Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1

The relevant influx pathway for stimulated Ca²⁺ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd³⁺, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca²⁺ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca²⁺ influx into CFPAC-1 cells was stimulated by either ATP (10^–4 and 10^–5 mol/l), carbachol (CCH, 10^–4 mol/l) or thapsigargin (TG, 10^–8 mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca²⁺]_i transient reflects Ca²⁺ influx into these cells, we investigated the influence of Flu and Gd³⁺ on the level of the stimulated [Ca²⁺]_i plateau. (2) The fura-2 Mn²⁺-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the refilling period after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd³⁺ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd³⁺ were potent inhibitors of the stimulated Ca²⁺ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca²⁺]_i plateau in a concentration dependent manner, with an IC₅₀ value of 33 mol/l (n = 6). Similar results were obtained for the CCH-(n = 5) and the TG-induced (n = 5) [Ca²⁺]_i plateau. Gd³⁺ concentration dependently inhibited the stimulated Ca²⁺ plateau. A complete block of the ATP-induced [Ca²⁺]_i plateau was seen at 0.5 mol/l (ATP 10^–5 mol/l, n = 8). The second approach showed that Flu (10^–4 mol/l) completely inhibited the ATP- (10^–5 mol/l, n = 3), CCH-(10^–4 mol/l, n = 4) and TG-(10^–8 mol/l, n = 3)-induced fura-2 Mn²⁺ quench. Gd³⁺ also inhibited the fura-2 Mn²⁺-quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd³⁺ (n = 8) inhibited the refilling of the ATP-sensitive intracellular Ca²⁺ store. These results show that inhibitors of non-selective cation currents in other epithelial preparations are potent inhibitors of stimulated Ca²⁺ influx in CFPAC-1 cells. Whether this inhibitory effect concerns a non-selective cation channel remains to be established.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Excess divalent cations activate Ca2+-mobilizing receptors in pancreatic acinar cells

In single, enzymatically dissociated, rat pancreatic acinar cells, external application of excess divalent cations (Ca²⁺, Sr²⁺, Ba²⁺, Ni²⁺ and Mg²⁺ over 50 mM) induced Ca²⁺-dependent current responses monitored with the whole-cell recording technique. Inclusion of either EGTA, heparin or GDP[S] in the internal solution or treatment of acinar cells with a phorbol ester abolished the divalent-cation-induced responses. In contrast, internal inositol trisphosphate (InsP ₃) or GTP[S] potentiated the responses. The results indicate that excess divalent cations activate membrane surface receptors or receptor/effector complexes, thereby inducing InsP ₃-mediated Ca²⁺ mobilization. The mechanism may be due to modulation of the receptors by changes in electrical profile through indirect action of divalent cations on membrane surface charges, i. e. neutralization of anionic charges. This proposal was supported by the evidence that the trivalent cation, La³⁺, and the polyvalent cation, protamine, both at much lower concentrations, could induce Ca²⁺-dependent responses, which were abolished by internal application of heparin, GDP[S] or a high concentration of EGTA or by protein kinase C activation with a phorbol ester.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

G-Protein- and capacitatively regulated Ca2+ entry pathways are activated by muscarinic receptor stimulation in a human submandibular ductal cell line

In the human submandibular ductal cell line (HSG) thapsigargin and carbachol stimulated Ca²⁺ release from the internal Ca²⁺ pool, resulting in the activation of capacitatively regulated Ca²⁺ entry (CRCE). This entry pathway was permeant to both Ca²⁺ and Mn²⁺, blocked by Ni²⁺ and insensitive to the muscarinic antagonist, atropine. Carbachol also stimulated an increase in cytosolic [Ca²⁺] in internal Ca²⁺-pool-depleted (i.e.thapsigargin-treated) cells which was dependent on the presence of external Ca²⁺ and blocked by Ni²⁺, demonstrating that it was due to Ca²⁺ entry. However, under the same experimental conditions, carbachol was unable to stimulate Mn²⁺ entry. Additionally, this latter carbachol-stimulated Ca²⁺ entry pathway was blocked by atropine. Pretreatment of HSG cells with AlF₄-increased basal rates of Mn²⁺ entry due to CRCE activation, but attenuated carbachol-stimulated Ca²⁺ entry into thapsigargin-treated cells. The data suggest that two distinct divalent cation entry pathways are activated in muscarinic-receptor-stimulated HSG cells; a CRCE mechanism, permeable to both Mn²⁺ and Ca²⁺, and a second entry mechanism, permeable only to Ca²⁺. The latter does not depend on internal pool depletion, but appears to be regulated via G-protein activation.We thank Dr. Bruce Baum for his encouragement and support during the course of this work. We also thank our colleagues for their cooperation and assistance.     

