Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells.

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.

STIM1, but not STIM2, is required for proper agonist-induced Ca2+ signaling.

The stromal interaction molecules STIM1 and STIM2 sense a decreasing Ca(2+) concentration in the lumen of the endoplasmic reticulum and activate Ca(2+) channels in the plasma membrane. In addition, at least 2 reports suggested that STIM1 may also interact with the inositol 1,4,5-trisphosphate (IP(3)) receptor. Using embryonic fibroblasts from Stim1(-/-), Stim2(-/-) and wild-type mice, we now tested the hypothesis that STIM1 and STIM2 would also regulate the IP(3) receptor. We investigated whether STIM1 or STIM2 would be the luminal Ca(2+) sensor that controls the loading dependence of the IP(3)-induced Ca(2+) release. Partial emptying of the stores in plasma-membrane permeabilized cells resulted in an increased EC(50) and a decreased Hill coefficient for IP(3)-induced Ca(2+) release. This effect occurred both in the presence and absence of STIM proteins, indicating that these proteins were not the luminal Ca(2+) sensor for the IP(3) receptor. Although Stim1(-/-) cells displayed a normal IP(3)-receptor function, agonist-induced Ca(2+) release was reduced. This finding suggests that the presence of STIM1 is required for proper agonist-induced Ca(2+) signaling. Our data do not provide experimental evidence for the suggestion that STIM proteins would directly control the function of the IP(3) receptor.

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum.

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.

Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2+ store.

Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10 microM thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation.

Additional fluxes of activator Ca2+ accompanying Ca2+ release from the sarcoplasmic reticulum triggered by insP3-mobilizing agonists.

Activation of phospholipase C (PLC)-linked receptors leads not only to Ca2+ release from the sarcoplasmic reticulum (SR) by inositol-1,4,5-trisphosphate (InsP3), but also to Ca2+ entry via opening of receptor-activated Ca2+ channels (RACCs) and store-operated Ca2+ channels (SOCs), in addition to possible contributions of Ca2+ release from non-SR stores. We review recent results on these non-SR Ca2+ fluxes. In A7r5 smooth-muscle cells (SMCs), high InSP3 concentrations release Ca2+ from a thapsigargin-insensitive store. Presumably this store corresponds to the Golgi and is filled by a Pmrl-type Ca2+ pump. Molecular candidates for RACCs and SOCs are found among the members of the TRPC channel family. Inoue and colleagues have recently demonstrated that in vascular SMCs TRPC6 is an essential part of a RACC that is activated by alpha-adrenergic stimulation via the diacylglycerol branch of phosphatidylinositol-4,5-bisphosphate hydrolysis. In TRPC4 knockout mice, contractility of SMCs appears unaffected. However, endothelium-dependent relaxation is impaired mainly due to lack of a SOC activity in endothelial cells. The best-characterized SOC current, mainly observed in blood cells, is Icrac Recently, it has been proposed that CaTI (TRPV5) forms at least part of the pore of CRAC. This view is challenged by data from our laboratory.

Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell.

Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca(2+)](i) oscillations in rat megakaryocytes, as in other cells, results from a Ca(2+)-dependent inhibition of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Moreover, we find that this inhibition becomes progressively less effective with higher IP(3) concentrations. We suggest that disinhibition, by increasing IP(3) concentration, of Ca(2+)-dependent inhibition is a common mechanism for the regulation of [Ca(2+)](i) oscillations in cells containing IP(3)-sensitive Ca(2+) stores.

Inositol 1,4,5-trisphosphate and adenophostin analogues induce responses in turtle olfactory sensory neurons.

Using the whole-cell mode of the patch-clamp technique, we recorded inward currents in response to inositol-1,4,5-trisphosphate (IP3) and adenophostin analogues in turtle olfactory sensory neurons. Dialysis of IP3 into the neurons induced inward currents with an increase in membrane conductance in a dose-dependent manner under the voltage-clamp conditions (holding potential -70 mV). The application of Ca2+-free Ringer solution to neurons previously dialysed with IP3 induced inward currents that were reversibly inhibited by application of Na+, Ca2+-free Ringer solution, normal Ringer solution or 10 microM ruthenium red. Dialysis of the adenophostin analogues, novel IP3 receptor ligands, also induced inward currents with an increase in membrane conductance. The magnitude of the responses to the adenophostin analogues varied among these derivatives. The application of Ca2+-free Ringer solution to neurons previously dialysed with the adenophostin analogues induced inward currents that were inhibited by the application of normal Ringer solution. The reversal potential of inward currents induced by an adenophostin analogue was similar to that induced by IP3, suggesting that inward currents induced by the adenophostin analogue were generated by a similar ionic mechanism to that induced by IP3. The present study demonstrated that IP3-mediated transduction pathways exist in turtle olfactory receptor neurons and that adenophostin analogues act as agonists of IP3.

Molecular recognition of adenophostin, a very potent Ca2+ inducer, at the D-myo-inositol 1,4,5-trisphosphate receptor.

