Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Calcium oscillations in gingival epithelial cells infected with Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis modulates epithelial cell signal transduction pathways including Ca2+ signaling, and internalizes within the host cell cytoplasm. Since nuclear and cytoplasmic [Ca2+] increases can induce different host cell responses, P. gingivalis-related [Ca2+] changes in these compartments were measured by digital fluorescent imaging microscopy. Non-deconvolved and deconvolved fura-2 images showed that P. gingivalis exposure caused human gingival epithelial cells cultured in physiologic [Ca2+] levels to undergo sustained oscillations of [Ca2+] in nuclear and cytoplasmic spaces. However, P. gingivalis invasion was not tightly correlated with intracellular [Ca2+] oscillations, since invasion could significantly precede, or even occur in the absence of, oscillations. [Ca2+] oscillations required a Ca2+ influx, which was completely inhibited by La3+ or 2-APB (2-aminoethoxydiphenyl borate), indicating Ca2+ entry was via a Ca(2+)-permeable channel. Ca2+ entry was likely not via a store-operated channel, since Ca2+ release from intracellular stores was not observed during cellular uptake of P. gingivalis. Hence, uptake of P. gingivalis in gingival epithelial cells induces oscillations in nuclear and cytoplasmic spaces by activating a Ca2+ influx through Ca2+ channels.     

The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Ag stimulation of rat basophilic leukemia (RBL-2H3) cells results in hydrolysis of inositol phospholipids, a transient increase in concentration of cytosol Ca2+ [( Ca2+]i), a gradual increase in cytosolic pH (pHi) and the activation of protein kinase C. To determine whether all these changes serve as signals for secretion, studies were conducted with cells permeabilized with streptolysin O in which pHi and [Ca2+]i could be varied independently of each other and enzyme activities could be manipulated. At resting pHi (approximately 7.0) and [Ca2+]i (0.1 microM), the permeabilized cells showed little secretory response to Ag. At resting pHi, elevated levels of Ca2+ (0.33 microM) were required for maximal secretory response to Ag. At a pHi of 7.4, however, 0.1 microM [Ca2+]i was sufficient to sustain near maximal responses to Ag. Therefore, a small increase of [Ca2+]i to 0.33 microM was required to initiate secretion, but once the pHi was elevated secretion could be sustained at near basal levels of [Ca2+]i. Since elevating the [Ca2+]i and pHi, by themselves promoted little secretion, another potentiating signal must have been generated by antigen stimulation. This signal was possibly transduced via hydrolysis of inositol phospholipids and protein kinase C. Even with an elevated [Ca2+]i (0.33 microM) the hydrolysis of the phospholipids and secretion stimulated by Ag were inhibited by guanosine 5'(2-O-thio)diphosphate and neomycin. Furthermore, both protein-kinase C and the secretory response to Ag were lost after permeabilized cells were washed but both were retained if cells were exposed to PMA before permeabilization.     

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.     

The actions of Ca2+ ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. A comparison with antigen stimulation

Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation.     

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.     

Aequorin detects increased cytoplasmic calcium in platelets stimulated with phorbol ester or diacylglycerol

A biochemical pathway to platelet activation involving protein kinase C has been deemed "Ca2+-independent", because the intracellular fluorophore quin2 indicates no rise in cytoplasmic [Ca2+] in platelets stimulated by certain agonists. However, unlike quin2, the Ca2+-sensitive photoprotein aequorin demonstrates a rise in [Ca2+] when platelet aggregation is induced by phorbol ester or diacylglycerol. Aequorin and quin2 appear to report different aspects of Ca2+ homeostasis, and the absence of a quin2 signal may not be sufficient to establish that a metabolic pathway is "Ca2+-independent".     

Measurement of intracellular Ca2+ in cultured arterial smooth muscle cells using Fura-2 and digital imaging microscopy

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.     

Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

Spatial and temporal characteristics of the increase in intracellular Ca2+ induced in cytotoxic T lymphocytes by cellular antigen

The increase in intracellular Ca2+ concentration [( Ca2+]i) in cytolytic T lymphocytes in response to target cell binding was investigated by ratio image fluorescence microscopy. Ca2+ mobilization from intracellular stores occurred at a site distal to target cell contact and was transient. Extracellular Ca2+ influx resulted in an increase in [Ca2+]i that was prolonged and distributed more proximal to the target cell. Oscillations in [Ca2+]i were observed after target cell contact, although the periodicity was dependent on the presence of extracellular Ca2+. The Ag-specific reorientation of cytoplasmic granules occurred well after [Ca2+]i had begun to decline to a resting level, but was dependent on extracellular Ca2+. These studies indicate that the Ag-stimulated increase in [Ca2+]i exhibits considerable spatial and temporal variation, and that these characteristics are altered by the availability of extracellular Ca2+. The results also suggest that these changes in [Ca2+]i may play a role in the cytoplasmic events that accompany T cell-mediated cytolysis.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.     

Delayed activation of large-conductance Ca2+-activated K channels in hippocampal neurons of the rat.