Induction of Ca2+ oscillations by selective,U73122-mediated,depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca²⁺ sequestering and releasing properties of internal Ca²⁺ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca²⁺ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P ₃]-sensitive, but not the Ins(1,4,5)P ₃-insensitive, Ca²⁺ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca²⁺ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av), which was largely independent of external Ca²⁺. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonistevoked [Ca²⁺]_i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca²⁺-ATPase activity resulted in a [Ca²⁺]_i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P ₃- casu quo agonist-sensitive internal Ca²⁺ store, whereas thapsigargin affects both the Ins(1,4,5)P ₃-sensitive and -insensitive Ca²⁺ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca²⁺]_i. The U73122-evoked oscillations were abolished in the absence of external Ca²⁺. The ability of U73122 to generate external Ca²⁺-dependent Ca²⁺ oscillations suggests that depletion of the agonistsensitive store leads to an increase in Ca²⁺ permeability of the plasma membrane and that the Ins(1,4,5)P ₃-insensitive Ca²⁺ pool is necessary for the Ca²⁺ oscillations.     

Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation

Two effects of Mn²⁺ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence of absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a latch-like state, and the other involves direct Mn²⁺ activation of contractile proteins. Cells activated by Mn²⁺ in the presence of ATP and the absence of Ca²⁺, Mg²⁺ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn²⁺ contracting solution to prevent protein oxidation by Mn²⁺, the cells still contracted when exposed to Mn²⁺, but relaxed rapidly when the Mn²⁺ was removed. In the presence of DTT both the Mn²⁺ activation and the relaxation following removal of Mn²⁺ were more rapid than normal Ca²⁺-activated contractions and relaxations. The skinned fibers activated by Mn²⁺ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50mM) and then contracted again in response to Ca²⁺ and relaxed normally. The Mn²⁺ activation was not associated with myosin light chain phosphorylation, in contrast to Ca²⁺-activated contractions.A preliminary report of this work was given at the Biophysical Society Meeting, February 1987: Hoar PE, Kerrick WGL (1987) Mn²⁺ activates skinned smooth muscle cells directly without myosin light chain phosphorylation and by reversible oxidation. Biophys J 51:332a     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

Multiple mechanisms of manganese-induced quenching of fura-2 fluorescence in rat mast cells

Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca²⁺]_i) were used to study Mn²⁺ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (I _CRAC for calcium release-activated calcium current), supports a small but measurable Mn²⁺ current. In the presence of intracellular BAPTA, a Mn²⁺ current through I _CRAC was recorded in isotonic MnCl₂ (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca²⁺. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn²⁺ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn²⁺ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn²⁺ was taken up into calcium stores and was subsequently co-released with Ca²⁺ by Ca²⁺-mobilizing agonists.     

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

 Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel. We tested whether a gradient of intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlies the horizontal polarization of K⁺ channel activity. [Ca²⁺]_i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca²⁺]_i indicated that a horizontal gradient exists, with [Ca²⁺]_i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca²⁺]_i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K⁺ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca²⁺]_i to test whether its maintenance is due to an uneven distribution of Ca²⁺ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La³⁺ alone or together with charybdotoxin, a specific blocker of Ca²⁺-sensitive K⁺ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca²⁺ influx into the cell body and the lamellipodium with La³⁺ was followed by a decrease of [Ca²⁺]_i at both cell poles. This points to Ca²⁺ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn²⁺ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca²⁺]_i. We hypothesize that the polarized distribution of intracellular Ca²⁺ stores contributes significantly to the generation of a gradient of [Ca²⁺]_i. Received: 22 July 1996 / Received after revision: 17 December 1996 / Accepted: 10 January 1997     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

Spatial distribution of intracellular,free Ca2+ in isolated rat parotid acini

The spatial distribution of intracellular, free Ca²⁺ ([Ca²⁺]_i) in rat parotid acini was measured by imaging fura-2 fluorescence from individual acinar cells by means of a digital imaging microscope. Upon cholinergic stimulation in a Krebs-Ringer bicarbonate buffer at (37° C), [Ca²⁺]_i increased synchronously at both the basolateral and luminal membranes as well as in all cells of the secretory endpiece, reaching peak [Ca²⁺]_i levels 1 s after stimulation. Atropine addition caused a rapid down-regulation of [Ca²⁺]_i, which, however, never reached prestimulatory levels. When acini were stimulated in a medium containing 5 nM Ca²⁺, the Ca²⁺ mobilization arising from internal pools caused an increase in [Ca²⁺]_i predominantly near the basolateral area, where the endoplasmic reticulum is located, and standing Ca²⁺ gradients were observed for up to 10 s. A mathematical model is developed to simulate the time courses of the Ca²⁺ profiles through the cytoplasm using estimated values of the Ca²⁺ diffusion coefficients and the cytosolic Ca²⁺ buffering capacity. It is concluded that under physiological conditions, the Ca²⁺ release from the endoplasmic reticulum is responsible for the activation of the basolaterally located K⁺ channels. Furthermore, Ca²⁺ influx from the interstitium is responsible for much of the rise in [Ca²⁺]_i near the luminal membranes, where the Cl^– channels are supposed to be located.     

