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.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Extracellular ATP has a potent effect to enhance cytosolic calcium and contractility in single ventricular myocytes

The effect of extracellular ATP on the contraction of single rat cardiac myocytes was investigated, together with the effect on the transient change in cytosolic Ca2+ (Cai) elicited by excitation and on the relationship between these two parameters. In unstimulated single myocytes, ATP caused a small increase in Cai (measured as the ratio of fluorescence of Indo-1 at 410 to that at 490 nm. In myocytes bathed in a medium containing 1.0 mM [Ca2+] at 23 degrees C and stimulated at 1 Hz, ATP (1 microM) resulted in a two-threefold increase in amplitude of contraction, as measured by video cinemicrographic techniques. The duration of the Cai-transient was not altered but its amplitude was markedly enhanced, as was the amplitude of contraction. The relation between Cai and contraction-amplitude was not altered by ATP, when measured over a range of extracellular [Ca2+], suggesting that ATP does not affect the myofilament-Ca2+ interaction. The primary site of action of ATP in increasing Cai is at the sarcolemma since the addition to suspensions of myocytes of caffeine (10 mM), which depletes the sarcoplasmic reticulum Ca2+ load, does not prevent the subsequent increase of Cai due to ATP. Further, lowering of the extracellular [Ca2+] to less than 1 microM with EGTA abolishes the response of Cai to ATP, though not the response to caffeine. Thus in rat cardiac myocytes ATP stimulates trans-sarcolemmal influx of Ca2+: ADP, AMP and adenosine are ineffective. ATP markedly augments the amplitude of the Cai transient elicited by electrical stimulation thus rendering it a potent inotropic agent.     

Ca entry at rest and during prolonged depolarization in dialyzed squid axons

Ca influx has been studied in squid axons under internal dialysis control. In axons dialyzed with "normal" physiological conditions (Nai = 40-50 mM, Cai2+ = 0.06-0.1 microM, ATP = 2 mM, Ki = 310 mM), 70% of the resting Ca influx is sensitive to external TTX (K0.5 congruent to 5 nM), 20% of it can be accounted by the reversal of the Na-Ca exchange, and the remaining fraction (10%) is insensitive to TTX, D-600, and Nai. The Ca antagonic drug D-600 (50-100 microM) has an inhibitory effect on the resting Ca influx. This compound was found to affect both the TTX sensitive and the Nai-dependent Ca influx components. In the presence of Nai and ATP, Cai2+ activates the carrier mediated Ca entry (Nai-dependent Ca influx). Most of the activation occurs in the submicromolar range of Cai2+ concentrations (K0.5 congruent to 0.6 microM). In the absence of Nai and/or ATP, no activation of Ca influx by Cai2+ was found up to about 5 microM Cai2+. Prolonged depolarization with high Ko causes an increase in Ca influx sustained for long time (minutes). Depolarizing the axons by removing Ki causes the same effect. This depolarization-induced Ca entry was only observed in axons containing Nai. In the absence of Nai, Ca influx decreases with increasing Ko. The activation of the carrier mediated Ca entry (electrogenic Na/Ca exchange) by membrane depolarization was found to be markedly dependent on the magnitude of Ca2+ i. Increasing the magnitude of Ca2+ i from 0.1 to 0.6 microM causes a ten fold increase in the extra Ca influx induced by a K-depolarization.     

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.     

Propagation of transient Ca2+ increase in sea urchin eggs upon fertilization and its regulation by microinjecting EGTA solution

Upon fertilization, the concentration of intracellular Ca2+ (Cai) in sea urchin eggs increased up to 3 microM when measured with fura-2, a fluorescent Ca indicator and the increase in Cai traversed from the sperm entry point as a wave over the entire egg at the mean propagation velocities of 5.0 microns/sec in C. japonicus egg and 5.3 microns/sec in H. pulcherrimus egg. However, the velocity was not uniform; i.e., it was rapid in the vicinity of the sperm entry point and the opposite point, but slow in the central region of the egg. Microinjecting a Ca-EGTA buffer and an IP3 solution into the C. japonicus egg induced the transient Cai increase more rapidly than that upon fertilization, due perhaps to the diffusion of the injectates. In order to investigate Ca2+ release during Cai increase upon fertilization, EGTA solutions were microinjected into unfertilized or fertilizing eggs. Microinjecting 100 mM EGTA (final concentration of 1 mM) not only suppressed the transient Cai increase, but also reduced the increased Cai rapidly, and never induced egg activation after insemination, whereas 10 mM EGTA (final concentration of 0.1 mM) did not significantly affect the Cai increase or the activation. Ca2+ released upon fertilization was estimated to be 150-170 microM in the egg cytoplasm from the amount of microinjected EGTA and fura-2. It was concluded that although more than 150 microM of Ca2+ was released intracellularly upon fertilization, Cai increased to only a few microM because most of the released Ca2+ was sequestered by intracellular Ca2+ binding substances.     

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.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).     

