Agonist-sensitive Stores Research Articles

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Inhibitors of Bcl-2 protein family deplete ER Ca2+ stores in pancreatic acinar cells

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca+2] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca+2] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca+2] and endoplasmic reticulum [Ca+2]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP3Rs and RyRs and substantially reduced by strong [Ca2+] buffering. Inhibition of IP3Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca+2] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.

Homocysteine induces caspase activation by endoplasmic reticulum stress in platelets from type 2 diabetics and healthy donors

Diabetes mellitus is a disease characterised by hyperglycaemia and associated with several cardiovascular disorders, including angiopathy and platelet hyperactivity, which are major causes of morbidity and mortality in type 2 diabetes mellitus. In type 2 diabetic patients, homocysteine levels are significantly increased compared with healthy subjects. Hyperhomocysteinaemia is an independent risk factor for macro- and microangiopathy and mortality. The present study is aimed to investigate the effect of homocysteine on platelet apoptosis. Changes in cytosolic or intraluminal free Ca(2+) concentration were determined by fluorimetry. Caspase activity and phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) were explored by Western blot. Our results indicate that homocysteine releases Ca(2+) from agonist sensitive stores, enhances eIF2alpha phosphorylation at Ser(51) and activates caspase-3 and -9 independently of extracellular Ca(2+). Homocysteine induced activation of caspase-3 and -9 was abolished by salubrinal, an agent that prevents endoplasmic reticulum (ER) stress-induced apoptosis. Homocysteine-induced platelet effects were significantly greater in type 2 diabetics than in healthy subjects. These findings demonstrate that homocysteine induces ER stress-mediated apoptosis in human platelets, an event that is enhanced in type 2 diabetic patients, which might be involved in the pathogenesis of cardiovascular complications associated with type 2 diabetes mellitus.

A small component of the endoplasmic reticulum is required for store-operated Ca2+ channel activation in liver cells: evidence from studies using TRPV1 and taurodeoxycholic acid

The question of whether the activation of SOCs (store-operated Ca(2+) channels) requires the whole or part of the ER (endoplasmic reticulum) has not been fully resolved. The role of a putative sub-compartment of the ER in SOC activation in liver cells was investigated using ectopically expressed TRPV1 (transient receptor potential vanilloid 1), a non-selective cation channel, and TDCA (taurodeoxycholic acid), an activator of SOCs, to release Ca(2+) from different regions of the ER. TRPV1 was expressed in the ER and in the plasma membrane. The amount of Ca(2+) released from the ER by a TRPV1 agonist, measured using fura-2, was the same as that released by a SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) inhibitor, indicating that TRPV1 agonist-sensitive stores substantially overlap with SERCA inhibitor-sensitive stores. In contrast with SERCA inhibitors, TRPV1 agonists did not activate store-operated Ca(2+) entry. These findings were confirmed by patch-clamp recording. Using FFP-18, it was shown that SERCA inhibitors release Ca(2+) from the ER located closer to the plasma membrane than the region from which TRPV1 agonists release Ca(2+). In contrast with SERCA inhibitors, TRPV1 agonists did not induce a redistribution of STIM1 (stromal interaction molecule 1). TDCA caused the release of Ca(2+) from the ER, which was detected by FFP-18 but not by fura-2, and a redistribution of STIM1 to puncta similar to that caused by SERCA inhibitors. It is concluded that in liver cells, Ca(2+) release from a small component of the ER located near the plasma membrane is required to induce STIM1 redistribution and SOC activation.

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H(2)O(2)) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H(2)O(2) evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H(2)O(2) resulted in a increase in [Ca(2+)]( i ), indicating the release of calcium from intracellular stores. H(2)O(2) abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H(2)O(2)-induced calcium release. We conclude that H(2)O(2) induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+] i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane.

