Intracellular calcium requirements for β1 integrin activation

Human polymorphonuclear leukocytes (PMNs) express β1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca⁺⁺]_i) to inside-out activation of β1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of β1 integrins was determined by measuring the expression of an activation-dependent epitope on the β1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase β1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C₈) at 100 μM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C₈ and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca⁺⁺]_i and stimulating PKC in β1 integrin activation. Chelation of [Ca⁺⁺]_i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca⁺⁺]_i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca⁺⁺]_i chelation on β1 integrin activation was reversed by repleting [Ca⁺⁺]_i with ionomycin in a Ca⁺⁺-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca⁺⁺]_i in inside-out activation of β1 integrins, probably through a synergistic effect with PKC activation. J. Cell. Physiol. 175:193–202, 1998. © 1998 Wiley-Liss, Inc.     

Influence of calcium on alginate production and composition in continuous cultures of Azotobacter vinelandii

Summary Azotobacter vinelandii strain E was cultivated in PO ₄ ^-- limited continuous cultures. The influence of growth medium Ca⁺⁺ levels on dry cell weight and alginate production and composition was examined. Low Ca⁺⁺ concentrations (<0.34 mM) were observed to inhibit growth, particularly in cultures maintained at a high dilution rate (D=0.32 hr^-1). In cultures with high levels of polysaccharide (>1.0 g l^-1), the production of alginate with a predominantly heteropolymeric structure was favoured by increasing Ca⁺⁺ levels. In cultures containing less polysaccharide (<1.0 g l^-1) increasing Ca⁺⁺ levels (0.068–0.34 mM Ca⁺⁺) resulted in the production of alginates high in polyguluronate. With further increases in Ca⁺⁺ levels (0.34–2.72 mM Ca⁺⁺) synthesis of alginates with a more heteropolymeric structure occurred. It is proposed that extracellular epimerisation of alginate is influenced by intermolecular associations, the formation of which is mediated by both Ca⁺⁺ concentration and the concentration of the polymer itself.     

Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical — red light promoted and subsequent far-red irradiation reversed an influx of Ca⁺⁺ ions into the protoplasts. These findings imply phytochrome regulation of Ca⁺⁺ fluxes across the plasma membrane. The influx of Ca⁺⁺ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16–18 min after illumination, after which a 6–8 min efflux process was triggered and the basal Ca⁺⁺ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca⁺⁺ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated.Abbreviations A difference in absorbance - CPZ chlorpromazine - FR far-red (light) - MX murexide - PI phosphatidylinositol - PIP₂ phosphatidylinositol 4, 5-bisphosphate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - POPOP 1, 4-bis [2-(5-phenyl-1, 3-oxazolyl)]-benzene - PPO 2, 5-diphenyl-1, 3-oxazole - R red (light) - SOV sodium orthovanadate     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Binding of [⁶⁵Zn⁺⁺] and [⁴⁵Ca⁺⁺] to the acetylcholine (ACh)-receptor, purified from the $\text{Torpedo}$ electric organ, was studied by equilibrium dialysis. Whereas [⁶⁵Zn⁺⁺] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10⁻⁶M, no binding of [⁴⁵Ca⁺⁺] at concentrations up to 10⁻³M could be detected with this method. However, the binding of [acetyl-³H]choline to the receptor was blocked equally by very high Zn⁺⁺ or Ca⁺⁺ concentrations, and the K_i for this low affinity binding was 7 × 10⁻³M. The high affinity binding of [⁶⁵Zn⁺⁺] to the receptor was blocked best by Cd⁺⁺ then Co⁺⁺ and Mn⁺⁺, but least by Mg⁺⁺ and Ca⁺⁺. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca⁺⁺ that could not be removed even by extensive dialysis. When Ca⁺⁺-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca⁺⁺. This was equivalent to 15 moles of Ca⁺⁺ for each mole of ACh bound at saturation. It is suggested that the source of this Ca⁺⁺ is endogenous, and that it is tightly bound to the ACh-receptor molecule.     

Lanthanum in heart cell culture. Effect on calcium exchange correlated with its localization

Correlation of the localization of La⁺⁺⁺ with its effects on Ca⁺⁺ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca⁺⁺ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca⁺⁺/kg wet cells or 43% of the exchangeable Ca⁺⁺ (cells perfused with [Ca⁺⁺]_o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca⁺⁺/kg wet cells or 57% of the exchangeable Ca⁺⁺. 0.5 mM [La⁺⁺⁺]_o displaces 0 52 mmoles Ca⁺⁺/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca⁺⁺ influx and efflux The presence of La⁺⁺⁺ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca⁺⁺ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La⁺⁺⁺ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle     

Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

Background  

The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca⁺⁺/Mg⁺⁺-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca⁺⁺/Mg⁺⁺-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.     

