Interactions between calcium and cAMP signaling.

The calcium ion is quite possibly the single most pervasive signaling molecule used by living organisms for the purpose of communicating internal and external states. It differs from other messengers in that it is neither created nor destroyed, but just moved around inside and outside the cell via transporters, pumps and channels to alter its concentration in specific cellular locations. These changes in free [Ca(2+)] are then detected by a wide array of Ca(2+)-binding effector proteins whose affinities are appropriately tuned to respond to a particular type of [Ca(2+)] change. This deceptively simple paradigm dominates the function of many cell types, for example in driving contraction of muscle, action potential generation in nerves, fluid, hormone, and enzyme secretion in secretory cells, and certain immune responses. However, the Ca(2+) signal does not work in strict isolation, but rather is fine-tuned by many other signals, not the least of which is the other major second messenger, cyclic AMP (cAMP). Conversely, the cAMP pathway is subject to modification by the calcium signal and its various effectors at many different levels. These two fundamental second messengers, used throughout eukaryotes and even prokaryotes, are thus inextricably intertwined. The purpose of the present article is to provide an update on some of the recently described forms of reciprocal regulation between Ca(2+) and cAMP signaling circuits, with emphasis on interactions that take place in localized domains of the cell.

Asymmetrical, agonist-induced fluctuations in local extracellular [Ca(2+)] in intact polarized epithelia.

We recently proposed that extracellular Ca(2+) ions participate in a novel form of intercellular communication involving the extracellular Ca(2+)-sensing receptor (CaR). Here, using Ca(2+)-selective microelectrodes, we directly measured the profile of agonist-induced [Ca(2+)]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid-secreting cells. The Ca(2+)-mobilizing agonist carbachol elicited a transient, La(3+)-sensitive decrease in basolateral [Ca(2+)] (average approximately 250 microM, but as large as 530 microM). Conversely, carbachol evoked an HgCl2-sensitive increase in [Ca(2+)] (average approximately 400 microM, but as large as 520 microM) in the lumen of single gastric glands. Both responses were significantly reduced by pre-treatment with sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump inhibitors or with the intracellular Ca(2+) chelator BAPTA-AM. Immunofluorescence experiments demonstrated an asymmetric localization of plasma membrane Ca(2+) 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 [Ca(2+)]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.

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.

Respiratory system of arachnids II: morphology of the tracheal system of Leiobunum rotundum and Nemastoma lugubre (Arachnida, Opiliones).

The tracheal system of two species of harvestmen with different life styles was investigated: the long-legged, Leiobunum rotundum (Phalangioidea, Phalangiidae) and the short-legged Nemastoma lugubre (Troguloidea, Nemastomatidae). The morphology of the tracheae is very similar in both species: The branching pattern is basically asymmetric and dichotomous, and the tracheae taper between branching points. The tracheal diameters range from 160 to 0.3 mum in L. rotundum (mean body mass 25.3 mg), and from 70 to 0.5 mum in N. lugubre (mean body mass 3.8 mg). Ultrastructurally, the tracheal walls are similar to those of insects, consisting of an outer hypodermal layer and an inner cuticular layer with taenidial structures. Tracheae of all diameters have close contact with organs and muscles, indicating that diffusive gas exchange may take place through the walls of all tracheae. The finest tracheae end freely in the hemolymph or at the surface of organs and muscles. Exceptions are tracheal penetration of the central nerve mass in the prosoma in both species, and of the muscles in L. rotundum. The latter may correlate with the active perambulatory life style of L. rotundum.

Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells.

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

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+].

Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+].

ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10-15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release-activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.

Quantification of intraluminal free [Ca] in the agonist-sensitive 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.

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

Under resting conditions, steady-state [Ca] in agonist-sensitive Ca stores reflects a balance between active uptake (usually mediated by a thapsigargin-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 InsP(3) 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 ATPgammaS) 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.

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.

Free [Ca] within organelles of permeabilized BHK-21 cells was measured using ratio imaging of compartmentalized mag-fura-2. In BHK-21 cells, this dye monitors free [Ca] in principally one type of ATP-dependent Ca-sequestering organelle in which intrastore Ca was released uniformly and entirely by 100 nM thapsigargin or removal of ATP or Ca from the bath, and was reduced by 85% upon treatment with a supramaximal dose of InsP3 (6 microM). Examination of the spatial distribution of InsP3-sensitive Ca stores showed that InsP3 released Ca throughout all regions of the cell, although we often noted a perinuclear region (which we speculate may correspond to the Golgi apparatus) with reduced responsiveness to InsP3. InsP3-induced changes of intraluminal Mg could not be detected. Cyclic ADP-ribose, ryanodine, caffeine, mitochondrial inhibitors, and GTP, agents known to influence intraorganellar Ca sequestration in other cell types, were all without effect on the mag-fura-2 ratio. In situ calibration of the mag-fura-2 ratio with Ca ionophores revealed that the average free intraorganellar [Ca] was initially 188 +/- 21 microM in the presence of 170 nM free Ca and 3 mM ATP, and was reduced to 25 +/- 5 microM upon stimulation with 6 microM InsP3. The ionic dependence of the release and reloading process was also investigated. The presence of either K, Na, or Cl could consistently support both InsP3-induced release and the refilling of stores with Ca, but physiological concentrations of HCO3 were effective in sustaining the response in only 24% of cells examined.

