Quin2-loaded Cells Research Articles

Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake.

Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca2+ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 micrograms/ml of compound 48/80, a rapid increase in intracellular Ca2+ was observed. At concentrations higher than 10(-6) M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca2+ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca2+ release from the intracellular Ca store.

Changes of plasma membrane permeability in neutrophils treated with polycations

The effects of the polycations poly-L-arginine, poly-L-lysine, and poly-ethyleneimine on rabbit neutrophil membrane permeability were compared. LDH release, quin2 release from quin2-loaded cells, and increase of indo 1 fluorescence were considered as measures for changes in membrane permeability. All polycations cause abundant LDH release. Quin2 release occurs more rapidly than LDH release, and the increase of indo 1 fluorescence is even faster. Apparently polycation-induced permeability changes occur gradually, allowing the influx (or efflux) of small molecules more rapidly than larger ones. A number of divalent and trivalent cations inhibit polycation-induced LDH and quin2 release in a way that resembles the inhibition of other cytotoxic agents described in literature. In the absence of extracellular Ca2+, the polycations induce little lysozyme release. In the presence of extracellular Ca2+, there is abundant lysozyme release, indicating that the influx of Ca2+ causes exocytosis. Exocytosis still occurs when Ca2+ is added some time after polycation addition, indicating that polycation treatment leaves the cells largely intact. All polycations tested have in common that they cause gradual changes in the permeability of the plasma membrane only, which opens the possibility to use them as membrane-permeabilizing agents for the study of Ca(2+)-induced exocytosis.

Extracellular ATP 4− permeabilizes thymocytes not only to cations but also to low-molecular-weight solutes

Extracellular ATP increases the free cytosolic calcium concentration ([Ca 2+] i) of thymocytes by changing the membrane permeability to divalent cations: (1) no [Ca 2+] i elevationis observed in the absence of extracellular calcium and (2) ATP also promotes Mn 2+ entry into thymocytes, as shown by a stimulated quench of the fluorescence of Quin2-loaded cells. Using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, a dye which usually does not pass through membranes, we showed that ATP permeabilizes thymocyte membranes not only to cations but also to low-molecular-weight solutes. The ATP effect is dose-dependent, specific and reversible; ATP 4− appears to be the active species and probably acts through specific receptor(s). Both medullary and cortical thymocytes respond to ATP whereas splenocytes do not; this shows that the ATP effect is restricted to certain populations of lymphocytes. The thymoma-derived cell line, EL-4, the permeability of which is also sensitive to ATP, could provide a useful model for studying the putative ATP 4− receptor(s) of thymocytes.

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages.

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.

The role of cytosolic free Ca2+ and protein kinase C in acetylcholine-induced insulin release in the clonal β-cell line, HIT-T15

We examined the contribution of signal-transduction pathways to acetylcholine-induced insulin release in the clonal beta-cell line HIT-T15. To assess the importance of changes in cytosolic free Ca2+ [( Ca2+]i), we studied time courses of the effects of glucose and acetylcholine on [Ca2+]i and insulin release in quin 2-loaded HIT cells. Incubation in the presence of glucose (2 mM) resulted in a sustained increase in [Ca2+]i in HIT cells from 98 +/- 7 nM to 195 +/- 12 nM measured after 9 min, whereas subsequent addition of acetylcholine (50 microM) produced a transient increase in [Ca2+]i which reached a peak after 30 s (at 274 +/- 10 nM), returning to pre-stimulus levels after 3 min. In contrast, incubation of HIT cells with acetylcholine in the presence of glucose produced a sustained increase in insulin release over and above that stimulated by glucose alone; after 10 min acetylcholine had potentiated glucose-stimulated insulin release by an additional increment of 135%. The transient increase in [Ca2+]i induced by acetylcholine was dose-dependent, and was prevented by omission of glucose or extracellular Ca2+ from the incubation medium. It was also inhibited by inclusion of 50 microM-verapamil in the incubation medium (by 87 +/- 3%) or by decreasing the Na+ concentration in the medium (by 73 +/- 6%). To evaluate the role of the protein kinase C pathway, we have pretreated HIT cells with the phorbol ester 12-O-tetradecanoylphorbol acetate (TPA), to deplete the protein kinase C activity, and have compared their secretory activity with that of control cells. Protein kinase C activity was decreased by 73% in HIT cells cultured in the presence of 200 nM-TPA for 22-24 h. TPA pre-treatment also significantly decreased the insulin content of HIT cells, but had no effect on cell number or the increases in [Ca2+]i induced by glucose or acetylcholine. TPA-pre-treated cells responded comparatively less well to secretagogues than did control cells: glucose-stimulated insulin release was decreased by 40%, whereas potentiation by TPA was significantly decreased by 50% in comparison with control cells (P less than 0.05, n = 24). Acetylcholine (50 microM) potentiated glucose-stimulated insulin release by 61% in control cells. This effect was abolished in HIT cells pre-treated with TPA, whereas these cells still retained their normal secretory response to stimulation by forskolin. These data suggest that an early increase in [Ca2+]i may be important for the initial increase in insulin release induced by acetylcholine in HIT cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced release of prostacyclin from quin 2-loaded endothelial cells