We applied a fast concentration jump system to produce step changes in Ca2+ concentration [( Ca2+]i) on the cytoplasmic side of the inside-out membrane patch, excised from isolated rat hippocampal pyramidal neurons, and examined the time course of the activation phase of the large-conductance K channel (the BK channel; approximately 266 pS) after a step rise in [Ca2+]i. Diffusion of Ca2+ from the electrode tip to the cytoplasmic surface of the patch was estimated to be almost completed in 10 ms. After a step increase in [Ca2+]i from 0.04 to 3.2-1,000 microM, the activation of the K channel started after a clear latency of 280-18 ms and proceeded along a sigmoidal function. This was in sharp contrast with the rapid deactivation that began without delay and that was completed within 50 ms. The latency in activation was not accounted for by the binding of Ca2+ to EGTA in unstirred layers in the patch, since this binding was reported to be slow, taking up to seconds at physiological pH. Calmodulin (1 microM) did not affect the delay, the activation rate, or the steady-state current level. The calmodulin inhibitors W-7 and W-5 caused flickering of the single-channel current. These results indicate a delayed activation of the BK channel after a step rise in [Ca2+]i, suggesting that the BK current does not contribute to the repolarization of the action potential. Calmodulin is probably not involved in the activation process of the channel.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.     

Increases in Cytosolic Ca2+ in Parsley Mesophyll Cells Correlate with Leaf Senescence

The ability to maintain the cytoplasmic Ca2+ concentration ([Ca2+]cyt) at homeostatic levels has been examined during leaf senescence in detached parsley (Petroselinum crispum) leaves. Fluorescence ratiometric imaging of mesophyll cells isolated from parsley leaves at various senescence stages and loaded with the Ca2+ indicator fura-2 has revealed a distinct elevation of [Ca2+]cyt, which was positively correlated with the progress of leaf senescence. This initial increase of [Ca2+]cyt, which was first observed in cells isolated from 3-d-senescent leaves, occurred 1 d before or in parallel with changes in two established senescence parameters, chlorophyll loss and lipid peroxidation. However, the [Ca2+]cyt elevation followed by 2 d the initial increase in the senescence-associated proteolysis. Whereas the [Ca2+]cyt of nonsenescent cells remained at the basal level, the elevated [Ca2+]cyt of the senescent cells was a long-lasting effect. Experimental retardation of senescence processes, achieved by pretreatment of detached leaves with the cytokinin benzyladenine, resulted in maintenance of homeostatic levels of [Ca2+]cyt in cells isolated from 3-d-senescent leaves. These observations demonstrate for the first time to our knowledge a correlation between elevated [Ca2+]cyt and the process of senescence in parsley leaves. Such senescence-associated elevation of [Ca2+]cyt, which presumably results from a loss of the cell's capability to extrude Ca2+, may serve as a signal inducing subsequent deteriorative processes.     

Effect of t-butyl hydroperoxide on Ca2+ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca2+ ([Ca2+]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca2+]i in a concentration-dependent fashion between 50-250 microM with an EC50 of 100 microM. The [Ca2+]i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca2+. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca2+]i increase. The 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase in Ca2+ medium was reduced by 42 +/- 5% by pretreatment with 0.1 microM nicardipine but not by 10 microM verapamil, nifedipine, nimodipine or diltiazem, or by 50 microM La3+ or Ni2+. Pretreatment with 10 microM t-butyl hydroperoxide for 40 min did not affect 10 microM ATP-induced [Ca2+]i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca2+]i increase in PC12 cells by causing store Ca2+ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ influx via a nicardipine-sensitive pathway.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Glucose sensing of individual pancreatic beta-cells involves transitions between steady-state and oscillatory cytoplasmic Ca2+.

Glucose stimulation of individual pancreatic beta-cells is associated with a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) manifested either as large amplitude oscillations (0.2-0.5/min) or as a sustained increase. Determinants for the transitions between the basal and the two stimulated states have now been studied using dual-wavelength fluorometric measurements on individual ob/ob mouse beta-cells loaded with the Ca2+ indicator Fura-2. The transition from the basal state to large amplitude oscillations was induced by raising the glucose concentration to 7 mM or above. The frequencies and shapes of the [Ca2+]i cycles remained largely unaffected when raising glucose as high as 40 mM. However, in some cells the oscillatory pattern was transformed into a sustained increase of [Ca2+]i at high glucose concentrations. Although the peak values for the oscillations exceeded the steady-state increase, the time average [Ca2+]i was higher during the latter phase. Both types of glucose-induced transitions were facilitated by the presence of 1-100 nM glucagon. Protein kinase C activation by 10 nM of the phorbol ester TPA resulted in a transformation of the glucose-induced oscillations into a sustained increase of [Ca2+]i but the levels reached were considerably lower than obtained with glucose alone. It is concluded that the glucose sensing of the individual beta-cell is based on sudden transitions between steady-state and oscillating cytoplasmic Ca2+. It is these transitions rather than alterations of the oscillatory characteristics which determine the average [Ca2+]i regulating insulin release.