Neuronal N-type calcium channel blockers: a series of 4-piperidinylaniline analogs with analgesic activity.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

Structure-activity relationship at the leucine side chain in a series of N,N-dialkyldipeptidyl-amines as N-type calcium channel blockers.

Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists.

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.

Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

N,N-dialkyl-dipeptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.

Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Characteristics of [125I]omega-conotoxin MVIIA binding to rat neocortical membranes.

[125I]omega-Conotoxin MVIIA (omega-CTM) binding to N-type voltage-sensitive calcium channels (VSCCs) was characterized using rat neocortical membranes. [125I]omega-CTM bound rapidly and with high affinity; these parameters were similar to binding using omega-conotoxin GVIA ([125I]omega-CTG). Unlike [125I]omega-CTG, however, [125I]omega-CTM readily dissociated from its binding site. Monovalent and divalent cations, polyamines, and aminoglycosides inhibited [125I]omega-CTM binding. Since [125I]omega-CTM appears to bind to the same site as [125I]omega-CTG in mammalian neurons, the reversibility of [125I]omega-CTM binding makes this ligand preferable for equilibrium binding analyses.

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils.

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Cyclic AMP inhibits secretion from electroporated human neutrophils.

It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.

Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins.

Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP-utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.

Protein kinase C is not involved in secretion by permeabilized human neutrophils.

The generally accepted sequence of intracellular signal transduction involves: (1) cell surface receptor-ligand interactions; (2) activation of G-proteins; (3) activation of phospholipase C, leading to inositol phosphate (IP3), and diacylglycerol production; (4) parallel mobilization of intracellular Ca2+ by IP3, and; (5) activation of protein kinase C (PKC) by diacylglycerol and Ca2+, leading to; (6) cellular responses. Human neutrophils appear to utilize this cascade, at least in general, and some, but not all, elements of the intracellular signal cascade known to be operating in intact cells also function in permeabilized cell systems. We have previously shown that permeabilized neutrophils can be induced to secrete lysosomal enzymes in response to elevated levels of Ca2+ alone and this secretion can be synergistically enhanced by the presence of guanine nucleotides. We now show that Ca2+, in the presence and absence of guanine nucleotides, can stimulate the production of soluble inositol phosphates. Furthermore, neomycin, a putative inhibitor of phospholipase C, can block Ca2(+)-induced secretion. These data thus suggest a role for phospholipase C activity or its products in the transduction process. The next enzymatic activity 'downstream' is PKC. Consequently, we looked at the role Mg-ATP, one of the substrates of PKC, plays in degranulation by permeabilized neutrophils, We found no obligatory role for this nucleotide in the secretory process. We then looked at the activity of oleoyl-acetyl-glycerol (OAG), a synthetic diacylglycerol and PKC agonist, on degranulation. We found that OAG was largely additive with Ca2+. Another PKC agonist, phorbol myristate acetate (PMA), also did not display notable synergy. Finally, inhibitors of PKC activity were not capable of blocking secretion, either in the presence or absence of guanine nucleotides. Thus, while circumstantial evidence seems to point towards a requirement for phospholipase C activation and diacylglycerol production in secretion, we were unable to demonstrate the next putative step in signal transduction, namely activation of PKC.

Osmotic forces are not critical for Ca2+-induced secretion from permeabilized human neutrophils.

In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP-stimulated, intact neutrophils and Ca2+-stimulated, permeabilized cells. We employed a HEPES-based buffer system which was supplemented with: a) permeant (KCl or NaCl) or impermeant (Na-isethionate or choline-Cl) ions, or b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP-stimulated release from intact cells was supported by NaCl or Na-isethionate greater than KCl greater than choline-Cl or sucrose greater than urea. In contrast, the rank order of Ca2+-stimulated release from permeabilized cells was choline-Cl greater than Na-isethionate, KCl, or NaCl greater than sucrose greater than urea. Hypo-osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo-osmolality inhibited both FMLP-stimulated degranulation from intact cells and Ca2+-induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca2+-induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo-osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.

The kinetics of secretion from permeabilized human neutrophils: release of elastase and correlations with other granule constituents and right angle light scatter.

We have previously reported that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin. These permeabilized cells can be induced to secrete lysosomal constituents when exposed to micromolar levels of free Ca2+, a process that is enhanced by certain guanine nucleotides. We examined the kinetics in this system by employing both direct and indirect measures of secretion. A continuous, fluorescent assay of elastase permits real-time monitoring of secretion from azurophil granules. The kinetics of elastase release proved to be rapid, beginning within 3-10 sec and reaching a maximum at 1-2 min. Changes in the Ca2+ concentration did not affect the "lag period" for release. A comparison of the Ca2+ dose-response curves for release of the various granule constituents indicated that elastase was being secreted along with other contents of the azurophil granules. Changes in right angle light scatter (RLS), which have been shown to correlate closely with secretion, also commenced rapidly after the addition of Ca2+; when measured simultaneously, both the Ca2+ dose-response characteristics for changes in RLS and elastase release were very similar. Changes in RLS could be halted within 5 sec by excess EGTA and restarted promptly by repletion with secretory concentrations of Ca2+. In addition, neomycin, a phospholipase C inhibitor, profoundly diminished degranulation as monitored by RLS and end-point techniques. A continuous assay employing 9-aminoacridine self-quenching as a measure of secretion proved far less satisfactory, but, nonetheless, produced similar kinetics and dose-response characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine nucleotides reduce the free calcium requirement for secretion of granule constituents from permeabilized human neutrophils.