The acetoxymethyl ester (AM) of quin-2 (quin-2/AM) enhanced the release of prostacyclin (PGI 2) from bovine aortic endothelial cells stimulated by ATP, bradykinin or ionophore A23187. It also increased the mobilization of free arachidonic acid in response to ATP. Ca 2+-clamping with a combination of EGTA and quin-2/AM abolished the response to ATP. The effect of quin-2/AM was mimicked by a structural analog, the acetoxymethyl ester of 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA/AM), but not by the heavy metal chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and only slightly by fura-2/AM. The mechanism of this pharmacological action of quin-2/AM and its potential for the design of PGI 2-stimulating drugs remain to be explored.

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2.

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.

Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal beta-cell line HIT-T15.

We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal beta-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mM increased Cai by 36%, from 89 +/- 4 to 121 +/- 6 nM (mean +/- S.E.M., n = 23). Further raising the medium glucose concentration to 10 mM increased Cai to 139 +/- 6 nM. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mM K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 +/- 48 nM. The increases in Cai in response to glucose of K+ were blocked by the simultaneous presence of verapamil (50 microM). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells

To determine whether increases in the cytosolic free Ca 2+ concentration ((Ca 2+] i) accompany agoniststimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca 2+] i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca 2+] i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca 2+] i had been reduced by loading cells with quinl in medium containing low [Ca 2+]. Ionomycin increased [Ca 2+] i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca 2+] i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca 2+] i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+] i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca 2+] i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca 2+] i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca 2+] i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca 2+] i than does TPA-stimulated secretion.

Free calcium and calmodulin levels in acinar carcinoma and normal acinar cells of rat pancreas

Exposure of acinar carcinoma cells and normal acinar cells of rat pancreas to the muscarinic agonist drug carbamylcholine stimulated 45Ca2+ outflux from 45Ca2+-labeled cells. More rapid outflux of 45Ca2+ was detected for carcinoma cells following muscarinic stimulation than for normal cells. Direct fluorometric measurement of cytosolic Ca2+ under basal (unstimulated) conditions in quin 2-loaded cells revealed significantly lower concentration of free Ca2+ in carcinoma cells (approximately 180 nM) than in normal cells (approximately 200 nM). Stimulation with 1 mM carbamylcholine increased the cytosolic Ca2+ concentration in carcinoma and normal cells to approximately 1900 nM, after which carcinoma cells removed cytosolic Ca2+ at a faster rate to a post-stimulation plateau concentration of approximately 140 nM, in comparison to normal cells which obtained a post-stimulation plateau concentration of approximately 300 nM. Essentially identical differences between carcinoma and normal cells were detected upon stimulation with the peptidergic agonist cholecystokinin octapeptide. Finally, carcinoma cells demonstrated approximately 3 times greater calmodulin concentration than normal acinar cells. Also, the calmodulin antagonist drug W7 (N-6-(aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibited the carbamylcholine-induced release of intracellular Ca2+ in acinar carcinoma cells. These results indicate that neoplastic pancreatic acinar cells have retained mechanisms of muscarinic- and peptidergic-stimulated intracellular Ca2+ release, and implicate calmodulin as a regulatory factor in secretagogue activation of intracellular Ca2+ release. We propose that the more rapid decline of intracellular Ca2+ concentration following muscarinic or peptidergic stimulation and the increased intracellular calmodulin concentration indicate calmodulin-mediated down-regulation of free cytosolic Ca2+ in acinar carcinoma cells to levels lower than those of normal cells.

Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes.

