Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.     

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment of the PMNs with pertussis toxin (PT) or 4-beta-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTP gamma[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 microM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTP gamma S binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTP gamma S alone at low concentrations of Ca2+ (0.1-1 microM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTP gamma S, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTP gamma S. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.     

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

Signal transduction in cells following binding of chemoattractants to membrane receptors

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

TPA inhibits insulin stimulated PIP hydrolysis in fat cell membranes: evidence for modulation of insulin dependent phospholipase C by proteinkinase C

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.     

IL-8 stimulates phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes.

IL-8 is a neutrophil-specific chemoattractant and cellular activator which exists in at least three forms, 69, 72, and 77 amino acids. The predominant monocyte product has 72 amino acids, whereas endothelial cells secrete the 77-amino acid form. The 72-amino acid form has been shown to increase intracellular calcium in neutrophils, but the exact biochemical pathways involved in stimulation of these cells is unknown. N-formyl peptide chemoattractants in neutrophils stimulate the formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a reservoir for second messenger molecules and regulator of actin assembly through its association with the actin-binding proteins, profilin, and gelsolin. The present study examined whether IL-8 altered the enzyme which synthesizes PIP2, phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact neutrophils with 10 nM IL-8 caused approximately a twofold increase in the activity of the enzyme. All forms of IL-8 stimulated PIP kinase activity in concentrations ranging from 1 to 50 nM, and the dose-response curves exactly correlated with the order of potency of these cytokines for interacting with the IL-8R on the surface of neutrophils. Lineweaver-Burk analysis of the kinetics of PIP kinase assayed in the presence of 0.03 to 0.7 mM ATP showed that 10 nM IL-8 increased the Vmax of the enzyme 38 to 70.5%, with no significant change in the apparent Km for ATP or for PIP. The stimulation of PIP kinase activity could not be explained by decreased degradation of PIP2 by phospholipase C or phosphomonoesterase activity in the membranes isolated from cells treated with IL-8 or by a decrease in the degradation of ATP. The microfilament disrupter, cytochalasin b, inhibited IL-8 induced stimulation of PIP kinase. These findings demonstrate that all forms of IL-8 stimulate PIP kinase in human neutrophils. This event may provide molecular signals to these cells that are necessary to maintain or change the state of microfilament assembly during cellular activation.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.     

PLC gamma 1, a possible mediator of T cell receptor function

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.     

Phospholipase C activation and diacylglycerol kinase inactivation lead to an increase in diacylglycerol content in spontaneously hypertensive rat

Activities of three kinases, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) kinases, and phospholipase C were measured in erythrocyte ghosts from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). PI kinase activity was significantly higher in SHR than WKY but there was no significant difference in PIP kinase activity between SHR and WKY. The activity of phospholipase C, which hydrolyzes PIP2, was also increased in SHR. However, DG kinase activity was, on the contrary, decreased in SHR. These results suggest that there is a tendency to accumulate DG in SHR. Indeed, DG content in erythrocytes of SHR increased 1.7-fold compared to that of WKY. Such DG accumulation may cause the sustained activation of protein kinase C in SHR, since DG is a physiological activator for protein kinase C.     

Mechanism of neutrophil activation by an unopsonized inflammatory particulate. Monosodium urate crystals induce pertussis toxin-insensitive hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Monosodium urate crystals are believed to trigger acute inflammation via the direct stimulation of leukocytes. Unopsonized urate crystals activate neutrophil (PMN) membrane G proteins in a pertussis toxin (PT)-sensitive manner, but induce PT-insensitive cytosolic [Ca2+]i elevation. Thus, we have further defined the mechanism of PMN responsiveness to urate crystals in this study. Though urate crystals can increase membrane permeability by lytic effects, we observed elevation of PMN cytosolic [Ca2+]i in the absence of extracellular [Ca2+]i. In addition, the early, crystal-induced cytosolic [Ca2+]i transient was buffered in cells loaded with a [Ca2+]i-chelator. This suggested mobilization of internal [Ca2+]i stores, which was supported by demonstrating rapid phosphatidylinositol bisphosphate (PIP2) hydrolysis, and the formation of inositol (1,4,5) trisphosphate (as well as phosphatidic acid) in a PT-insensitive manner. Importantly, PMN activation by urate crystals was discriminatory, as evidenced by the absence of phosphatidylinositol trisphosphate formation, a PT-sensitive event triggered by chemotactic factors. Urate crystal-induced PIP2 hydrolysis was not a nonspecific consequence of the early cytosolic [Ca2+]i transient itself, and it did not require phagocytosis. However, crystal-induced O2- release was markedly inhibited by buffering of the early cytosolic [Ca2+]i transient under conditions where crystal phagocytosis and PMA-induced O2- release were unaffected. We conclude that urate crystals activate PT-insensitive PIP2 hydrolysis, resulting in IP3 generation, and early urate crystal-induced mobilization of cytosolic [Ca2+]i. This pathway appears to modulate crystal-induced O2- release.     

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.     

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.     

Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.     

Modulation of phosphatidylinositol-4,5-bisphosphate hydrolysis in rat aorta by guanine nucleotides, calcium and magnesium

Rat aortic smooth muscle homogenates and membrane preparations contain a phospholipase C which hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2). We discovered that guanyl-5'yl-imidodiphosphate (Gpp(NH)p) activated the hydrolysis of exogenous PIP2 but not of phosphatidylinositol (PI) in rat aortic membranes. Further, maximal Gpp(NH)p-dependent hydrolysis was dependent on physiological levels of calcium. Also, magnesium inhibited PIP2 hydrolysis and catalyzed the dephosphorylation of PIP2 to phosphatidylinositol-4-phosphate (PIP). The results imply that PIP2 is the primary substrate of the nucleotide-regulated phospholipase C in rat aorta and that calcium and magnesium are physiological regulators of this activity.     

Effects of the wasp venom peptide, mastoparan, on a phosphoinositide-specific phospholipase C purified from rabbit brain membranes

The wasp venom peptide, mastoparan (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2), activated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis as catalyzed by a phosphoinositide-specific phospholipase C (PLC-Im) purified from rabbit brain membranes. This activation was found when the molar ratio of mastoparan to PIP2 was less than 1 and when the concentration of PIP2 exceeded 10 microM. PIP2 breakdown was inhibited at both high and low substrate concentrations if the molar ratio of mastoparan to PIP2 was greater than 1. The stimulatory effect of mastoparan correlated with its ability to restrict aggregation of PIP2 into higher order structures (liposomes or mixed deoxycholate/phospholipid micelles) as the concentration of PIP2 was increased to 10 microM or greater. Mastoparan stimulation of PIP2 breakdown required the presence of a higher calcium concentration than was necessary for detection of enzyme activity. Both the stimulatory and inhibitory effects of mastoparan on PIP2 hydrolysis were lost if 2.5 mM deoxycholate was present in the assays. Hydrolysis of phosphatidylinositol (PI) by PLC-Im was inhibited at all concentrations of mastoparan tested. These results show that both PIP2 and PI are suitable substrates for PLC-Im, depending on the physical characteristics of their aggregates in aqueous suspension. An amphiphilic alpha-helix-forming peptide such as mastoparan may modulate phospholipase C activity due to the peptide's interaction with phospholipid substrates.     

Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.