Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells.

The objective of this study was to evaluate the role of mitochondrial Ca2+ uptake (MCU) in modulation (shaping) of the glutamate (Glu)-induced changes in neuronal cytoplasmic Ca2+ ([Ca2+]i). In order to block MCU, nerve cells were treated with mitochondrial inhibitors (MI) inducing collapse of the mitochondrial potential (Delta Psim). Measurements of changes in [Ca2+]i were performed using either the low-affinity (fura-2FF) or high-affinity (fura-2) Ca2+ indicators. Loading of nerve cells with rhodamine 123 made it possible to monitor changes in Delta Psim. In the first series of experiments it was shown that blockade of MCU in fura-2FF-loaded cells with a cocktail of rotenone (2 microM)+oligomycin (2.5 microg/ml) greatly (2.53+/-0.4 times, n=61) increased the [Ca2+]i response to a 1-min Glu (100 microM) pulse. In fura-2-loaded cells, this increase was small (less than 1.3 times) or absent. In the second series of experiments, cocktails of rotenone+oligomycin or FCCP (1 microM)+oligomycin were applied during a prolonged Glu application. This produced strong mitochondrial depolarisation and an additional [Ca2+]i increase. In most cells the latter could be reversed or prevented by a removal of external Ca2+. The MI-induced additional [Ca2+]i increase was especially pronounced in cells loaded with fura-2FF. In some neurones a removal of external Ca2+ did not produce a decrease in [Ca2+]i during combined Glu+MI application, suggesting an impairment of [Ca2+]i extrusion mechanisms of these cells. The conclusion is drawn that MCU makes a considerable contribution to regulation of [Ca2+]i responses caused by Ca2+ influx via Glu-activated ionic channels. The reasons for a quantitative difference between [Ca2+]i responses observed in fura-2- and fura-2FF-loaded neurones are discussed.     

Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering

Mitochondrial Ca(2+) uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca(2+) uptake, changes in mitochondrial matrix free Ca(2+), and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca(2+)](out) and [Ca(2+)](mito) during Ca(2+) uptake in isolated cardiac mitochondria and identify two components of Ca(2+) influx. [Ca(2+)](mito) recordings revealed that the first, MCU(mode1), required at least 1 μM Ru360 to be completely inhibited, and responded to small Ca(2+) additions in the range of 0.1 to 2 μM with rapid and large changes in [Ca(2+)](mito). The second component, MCU(mode2), was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca(2+) uptake for large Ca(2+) additions in the range of 2 to 10 μM; however, it had little effect on steady-state [Ca(2+)](mito). MCU(mode1) mediates changes in [Ca(2+)](mito) of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca(2+) buffering in the matrix associated with this pathway. In contrast, the much higher Ca(2+) loads evoked by MCU(mode2) activate a secondary dynamic Ca(2+) buffering system consistent with calcium-phosphate complex formation. Increasing P(i) potentiated [Ca(2+)](mito) increases via MCU(mode1) but suppressed [Ca(2+)](mito) changes via MCU(mode2). The results suggest that the role of MCU(mode1) might be to modulate oxidative phosphorylation in response to intracellular Ca(2+) signaling, whereas MCU(mode2) and the dynamic high-capacity Ca(2+) buffering system constitute a Ca(2+) sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca(2+)](mito) itself but rather a downstream byproduct of total mitochondrial Ca(2+) loading.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death

Mitochondrial calcium uniporter (MCU) is a conserved Ca²⁺ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stressinduced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca²⁺ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca²⁺ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.     

Transport and control of Ca2+ by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca2+ and evidence for the involvement of more than one pool

Pigeon erythrocytes did not behave as expected from simple feedback mechanisms. The pool size for exchangeable cell Ca2+ was approximately proportional to the A23187-induced apparent 45Ca2+ influx ("J(in,app)") from 0.4 to 14 mumoles/min X l cell water at 184 microM external [Ca2+]. From earlier data, total cell 45Ca2+ was approximately proportional to J(in,app) from 10 to 120 mumoles/l X min. Thus there was no influx range where cell 45Ca2+ was held approximately constant. External [Ca2+] affected Ca2+ pool size independently of its effect of J(in,app). Trifluoperazine did not increase cell 45Ca2+ with or without A23187. In the presence of A23187, 45Ca2+ entered a pool early in the incubation which later became inaccessible to 45Ca2+ entry and exit. Lysolecithin addition produced an abrupt rise in cell 45Ca2+, much of which occupied a pool that quickly became inaccessible. The increased 45Ca2+ influx induced by lysolecithin dropped quickly and markedly with time. It is hard to explain inaccessible pool(s), especially in the presence of A23187 by membrane-bounded compartments. We suggest that nonexchangeable 45Ca2+ might be held by an energy-dependent binding protein(s).     