It has previously been shown that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GM1, stimulates the proliferation of rat thymic lymphocytes (Spiegel, S., P. H. Fishman, and R. J. Weber, 1985, Science [Wash. DC], 230:1285-1287). The purpose of this study was to identify which transmembrane signaling system(s) are activated by the B subunit of cholera toxin. We compared the effects of B subunit and concanavalin A (Con A), a potent mitogenic lectin, on a number of second messenger systems that are putative mediators of T cell activation. Changes in the fluorescence of quin2-loaded cells revealed that mitogenic doses of either B subunit or Con A induced rapid and sustained increases in cytoplasmic free Ca2+ ([Ca2+]i). Within 5 min, [Ca2+]i increased from a basal level of 69 +/- 4 to 136 +/- 17 and 185 +/- 24 nM, respectively. The effects of B subunit and Con A were additive and largely dependent on the presence of extracellular Ca2+, though release of Ca2+ from intracellular stores could be detected for Con A, but not B subunit, using indo-1. The B subunit had no effect on either inositol phosphate levels or on the distribution of protein kinase C, indicating that, unlike Con A, the B subunit does not activate phosphoinositide hydrolysis. Fluorimetric measurements on cells loaded with bis(carboxyethyl)-5,6-carboxyfluorescein revealed that Con A induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas B subunit had no effect on intracellular pH. Finally, by monitoring bis-oxonol fluorescence, we found that Con A induced a small hyperpolarization of the membrane potential, whereas B subunit had no acute effect. These data suggest that the biological effects of B subunit are mediated by an increase in [Ca2+]i resulting from a net influx of extracellular Ca2+.

Calcium ionophore-induced changes in HCO 3− secretion and Cl − absorption in turtle bladder: relation to action of 3-isobutyl-1-methylxanthine

The calcium ionophore A23187 stimulates luminal alkalinization and inhibits Cl − absorption in short-circuited urinary bladders of postprandial or alkalotic turtles. The ionophore appears to mimic the action of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) by its similar effects on HCO 3 − secretion and Cl − absorption and by increasing cytosolic cAMP levels of isolated bladder epithelial cells. However, only A23187 (or ionomycin), but not IMBX or cAMP, elevated cytosolic Ca 2+ of aequorin- or quin2-loaded cells. Since A23187, but not IBMX or cAMP inhibits luminal acidification, we postulate that cytosolic Ca 2+ (1) regulates the acidification process by a cAMP-independent mechanism and (2) controls HCO 3 − secretion as well as Cl − absorption, at least in part, via cAMP-mediated pathways.

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells.

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.

Origin of intracellular calcium and quantitation of mobilizable calcium in neutrophils stimulated with chemotactic peptide

The origin and amount of mobilized Ca 2+ in chemotactic peptide-stimulated guinea pig neutrophils were examined using biochemical techniques. The total amount of releasable Ca 2+ by 20 μM A23187 from the unstimulated intact cells was 0.91 nmol 4·10 6 cells , as assessed by change in absorbance of the antipyrylazo III-Ca 2+ complex. Two types of internal vesicular Ca 2+ pool, mitochondrial and non-mitochondrial pool were identified in the saponin-permeabilized cells. The total amount of releasable Ca 2+ was comparable to that accumulated by the non-mitochondrial pool at (1−2)·10 −7 M of a free Ca 2+ concentration. The mitochondrial uncoupler, capable of releasing Ca 2+ from the mitochondrial pool, neither modified the basal cytosolic free Ca 2+ in quin 2-loaded cells nor caused a Ca 2+ efflux from the intact cells. These results suggest that the releasable Ca 2+ may be located in the non-mitochondrial pool of unstimulated intact cells, and the mitochondrial pool contains little releasable Ca 2+. The addition of fMet-Leu-Phe increased the cytosolic free Ca 2+ by two processes: Ca 2+ mobilization from internal stores and Ca 2+ influx through the surface membrane. The Ca 2+ mobilized and effluxed from the intact cells by stimulation with the maximal doses of fMet-Leu-Phe was estimated to be 0.27 nmol 4·10 6 cells . Almost equal amounts were released by the maximal doses of inositol 1,4,5-trisphosphate from the non-mitochondrial pool of saponin-treated cells that had accumulated Ca 2+ at a free Ca 2+ concentration of 1.4·10 −7 M. The mechanism related to the Ca 2+ influx by fMet-Leu-Phe stimulation was also examined. The addition of nifedipine or phosphatidic acid did not affect the change in the cytosolic free Ca 2+ induced by fMet-Leu-Phe, thereby suggesting that the receptor-mediated Ca 2+ channel may be involved in the Ca 2+ influx.