Three types of cytoplasmic Ca2+ oscillations in stimulated pancreatic beta-cells

Oscillations of cytoplasmic Ca2+ (Ca2+i) involved in cell regulation have recently attracted considerable attention. In the pancreatic beta-cells an intermediate concentration of glucose (11 mM) induces large oscillations of Ca2+i with periods of 2 to 6 min. Procedures stimulating insulin secretion further, such as raising glucose to 20-30 mM or adding carbachol, ATP, theophylline, glucagon, or forskolin, often changed these oscillations into a steady increase of Ca2+i. In addition, forskolin and glucagon triggered prominent 9- to 14-s Ca2+i spikes during the intervals of increased Cai2+, whereas carbachol and ATP initiated a series of rapid spikes of decreasing magnitude and increasing duration (6-11 s). All types of oscillations depended on the presence of extracellular Ca2+i, but carbachol and ATP also induced single Cai2+ transients in the absence of the cation. The results demonstrate hitherto unknown oscillations of Ca2+i in the pancreatic beta-cell which are dependent in different ways on Ca2+ entry.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiological characterization of 45Ca2+ and 65Zn2+ transport by lobster hepatopancreatic endoplasmic reticulum

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.     

The mechanism of the calcium signal and correlation with histamine release in 2H3 cells

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.     

Regulation of human eosinophil migration across lung epithelial monolayers by distinct calcium signaling mechanisms in the two cell types.

In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca2+ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca2+ in eosinophils with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca2+ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca2+ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La3+ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La3+ to eosinophils did not. Taken together, these results show the importance of Ca2+ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca2+ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca2+ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca2+.     

Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration ([Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of [Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of [Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal [Ca2+]i but had no effect on the rate of agonist-induced [Ca2+]i increase or its absolute amount. In contrast, the kinetics of [Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in [Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second [Ca2+]i rise in response to the agonists occurred. The decrease in [Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in [Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.     

Simultaneous exposure to ATP and phenylephrine induces a sustained elevation in the intracellular calcium concentration in supraoptic neurons

Vasopressin (VP) release from the hypothalamo-neurohypophyseal system (HNS) is stimulated by ATP activation of P2X purinergic receptors and by activation of 1-adrenergic receptors by phenylephrine (PE). These responses are potentiated by simultaneous exposure to ATP+PE. Potentiation was blocked by depleting intracellular calcium stores with thapsigargin. To test the hypothesis that the synergistic response to ATP+PE reflects alterations in the intracellular calcium concentration ([Ca2+]i), [Ca2+]i was monitored in supraoptic neurons in HNS explants loaded with fura 2-AM. Both ATP and PE induced rapid, but transient, elevations in [Ca2+]i. In 0.3 mM Ca2+, the peak response to ATP was greater than to PE but did not differ from the peak response to ATP+PE. A sustained elevation in [Ca2+]i was induced by ATP+PE, that was greater than ATP or PE alone. In 2 mM Ca2+, the peak response to ATP+PE was significantly greater than to either ATP or PE alone, and the sustained response to ATP+PE was greater than to either agent alone. Responses were comparable in the presence of TTX. The sustained elevation in [Ca2+]i was also observed when ATP+PE was removed after 1 min, but it was eliminated by either thapsigargin or removing external calcium, indicating that both calcium influx and calcium release from internal stores are required. Some cells were vasopressinergic based on a VP-induced increase in [Ca2+]i. These observations support the hypothesis that simultaneous exposure to ATP+PE induces a different pattern of [Ca2+]i than either agent alone that may initiate events leading to synergistic stimulation of VP release.     

Calcium fluxes across the membrane of sarcoplasmic reticulum vesicles

The relationship between calcium exchange across the membrane of sarcoplasmic reticulum vesicles and phosphoenzyme (EP) was examined in calcium transport reactions using a limited amount of ATP as substrate. Rapid calcium influx and efflux (approximately 385 nmol.(mg.min)-1), measured in reactions in which ATP concentration fell from 20 microM, was accompanied by a shift in the equilibrium between an ADP-sensitive EP and an ADP-insensitive EP toward the former. Rapid exchange between ATP and ADP (approximately 1500 nmol.(mg.min)-1) was also observed under conditions where no significant incorporation of Pi into ATP took place, suggesting that ATP in equilibrium ADP exchange can occur without Cao in equilibrium Cai exchange. Ca2+ permeability during the calcium transport reaction was estimated in reactions carried out with acetylphosphate, which produces a hydrolytic product that does not participate in the backward reaction of the calcium pump. Under conditions where the calcium content exceeded 43 nmol.mg-1, a level that may reflect the binding of calcium ions to sites inside the sarcoplasmic reticulum, the rate constant for Ca2+ efflux was 0.33 min-1. These data allow the rate of passive Ca2+ efflux to be estimated as approximately 17 nmol.(mg.min)-1 at the time when calcium content was maximal and a rapid Cao in equilibrium Cai was observed. It is concluded that the majority of the rapid Ca2+ efflux is mediated by partial backward reactions of the calcium pump ATPase.     

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.     

The calcium mobilizing tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion

The ability of the platelet agonists thapsigargin (Tg) and thrombin to elevate the cytoplasmic free calcium level ([Ca2+]i) was examined. Both agonists induced a transient increase of [Ca2+]i with a different time-course, however. Thus, the maximal [Ca2+]i was reached 15 sec and 2 min after stimulation with thrombin and Tg, respectively. The thrombin induced rise of [Ca2+]i was reversible, which indicates that active calcium sequestration and/or extrusion is operating. Tg affected [Ca2+]i in a divergent manner, thus, [Ca2+]i was stabilized on a elevated level without initial formation of a pronounced peak. The decline in [Ca2+]i observed after thrombin stimulation was not impaired by the calmodulin binding drug trifluoperazine but it was strongly reduced by vanadate, which suggests the active calcium transport systems to be insensitive to calmodulin. We put forward the hypothesis that the tumor promoting activity of Tg is attributable to its ability to stabilize [Ca2+]i on a new elevated steady state level.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.