A guanine nucleotide regulatory protein controls polyphosphoinositide metabolism, Ca2+ mobilization, and cellular responses to chemoattractants in human monocytes

Previous studies demonstrated that oligopeptide chemoattractant receptors on PMN and macrophages exist in high and low affinity states which are interconvertible by guanosine di- and triphosphates. These observations suggest that guanine nucleotide regulatory (N) proteins play a role in phagocyte activation by chemotactic factors. The data presented here indicate that chemotactic factor receptors on monocytes utilize an N protein to activate phospholipase C and subsequent biologic responses by the cells. This conclusion is based on the findings that inactivation of an N protein of 41,000 m.w. by Bordetella pertussis toxin (PT) treatment abolishes monocyte responsiveness to chemoattractants but not to lectins, PMA, or the Ca2+ ionophore A23187. Treatment with PT inhibited IP3 production, Ca2+ mobilization, and cellular activation as assessed by chemotaxis and changes in forward light scattering in response to the chemoattractants by at least 80%. Therefore, a PT-sensitive N protein plays an important role in the activation of monocytes by chemoattractants.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

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 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.     

A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction

Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C.     

Chemoattractants stimulate phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes

Chemoattractant receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C is instrumental for leukocyte activation. Previous studies have demonstrated that chemoattractant treatment of intact polymorphonuclear leukocytes (PMN) causes a transient decrease in PIP2 due to phospholipase C activation, followed by an increase in cellular PIP2 levels. The present study determined whether chemoattractants altered the activities of the two enzymes responsible for the synthesis of PIP2, phosphatidylinositol kinase, and phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact PMN with the N-formylated peptide chemoattractant formyl-methionyl-leucyl-phenylalanine at 37 degrees C caused a rapid (3 min), 2-fold stimulation of PIP kinase activity isolated from a particulate membrane fraction. The increase in PIP kinase was dose-dependent for a variety of N-formylated chemoattractants as well as leukotriene B4. Lineweaver-Burk analysis showed that the Vmax of PIP kinase was increased 2-fold by formyl-methionyl-leucyl-phenylalanine, without a significant change in the apparent Km of the enzyme for ATP. Phosphatidylinositol kinase was, however, not altered by any chemoattractants tested. Nonchemotactic activators of the oxidative burst in leukocytes such as phorbol myristate acetate and ionophore A23187 did not significantly alter PIP kinase, suggesting a specificity for chemotactic agents. These findings demonstrate direct, chemoattractant-induced stimulation of PMN PIP kinase which may serve to replenish the important phospholipid, PIP2, in the membrane following its hydrolysis by phospholipase C.     

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.     

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.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase

The affinity of the chemoattractant receptor for N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) on human polymorphonuclear leukocytes (PMNs) is regulated by guanine nucleotides, and chemoattractants stimulate increased intracellular cAMP levels in PMNs. Our data, however, indicate that this receptor does not activate membrane-bound adenylate cyclase via direct nucleotide regulatory protein (N) coupling but instead raises cAMP levels indirectly via a mechanism which appears to require Ca2+ mobilization. This conclusion is based on the following data: 1) prostaglandin E1 (PGE1) activated and alpha 2-adrenergic treatment inhibited adenylate cyclase activation in PMN plasma membranes; fMet-Leu-Phe, however, neither activated nor inhibited adenylate cyclase in these membranes; 2) depletion of extracellular Ca2+ had no effect on isoproterenol and PGE1 elicited cAMP responses in intact PMNs while peak fMet-Leu-Phe and A23187-induced responses were reduced by approximately 50 and 80%, respectively; 3) 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, a purported Ca2+ antagonist, caused almost complete inhibition of fMet-Leu-Phe and ionophore-induced cAMP responses in intact cells but had no effect on PGE1 and isoproterenol; 4) alpha 2-adrenergic agonists inhibited PGE1 but not chemoattractant- or A23187-elicited cAMP responses in intact PMNs; and 5) pretreatment of cells with a phosphodiesterase inhibitor (isobutylmethylxanthine) greatly potentiated the PGE1 and isoproterenol cAMP responses but nearly abolished the peak fMet-Leu-Phe response. Thus, chemoattractants appear to utilize a novel mechanism to raise cAMP levels which appear to require Ca2+ mobilization and could be mediated in part through a transient inhibition of phosphodiesterases. We suggest that stimulation of PMN functions by chemoattractants may utilize an N-coupled process to generate a Ca2+ signal which could in turn raise intracellular cAMP levels indirectly and thereby provide negative regulation.     

TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.     

Metabolism and functions of inositol phosphates

Activation of Ca2+-mobilizing receptors rapidly increases the cytoplasmic Ca2+ concentration both by releasing Ca2+ stored in endoplasmic reticulum and by stimulating Ca2+ entry into the cells. The mechanism by which Ca2+ release occurs has recently been elucidated. Receptor activation of phospholipase C results in the hydrolysis of the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield two intracellular messengers, diacylglycerol (DAG) and (1,4,5)inositol trisphosphate [(1,4,5)IP3]. DAG remains in the plasma membrane where it stimulates protein phosphorylation via the phospholipid-dependent protein kinase C. (1,4,5)IP3 diffuses to and interacts with specific sites on the endoplasmic reticulum to release stored Ca2+. Receptor stimulation of phospholipase C appears to be mediated by one or more guanine nucleotide-dependent regulatory proteins by a mechanism analogous to hormonal activation of adenylyl cyclase. The actions of (1,4,5)IP3 on Ca2+ mobilization are terminated by two metabolic pathways, sequential dephosphorylation to inositol bisphosphate (IP2), inositol monophosphate (IP) and inositol or by phosphorylation to inositol tetrakisphosphate (IP4) and sequential dephosphorylation to different inositol phosphates. A sustained cellular response also requires Ca2+ entry into the cell from the extracellular space. The mechanism by which hormones increase Ca2+ entry is not known; a recent proposal involving movement of Ca2+ through the endoplasmic reticulum, possibly regulated by IP4, will be considered here.     