Cell-specific patterns of oscillating free Ca2+ in carbamylcholine-stimulated insulinoma cells

The effect of the muscarinic agonist carbamylcholine on cytoplasmic Ca2+ concentration ([Ca2+]i was examined at the single cell level in clonal pancreatic beta-cells (HIT). Cells were loaded with the indicator dye fura 2, and [Ca2+]i was measured by microfluorimetry. Carbamylcholine induced changes in Ca2+ that differed from cell to cell and provoked in some cells oscillatory Ca2+ fluctuations. During a transient, free Ca2+ rose to a peak within 1-3 s. The frequency of the oscillations increased with agonist concentration. Oscillations in [Ca2+]i occurred in the absence of external Ca2+. When cells were perifused for a sufficient period of time without carbamylcholine, near identical Ca2+ responses were elicited in each cell by successive applications of the agonist. Thus, individual cells displayed characteristic and reproducible Ca2+ responses with respect to amplitude, frequency, and shape of the transients as well as latency in onset of the initial Ca2+ rise. We propose that the biological response to a Ca2+ agonist in a given cell is not only determined by the frequency and amplitude of Ca2+ oscillations but is governed by the unique pattern of the Ca2+ signal of each cell, which may be termed "Ca2+ fingerprint."     

Trypanosomes and the solution to a 50-year mitochondrial calcium mystery

The ability of mitochondria to take up Ca(2+) was discovered 50 years ago. This calcium uptake, through a mitochondrial calcium uniporter (MCU), is important not only for the regulation of cellular ATP concentration but also for more complex pathways such as shaping Ca(2+) signals and the activation of programmed cell death. The molecular nature of the uniporter remained unknown for decades. By a comparative study of mitochondrial protein profiles of organisms lacking or possessing MCU, such as yeast in the former case and vertebrates and trypanosomes in the latter, two groups recently found the protein that possesses all the characteristics of the MCU. These results add another success story to the already substantial contributions of trypanosomes to mammalian biochemistry.     

Ca2+]i as a potential downregulator of alpha2beta1-integrin-mediated A2058 tumor cell migration to type IV collagen

We have investigated cellular Ca2+ regulation during A2058 human melanoma cell chemotaxis to type IV collagen (CIV). We have identified alpha2beta1-integrin as the primary mediator of A2058 cell response to CIV in vitro. Integrin ligation initiated a characteristic intracellular Ca2+ concentration ([Ca2+]i) response consisting of an internal release and a receptor-mediated Ca2+ entry. Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible internal Ca2+ stores while initiating a store-operated Ca2+ release (SOCR). CIV-mediated Ca2+ entry was additive to TG-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca2+ initiated a sustained influx. Elevated [Ca2+]i from TG-SOCR or ionophore significantly attenuated cell migration to CIV by recruiting the Ca2+/calcineurin-mediated signaling pathway. Furthermore, low [Ca2+]i induced by EGTA application in the presence of ionophore fully restored cell motility to CIV. Together, these results suggest that [Ca2+]i signaling accompanying A2058 cell response to alpha2beta1-integrin ligation is neither necessary nor sufficient and that elevated [Ca2+]i downregulates cell motility via a calcineurin-mediated mechanism in A2058 cell chemotaxis to CIV.     

Effect of methylglyoxal on intracellular calcium levels and viability in renal tubular cells