Simplified calcium transport and storage pathways

The control of calcium concentration in the cytoplasm of most cells involves both the influx and efflux of Ca⁺⁺ from extracellular fluid and the release and uptake of Ca⁺⁺ from two separate, but interacting intracellular membrane-bound Ca⁺⁺ stores: (1) the ryanodine receptor-activated calcium store (RyR) and (2) the inositol-trisphosphate (IP₃) receptor calcium store (Golovina and Blaustein, 1997, Spatially and functionally distinct Ca²⁺ stores in sarcoplasmic and endoplasmic reticulum. Science 275, 1643–1648). A more complete understanding of calcium pathways may lead to the development of new strategies to reduce the pathophysiology induced by severe hyperthermia, exercise, hypoxia, and other stresses. This review discusses the fundamental mechanisms involved in the control of Ca_i, the main regulator of biochemical processes, and ultimately, of physiological responses to moderate and severe physical exercise and stress.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Intraerythrocytic plasmodial calcium metabolism

Both prokaryotes and eukaryotes require Ca⁺⁺ for a variety of cellular functions. Intraerythrocytic plasmodia, however, exist within a cell ordinarily impermeable to external Ca⁺⁺. Our investigations of Ca⁺⁺ homeostasis in murine Plasmodium berghei reveal that (1) infected erythrocytes contain 10 – 15 times as much Ca⁺⁺ as do uninfected red cells, (2) these large amounts of Ca⁺⁺ are located almost exclusively within the parasite, and (3) the parasite obtains at least a portion of this Ca⁺⁺ through causing increased permeability of the host cell membrane to external Ca⁺⁺.     

The effects of intracellular iontophoretic injection of calcium and sodium ions on the light response of Limulus ventral photoreceptors

During intracellular iontophoretic injection of Ca⁺⁺ into Limulus ventral photoreceptor cells, there is a progressive diminution of the light response. Following Ca⁺⁺ injection, the size of the light response slowly recovers. Similarly, there is a progressive diminution of the light response during intracellular injection of Na⁺ and recovery after the injection is stopped. The rate of diminution during Na⁺ injection is greater for higher [Ca⁺⁺]_out. In solutions which contain 0.1 mM Ca⁺⁺, there is nearly no progressive decrease in the size of the light response during Na⁺ injection. Intracellular injections of Li⁺ or K⁺ do not progressively decrease the size of the light response. We propose that an increase in [Na⁺]_in leads to an increase in [Ca⁺⁺]_in and that an increase in [Ca⁺⁺]_in by any means leads to a reduction in responsiveness to light.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

Theoretical description of the calcium channel in rat spinal ganglion neurons

The surface charge density (σ′₀) and the binding constant of Ca⁺⁺ with charged groups on the outer surface of the membrane (K_Ca) were calculated from experimentally determined values of the shift of the current-voltage characteristic curves of calcium currents in the membrane of rat spinal ganglion neurons: σ′₀ = 0.15 ± 0.05 e/nm² and K_Ca = 70 ± 10 liters/mole. Using a three-barrier model the energy profile of the calcium channel of the membrane of these neurons was calculated for Ca⁺⁺, Ba⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and verapamil. The calcium current was shown to be determined mainly by the depth of the potential hole corresponding to the outer binding site of the calcium channel. It is concluded from the results that the outer binding site of the calcium channel contains only one carboxyl group.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins

Background  

A wide range of stimuli evoke rapid and transient increases in [Ca²⁺]_cyt in plant cells which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca²⁺-binding EF-hand motifs was analyzed.     

Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene

Background  

Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca²⁺ level ([Ca²⁺]_i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca²⁺/calmodulin-dependent protein kinase.     

Mitogen-activated Ca++ channels in human B lymphocytes

Two complementary experimental methods have been used to examine mitogen-induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen-induced changes in [Ca⁺⁺]_i and transmembrane potential. Activation of human B cells with anti-μ antibodies resulted in a biphasic rise in [Ca⁺⁺]_i, the second phase being mediated by the influx of extracellular Ca⁺⁺. Ca⁺⁺ influx was inhibited by high [K⁺]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti-μ antibodies to the bath solution, inward currents were observed at negative voltages. Whole-cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than ?40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP₃ into the intracellular solution, suggesting that IP₃ generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti-μ and IP³ induced currents were blocked by 1 mM La⁺⁺⁺, which is known to block Ca⁺⁺ channels. These results strongly support the presence of membrane Ca⁺⁺ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca⁺⁺ influx. These mechanisms work in concert to regulate the level of [Ca⁺⁺]_i during the early phases of human B cell activation. © 1993 Wiley-Liss, Inc.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.