Direct measurement of free Ca in organelles of gastric epithelial cells.

When loaded as the acetoxymethyl ester (AM) derivative, the fluorescent probe mag-fura 2 accumulates in both the cytoplasm and the subcellular compartments. The relatively high dissociation constant of this dye for Ca (53 microM) permits the measurement of changes in the free concentration of this ion in a variety of organelles where Ca concentration ([Ca]) is high. To characterize Ca stores in gastric cells, we used digitonin to permeabilize cells within isolated rabbit gastric glands loaded with mag-fura 2-AM. This allowed cytosolic dye to leak out, leaving only compartmentalized dye behind. It appears that mag-fura 2 monitors [Ca] changes in several ATP-dependent Ca-sequestering pools; an inositol 1,4,5-trisphosphate (IP3)-releasable and thapsigargin-sensitive store (which probably includes the endoplasmic reticulum), a pool that is released by the mitochondrial inhibitors oligomycin+azide, valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and ruthenium red (and therefore likely represents mitochondria), and a residual pool that was resistant to release by mitochondrial inhibitors and thapsigargin. Ca sequestration into all pools was sensitive to changes in [ATP], indicating that treatments that reduce cellular [ATP] will cause certain organelles to lose their Ca to the cytoplasm. Caffeine and ryanodine, which mobilize Ca from internal stores in many cell types, induced Ca sequestration into an IP3-insensitive store of gastric cells, and caffeine caused a reduction in cytoplasmic [Ca] (as measured with fura 2). We also show that the quantitation of free [Ca] in a given pool is complicated by a nonlinearity in the relationship between the mag-fura 2 ratio and [Ca]. This effect is likely a consequence of monitoring the fluorescence from multiple pools simultaneously. However, this limitation does not detract from the ability of this method to yield important qualitative information about the nature and number of Ca stores within single gastric cells.

Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2.

Stimulation of cells with calcium-mobilizing agonists frequently results in inositol 1,4,5-trisphosphate (InsP3)-mediated discharge of Ca from an internal store. We report here a technique for directly monitoring Ca within this and other stores in gastric epithelial cells. This technique takes advantage of the propensity of the acetoxymethyl ester derivative of the fluorescent dye mag-fura-2 (which is sensitive to Ca concentrations above 5 microM) to accumulate in subcellular compartments where it can report changes in the free Ca concentration. Intact dye-loaded cells responded to cholinergic stimulation with a decrease in the 350 nm/385 nm excitation ratio, as measured in individual cells with a digital imaging microscope, consistent with reduced Ca concentration in one or more cellular compartments. When cells were permeabilized with digitonin and incubated in an "intracellular buffer," the cytoplasmic dye was released, leaving the mag-fura-2 in the internal store. InsP3 caused the ratio from the trapped indicator to decrease (i.e., Ca was released) in a dose-dependent manner, and this effect was blocked by the InsP3 receptor antagonist heparin. Ca sequestration into the internal store was ATP-dependent, and reuptake into the InsP3-sensitive pool was blocked by thapsigargin, a specific inhibitor of the Ca-ATPase of the internal store. We used this technique to investigate the role of Cl on the release and reloading of the InsP3-sensitive internal store and found that Ca uptake was reduced in Cl-free solutions, suggesting an important function for Cl in the refilling of this pool.

K-induced alkalinization in all cell types of rabbit gastric glands: a novel K/H exchange mechanism.

Digital image processing of the pH-sensitive dye BCECF was used to examine the effects of high [K] media on cytoplasmic pH (pHi) of individual cells within isolated rabbit gastric glands. When cells were acidified to pHi 6.5 from the resting pHi of 7.2-7.3 and then exposed to solution containing 77 mM K plus amiloride (to block Na/H exchange), recovery to pHi 7.0 was observed. This K-induced alkalinization occurred in all cell types of the gland, including cells within antral glands that were devoid of parietal cells (PC). This process was independent of extracellular Na and Cl and was unaffected by: 5 mM Ba or 200 microM bumetanide, or acute treatment with either 500 microM ouabain or 100 microM cimetidine, histamine or carbachol. SCH28080, which inhibits the PC H/K-ATPase when used in the low microM range of concentrations, blocked the K effect on pHi at 100 microM but was ineffective at 1 microM. A similar pHi recovery was also stimulated by Li, Cs (both 72 mM), and Tl (10 mM), in the order Li greater than K greater than Cs greater than Tl (all in the presence of amiloride), and these alkalinizations were also blocked by 100 microM SCH28080. Parallel experiments were performed to test the effect of these ions on 14[C]-aminopyrine accumulation, an index of acid secretion by the H/K-ATPase at the lumenal membrane of the PC. There was no correlation between the rates of cation-induced pHi recovery from an acid load and H secretion as measured by the accumulation of aminopyrine. We conclude that the K- (and Cs- and Li-) dependent pHi recovery is mediated by a novel cation/H exchange mechanism that is distinct from the PC H/K-ATPase.