Methylglyoxal (2-oxopropanal), a physiological glucose metabolite, is a highly reactive dicarbonyl compound that can induce stress in cells and cause apoptotic cell death. This study examines the early signaling effects of methylglyxal on renal cells. It was found that methylglyoxal caused a slow and sustained rise of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (EC50=1.8 mM). Methylglyoxal also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 80%. Depletion of intracellular Ca2+ stores with thapsigargin (TG), an endoplasmic reticulum (ER) Ca2+ pump inhibitor, did not affect methylglyoxal's effect. In Ca2+-free medium, the methylglyoxal-induced [Ca2+]i rise was abolished by depleting stored Ca2+ with carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Methylglyoxal-caused [Ca2+]i rise in the Ca2+-containing medium was not affected by modulation of protein kinase C activity, presence of voltage-gated Ca2+ channel blockers, or preincubation with thiol-containing antioxidants. U73122, an inhibitor of phospholipase C, abolished ATP (but not methylglyoxal)-induced [Ca2+]i rise. Furthermore, the [Ca2+]i-elevating effect of methylglyoxal was cell type-dependent, because methylglyoxal failed to cause [Ca2+]i rises in CHO-K1, neutrophils, or platelets. Pretreatment with methylglyoxal for 0-24 h decreased cell viability in a concentration- and time-dependent manner. Meanwhile, methylglyoxal-induced cell death involved apoptotic and necrotic events, the former being the dominant. These findings suggest that methylglyoxal induced a significant rise in [Ca2+]i in Madin-Darby canine kidney (MDCK) renal tubular cells by stimulating both extracellular Ca2+ influx and CCCP-sensitive intracellular Ca2+ release via as yet unidentified mechanisms. The cell type-specific Ca2+ signaling may play an important role in the early process of cytotoxic action of methylglyoxal.     

Comparative studies of intracellular Ca2+ in strongly and weakly metastatic rat prostate cancer cell lines

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca2+ measurements, we investigated Ca2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca2+ channels in either cell line. A Ca2+-dependent component was also absent in the voltage-activated outward K+ currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca2+ and the ability of the two cell lines to regulate intracellular Ca2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K+ decreased intracellular Ca2+ in the AT-2 but had no effect on intracellular Ca2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca2+ increased intracellular Ca2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca2+ into the AT-2 cells was suppressed by both CdCl2 (100-300 microM) and SKF-96365 (10-30 microM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca2+ influx mechanism that is present in the weakly metastatic AT-2 cells.     

Diethylstilbestrol-induced estrogen receptor-dependent [Ca2+]i rises and apoptosis in Chinese hamster ovary (CHO) cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations>or=1 proportional, variant increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. In Ca2+-free medium, after pretreatment with 50 proportional, variant DES, 1 proportional, variant thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca2+]i rises. At a concentration of 5 proportional, variant, DES increased cell viability. At concentrations of 100-200 microM, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 microM DES on viability was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 microM)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 microM). ICI-182,780 did not affect 5 microM DES-induced increase in viability but partly reversed 100 microM DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca2+]i rises by stimulating estrogen receptors leading to Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca2+-dependent pathway.     

Effects of Cd2+ upon Ca2+ fluxes and proliferation in concanavalin A-stimulated lymphocytes

The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.     

Sites and mechanisms of Ca2+ movement in non-excitable cells

The level of free cytosolic Ca2+ ([Ca2+]i) in cells is firmly established as a second messenger alternative to the cyclic nucleotides. Regulation of the activity of Ca2+ requires the use of membrane transporters of various types which can be classified in terms of their transport rate; channels (fast), carriers (intermediate) and pumps (slow). In general channels are used to elevate [Ca2+]i whereas pumps decrease [Ca2+]i. At physiological membrane potential and Na+ gradients, carriers such as the 3Na+/Ca2+ exchanger also deplete the cell of Ca2+. The carriers could also function in a reverse mode especially with plasma membrane depolarization. Intracellular organelles which can incorporate Ca2+ from and return Ca2+ to the cytosol play a central role in determining [Ca2+]i in resting and stimulated cells. In the resting cell they function as the major Ca2+ buffering system while in the stimulated cell they participate in the dynamic control of [Ca2+]i. The collection of papers in this volume discusses the mechanisms of modulation of cell Ca2+ by these organelles.     

Evidence of reciprocal regulation between the high extracellular calcium and RANKL signal transduction pathways in RAW cell derived osteoclasts

During bone resorption, osteoclasts are exposed to high Ca2+ concentrations (up to 40 mM). The role of high extracellular Ca2+ in receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast survival and their functional interrelationship is unclear. In this study, we show that RANKL enhances osteoclast tolerance to high extracellular Ca2+ by protecting the cell from cell death in a dose dependent manner. We have provided evidence that RANKL does this by attenuating high extracellular Ca2+-induced Ca2+ elevations. Moreover, we have found that high extracellular Ca2+-induced cell death was partially inhibited by a caspase-3 inhibitor, suggesting caspase-3-mediated apoptosis is involved. Conversely, using reporter gene assays and Western blot analysis, we have demonstrated that high extracellular Ca2+ desensitizes the RANKL-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK), and inhibits constitutive and RANKL-stimulated ERK phosphorylation, indicating a negative feed-back mechanism via specific RANKL signaling pathways. Taken together, this study provides evidence for a reciprocal regulation between high extracellular Ca2+ and RANKL signaling in RAW cell derived osteoclasts. Our data imply a cross talk mechanism of extracellular Ca2+ on osteoclast survival through the regulation of RANKL.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.     

