Aldebaran M. Hofer

Extracellular calcium sensing and signalling

Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+ — the extracellular calcium-sensing receptor — has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular calcium-sensing receptor.

Critical role for calcium mobilization in activation of the NLRP3 inflammasome

The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome mediates production of inflammatory mediators, such as IL-1β and IL-18, and as such is implicated in a variety of inflammatory processes, including infection, sepsis, autoinflammatory diseases, and metabolic diseases. The proximal steps in NLRP3 inflammasome activation are not well understood. Here we elucidate a critical role for Ca2+ mobilization in activation of the NLRP3 inflammasome by multiple stimuli. We demonstrate that blocking Ca2+ mobilization inhibits assembly and activation of the NLRP3 inflammasome complex, and that during ATP stimulation Ca2+ signaling is pivotal in promoting mitochondrial damage. C/EPB homologous protein, a transcription factor that can modulate Ca2+ release from the endoplasmic reticulum, amplifies NLRP3 inflammasome activation, thus linking endoplasmic reticulum stress to activation of the NLRP3 inflammasome. Our findings support a model for NLRP3 inflammasome activation by Ca2+-mediated mitochondrial damage.

Calcium: Extracellular calcium sensing and signalling

Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+ — the extracellular calcium-sensing receptor — has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular calcium-sensing receptor.

Intercellular communication mediated by the extracellular calcium-sensing receptor

Agonist-evoked, intracellular Ca2+-signalling events are associated with active extrusion of Ca2+ across the plasma membrane, implying a local increase in Ca2+ concentration ([Ca2+]) at the extracellular face of the cell. The possibility that these external [Ca2+] changes may have specific physiological functions has received little consideration in the past. Here we show that, at physiological ambient [Ca2+], Ca2+ mobilization in one cell produces an extracellular signal that can be detected in nearby cells expressing the extracellular Ca2+-sensing receptor (CaR), a cell-surface receptor for divalent cations with a widespread tissue distribution. The CaR may therefore mediate a universal form of intercellular communication that allows cells to be informed of the Ca2+-signalling status of their neighbours.

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+].

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 [Ca] 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 [Ca] 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 [Ca] was below the detection limit for the dye (< 5 microM). In situ calibrations with ionophores indicated that the apparent free resting intraluminal [Ca] was 260 +/- 43 microM (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 [Ca] in the agonist-sensitive store was calculated to be as high as 539 +/- 92 microM, assuming 12% of the Mag-Fura-2 to be in compartments where [Ca] was below 5 microM. In comparison, perturbations arising from the presence of Mg in stores are predicted to be relatively minor.

The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

FRET-based sensors for cAMP and PKA activity reveal that mitochondrial subcompartments host segregated cAMP cascades with distinct functional and kinetic signatures.

ATP regulates calcium leak from agonist-sensitive internal calcium stores.

ABSTRACT: Under resting conditions, steady‐state [Ca] in agonist‐sensitive Ca stores reflects a balance between active uptake (usually mediated by a thap‐sigargin‐sensitive Ca‐ATPase of the SERCA family) and passive efflux of Ca. Even though this pump‐leak cycle appears to be a common property of Ca‐storing organelles, little is known about the nature of the leak pathway. Ca homeostasis in thapsigargin‐sensitive internal Ca stores of single permeabilized BHK‐21 fibroblasts was examined using digital image processing of compartmentalized mag‐fura‐2 (a low‐affinity Ca indicator). It is shown here that the leak of Ca from internal stores is regulated specifically by the cytosolic ATP concentration. The rate of leak was 3.6 times slower in 0.375 mM [ATP] than in 4 mM [ATP] (Na or Mg salt). These effects were observed in the presence of 0 Ca/EGTA, thapsigargin, heparin, and ruthenium red, and therefore appear to be independent of the Ca‐ATPase, the InsP3 receptor and the ryanodine receptor. The ATP‐stimulated leak was seen in a variety of cell types, including rat basophilic leukemia cells and mouse pancreatic acinar cells. Other nucleotides (ADP, GTP, CTP, and UTP) and nonhydrolyzable ATP analogs (AMP‐PNP and ATPγS) did not reproduce the action of ATP. Changes in cellular metabolism and ensuing alterations in [ATP] will be expected to influence the filling state of internal Ca stores through effects on the passive leak pathway, potentially leading to modulation of Ca signaling and organellar function.—Hofer, A. M., Curci, S., Machen, T. E., Schulz, I. ATP regulates calcium leak from agonist‐sensitive internal calcium stores. FASEB J. 10, 302‐308 (1996)

Another dimension to calcium signaling: a look at extracellular calcium.

Cell biologists know the calcium ion best as a vital intracellular second messenger that governs countless cellular functions. However, the recent identification of cell-surface detectors for extracellular Ca2+ has prompted consideration of whether Ca2+ also functions as a signaling molecule in the extracellular milieu. The cast of Ca2+ sensors includes the well-characterized extracellular-Ca2+-sensing receptor, a G-protein-coupled receptor originally isolated from the parathyroid gland. In addition, other receptors, channels and membrane proteins, such as gap junction hemichannels, metabotropic glutamate receptors, HERG K+ channels and the receptor Notch, are all sensitive to external [Ca2+] fluctuations. A recently cloned Ca2+ sensor (CAS) in Arabidopsis extends this concept to the plant kingdom. Emerging evidence indicates that [Ca2+] in the local microenvironment outside the cell undergoes alterations potentially sufficient to exert biological actions through these sensor proteins. The extracellular space might therefore constitute a much more dynamic Ca2+ signaling compartment than previously appreciated.

Asymmetrical, agonist‐induced fluctuations in local extracellular [Ca2+] in intact polarized epithelia

We recently proposed that extracellular Ca2+ ions participate in a novel form of intercellular communication involving the extracellular Ca2+‐sensing receptor (CaR). Here, using Ca2+‐selective microelectrodes, we directly measured the profile of agonist‐induced [Ca2+]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid‐secreting cells. The Ca2+‐mobilizing agonist carbachol elicited a transient, La3+‐sensitive decrease in basolateral [Ca2+] (average ≈250 μM, but as large as 530 μM). Conversely, carbachol evoked an HgCl2‐sensitive increase in [Ca2+] (average ≈400 μM, but as large as 520 μM) in the lumen of single gastric glands. Both responses were significantly reduced by pre‐treatment with sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) pump inhibitors or with the intracellular Ca2+ chelator BAPTA‐AM. Immunofluores cence experiments demonstrated an asymmetric localization of plasma membrane Ca2+ ATPase (PMCA), which appeared to be partially co‐localized with CaR and the gastric H+/K+‐ATPase in the apical membrane of the acid‐secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca2+]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.