Effect of hydrogen peroxide on Ca 2+ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms

Using Fura-2-loaded human platelets we studied the nature of the mechanisms involved in Ca 2+ signalling mediated by H 2O 2. In a Ca 2+-free medium, H 2O 2 (10 μM–100 mM) induced a concentration-dependent increase in [Ca 2+] i. Depletion of either agonist-sensitive or mitochondrial Ca 2+ pools reduced this effect while depletion of both stores abolished it. Xestospongin C, an inositol 1,3,5-trisphosphate (IP 3) receptor inhibitor, reduced Ca 2+ release evoked by 1 mM H 2O 2 by 45%, indicating that H 2O 2-induced Ca 2+ release involves interaction with IP 3 receptors. Blockade of the IP 3 turnover by lithium or treatment with U-73122 did not modify H 2O 2-induced Ca 2+ release from the agonist-sensitive pool, suggesting the involvement of a mechanism independent of IP 3 generation. H 2O 2 inhibited Ca 2+ reuptake into the agonist-sensitive stores mediated by the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Thimerosal (5 μM), a sulphydryl reagent, induced Ca 2+ release from the agonist-sensitive stores. This event was impaired by treatment with 2 mM DTT, which also inhibited H 2O 2-induced Ca 2+ release from the agonist-sensitive pool but not from mitochondria. H 2O 2 reduced the ability of the plasma membrane Ca 2+ ATPase (PMCA) to extrude Ca 2+ by 75%, an effect that was unaffected by DTT. Consistent with this, thimerosal did not modify the PMCA activity. Finally, exposure to H 2O 2 triggered platelet aggregation, which was slower than that observed after agonist stimulation. We conclude that H 2O 2 induced Ca 2+ release from agonist-sensitive stores by oxidation of sulphydryl groups in SERCA and the IP 3 receptors independently of IP 3 generation. In addition, H 2O 2 induced Ca 2+ release from mitochondria and inhibited the PMCA activity by different mechanisms in human platelets.

Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Comparison of calcium mobilization in response to noradrenaline and acetylcholine in submandibular cells of newborn and adult rats

The response of mature and immature rat submandibular cells to α-receptor stimulation was compared in terms of the generation of inositol triphosphate (IP 3) and Ca 2+ mobilization, and of how the calcium mobilization response affects acetycholine (ACh)-induced Ca 2+ mobilization. In mature cells, noradrenaline (NA) caused much smaller IP 3 and Ca 2+ responses than ACh. However, the Ca 2+ release induced by NA was enough to partially discharge an agonist-sensitive store and to reduce Ca 2+ release by a subsequent ACh stimulus. Exposure to NA also caused an influx of Ca 2+ in the mature cells, which was largely associated with Ca 2+ entry induced by store depletion (i.e. capacitative entry). In the immature submandibular cells of newborn rats, NA caused essentially no IP 3 response and a small Ca 2+ release, which only partially affected the Ca 2+ released by a subsequent exposure to ALCh. In contrast to adult cells, immature cells did not show an increased Ca 2+ influx after exposure to NA. However, prestimulation with this agonist potentiated the Ca 2+ influx activated by ACh in the calls of newborn rats, but not in cells of adult rats. As both mature and immature submandibular cells have a well-developed phosphoinositide turnover response to ACh, the findings in mature cells suggest a less efficient coupling between α-receptors and phospholipase C, while those in immature cells suggest that this coupling is even less functional in the early stages of postnatal development. In permeabilized and 45Ca 2+-loaded mature cells, cyclic ADP-ribose (cADPR) released 13.4% of loaded 45Ca 2+ and this release was significantly reduced by pre-exposure to IP 3. Similarly, pre-exposure to cADPR also reduced the IP 3-induced 45Ca 2+ release. It is concluded that: (I) stimulation with NA induces a smaller Ca 2+ release in mature and immature submandibular cells than ACh; (2) the mediator for this small Ca 2+ mobilization may be cADPR; and (3) NA stimulates capacitative Ca 2+ entry in mature cells, but not in immature cells, and it also activates a Ca 2+ entry pathway distinct from the one induced by store depletion, particularly in immature cells.

