Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration

Transient increases in cytosolic free calcium concentration, [Ca2+]i, appear to be required for the migration of human neutrophils on poly-D-lysine-coated glass in the presence of dilute serum (Marks, P. W., and F. R. Maxfield. 1990. J. Cell Biol. 110:43-52). In contrast, no requirement for [Ca2+]i transients exists when neutrophils migrate on albumin-coated glass in the absence of serum. To determine the mechanism that necessitates [Ca2+]i transients on poly-D-lysine in the presence of serum, migration was examined on substrates consisting of purified adhesive glycoproteins. In the absence of external Ca2+, a treatment which causes the cessation of [Ca2+]i transients, migration on fibronectin (fn) and vitronectin (vn) was significantly inhibited. Migration was also inhibited in Ca2(+)-buffered cells on these substrates, indicating that this effect was the result of an alteration of [Ca2+]i. In the absence of external Ca2+, the inhibition of migration on fn or vn was more pronounced when soluble fn or vn was added to cells migrating on these substrates. This effect of soluble adhesive glycoprotein was specific: in the absence of external Ca2+, soluble fn did not affect the migration of cells on vn, and soluble vn did not affect the migration on fn. No additional inhibition of migration was observed in Ca2(+)-buffered cells with the addition of soluble adhesive glycoprotein. These data indicate that [Ca2+]i transients are involved in continued migration of human neutrophils on fn or vn, proteins which are part of the extracellular matrix that neutrophils encounter in vivo.     

Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.     

Evidence that phorbol ester interferes with stimulated Ca2+ redistribution by activating Ca2+ efflux in neutrophil leucocytes.

The free calcium ion concentration, [Ca2+]i, in the cytoplasmic matrix of quin2-loaded neutrophil leucocytes increases rapidly after addition of concanavalin A. This increase is effectively abolished by a short (3 min) preincubation with 10 nM-TPA (12-O-tetradecanoylphorbol 13-acetate). TPA also inhibits a [Ca2+]i rise of similar magnitude induced by low concentrations (10 nM) of calcium ionophore A23187, suggesting that phorbol ester does not interfere with a physiological influx mechanism. To investigate the effects of TPA further, cells were depleted of Ca2+ during quin2 loading and then re-equilibrated with normal extracellular [Ca2+]. The return to a stable [Ca2+]i value was preceded by a transient overshoot in [Ca2+]i, implying delayed activation of an efflux mechanism by rising [Ca2+]i. TPA abolished the transient, suggesting preactivation by TPA of the efflux mechanism before Ca2+ influx. TPA also stimulates net Ca2+ efflux from neutrophils and neutrophil cytoplasts. These observations are consistent with the thesis that TPA stimulates a Ca2+-efflux mechanism in these cells.     

ATP-induced calcium mobilization in human neutrophils

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Role of intracellular calcium in Pasteurella haemolytica leukotoxin-induced bovine neutrophil leukotriene B4 production and plasma membrane damage

Isolated neutrophils were used to study the intracellular calcium ([Ca2+]i) dependency of Pasteurella haemolytica leukotoxin-induced production of leukotriene B4 and plasma membrane damage. Exposure of neutrophils to leukotoxin caused a rapid and concentration-dependent increase in [Ca2+]i, followed by simultaneous plasma membrane damage and production of leukotriene B4. Removal of extracellular Ca2+, replacement of Ca2+ with other divalent cations, or exposure to high concentration of verapamil, an inhibitor of voltage-dependent calcium channels, inhibited leukotoxin-induced increases in [Ca2+]i, leukotriene B4 production, and membrane damage, thus indicating that influx of extracellular Ca2+ is necessary to produce these leukotoxin-induced neutrophil responses.     

The role of extracellular Ca2+ in the response of the hepatocyte to Ca2+-dependent hormones

The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.     

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functions. Comparison with the role of phosphotidylinositol-3 kinase.

In human neutrophils, both changes in intracellular Ca(2+) concentrations, [Ca(2+)]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca(2+)]i and its effector molecule calmodulin in human neutrophils. Increased [Ca(2+)]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3alpha (GSK-3alpha). Inhibition of calmodulin using a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3alpha phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca(2+)/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca(2+)]i. Taken together, our data indicate critical roles for changes in [Ca(2+)]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca(2+)/calmodulin sensitive pathway in human neutrophils.     

Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family

Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.     

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Intracellular calcium levels correlate with speed and persistent forward motion in migrating neutrophils.

The relationship between cytosolic free calcium concentration ([Ca2+]i) and human neutrophil motility was studied by video microscopy. Neutrophils stimulated by a uniform concentration of an N-formylated peptide chemoattractant (f-Met-Leu-Phe) were tracked during chemokinetic migration on albumin, fibronectin, and vitronectin. [Ca2+]i buffering with quin2 resulted in significant decreases in mean speed on albumin. To further characterize the relationship between [Ca2+]i changes and motility we carried out a cross-correlation analysis of [Ca2+]i with several motility parameters. Cross-correlations between [Ca2+]i and each cell's speed, angle changes, turn strength, and persistent forward motion revealed (i) a positive correlation between [Ca2+]i and cell speed (p < 0.05), (ii) no significant correlation between turns and calcium spikes, and (iii) the occurrence of turns during periods of low speed. Significant negative correlations between [Ca2+]i and angle change were noted on the high adhesion substrates vitronectin and fibronectin but not on the low adhesion substrate albumin. These data imply that there is a general temporal relationship between [Ca2+]i, speed, and persistent motion. However, the correlations are not sufficiently strong to imply that changes in [Ca2+]i are required proximal signals for velocity changes.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Ca2+]i independent mitogenesis in cultured human fibroblasts revealed by single cell microfluorimetry

A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i.     

Melanin has a role in Ca2+ homeostasis in human melanocytes

We have examined whether melanin affects Ca2+ homeostasis in cultured normal human melanocytes. Intracellular Ca2+ concentrations ([Ca2+]i), were measured in four Caucasian and in three Negroid melanocyte cultures. Under resting conditions [Ca2+]i was around 100 nM in all cultures, but differences between cells within cultures were observed. All cultures responded to endothelin-1 (ET-1) with increases in [Ca2+]i and there were no differences between Caucasian and Negroid cultures. However, large differences in responses between cells within cultures were observed, indicating that melanocyte cultures are very heterogeneous. The addition of 2.5 mM CaCl2 to melanocytes kept in Ca2+-free medium resulted in rapid and transient increases in [Ca2+]i of up to 1500 nM. These increases were on average more than two times smaller in melanocyte cultures established from Negroid donors compared with Caucasian cultures. In addition, well melanized Caucasian melanocytes, cultured in the presence of 400 microM tyrosine and 10 mM NH4Cl, showed a reduced increase in cytoplasmic Ca2+ concentration upon the addition of extracellular Ca2+. The difference in maintaining Ca2+ homeostasis between poorly and well melanized melanocytes may be the result of the clearance of cytoplasmic Ca2+ into melanosomes and the greater capacity for this in the more pigmented melanocytes.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor- mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.