Calcium channel blockers inhibit galvanotaxis in human keratinocytes

Directed migration of keratinocytes is essential for wound healing. The migration of human keratinocytes in vitro is strongly influenced by the presence of a physiological electric field and these cells migrate towards the negative pole of such a field (galvanotaxis). We have previously shown that the depletion of extracellular calcium blocks the directional migration of cultured human keratinocytes in an electric field (Fang et al., 1998; J Invest Dermatol 111:751-756). Here we further investigate the role of calcium influx on the directionality and migration speed of keratinocytes during electric field exposure with the use of Ca(2+) channel blockers. A constant, physiological electric field strength of 100 mV/mm was imposed on the cultured cells for 1 h. To determine the role of calcium influx during galvanotaxis we tested the effects of the voltage-dependent cation channel blockers, verapamil and amiloride, as well as the inorganic Ca(2+) channel blockers, Ni(2+) and Gd(3+) and the Ca(2+) substitute, Sr(2+), on the speed and directionality of keratinocyte migration during galvanotaxis. Neither amiloride (10 microM) nor verapamil (10 microM) had any effect on the galvanotaxis response. Therefore, calcium influx through amiloride-sensitive channels is not required for galvanotaxis, and membrane depolarization via K(+) channel activity is also not required. In contrast, Sr(2+) (5 mM), Ni(2+) (1-5 mM), and Gd(3+) (100 microM) all significantly inhibit the directional migratory response to some degree. While Sr(2+) strongly inhibits directed migration, the cells exhibit nearly normal migration speeds. These findings suggest that calcium influx through Ca(2+) channels is required for directed migration of keratinocytes during galvanotaxis and that directional migration and migration speed are probably controlled by separate mechanisms.     

TRPC6 contributes to the Ca(2+) leak of human erythrocytes.

Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), Na(+) and K(+). Excessive activation of these channels upon energy depletion, osmotic shock, Cl(-) depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6(-/-) mice. According to flow-cytometry, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca(2+) entry. Moreover, free Ca(2+) concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl(-)-depleted TRPC6(-/-) erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca(2+)-induced suicidal death.     

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

A protein kinase G-sensitive channel mediates flow-induced Ca(2+) entry into vascular endothelial cells.

The hemodynamic force generated by blood flow is considered to be the physiologically most important stimulus for the release of nitric oxide (NO) and prostacyclin (PGI(2)) from vascular endothelial cells (1). NO and PGI(2) then act on the underlying smooth muscle cells, causing vasodilation and thus lowering blood pressure (2, 3). One critical early event occurring in this flow-induced regulation of vascular tone is that blood flow induces Ca(2+) entry into vascular endothelial cells, which in turn leads to the formation of NO (4, 5). Here we report a mechanosensitive Ca(2+)-permeable channel in vascular endothelial cells. The activity of the channel was inhibited by 8-Br-cGMP, a membrane-permeant activator of protein kinase G (PKG), in cell-attached membrane patches. The inhibition could be reversed by PKG inhibitor KT5823 or H-8. A direct application of active PKG in inside-out patches blocked the channel activity. Gd(3+), Ni(2+), or SK&F-96365 also inhibited the channel activity. A study of fluorescent Ca(2+) entry revealed a striking pharmacological similarity between the Ca(2+) entry elicited by flow and the mechanosensitive Ca(2+)-permeable channel we identified, suggesting that this channel is the primary pathway mediating flow-induced Ca(2+) entry into vascular endothelial cells.     

A mechanosensitive cation channel in endothelial cells and its role in vasoregulation

Ca2+ is an important intracellular second messenger in signal transduction of endothelial cells. It has long been recognized that a mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel may increase intracellular Ca2+ level in endothelial cells. A recent finding is that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells. Another important finding is that the opening of this mechanosensitive channel by KT5823 leads to endothelium-dependent vascular dilation. Therefore, it appears that this channel may play a crucial role in the regulation of vascular tone.     