Quantification of intraluminal free [Ca] in the agonistmsensitive internal calcium store using compartmentalized fluorescent indicators: some considerations

Many fluorescent Ca indicators, particularly those loaded as acetoxymethyl (AM)-ester derivatives, are known to become compartmentalized into organelles. This property can be exploited to measure changes in free [Ca] in subcellular compartments, including the inositol (1,4,5)-trisphosphate-sensitive store. However, quantitative measurement of free [Cal within a particular compartment is complicated by the fact that dye may accumulate in a variety of organelles and, in many cases, by the Mg sensitivity of the indicator. Here the issue of the quantification of free [Cal within the thapsigargin-sensitive store in BHK-21 fibroblasts using the low affinity Ca indicator, Mag-Fura-2, has been re-examined. At least 88 ± 1.3% (SEM) of the compartmentalized dye was determined to be confined to the thapsigargin-sensitive store, with the remaining fraction accounted for by other compartments where [Cal was below the detection limit for the dye (< 5 pM). In situ calibrations with ionophores indicated that the apparent free resting intraluminal [Cal was 260 ± 43 μM (SEM). Our analysis shows, however, that dye reporting from regions of low [Ca] contributes disproportionately to the Mag-Fura-2 ratio measured over the whole cell, potentially resulting in large underestimations of intraluminal [Ca] in agonist-sensitive stores. Free [Cal in the agonist-sensitive store was calculated to be as high as 539 ± 92 μM, assuming 12% of the Mag-Fura-2 to be in compartments where [Cal was below 5 μM. In comparison, perturbations arising from the presence of Mg in stores are predicted to be relatively minor.

Arachidonic Acid Activates the Noncapacitative Entry of Ca2+ during [Ca2+]i Oscillations

Current models for agonist-activated Ca2+ entry in nonexcitable cells focus on the capacitative mechanism where entry is activated as a downstream result of the sustained depletion of agonist-sensitive stores without any direct requirement for inositol phosphates. This mechanism has been shown to be important for the sustained Ca2+ signals seen in a variety of nonexcitable cells under conditions of maximal stimulation. In contrast, relatively little attention has been given to Ca2+ entry under more physiological levels of agonist where, for example, oscillating Ca2+ responses are common. In recent studies using cells from the exocrine avian nasal gland, we have shown that agonist-activated Ca2+ entry under these conditions demonstrates properties that are inconsistent with current versions of the capacitative model. We now report that activation of this novel noncapacitative Ca2+ entry is via a distinct signaling pathway involving an agonist-induced, phospholipase A2-mediated generation of arachidonic acid.

Evidence for a non-capacitative Ca2+ entry during [Ca2+] oscillations.

Current models for the agonist-induced activation of Ca2+ entry from the extracellular medium in non-excitable cells generally emphasize a capacitative mechanism whereby Ca2+ entry is activated simply as a result of the emptying of intracellular Ca2+ stores, without any direct involvement of inositol phosphates. To date, the activation and control of Ca2+ entry have generally been studied under conditions where the agonist-sensitive stores undergo a profound and sustained depletion. However, responses under more normal physiological conditions typically involve the cyclical release and refilling of the stores associated with oscillations in [Ca2+], and the nature and control of entry under these conditions has received relatively little attention. In this study, using isolated cells from the exocrine avian nasal gland as a model system, we show that: (a) the agonist-enhanced rate of Mn2+ quench is independent of the cyclical emptying and refilling of the agonist-sensitive Ca2+ pool during oscillations; (b) the Ca2+ entry pathway is maintained in an activated state for extended periods following inhibition of oscillations under conditions in which agonist-sensitive stores can be shown to be full; (c) no Ca2+ entry could be detected in oscillating cells in experiments that followed a definitive protocol for the demonstration of capacitative entry; and (d) on initial exposure to low agonist concentrations, activation of Ca2+ entry preceded any detectable release of Ca2+ from the stores. We conclude that the essential characteristics of the control of Ca2+ entry during oscillations are incompatible with current capacitative models.

Agonist-evoked calcium efflux from a functionally discrete compartment in Xenopus oocytes

Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45CaC1 2 resulted in a typical Ca-activated chloride current, but not in 45Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP 3), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP 3 induced efflux. Emptying the agonist sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP 3 sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.

