Molecular characterization of a phospholipase D generating anandamide and its congeners

Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.     

Hypoxic vasorelaxation: Ca2+-dependent and Ca2+-independent mechanisms

The mechanisms of oxygen sensing in vascular smooth muscle have been studied extensively in a variety of tissue types and the results of these studies indicate that the mechanism of hypoxia-induced vasodilation probably involves several mechanisms that combined to assure the appropriate response. After a short discussion of the regulatory mechanisms for smooth muscle contractility, we present the evidence indicating that hypoxic vasorelaxation involves both Ca2+-dependent and Ca2+-independent mechanisms. More recent experiments using proteomic approaches in organ cultures of porcine coronary artery reveal important changes evoked by hypoxia in both Ca2+-dependent and Ca2+-independent pathways.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

Ca2+依赖和Ca2+不依赖的囊泡分泌研究进展

Ca2 是促发囊泡胞吐的关键调节因子.最近的研究表明,分泌囊泡和通道之间的空间距离调节囊泡分泌的过程和性质.Ca2 通道开口附近形成的Ca2 微区和Ca2 钠区和囊泡快速递质释放有非常紧密的联系.SNARE蛋白和钙离子传感器synaptotagmins等在触发分泌中起调控作用.同时另有一类不依赖于Ca2 的囊泡分泌存在.Latrotoxin和mastoparan等可以激活这一类不依赖于Ca2 的信号通路,从而触发囊泡释放.本文主要从ca2 对囊泡胞吐的调控作用着手,综述了Ca2 依赖和Ca2 不依赖的囊泡分泌过程和可能的调控机制.     

Identification and characterization of a phosphorylation-activated, cyclic AMP and Ca2+-independent protein kinase in the brain

A cyclic AMP and calcium-independent protein kinase has been identified and purified from pig brain to near homogeneity. This independent protein kinase was isolated in an inactive form, and activation required ATP and Mg2+. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme contains 1 subunit with a molecular mass of about 36 kDa. Although there was no significant phosphorylation of phosphorylase, phosphorylase b kinase, casein, phosvitin, and protamine, this kinase was found to be very active toward myelin basic protein and histones H1, 2A, and 2B. Trypsinolysis completely destroyed the kinase activity, indicating that this is not a protease-activated protein kinase. More interesting, this cAMP and calcium-independent protein kinase can be regulated by its state of phosphorylation. In its non-phosphorylated state, the kinase was essentially inactive but could be fully activated when the enzyme was phosphorylated up to a 1:1 molar ratio. Conversely, partial dephosphorylation of the phosphorylated enzyme was associated with a time-dependent decrease in the kinase activity and a loss of 32P. All the results taken together point out that this kinase is distinguished from all the reported protein kinases and may represent a previously undiscovered protein kinase. The results also provide initial evidence that a cascade activation mechanism may possibly be involved in the regulation of a protein kinase activity which is independent of cAMP and calcium.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner

The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa²⁺-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca²⁺ concentration([Ca²⁺]_i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca²⁺concentration. The elevation in[Ca²⁺]_iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa²⁺ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa²⁺-independent mechanism.

     

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2+-ATPase isolated from goat spermatozoa

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg²⁺-independent Ca²⁺-ATPase without any significant effect on Mg²⁺-dependent Ca²⁺-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.     

Microautophagic vacuole invagination requires calmodulin in a Ca2+-independent function

Microautophagy is the uptake of cytosolic compounds by direct invagination of the vacuolar/lysosomal membrane. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. Autophagic tubes are topologically equivalent to the invaginations at multivesicular endosomes. At the tip of an autophagic tube, vesicles (autophagic bodies) pinch off into the vacuolar lumen for degradation. In this study we have identified calmodulin (Cmd1p) as necessary for microautophagy. Temperature-sensitive mutants for Cmd1p displayed reduced frequencies of vacuolar tube formation and/or abnormal tube morphologies. Microautophagic vacuole invagination was sensitive to Cmd1p antagonists as well as to antibodies to Cmd1p. cmd1 mutants with substitutions in the Ca2+-binding domains showed full invagination activity, and vacuolar membrane invagination was independent of the free Ca2+ concentration. Thus, rather than acting as a calcium-triggered switch, Cmd1p has a constitutive Ca2+-independent role in the formation of autophagic tubes. Kinetic analysis indicates that calmodulin is required for autophagic tube formation rather than for the final scission of vesicles from the tip of the tube.     

Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

We reported previously that a Ca²⁺-ATPase in rat testes and goat spermatozoa could be activated by Ca²⁺ alone without Mg²⁺, though it has a lot of similarities with the well known Ca²⁺, Mg²⁺-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca²⁺ and at low temperature. Increase of the concentration of Ca²⁺ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca²⁺, Mg²⁺-ATPase. The findings strengthened our previous report that Mg²⁺-independent Ca²⁺-ATPase is involved in Ca²⁺ transport and Ca²⁺ uptake like Ca²⁺, Mg²⁺-ATPase.     

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.     

Mutations at lysine 525 of inducible nitric-oxide synthase affect its Ca2+-independent activity

Calmodulin binding to inducible nitric-oxide synthase may play an important role in its Ca(2+)-independent activity. Studies of inducible nitric-oxide synthase chimeras containing the calmodulin binding sequence of neuronal or endothelial nitric-oxide synthases show that the calmodulin binding sequence of inducible nitric-oxide synthase is necessary but not sufficient for the Ca(2+)-independent activity. The mutations at lysine 525 located at the C terminus of the calmodulin binding sequence of inducible nitric-oxide synthase were examined for the effects on the Ca(2+)-independent activity with chimeras containing the oxygenase or reductase domains of inducible or neuronal nitric-oxide synthases. Results show that the Ca(2+)-independent binding of calmodulin is not solely responsible for maximal Ca(2+)-independent activity of inducible nitric-oxide synthase. Lysine 525 of inducible nitric-oxide synthase may also play an important role in coordinating the maximal Ca(2+)-independent activity.     

Pore formation and uncoupling initiate a Ca2+-independent degradation of mitochondrial phospholipids

Mitochondria contain a type IIA secretory phospholipase A(2) that has been thought to hydrolyze phospholipids following Ca(2+) accumulation and induction of the permeability transition. These enzymes normally require millimolar Ca(2+) for optimal activity; however, no dependence of the mitochondrial activity on Ca(2+) can be demonstrated upon equilibrating the matrix space with extramitochondrial Ca(2+) buffers. Ca(2+)-independent activity is seen following protonophore-mediated uncoupling, when uncoupling arises through alamethicin-mediated pore formation, or upon opening the permeability transition pore. Under the latter conditions, activity continues in the presence of excess EGTA but is somewhat enhanced by exogenous Ca(2+). The Ca(2+)-independent activity is best seen in media of high ionic strength and displays a broad pH optimum located between pH 8 and pH 8.5. It is strongly inhibited by bromoenol lactone but not by arachidonyl trifluoromethyl ketone, dithiothreitol, and other inhibitors of particular phospholipase A(2) classes. Immunoanalysis of mitochondria and mitochondrial subfractions shows that a membrane-bound protein is present that is recognized by antibody against an authentic iPLA(2) that was first found in P388D(1) cells. It is concluded that mitochondria contain a distinct Ca(2+)-independent phospholipase A(2) that is regulated by bioenergetic parameters. It is proposed that this enzyme, rather than the Ca(2+)-dependent type IIA phospholipase A(2), initiates the removal of poorly functioning mitochondria by processes involving autolysis.     

Ca2+-induced fusion of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles.

The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3-sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl2 were studied by mixing membrane lipids based on an assay of fluorescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with increasing molar concentration of sulfatide. This inhibitory effect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 - 9.0. Similar inhibitory effect was also observed in Ca2+-induced fusion of DOPE/ganglioside GM1 vesicles but at a lower concentration of the glycosphingolipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca2+-induced lipid mixing and the pH sensitivity of this system was not affected.These results are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was retained when they were allowed to fuse with synaptosomes in the absence of Ca2+ by a mechanism involving protein mediation.     

The Na(+)-independent Ca2+ efflux mechanism of liver mitochondria is not a passive Ca2+/2H+ exchanger

Whether the Na(2+)-independent Ca2+ efflux mechanism of liver mitochondria is a Ca2+/2H+ exchanger and whether this exchanger is a passive mechanism have been controversial since shortly after the discovery of this mechanism. Here, a new approach to determining if the mechanism is passive is developed based on the energy available to a passive Ca2+/2H+ exchanger. Conditions are identified in which the Na(+)-independent Ca2+ efflux mechanism transports Ca2+ out of mitochondria against a Ca2+ gradient many times greater than that possible for a passive Ca2+/2H+ exchanger, thus ruling this out as a possible mechanism.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.