Muscarinic receptor-stimulated Ca 2+ signaling and inositol lipid metabolism in avian salt gland cells

Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca 2+] i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca 2+] i, while incorporation of [ 32P]P i into phosphatidylinositol (PI) and phosphatidate are similarly increased. [ 3H]Inositol phosphates, measured in the presence of Li +, increased 13-fold. The stimulated increment in [Ca 2+] i required extracellular Ca 2+, whereas [ 3H]inositol phosphate accumulation was independent of external Ca 2+. Dose-response curves for carbachol-induced increments in [Ca 2+] i, PI labeling, and labeled inositol phosphate release are similar, with EC 50 values of 6, 4.5 and 8 μM, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 ± 0.3 nM and 0.48 ± 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.

4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes.

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.

Comparative Effect of Angiotensin II, Potassium, Adrenocorticotropin, and Cyclic Adenosine 3′,5′-Monophosphate on Cytosolic Calcium in Rat Adrenal Cells

The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells.

Role of a pertussis toxin substrate in the control of lectin-induced cap formation in human neutrophils.

We have examined the role of GTP-binding proteins and the associated cyclic AMP- and calcium-related transduction mechanisms in the regulation of capping in human neutrophils. Pertussis toxin (PT), a probe for the GTP-binding protein Ni, abolished capping induced by fluorescein isothiocyanate-conjugated concanavalin A (Con-A), whereas cholera toxin, a probe for the GTP-binding protein Ns, was without effect. Consistent with the latter finding, ligands acting at receptors associated with the Ns protein, namely the prostaglandin E1 and beta-adrenergic agonists, were without effect on the capping reaction. The possible role of mobilization of internal calcium was evaluated by using Quin2-loaded cells. Calcium mobilization was observed at concentrations of Con-A which yielded optimal capping (10 micrograms/ml). Treatment with PT, phorbol myristrate acetate or 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) abolished both calcium mobilization and capping. Colchicine, which substantially enhanced capping, had no effect on calcium mobilization. At concentrations of the lectin above those required for capping, superoxide generation and enzyme release were noted. These reactions were less susceptible to inhibition by PT, effects being observed only on the Kact. for Con-A-mediated superoxide generation with little effect on the Vmax. The degree of PT-mediated inhibition for enzyme release with Con-A was much lower than that observed with fMet-Leu-Phe. Our results imply that a step involving Ni-mediated calcium mobilization, sensitive to phorbol myristate acetate, is essential to the regulation of capping; a distinct mechanism may be involved in colchicine-mediated enhancement of capping; and Ni may play a relative minor role in the regulation of lectin-mediated exocytosis.

Synergy between the T3/antigen receptor complex and Tp44 in the activation of human T cells.

In addition to the T3/antigen receptor complex (T3/Ti), other T cell surface molecules participate in early events involved in human T cell activation. In this report we document that monoclonal antibody 9.3, which recognizes a 90,000 dalton homodimer expressed on human T cells, synergizes with ligands reacting with T3/Ti to activate purified T cells and Jurkat, a human T cell leukemic line. Unlike phorbol myristate acetate (PMA), 9.3 was able to synergize only with anti-T3 or anti-Ti if these antibodies were immobilized. Moreover, 9.3 failed to synergize with the calcium ionophore ionomycin. At high concentrations only, 9.3 could synergize with PMA in the activation of Jurkat and a T3/Ti negative mutant of Jurkat. At such high concentrations of 9.3, small transient increases in cytoplasmic free calcium ((Ca++)i) were detected in quin 2-loaded Jurkat cells. This increase in (Ca++)i was the result of release of internal stores of calcium. 9.3 induced the hydrolysis of polyphosphoinositides, albeit the magnitude of inositol phosphates generated in response to 9.3 was substantially less than that observed with anti-Ti. No effect on pkC translocation was observed in Jurkat cells stimulated with 9.3. Although the small increase in (Ca++)i induced by 9.3 may account for its synergy with PMA, this effect is unlikely to account for the more potent synergistic effect observed with 9.3 and phytohemagglutinin or immobilized anti-T3 and anti-Ti antibodies.