The recognition mode of adenophostin A at the D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor was investigated. Comparison of conformations of Ins(1,4,5)P(3) and adenophostin A by using the combination of NMR and molecular mechanics (MM) calculations demonstrated that adenophostin A adopted a moderately extended conformation regarding the distance between the 2'-phosphoryl group and the 3' ',4' '-bisphosphate motif, as suggested previously [Wilcox, R. A. et al. (1995) Mol. Pharmacol. 47, 1204-1211]. Based on the nuclear Overhauser effect (NOE) observed between 3'-H and 1' '-H and on MM calculations, the molecular shape of adenophostin A proved to be an extended form at least in solution, in contrast to Wilcox's compactly folded, preliminary hairpin model. GlcdR(2,3',4')P(3), an adenophostin analogue without adenine moiety, was found to be less potent than adenophostin A and almost equipotent to Ins(1,4,5)P(3). We propose the possibility that (i) the optimal spatial arrangement of the three phosphoryl groups and/or (ii) the interaction of the adenine moiety of adenophostin A with the putative binding site, if it exists in the vicinity of the Ins(1,4,5)P(3)-binding site, might account for the exceptional potency of adenophostin A.

Expression of the ryanodine receptor type 3 in skeletal muscle. A new partner in excitation-contraction coupling?

Mobilization of Ca2+ from the endoplasmic reticulum (ER) is mediated by two related groups of Ca2+ release channels, the inositol 1,4,5 trisphosphate (InsP3) receptors and the ryanodine receptors. The InsP3 receptors have been studied in a large number of cells where they regulate many different activities upon stimulation with a variety of agonists. Ryanodine receptors have been essentially studied with respect to their role in regulating muscle contraction in both cardiac and skeletal muscles. In the recent years, InsP3 receptors and ryanodine receptors have been found to be co-expressed in neurons and other cell types, including smooth muscle cells. This emerging picture reveals that within one cell different combinations of two or more isoforms of Ca2+ release channels (i.e., multiple InsP3 receptors and/or ryanodine receptors) can be expressed at the same time. New data on the expression of two isoforms of ryanodine receptors in developing skeletal muscles or in specialized adult muscles have provided initial ground to test the hypothesis that combinations of various Ca2+ release channels may be relevant to adapt the modality of Ca2+ release to regulation of specific cellular functions.

Agonist-evoked Ca2+ mobilization from stores expressing inositol 1,4,5-trisphosphate receptors and ryanodine receptors in cerebellar granule neurones.

The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-D-aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 +/- 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 +/- 6% of the control). CCh-evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 microM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 microM), ryanodine (10 microM), or dantrolene (25 microM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca2+]i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 microM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 +/- 14%; ACPD, 27 +/- 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR-mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP3-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA-evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.

Crosstalk between cellular morphology and calcium oscillation patterns. Insights from a stochastic computer model.

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca2+]i) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca2+ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such "Ca2+ fingerprints' can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca2+ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca(2+)-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461-1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca2+ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca2+ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.

Protein kinase C activation inhibits receptor-evoked inositol trisphosphate formation and induction of cytosolic calcium oscillations by decreasing the affinity-state of the cholecystokinin receptor in pancreatic acinar cells.

Digital-imaging microscopy of Fura-2-loaded pancreatic acinar cells revealed that the C-terminal octapeptide of cholecystokinin (CCK8) dose-dependently recruited 94% of freshly isolated acinar cells in terms of receptor-evoked Ca2+ mobilization. Maximal and half-maximal cell-recruitment were reached with 0.1 nM and 16.8 pM CCK8, respectively. The upstroke of the dose-recruitment curve consisted of cells displaying oscillatory changes in free cytosolic Ca2+ concentration ([Ca2+]i). After having reached its maximum, the percentage oscillating cells dose-dependently decreased upon further increasing of the CCK8 concentration. Pretreatment of the acinar cells with 0.1 microM TPA caused a rightward shift of the dose-recruitment curve but did not change the maximal effect of CCK8 on the recruitment of oscillating cells. Half-maximal recruitment was obtained with 287 pM CCK8. This observation demonstrates that high levels of protein kinase C activation do not inhibit Ca2+ oscillations at a level downstream to receptor activation. Moreover, this observation demonstrates that protein kinase C-mediated inhibition of Ca2+ oscillations evoked by submaximal CCK8 concentrations occurs at the receptor level, converting it from a high-affinity state into a low-affinity state. This conclusion is supported by the observation that TPA completely inhibited the recruitment of acinar cells in response to the high-affinity receptor agonist JMV-180. The inhibitory action of TPA on CCK8-evoked cell-recruitment was paralleled by an inhibitory effect of the phorbol ester on the CCK8-evoked peak increase in average inositol trisphosphate concentration in a population of acinar cells. This observation indicates that low concentrations of CCK8 interact with the high-affinity CCK receptor to increase [Ca2+]i through the intermediation of inositol trisphosphate.