Two classes of agonist-sensitive Ca2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin

In the absence of extracellular Ca2+, extensive Ca2+ release from the platelet intracellular stores [monitored as an increase of intracellular Ca2+ concentration ([Ca2+]i)] is produced by the combined action of the endomembrane Ca(2+)-ATPase inhibitor thapsigargin and 2 nM ionomycin. The titration of Ca2+ unloading with thapsigargin (plus ionomycin) shows that a substantial fraction of the store-associated Ca2+ is released by 8-10 nM thapsigargin, but that 100-200 nM thapsigargin is required for the complete release. The store depletion obtained in similar conditions with a different endomembrane Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ), is always incomplete. It is completed by thrombin or by 10 nM thapsigargin. We conclude that two different types of Ca2+ pumps exist in platelets, one sensitive to TBHQ and to high thapsigargin, the other insensitive to TBHQ and sensitive to low thapsigargin. They are distributed separately in discrete subpopulations of the agonist-sensitive stores. The influx of external Ca2+ is maximal when both types of stores are Ca(2+)-depleted, either by high thapsigargin or by the combined action of low thapsigargin and TBHQ.

Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides

In aspirin-treated platelets the thrombin-induced increase of cytosolic Ca2+ ([Ca2+]i) associated with the release from the intracellular stores is followed by a decrease to the baseline which is largely dependent on the re-uptake into the stores. This is shown by the further increase of [Ca2+]i upon inhibition of the endomembrane Ca(2+)-ATPase with thapsigargin. The re-uptake of Ca2+ into the stores is accelerated by sodium nitroprusside (SNP) or prostacyclin (PGI2). In all cases, after store depletion with thapsigargin the influx of external Ca2+ is maximal. After a thrombin-induced cycle of Ca(2+)-release re-uptake the stores are partly full: in these conditions the addition of external Ca2+ elicits a significant increment of [Ca2+]i and a further filling of the stores. Both are strongly reduced if Ca2+ addition is preceded by SNP or PGI2. Similar results are obtained also if (by supplementing and then cheleting Ca2+) the stores are as full as in native platelets at the moment of adding Ca2+. The thrombin-activated Ca2+ influx is reversed by hirudin. A PGI2- and SNP-sensitive Mn2+ influx is observed if Mn2+ is added in place of Ca2+. It is concluded that thrombin activates a cyclic nucleotide-sensitive Ca2+ (and Mn2+) influx pathway dependent on the occupancy of the thrombin receptor and independent of the filling state of the stores. In the absence of thrombin, thapsigargin releases Ca2+ relatively rapidly from a fraction of the stores; the remaining deposits are discharged much more slowly. This may indicate that platelets contain two distinct classes of agonist-sensitive stores. The addition of external Ca2+ (or Mn2+) at short or long incubation times with thapsigargin monitors the influx of Ca2+ activated by the depletion of one or both types of stores. The depletion of each type of store activates Ca2+ (Mn2+) influx. This type of cation influx is not inhibited by the cyclic nucleotides.

