Silvana Curci

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+].

Determination of intracellular K+ activity in rat kidney proximal tubular cells.

The intracellular K+ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K+-sensitive microelectrodes, which were filled with a liquid K+-exchanger resin that has also a small sensitivity to Na+. Both electrodes had tip diameters of 0.2 μm or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was −76.3±8.1 mV (n=81) and the potential difference measured with the K+-sensitive microelectrode was −7.2±5.8 mV (n=32). Based on the activity of K+ in the extracellular fluid of ∼3 mmol/l the intracellular K+ activity was estimated to be ∼82 mmol/l. Assuming equal K+-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K+ concentration is ∼113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K+-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K+ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K+ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na+/K+-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.

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)

Evidence for rheogenic sodium bicarbonate cotransport in the basolateral membrane of oxyntic cells of frog gastric fundus

AbstractIonic conductance properties of the basolateral cell membrane of oxyntic cells were studied in frog gastric fundus in vitro. After mounting the fundus in a modified Ussing chamber the serosal connective tissue was dissected off and individual oxyntic cells were punctured from the serosal surface with microelectrodes. Under resting conditions the membrane potential averaged −56.9, SD±9.5 mV (n=63), cytoplasm negative. Lowering or raising serosal HCO3− concentration from 17.8 to 6 or 36 mmol/l respectively at constant

Spatial distribution and quantitation of free luminal [Ca] within the InsP3-sensitive internal store of individual BHK-21 cells: ion dependence of InsP3-induced Ca release and reloading.

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Extracellular calcium acts as a “third messenger” to regulate enzyme and alkaline secretion

depolarized or hyperpolarized the cell membrane by +16.7 or −18.2 mV respectively. Sudden removal of serosal Na+ also depolarized the cell membrane (anomalous Nernst response). Since both the HCO3− dependent and the Na+ dependent potential changes were strongly depressed by the disulfonic stilbene SITS and since the potential response to HCO3− was virtually abolished in Na+-free solution we conclude that a rheogenic Na+ (HCO3−)n-cotransport system (n>1) is present in the basolateral cell membrane of oxyntic cells. Its possible role in base transfer during HCl-secretion or HCO3− secretion remains to be elucidated.

Alkaline secretion by frog gastric glands measured with pH microelectrodes in the gland lumen

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.