Signal transduction and ligand-receptor dynamics in the neutrophil. Ca2+ modulation and restoration

Intracellular Ca2+ rises when neutrophils are stimulated with formyl peptide ligands. There is enough Ca2+ released to complex approximately 200 microM Quin 2, (220 +/- 90 microM, 7 donors). This result is interpreted in terms of a fixed storage pool of Ca2+ of 44 pmol/10(6) cells. When extracellular Ca2+ is removed from the medium with 5 mM EGTA (final pH 7.4) just prior to cell stimulation, neither the magnitude nor the early time course of the Quin 2 response to formyl peptide is dramatically influenced. This result supports the concept that neither Ca2+ influx nor efflux, which are elevated in stimulated cells, contributes in a major way to the free Ca2+ pool which is monitored by Quin 2 during the early activation phase of cell responses. We have used intracellular Quin 2, and extracellular Ca2+ without the use of EGTA or ionophores to manipulate the levels of intracellular Ca2+. This is accomplished by depleting cells of intracellular Ca2+ by loading with Quin 2 in the absence of Ca2+. Intracellular Ca2+ is modulated by adding back Ca2+ to the medium. Using simultaneous analyses of cell function and Quin 2 fluorescence, we find that at least two aspects of cellular responsiveness (degranulation and O2- production) depend upon the level of available Ca2+. In contrast, the first phase, at least, of a biphasic rapid light scattering response which is related to actin polymerization is independent of Ca2+. We find that the Ca2+- sensitive cell responses can be partially restored in Ca2+-depleted cells if Ca2+ is provided within 30 s, a period which may reflect the putative lifetime of the transiently active ligand-receptor complex.     

Calcium homeostasis in trigeminal ganglion cell bodies

In primary sensory afferent neurons, Ca2+ plays a vital role in the regulation of cellular processes including receptor and synaptic plasticity, neurotransmitter and trophic factor release and gene regulation. Current understanding of the mechanisms underlying Ca2+ homeostasis of primary sensory afferent neurons is mostly derived from studies on dorsal root ganglia and nodose ganglia neuron cell bodies. Little is known about Ca2+ homeostasis in trigeminal ganglion neurons (TGNs). To determine what cellular processes contribute to electrically-evoked Ca2+ transients in TGNs, we probed Ca2+ regulatory mechanisms in TGN cell bodies from the ophthalmic division with a panel of pharmacological reagents. Ca2+ transients were evoked in fura-2 loaded TGNs by depolarizing the plasma membrane with brief (500 ms) puffs of 50 mM KCl. Cyclopiazonic acid (CPA; 5 microM), an inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), significantly decreased the peak amplitude, and slowed the decay, of the KCl-evoked Ca2+ transients in TGNs. The mitochondrial protonophore, carbonyl cyanide 3-chloro-phenylhydrazone (CCCP; 5 microM) significantly increased the peak amplitude of KCl-evoked Ca2+ transients. These data demonstrate that Ca2+ stores do play a major role in Ca2+ homeostasis in TGN cell bodies. To determine the role of the sodium-calcium exchanger (NCX) in KCl-evoked Ca2+ transients in TGNs, we inhibited the exchanger with KB-R7943 (10 microM), or by replacing Na+ with Li+. NCX inhibition did not affect either the peak amplitude or the decay kinetics of the KCl-evoked Ca2+ transients. Therefore, the NCX does not play a significant role in removing cytosolic Ca2+ from TGNs. To test whether the plasma membrane calcium-ATPase (PMCA) contributes to Ca2+ extrusion, we inhibited its activity by a shift to alkaline pH (9.0). At pH 9.0, both the peak amplitude and decay time of the KCl-evoked Ca2+ transient were increased significantly. These data suggest that, in TGNs, the PMCA is the major mechanism for removing cytosolic Ca2+ following electrical activity.     

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.