1) was inhibited by somatostatin (SRIH) in a dose-dependent manner (ED50 nM). The inhibition (20% of control levels) was detectable within 50 s and maximal within 90 s. Thyroliberin (TRH) enhancement of PRL secretion was biphasic. SRIH inhibited both phases equally. Ionomycin in combination with the phorbol ester, TPA, mimics the TRH-elicited PRL release, and SRIH partly inhibited this effect. SRIH had no effect on TRH-stimulated formation of inositol trisphosphate, and only small effects on TRH-activated adenylate cyclase. Vasoactive intestinal peptide (VIP) and forskolin stimulated cAMP formation and PRL release potently. SRIH inhibited both effects of VIP and forskolin, and there was a close correlation between the inhibition of PRL secretion and cAMP accumulation. 8-Bromo-cAMP enhanced PRL release, an effect that was also partly reduced by SRIH. The Ca2+ channel activator, BAY-K-8644 and high extracellular K+ increased PRL release, and SRIH caused a partial reduction in the release response to both secretagogues. SRIH lowered [Ca2+]i, and markedly reduced the rise in [Ca2+]i elicited by TRH, VIP and K+. SRIH did not influence the Ca2+ spikes recorded in Na+-free solution, and had no effect on the TRH-induced membrane potential changes. Our results demonstrate that SRIH may inhibit PRL release from GH4C1 cells by (1) inhibiting hormone-sensitive adenylate cyclase, (2) blocking the effect of cAMP and (3) lowering [Ca2+]i. None of these effects is, however, sufficient to explain all the effects of SRIH, suggesting that SRIH also exerts a major action at a step subsequent to cAMP accumulation and [Ca2+]i elevation. Since the GH4C1 cells possess one single class of binding sites, this implies that the same SRIH receptor is coupled to several cellular signalling systems.     

Simultaneous recordings of cytosolic Ca2+ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH4C1 clonal cells was studied using fura-2 to monitor the cytosolic Ca2+ level ([Ca2+]i) in single cells, combined with recordings of membrane potential and current. The average value of [Ca2+]i was 109 nM (mean +/- SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca2+]i, peaking at 795 +/- 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca2(+)-activated K+ channels. In 75% of the cells the initial peak in [Ca2+]i was followed by a prolonged plateau phase at 247 +/- 76 nM (n = 84). In current clamp the second-phase elevation of [Ca2+]i was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization in silent cells. This elevation of [Ca2+]i was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca2+]i was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca2(+)-free conditions eliminated the second-phase elevation in [Ca2+]i, whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca2+]i in the GH4C1 cells. The second-phase elevation of [Ca2+]i caused by thyroliberin is the result of influx through voltage-sensitive Ca2+ channels, without involving agonist-gated channels.     

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.     

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.     

Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.     

Signaling requirements for the expression of the transactivating factor NF-AT in human T lymphocytes.

A protein called nuclear factor of activated T cells (NF-AT) binds to DNA sequences within the enhancer region of the interleukin 2 (IL 2) gene and appears necessary for both the inducibility and T cell specificity of IL 2 expression. IL 2 production is regulated by multiple signals including those generated via activation of the T cell antigen receptor complex (TcR/CD3), CD2 antigen, protein kinase C (PKC) or elevation of intracellular free calcium concentration ([Ca2+]i). We have, therefore, explored the role of these different stimuli in regulating the nuclear expression of NF-AT in human peripheral blood-derived lymphocytes. Results presented herein indicate that maximal expression of NF-AT in T cells requires at least two signals: PKC activation and TcR/CD3 or CD2 triggering, [Ca2+]i increases and TcR/CD3 or CD2 triggering. Data are presented that indicate that either the [Ca2+]i or PKC signal generated via the TcR/CD3 complex would not alone induce NF-AT expression, and that the TcR/CD3 complex probably regulates NF-AT expression because of its ability to regulate multiple intracellular signals in T cells, and not via any single biochemical event. The combination of CD2 mAb GT2/OKT11 used in the present study to trigger the CD2 antigen is able to act in synergy with phorbol 12,13-dibutyrate or ionomycin to induce NF-AT expression. However, these CD2 mAb do not elevate [Ca2+]i or activate PKC, suggesting that signals other than [Ca2+]i or PKC can regulate NF-AT expression in peripheral blood-derived T cells.     

ATP-induced [Ca2+]i changes in the human corneal epithelium

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.     

Response of human B cells to different anti-immunoglobulin isotypes: absence of a correlation between early activation events and cell proliferation

Cross-linking of surface immunoglobulin (sIg) by antibodies against IgM, IgG and IgD activates B cells and in some circumstances can induce cell proliferation. We studied the potential link between anti-Ig-induced changes in the cytosolic free Ca2+ concentration ([Ca2+]i), inositol phosphate production and the ability to induce cell proliferation in the presence or absence of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Anti-IgM, but not anti-IgD or anti-IgG, induced cell proliferation in the presence but not the absence of TPA. Each of the antibodies induced a rapid increase in [Ca2+]i which appeared to be due to release of Ca2+ from internal stores. This was followed by a sustained increase in [Ca2+]i, apparently due to Ca2+ uptake from the extracellular medium. Anti-IgD induced the greatest increase in [Ca2+]i, anti-IgM induced intermediate changes and anti-IgG the lowest change. Since inositol 1,3,5-trisphosphate (IP3) can release Ca2+ from internal stores, we tested the ability of each anti-Ig isotype to increase concentrations of IP3. In contrast to the change in [Ca2+]i and proliferation, anti-IgG induced the most significant increase in IP3 concentrations. Taken together these data indicate that changes in [Ca2+]i, inositol phosphate production and anti-Ig-induced human B cell proliferation are not directly linked. They also demonstrate that changes in [Ca2+]i, inositol phosphate production and activation of protein kinase C are not sufficient to induce proliferation of human B cells. It appears that anti-IgM induces an additional Ca2+-independent, inositol phosphate-independent and protein kinase C-independent activation signal which can collaborate with TPA to induce B cell proliferation. The molecular events involved in this signal remain to be identified.     

Response to ATP is accompanied by a Ca2+ influx via P2X purinoceptors in the coronary arterioles of golden hamsters

In the vascular wall, adenosine-5'-triphosphate (ATP) released along with noradrenaline from sympathetic nerve terminals is considered to play an important role in controlling intracellular calcium ion ([Ca2+]i) levels in arteries. The present study examined how vascular smooth muscle cells in coronary arterioles respond to ATP in relation to [Ca2+]i dynamics. For this purpose, the dynamics of [Ca2+]i in the coronary arterioles of golden hamsters was examined by real-time laser scanning confocal microscopy. This technique enabled the visualization of [Ca2+]i changes in response to ATP in the intact coronary arterioles, the ultrastructure of which was well preserved. It was shown that an increase in [Ca2+]i in the arteriole smooth muscle cells was elicited by ATP. While P1 purinoceptor agonists have no effect on this process, P2 purinoceptor agonists were found to induce a [Ca2+]i increase in the smooth muscle cells. Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca2+]i dynamics, but reactive blue 2 (a P2Y receptor antagonist) did not. Uridine-5'-triphosphate (a P2Y receptor agonist) had no effect on [Ca2+]i, but alpha,beta-methylene ATP (a P2X receptor agonist) caused a strong increase in [Ca2+]i. We conclude that smooth muscle cells of the hamster coronary arterioles possess P2X, but not P1 or P2Y purinoceptors. The smooth muscle cells probably respond to extracellular ATP via P2X purinoceptors, resulting in the contraction of the coronary arterioles.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.