Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.     

Double stimulation with FMLP and Con A restores the activation of the respiratory burst but not of the phosphoinositide turnover in Ca2+-depleted human neutrophils. A further example of dissociation between stimulation of the NADPH oxidase and phosphoinositide turnover

The results reported here show that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A) may occur with a stimulus response coupling sequence that bypasses the activation of phosphoinositide hydrolysis, monitored as accumulation of inositol phosphates and glycerophosphoinositol, and the increase in [Ca2+]i. In fact: in Ca2+-depleted neutrophils FMLP and Con A do not induce the respiratory burst and the activation of phosphoinositide hydrolysis. The addition of Ca2+ restores both the respiratory and the phosphoinositide responses; the double treatment of Ca2+-depleted neutrophils with FMLP and Con A in sequence, before FMLP and then Con A and vice versa, or simultaneously, restores the capacity to respond to the second stimulus with the respiratory burst but not with the activation of phosphoinositide hydrolysis. These findings suggest that, for the activation of the NADPH oxidase by FMLP and by Con A: the transduction pathway including the stimulation of phosphoinositide turnover, the Ca2+ changes and the activity of the protein kinase C is not required, or is not the unique, and one stimulus may trigger more than one transduction pathway. Possible transduction pathways are discussed.     

Diacylglycerol kinase inhibitors R59022 and dioctanoylethylene glycol potentiate the respiratory burst of neutrophils by raising cytosolic Ca2+

The diacylglycerol kinase inhibitors, R59022 and dioctanoylethylene glycol (diC8-eg), potentiate stimulation of the respiratory burst by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. However, in contrast to the potentiation observed in intact cells, neither R59022 nor diC8-eg enhanced the effect of fMLP on O2 consumption in electropermeabilized neutrophils, under conditions where cytosolic [Ca2+] was held constant using EGTA. In unstimulated, intact cells treatment with the diacylglycerol kinase inhibitors elicited an increase in cytosolic Ca2+ ([Ca2+]i). The results suggest that enhancement of the respiratory burst by diC8-eg and R59022 is mediated by a rise in [Ca2+]i, rather than by inhibition of diacylglycerol kinase.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate and increase in cytosolic free Ca2+-concentration induced in a human T cell leukemia line, JURKAT, by monoclonal antibodies against the T3 complex

The monoclonal antibodies against the T3 complex on human T lymphocytes, anti-Leu-4, OKT3, and T3, induced an accumulation of inositol phosphates in a human T cell leukemia line, JURKAT, in the presence of LiCl. The monoclonal antibodies also induced an increase in the cytosolic free Ca2+-concentration ([Ca2+]i) in JURKAT. The accumulation of inositol phosphates and the increase in [Ca2+]i were specifically induced by the monoclonal antibodies against the T3 complex. Other monoclonal antibodies against differentiation antigens on human T lymphocytes were not active in inducing these responses in JURKAT. Stimulation of JURKAT by anti-Leu-4 induced a rapid and immediate decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and an increase in the 32P-labeling of phosphatidic acid, which occurred after a short lag period. An analysis of inositol phosphates formed in the anti-Leu-4-stimulated JURKAT indicated the formation of inositol trisphosphate. These results strongly suggested that the T3 complex or T3/antigen receptor (Ti) complex functions as a receptor which transduces antigen signal, presented by either antigen-presenting cells or target cells, into the hydrolysis of PtdIns(4,5)P2. Fetal bovine serum at a dose of 1-20 microliters/ml induced a marked and transient [Ca2+]i increase in JURKAT immediately after addition. However, the level of formation of inositol phosphates was very small in cells stimulated by fetal bovine serum. Fetal bovine serum induced an immediate increase in the 32P-labeling of phosphatidic acid in JURKAT. These and other results suggested that serum increased [Ca2+]i in JURKAT by a mechanism different from that for the anti-Leu-4-induced [Ca2+]i response.     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.     

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.     

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.     

Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium.

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.     

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.     

Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.     

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.     

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phosphatidylinositol turnover measured as modification of incorporation of [32P]Pi and [3H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca2+, without a decrease in the total quantity of Ca2+ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca2+ concentration [Ca2+]i, without a decrease in the magnitude of the final increase of [Ca2+]i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca2+-phospholipid-dependent protein kinase C.     

Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils [published erratum appears in J Cell Biol 1990 Mar;110(3):861]

Human neutrophils exhibit multiple increases in cytosolic free calcium concentration [( Ca2+]i) spontaneously and in response to the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Jaconi, M. E. E., R. W. Rivest, W. Schlegel, C. B. Wollheim, D. Pittet, and P. D. Lew. 1988. J. Biol. Chem. 263:10557-10560). The function of these repetitive increases in [Ca2+]i, as well as the role of Ca2+ in human neutrophil migration, remain unresolved. We have used microspectrofluorometry to measure [Ca2+]i in single fura-2-loaded human neutrophils as they moved on poly-D-lysine-coated glass in the presence of serum. To investigate the role of Ca2+ in human neutrophil migration, we examined cells in the presence and absence of extracellular Ca2+, as well as intracellular Ca2(+)-buffered and Ca2(+)-depleted cells. In the presence of extracellular Ca2+, multiple increases and decreases in [Ca2+]i were frequently observed, and at least one such transient increase in [Ca2+]i occurred in every moving cell during chemokinesis, chemotaxis, and phagocytosis. In addition, neutrophils that extended pseudopodia and assumed a polarized morphology after plating onto a surface were always observed to exhibit [Ca2+]i transients even in the absence of chemoattractant. In contrast, a [Ca2+]i transient was observed in only one of the nonpolarized stationary cells that were examined (n = 15). Although some cells exhibited relatively periodic increases and decreases in [Ca2+]i, resembling the regular oscillations that have been observed in some cell types, many others exhibited increases and decreases in [Ca2+]i that varied in their timing, magnitude, and duration. Buffering of [Ca2+]i or removal of extracellular Ca2+ damped out or blocked transient increases in [Ca2+]i and reduced or inhibited the migration of neutrophils. Under these conditions, polarized cells were often observed to make repeated attempts at migration, but they remained anchored at their rear. These data suggest that transient increases in [Ca2+]i may be required for the migration of human neutrophils on poly-D-lysine-coated glass in the presence of serum by allowing them to release from previous sites of attachment.     

Direct evidence that burst but not sustained secretion of prolactin stimulated by thyrotropin-releasing hormone is dependent on elevation of cytoplasmic calcium

Thyrotropin-releasing hormone stimulation of prolactin secretion from rat pituitary (GH3) cells is biphasic with a secretory burst (0-2 min) at a higher rate, followed by sustained secretion (beyond 2 min) at a lower rate. Based on the effects of calcium ionophores, K+ depolarization, and diacylglycerol (or phorbol esters), it was suggested that the secretory burst is dependent on elevation of cytoplasmic free calcium concentration [( Ca2+]i) whereas sustained secretion is mediated by lipid-activated protein phosphorylation. In this study, we pretreated GH3 cells with 0.03 mM arachidonic acid to abolish thyrotropin-releasing hormone-induced elevation of [Ca2+]i (Kolesnick, R. N., and Gershengorn, M. C. (1985) J. Biol. Chem. 260, 707-713). In control cells, basal secretion was 0.7 +/- 0.2 ng/10(6) cells/min which increased to 8.3 +/- 0.8 between 0 and 2 min after TRH and remained elevated at 3.3 +/- 0.2 between 2-10 min. In cells pretreated with arachidonic acid, TRH stimulated prolactin secretion to only 2.6 +/- 0.3 ng/10(6) cells/min between 0 and 2 min and to 3.2 +/- 0.2 between 2 to 10 min; these values are not different from each other nor from the response between 2 and 10 min in control cells. K+ depolarization, which elevates [Ca2+]i even in arachidonic acid-pretreated cells but does not affect lipid metabolism, caused only a secretory burst. Bovine serum albumin, which binds free arachidonic acid and reverses arachidonic acid inhibition of TRH-induced elevation of [Ca2+]i, reversed the inhibition of the secretory burst stimulated by TRH. These studies present direct evidence that the burst of prolactin secretion stimulated by TRH is dependent on an elevation of [Ca2+]i whereas the sustained phase of secretion is independent of such elevation.     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)