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.     

Identification of neutrophil granule protein cathepsin G as a novel chemotactic agonist for the G protein-coupled formyl peptide receptor

The antimicrobial and proinflammatory neutrophil granule protein cathepsin G (CaG) has been reported as a chemoattractant for human phagocytic leukocytes by using a putative G protein coupled receptor. In an effort to identify potential CaG receptor(s), we found that CaG-induced phagocyte migration was specifically attenuated by the bacterial chemotactic peptide fMLP, suggesting these two chemoattractants might share a receptor. In fact, CaG chemoattracts rat basophilic leukemia cells (RBL cells) expressing the high affinity human fMLP receptor FPR, but not parental RBL cells or cells transfected with other chemoattractant receptors. In addition, a specific FPR Ab and a defined FPR antagonist, cyclosporin H, abolished the chemotactic response of phagocytes and FPR-transfected cells to CaG. Furthermore, CaG down-regulated the cell surface expression of FPR in association with receptor internalization. Unlike fMLP, CaG did not induce potent Ca(2+) flux and was a relatively weaker activator of MAPKs through FPR. Yet CaG activated an atypical protein kinase C isozyme, protein kinase Czeta, which was essential for FPR to mediate the chemotactic activity of CaG. Thus, our studies identify CaG as a novel, host-derived chemotactic agonist for FPR and expand the functional scope of this receptor in inflammatory and immune responses.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Potential role for a guanine nucleotide regulatory protein in chemoattractant receptor mediated polyphosphoinositide metabolism, Ca++ mobilization and cellular responses by leukocytes

Islet activating protein from Bordetella pertussis toxin which ribosylates certain guanine nucleotide regulatory proteins causes a marked reduction of chemoattractant-elicited responses such as chemotaxis, O2 production and cAMP elevations in human polymorphonuclear leukocytes. The toxin appears to exert its effects by preventing the rapid breakdown of phosphatidylinositol 4,5-bisphosphate induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, thereby inhibiting the increase in intracellular [Ca++] which normally follows chemoattractant stimulation. Responses of leukocytes exposed to Concanavalin A, the Ca++ ionophore A23187, or phorbol myristate acetate were not affected by the toxin. Thus the chemoattractant receptor appears to be coupled to a phosphoinositide specific phospholipase C through a guanine nucleotide regulatory protein. We propose that this complex of receptor-guanine nucleotide regulatory protein-phospholipase C may be applicable to the class of receptors which mobilize intracellular Ca++ by stimulating polyphosphoinositide breakdown.     

Carbachol and oxytocin stimulate the generation of inositol phosphates in the guinea pig myometrium

In the guinea pig myometrium prelabelled with myo-[2-3H]inositol, carbachol and oxytocin enhanced a concentration-dependent and rapid release of IP3 which preceded that of IP2 and IP1. The specific receptor-mediated phospholipase C activation degrading PIP2 to IP3 did not require the presence of extracellular Ca2+. The ionophore A23187 as well as K+ depolarization failed to increase inositol phosphate accumulation. It is proposed that IP3 could have a role in the contraction of uterine smooth muscle elicited by the activation of muscarinic as well as of oxytocin receptors.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Calcium and abnormal reactivity of vascular smooth muscle in hypertension

It has been well documented that vascular smooth muscle (VSM) reactivity, as well as calcium sensitivity in response to neurotransmitters is increased in a number of blood vessels in established hypertension. Regulation of VSM reactivity involves the interaction of neurotransmitters and blood-borne hormones with specific receptors on target cell membranes. This results in phospholipase-C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and the generation of two second messengers: inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) both of which act synergistically to produce muscle contraction. We will summarize recent findings in this review which suggest that in essentially hypertensive patients and spontaneously hypertensive rats (SHR), the activation of phospholipase C in response to hormones is increased. Further, we will discuss how increases in phospholipase C activation via GTP-binding proteins may explain the observed increases in Ca2+ influx through potential- and receptor-operated Ca2+ channels, increased activation of protein kinase-C and increased [Ca2+]i in hormone-stimulated blood platelets and VSM cells in the hypertensive state. In addition to these defects, a decrease in the plasma membrane Ca2+ pump and Ca2+-binding proteins has been demonstrated in hypertension. Thus, it appears that the defect in Ca2+ metabolism in the hypertensive vessels is multifocal. All these defects in Ca2+ metabolism together may lead to an increase in peripheral vascular resistance with a concomitant increase in blood pressure.     

Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein. Evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release.

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca2+ mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the G alpha i2, G alpha i3, G alpha s, G beta 35, and G beta 36 protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca2+ mobilization, we investigated the effect of PT on Ca2+ responses using fluorescence spectroscopy and the Ca2+ indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 microgram/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca2+ from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca2+ release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca2+ response was inhibited by approximately 72%, suggesting that the major fraction of the Ca2+ response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the beta-subunit of G proteins because quantitative immunoblots showed that levels of the beta-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca2+ pool because Ca2+ release induced by agents that release Ca2+ directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [3H]inositol trisphosphate (IP3) formation because the level of inhibition of IP3 formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT-sensitive species is the major G protein pathway that couples thrombin-receptor activation to Ca2+ mobilization. This G protein appears to be involved not in the mechanisms that generate IP3 but rather possibly in coupling at the level of the intracellular Ca2+ store.