Human neutrophils can be permeabilized with the cholesterol complexing agent digitonin and then induced to secrete lysosomal constituents by increases in free Ca2+ alone. In order of increasing requirements for Ca2+, vitamin B-12 binding protein, lysozyme and beta-glucuronidase were released. A variety of guanine nucleotides were examined with respect to their abilities to modulate this response. GTP, along with its analogues 5'-guanylylimidodiphosphate (Gpp[NH]p) and guanosine-5'-O-[3-thio]-triphosphate (GTP[gamma S]) decreased the Ca2+ requirements for secretion of all three granule constituents by one third to one order of magnitude. This synergy was dependent upon the concentration of guanine nucleotides employed. The effects of Gpp[NH]p could be blocked with the inactive derivative GDP[beta-S]. The active guanine nucleotides, particularly GTP, served as stimuli in their own right. At high concentrations of Ca2+ and GTP, degranulation was strikingly inhibited; inhibition was also achieved with high concentrations of guanylyl[beta, gamma-methylene]diphosphate (Gpp[CH2]p). Both GDP and GMP were without any effect. When neutrophils were pretreated with pertussis toxin, granule discharge induced by fMet-Leu-Phe was almost completely blocked, as reported by others. If the neutrophils pretreated with pertussis toxin were then permeabilized with digitonin, the synergy between Ca2+ and the stimulatory guanine nucleotides was maintained. These data suggest the involvement of G-proteins in secretion induced by Ca2+; however, this response either uses a different G-protein or a different pool of G-proteins from those responses triggered by fMet-Leu-Phe.

Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium.

We have recently reported that human neutrophils can be permeabilized with the cholesterol complexing agent saponin and that these cells can be induced to secrete the granule enzyme lysozyme in response to micromolar levels of free calcium. We now report that digitonin can be used in place of saponin and that it has several advantages. Permeabilization of human neutrophils was accomplished with 10 micrograms/ml digitonin in a high potassium medium. Normally impermeant solutes such as [14C]sucrose and inulin [14C]carboxylic acid gained access to one half of the intracellular water space marked with [3H]H2O. Between 30 and 100% of the cytoplasmic enzyme, lactate dehydrogenase, leaked from the intracellular space. The permeabilization process and calcium-triggered granule secretion were critically dependent upon temperature, time and digitonin concentration. Permeabilized neutrophils secreted beta-glucuronidase, lysozyme and vitamin B-12 binding-protein, constituents of both azurophil and specific granules, when exposed to micromolar levels of free calcium. Release of specific granule constituents appeared to be more sensitive to free calcium than release from azurophil granules. Although the amount of permeabilization varied considerably with each batch of cells, release of these granule markers was a consistent finding. Release of granule markers was accompanied by resealing of the cells to high-molecular-weight (Mr greater than 5000) solutes. Electron microscopic evidence also suggested that granule and plasma membranes were intact following digitonin treatment and that fusion of these membranes occurred in response to calcium. These results suggest that elevation of intracellular free-calcium levels is a sufficient condition for lysosomal enzyme release.

Inositol trisphosphate mobilizes intracellular calcium in permeabilized adrenal chromaffin cells.

Using permeabilized chromaffin cells and the fluorescent probe Quin 2 (an indicator of free Ca2+), we found that inositol trisphosphate (IP3) specifically triggered an immediate and dose-dependent release of Ca2+ from intracellular stores. Desensitization of the response was observed at nonsaturating concentrations of inositol trisphosphate and resequestration of Ca2+ was not observed. While representing only a small fraction of the total cellular Ca2+, the amount released by IP3 could significantly raise cytosolic Ca2+ and may account for muscarinic effects on Ca2+ metabolism in chromaffin cells.

Micromolar concentrations of free calcium provoke secretion of lysozyme from human neutrophils permeabilized with saponin.

Permeabilization of human neutrophils has been accomplished by using saponin, a cholesterol complexing agent, permitting experimental manipulation of the intracellular milieu. Access of ordinarily impermeable solutes, such as [14C]-inulin or [14C]-sucrose, to the water space of the cells was considered the main criterion for permeabilization. Other criteria were substantial (50 to 80%) release of cytoplasmic lactate dehydrogenase and permeability to trypan blue. Successful permeabilization did not cause substantial release of the granule enzymes lysozyme or beta-glucuronidase. Washing the neutrophils, to remove soluble saponin and released cytoplasmic contents, and resuspension did not alter their permeabilized character. By supplementing the medium with CaCl2, thereby obtaining free Ca2+ concentrations of 1.5 X 10(-7) M to 10(-4) M, it was possible to stimulate lysozyme secretion from washed or unwashed permeabilized neutrophils. A total of 20 to 30% of the total cellular lysozyme was released during an incubation of 5 min at 37 degrees C. Secretion was inversely related to cell concentration. No beta-glucuronidase was secreted under these conditions and no response was obtained by using unpermeabilized cells. Thus, permeabilized neutrophils respond to increases in free Ca2+ alone, without resorting to conventional secretagogues. This system also permits the manipulation of intracellular constituents important for stimulus-response coupling.