Ca2+ efflux from platelets

Large amounts of Ca2+ (almost 20 nmol/10(8) cells) are released from platelets by exocytosis. This secretory-granule-associated Ca2+ does not contribute to the cytosolic free Ca2+ ([Ca2+]i), which is controlled by the much smaller agonist-sensitive Ca2+ pool, unless high (1 microM), but not low (0.04 microM) concentrations of ionomycin are present. Low concentrations of ionomycin release Ca2+ almost exclusively from the agonist-sensitive stores. In aspirinated platelets incubated in the presence of 0.5 mM EGTA the extensive depletion of the agonist-sensitive stores is obtained by the combined action of low ionomycin and the endomembrane Ca(2+)-ATPase inhibitor thapsigargin (which individually promote only a partial depletion). The subsequent decay of [Ca2+]i is increased by phorbol-myristate acetate, confirming that Ca2+ efflux from platelets is potentiated by the activation of protein kinase C [Pollock, W. K., Sage, S. O. & Rink, T. J. (1987) FEBS Lett. 210, 132-140]. A novel type of control of Ca2+ efflux appears to be exerted by the filling state of the stores. Treatment with low ionomycin or thapsigargin determines the release of a fraction of the stores-associated Ca2+; the subsequent decay of [Ca2+]i is slow. The decay rate of [Ca2+]i accelerates after extensive depletion of the stores following the addition of thapsigargin or ionomycin. If the depletion of the stores is induced by thrombin, added alone or in combination with thapsigargin, the increases of [Ca2+]i are the same and the subsequent decay rates are largely superimposable; however a large fraction of [Ca2+]i is reaccumulated into the stores in the absence, but not in the presence of thapsigargin, indicating that Ca2+ efflux is activated when the stores are empty. Ca2+ efflux can proceed against a concentration gradient. In 45Ca-loaded platelets, the thrombin-promoted 45Ca efflux is potentiated by thapsigargin. The protein-kinase-C-dependent and store-depletion-dependent stimulations of 45Ca efflux are additive. These observations indicate that, in addition to being activated by protein kinase C, Ca2+ efflux from platelets is activated by the depletion of the stores. The two activations appear to be additive.

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)P3]-sensitive, but not the Ins(1,4,5)P3-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 agonist-evoked [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-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)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-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 Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.

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 Mn(2+)-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 delta 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 Ca(2+)-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ oscillations and Ca2+ influx in Xenopus oocytes expressing a novel 5‐hydroxytryptamine receptor.

1. We expressed a novel 5-hydroxytryptamine receptor (SRL) in Xenopus oocytes and monitored cytosolic Ca2+ through the endogenous Ca(2+)-dependent Cl- channel activity using the double electrode voltage-clamp technique. 2. 5-Hydroxytryptamine (5-HT; 200 nM) led to an initial rapid oscillatory current followed by a pronounced secondary one, which lasted long after 5-HT wash-out (20-40 min) and was not affected by the receptor antagonist yohimbine. 3. Both phases of the current were abolished by heparin demonstrating a key role for IP3-induced Ca2+ release. 4. Caffeine (10 mM) alone did not evoke a current but reduced both phases of the current evoked by 5-HT. Ryanodine had no effect. No evidence for Ca(2+)-induced Ca2+ release was found. 5. The secondary current activated by 5-HT was sensitive to changes in extracellular Ca2+, suggesting it was evoked by Ca2+ influx. Reducing external Na+ did not affect this current, demonstrating that it was rather specific for Ca2+. 6. The Ca2+ influx pathway was much more sensitive to Cd2+ than other divalent ions (Co2+, Mn2+, Sr2+, Ba2+). It was insensitive to verapamil. 7. Injection of D-myo-inositol 1,4,5-trisphosphate, 3-deoxy-3-fluoro (IP3-F; an analogue not metabolized to D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4)), evoked either an oscillatory current or a rapid current followed by a sustained secondary one. The latter was sensitive to external Ca2+ and was blocked by Cd2+. Heparin dramatically reduced the IP3-F-evoked current. 8. Perfusion in Ca(2+)-free solution, once a secondary current had been generated, significantly decreased the amount of intracellular Ca2+ mobilized by 5-HT, indicating that the Ca2+ influx pathway plays an important role in pool refilling. 9. Block of Ca2+ influx by Cd2+ in cells that were oscillating transiently increased the amplitude and then either abolished the oscillations or made them irregular. This effect was also elicited by increasing external Ca2+. 10. These results demonstrate that 5-HT, acting via IP3, both releases Ca2+ from internal stores and evokes a pronounced Ca2+ influx. This last step is activated by pool depletion and is important for both refilling of the agonist-sensitive stores and modifying the oscillatory pattern.

Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy

The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH4Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH4Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na+, suggesting the presence of Na+/H+ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na+ from the bathing medium results in a decrease in resting [Ca2+]i, paralleled by a reduction in pHi. This suggests a role for Na+/H+ exchange in determining resting [Ca2+]i.