Inhibition by cytoplasmic nucleotides of a new cation channel in freshly isolated human and rat type II pneumocytes

Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with beta-escin. Excised patch-clamp experiments revealed activation by Ca(2+) concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (>10 microM) at the extracellular side did not inhibit. The channel was equally permeable for K(+) and Na(+) but was essentially impermeable for Cl(-), Ca(2+), and Mg(2+). It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP approximately ADP (EC(50) ATP > adenosine > cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-gamma-S. GTP did not inhibit. Cd(2+) blocked the channel with an EC(50) approximately 55.5 nM. We conclude that type II cells express a Ca(2+)-dependent, nucleotide-inhibited, nonselective, and Ca(2+)-impermeable cation channel (NSC(Ca/AMP)) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NSC(Ca/AMP). Potential physiological roles are discussed.     

A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat

To determine the transporters responsible for toxic Na(+) influx in wheat (Triticum aestivum), root plasma membrane preparations were screened using the planar lipid bilayer technique as an assay for Na(+)-permeable ion channel activity. The predominant channel in the bilayer was a 44-pS channel that we called the nonselective cation (NSC) channel, which was nonselective for monovalent cations and weakly voltage dependent. Single channel characteristics of the NSC channel were compared with (22)Na(+) influx into excised root segments. Na(+) influx through the NSC channel resembled (22)Na(+) influx in its partial sensitivity to inhibition by Ca(2+), Mg(2+), and Gd(3+), and its insensitivity to all other inhibitors tested (tetraethylammonium, quinine, Cs(+), tetrodotoxin, verapamil, amiloride, and flufenamate). Na(+) influx through the NSC channel also closely resembled an instantaneous current in wheat root protoplasts (S.D. Tyerman, M. Skerrett, A. Garill, G.P. Findlay, R. Leigh [1997] J Exp Bot 48: 459-480) in its permeability sequence, selectivity for K(+) over Na(+) (approximately 1.25), insensitivity to tetraethylammonium, voltage independence, and partial sensitivity to Ca(2+). Comparison of tissue, protoplast (S.D. Tyerman, M. Skerrett, A. Garill, G.P. Findlay, R. Leigh [1997] J Exp Bot 48: 459-480), and single- channel data indicate that toxic Na(+) influx is catalyzed by a single transporter, and this is likely to be the NSC channel identified in planar lipid bilayers.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

The externally derived portion of the hyperosmotic shock-activated cytosolic calcium pulse mediates adaptation to ionic stress in suspension-cultured tobacco cells

The influx of Ca(2+) into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca(2+)(cyt)) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and (45)Ca influx measurements using inhibitors of Ca(2+) influx (Gd(3+) and the Ca(2+)-selective chelator EGTA), and modulators of organellar Ca(2+) release (phospholipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca(2+) within 10 min) and complex Ca(2+)(cyt) pulse in tobacco cells, deriving both from Gd(3+)-sensitive externally derived Ca(2+) influx and from U73122- and neomycin-sensitive Ca(2+) release from an organelle. To determine whether each of the two components of this brief Ca(2+) signal regulate adaptation to hyperosmotic shock, the Ca(2+) pulse was modified by the addition of Gd(3+), U73122, neomycin, or excess Ca(2+), and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd(3+) treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca(2+) increased growth in salt but not in sorbitol. In contrast, exposure to inhibitors of IP(3) formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca(2+) influx and Ca(2+) release from an internal Ca(2+) depot, only Ca(2+) influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca(2+) signaling.     

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.     