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca²⁺ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10^–5 mol/l)-induced intracellular Ca²⁺ transient, (2) a reduced fura-2 Mn²⁺-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca²⁺ as a function of membrane voltage, we measured simultaneously the intracellular Ca²⁺ activity ([Ca²⁺]_i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT₂₉ cells. Ca²⁺ influx was either stimulated by ATP (10^–5 mol/l) or thapsigargin (TG, 10^–8 mol/l). After [Ca²⁺]_i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca²⁺]_i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca²⁺]_i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca²⁺]_i (n=9). Under extracellular Ca²⁺-free conditions [Ca²⁺]_i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca²⁺ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca²⁺]_i. This may be due either to a change of driving force for Ca²⁺ into the cell, or may reflect voltage-dependent regulation of the respective Ca²⁺ entry mechanism.     

Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2+]i oscillations in rat glioma cells

 The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca²⁺ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca²⁺]_i oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2–20 μM), bisindolylmaleimide (1 μM) or chelerythrine (1 μM), irreversibly suppressed thrombin-induced [Ca²⁺]_i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 μM) or thapsigargin (1 μM) (inhibitors of sarco/endoplasmic reticulum Ca²⁺-ATPase) caused no [Ca²⁺]_i response, indicating that intracellular Ca²⁺ stores were completely empty. We tested whether PKC affects the refilling of internal Ca²⁺ stores in thrombin-stimulated cells, by monitoring the amount of Ca²⁺ release caused by t-BuBHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca²⁺ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca²⁺ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator oleoyl acetyl glycerol. Furthermore, the capacitative Ca²⁺ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca²⁺ entry was induced in these experiments by depletion of Ca²⁺ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of intracellular stores did not influence the Ca²⁺ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor stimulation activation of PKC is required to maintain the refilling of intracellular Ca²⁺ stores for sustained [Ca²⁺]_i oscillations. Thus, the control by PKC of the capacitative Ca²⁺ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca²⁺-ATPase inhibition or by activation of the thrombin receptor. Received: 5 July 1996 / Received after revision and accepted: 7 October 1996     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells

There is increasing evidence that some agonists not only induce intracellular Ca²⁺ increases, due to store release and transmembranous influx, but also that they stimulate Ca²⁺ efflux. We have investigated the agonist-stimulated response on the intracellular Ca²⁺ activity ([Ca²⁺]_i) in the presence of thapsigargin (10^–8 mol/l, TG) in HT₂₉ and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10^–7–10^–3 mol/l, n=9), carbachol (10^–6–10^–3 mol/l, n=5) and neurotensin (10^–10–10^–7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca²⁺]_i in the presence of TG. Similar results were obtained with HT₂₉ cells. This decrease of [Ca²⁺]_i could be caused by a reduced Ca²⁺ influx, either due to a reduced driving force for Ca²⁺ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca²⁺ permeability. Using the fura-2 Mn²⁺ quenching technique we demonstrated that ATP did not slow the TG-induced Mn²⁺ quench. This indicates that the agonist-induced [Ca²⁺]_i decrease in the presence of TG was not due to a reduced influx of Ca²⁺ into the cell, but rather due to stimulation of Ca²⁺ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of –49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl^– conductance. This was expected, since at this time after TG stimulation all Ca²⁺ agonists still liberated some [Ca²⁺]_i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca²⁺]_i decrease cannot be explained by the reduction of the driving force for Ca²⁺ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca²⁺]_i increase in the presence of TG and concomitantly induced Cl^– and K⁺ conductances. We conclude that the agonist-induced decrease of [Ca²⁺]_i in the presence of TG probably unmasks a stimulation of [Ca²⁺]_i export.     

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile

Here, we describe novel mechanisms limiting a toxic cytosolic Ca²⁺ rise during adenosine 5′-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca²⁺ signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca²⁺ extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca²⁺ influx. Alternative inhibition of Ca²⁺ extrusion by carboxyeosin had a much smaller effect on Ca²⁺ influx. The coordinated metabolic inhibition of Ca²⁺ influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca²⁺ oscillations, which was due to the inhibition of Ca²⁺ release from internal stores. This could be particularly important for limiting Ca²⁺ toxicity during periods of hypoxia. In contrast, metabolic control of Ca²⁺ influx and Ca²⁺ release from internal stores spectacularly failed to prevent large toxic Ca²⁺ responses induced by bile acids—activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca²⁺ release from internal stores nor Ca²⁺ influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.