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.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

Regulation of intracellular calcium in squid axons

Internal dialysis and metallochromic indicators were used to determine the free calcium concentration and calcium buffering properties of squid axoplasm. The free calcium concentration in fresh unloaded squid axons is about 30 to 50 nM. About 6% of the calcium content (ca. 50 mumol/kg axoplasm) of a fresh squid axon is held in a metabolically labile, presumably mitochondrial, component. A morphological consequence of this finding is that there should be no accumulation of calcium in mitochondria of fresh squid axons unless there is a large component of nonlabile calcium. The physiological implication is that the mitochondria are probably not buffers for physiological perturbations in free calcium concentration. When an exogenous load of several hundred mumol/kg axoplasm with an ambient ionized calcium concentration above a few hundred nanomolar is applied to axoplasm, all of it goes into organelles. About one-third of that load is found in the mitochondria and about two thirds in some other organelles. When axoplasm is poisoned with carbonyl cyanide-p-trifluoromethonyphenylhydrazone (FCCP), around 70% of the load remains in the nonmitochondrial fraction.     

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Summary Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca _f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca _f has been measured using the intracellular Casensitive fluorescent dyes Fura 2 and Quin. 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Ca _f was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Ca _f was 300–380 nM and, as dye content was increased, Ca _f progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na–Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Ca _f , prolonged treatment with ouabain had no effect on Ca _f despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na–Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Ca _f . However, exchange-mediated Ca efflux may play a role in Ca _f regulation when cytosolic calcium is elevated.     

Synchronous transporting activity in epithelial cells in relation to intracellular calcium concentration.

Cultured monolayers of human sweat-gland epithelia have been used to measure electrogenic sodium transport, as short-circuit current, and intracellular Ca2+ concentration ([Ca]i) from Fura-2 fluorescence. The sodium currents in response to the agonists lysylbradykinin, histamine and carbachol show oscillatory behaviour in the 1-2 per minute frequency range. The oscillations can be terminated either by using specific antagonists or with amiloride, which prevents sodium entry into the epithelium. Oscillatory behaviour is also seen when [Ca]i is measured and occurs in the same frequency range. Sodium transport in these cultured epithelia is thought to result from an increase in [Ca]i, which in turn activates calcium-sensitive potassium channels, so increasing the electrochemical gradient for sodium entry. The oscillatory behaviour implies that the epithelial cells behave in synchrony to increase [Ca]i, so inducing synchronous changes in sodium current. It is shown that the behaviour is not unique to sodium-absorbing epithelia, and the possible utility of synchronous behaviour in epithelial tissues is discussed.     

UVB irradiation-induced dysregulation of plasma membrane calcium ATPase1 and intracellular calcium homeostasis in human lens epithelial cells

Ultraviolet B (UVB) could lead to the apoptosis of human lens epithelial cell and be hypothesized to be one of the important factors of cataractogenesis. In the human lens, Ca²⁺-ATPase is a major determinant of calcium homeostasis. Plasma membrane calcium ATPase1 (PMCA1) is a putative “housekeeping” isoform and is widely expressed in all tissues and cells, which plays an important role in calcium homeostasis. However, the effects of UVB-irradiation on the expression of PMCA1 and the cellular calcium homeostasis are still unclear. In the present study, we cultured human lens epithelial cells (HLE B-3) in vitro and investigated the effects of UVB irradiation on the expression of PMCA1 and the intracellular calcium homeostasis using real-time cell electronic sensing system, flow cytometry, fluo-3/AM probes, real-time quantitative PCR, and enzyme-linked immunosorbent assay techniques. We found that UVB irradiation could induce human lens epithelial cell death, cause intracellular calcium ion (Ca²⁺) elevation, inhibit Ca²⁺-ATPase activity and decrease the expression of PMCA1 at gene and protein levels, suggesting that the downregulation of PMCA1 and the disruption of calcium homeostasis may play important roles in UVB-induced HLE B-3 cell apoptosis.     

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (?log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.     

Regulation of intracellular calcium in human esophageal smooth muscles

We have investigated sources ofCa²⁺ contributing to excitation ofhuman esophageal smooth muscle, using fura 2 to study cytosolic freeCa²⁺ concentration([Ca²⁺]_i)in dispersed cells and contraction of intact muscles. Acetylcholine (ACh) caused an initial peak rise of[Ca²⁺]_ifollowed by a plateau accompanied by reversible contraction. Removal ofextracellular Ca²⁺ or addition ofdihydropyridine Ca²⁺ channelblockers reduced the plateau phase but did not prevent contraction.Caffeine also caused elevation of[Ca²⁺]_iand blocked responses to ACh. Undershoots of[Ca²⁺]_iwere apparent after ACh or caffeine. Blockade of the sarcoplasmic reticular Ca²⁺-ATPase bycyclopiazonic acid (CPA) reduced the ACh-evoked increase of[Ca²⁺]_iand abolished the undershoot, indicating involvement ofCa²⁺ stores. When contraction wasstudied in intact muscles, removal ofCa²⁺ or addition of nifedipinereduced, but did not abolish, carbachol (CCh)-induced contraction.Elevation of extracellular K⁺caused contraction that was inhibited by nifedipine, although CCh stillelicited contraction. CPA caused contraction and suppressed theCCh-induced contraction, whereas ryanodine reduced CCh-induced contraction. Our studies provide evidence that muscarinic excitation ofhuman esophagus involves both release ofCa²⁺ from intracellular stores andinflux of Ca²⁺.

     

Regulation of TRPM2 by extra- and intracellular calcium

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.