The voltage-independent cation channel in the plasma membrane of wheat roots is permeable to divalent cations and may be involved in cytosolic Ca2+ homeostasis

A voltage-independent cation (VIC) channel has been identified in the plasma membrane of wheat (Triticum aestivum) root cells (P.J. White [1999] Trends Plant Sci 4: 245-246). Several physiological functions have been proposed for this channel, including roles in cation nutrition, osmotic adjustment, and charge compensation. Here, we observe that Ca(2+) permeates this VIC channel when assayed in artificial, planar lipid bilayers, and, using an energy barrier model to describe cation fluxes, predict that it catalyzes Ca(2+) influx under physiological ionic conditions. Thus, this channel could participate in Ca(2+) signaling or cytosolic Ca(2+) homeostasis. The pharmacology of (45)Ca(2+) influx to excised wheat roots and inward cation currents through the VIC channel are similar: Both are insensitive to 20 microM verapamil or 1 mM tetraethylammonium, but inhibited by 0.5 mM Ba(2+) or 0.5 mM Gd(3+). The weak voltage dependency of the VIC channel (and its lack of modulation by physiological effectors) suggest that it will provide perpetual Ca(2+) influx to root cells. Thus, it may effect cytosolic Ca(2+) homeostasis by contributing to the basal Ca(2+) influx required to balance Ca(2+) efflux from the cytoplasm through ATP- and proton-coupled Ca(2+) transporters under steady-state conditions.     

Effect of cell ageing on Ca2+ influx into human red cells

The effect of cell ageing on Ca2+ entry was studied in this work, using sub-populations of young and old human red cells, separated by stringent percoll density gradients. Additionally, the influence of an osmotic gradient was investigated as a model for shear stress. Ca2+ entry was assessed at 37 degrees C, under conditions where the Ca2+ pump was either inhibited by NaVO3 (0.5-10 mM) or inactivated by ATP depletion. The entry was linear with time up to 1 h. No differences in Ca2+ influx between the two sub-populations were detected in isotonic Na(+)-medium. In contrast, after incubation in anisosmotic media, Ca2+ entry into old cells was significantly higher than into younger cells. In hypotonic Na(+)-medium, the entry into old cells was not affected by La3+ (10 microM) whilst it was partially blocked by Gd3+ at a similar level (half-maximal effect attained with about 1 microM Gd3+). The entry into young cells was only slightly stimulated by these lanthanides at low concentrations (10 microM), regardless of the tonicity of incubation medium. Further increasing Gd3+ levels above 10 microM markedly enhanced Ca2+ entry into both cell types. The selective blockade of Ca2+ influx by low Gd3+ concentrations suggests presence of mechano-sensitive channels, that become preferentially activated in old cells. Activation of these channels during in-vivo microcirculation may help to explain the increased Ca2+ content of senescent cells.     

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.     

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Mitochondria play a critical role in shaping the exocytotic response of rat pancreatic acinar cells

We have previously demonstrated [M. Campos-Toimil, T. Bagrij, J.M. Edwardson, P. Thomas, Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry, Curr. Biol. 12 (2002) 211-215] that in rat pancreatic acinar cells, Gd(3+)-sensitive Ca(2+) entry is instrumental in governing which second messenger pathways control secretory activity. However, in those studies, we were unable to demonstrate a significant increase in cytoplasmic [Ca(2+)] during agonist application as a result of this entry pathway. In the present study, we combined pharmacology with ratiometric imaging of fura-2 fluorescence to resolve this issue. We found that 2 microM Gd(3+) significantly inhibits store-mediated Ca(2+) entry. Furthermore, both the protonophore, CCCP (5 microM) and the mitochondrial Ca(2+)-uptake blocker, RU360 (10 microM), led to an enhancement of the plateau phase of the biphasic Ca(2+) response induced by acetylcholine (1 microM). This enhancement was completely abolished by Gd(3+); and as has been previously shown for Gd(3+), RU360 led to a switch to a wortmannin-sensitive form of exocytosis. Using MitoTracker Red staining we found a close association of mitochondria with the lateral plasma membrane. We propose that in rat pancreatic acinar cells, capacitative Ca(2+) entry is targeted directly to mitochondria; and that as a result of Ca(2+) uptake, these mitochondria release "third" messengers which both enhance exocytosis and suppress phosphatidylinositol 3-kinase-dependent secretion.