Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.     

Response of neutrophils to stimulus infusion: differential sensitivity of cytoskeletal activation and oxidant production

The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.02-0.2 nM/min). Under conditions of stimulus infusion intracellular calcium elevation, superoxide generation, and right angle light scatter and F-actin formation were measured. As the infusion rate was decreased into the range of 10 pM/min, lowering the rate of increase of receptor occupancy to approximately 0.5% per min, the calcium and right angle light scatter responses elongated in time and decreased in magnitude. Superoxide generation decreased below infusion rates of approximately 100 pM/min (occupancy increasing at a rate in the range of 5% per min). This behavior could contribute to differences between chemotactic responses, which appear to require low rates of receptor occupancy over long periods, and bactericidal or inflammatory responses (free radical generation and degranulation), which require bursts of occupancy of several percent of the receptors.     

A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors

Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca2+ approximately 5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate- and cAMP-induced Ca2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2(+)-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenyl-hydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca2+ uptake, suggest that different chemoreceptors activate a single Ca2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca2+ entry probably does not involve a Na+/Ca2+ exchanger or voltage-activated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2(+)-uptake response. Analysis of putative signaling mutants suggests that Ca2+ entry is not regulated by the guanine nucleotide-binding (G) protein subunits G alpha 1 or G alpha 2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).     

Co-expression of chemotactic ligand receptors on human peripheral blood monocytes

Directed migration of monocytes is dependent upon interaction of cell surface receptors and specific chemotactic ligands. To determine whether circulating human monocytes express multiple chemotactic ligand receptors or whether subpopulations of monocytes exist with a single receptor specificity, nonoverlapping fluorescent probes for two chemotactic ligands, N-formyl methionyl leucyl phenylalanine (FMLP) and C5a, were developed to simultaneously evaluate the expression of receptors for these ligands on individual monocytes. The subsequent incubation with different fluorochrome labeled C5a and FMLP probes and monoclonal antibodies specific for antigenic determinants on distinct subsets of mononuclear cells followed by analysis with dual parameter flow microfluorometry indicated that cells that express C5a and FMLP receptors are the OKM1, Mac-1, and Fc gamma receptor positive population. Furthermore, it was demonstrated that approximately 90% of peripheral blood monocytes expressed FMLP receptors, and the majority of FMLP+ cells were also C5a receptor positive. In addition, a parallel spectrum of chemotactic ligand receptor density from low to high levels was demonstrated for both C5a and FMLP. Additional analysis revealed that the density of chemotactic ligand receptors on resting peripheral blood monocytes did not correlate with monocyte maturation levels measured by HLA-DR expression. Elucidation of the monocyte chemotactic receptor-ligand interactions that lead to migration and/or activation may provide insight into the regulation of monocyte function in inflammation.     

Multiple receptor states are required to describe both kinetic binding and activation of neutrophils via N-formyl peptide receptor ligands

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).     

Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.     

Two cAMP receptors activate common signaling pathways in Dictyostelium.

Multiple signal transduction pathways within a single cell may share common components. In particular, seven different transmembrane helix receptors may activate identical pathways by interacting with the same G-proteins. Dictyostelium cells respond to cAMP using one such receptor, cAR1, coupled by a typical heterotrimeric G-protein to intracellular effectors. However, cells in which the gene for cAR1 has been deleted are unexpectedly still able to respond to cAMP. This implies either that certain responses are mediated by a different receptor than cAR1, or alternatively that a second, partially redundant receptor shares some of the functions of cAR1. We have examined the dose response and ligand specificity of one response, cAMP relay, and the dose response of another, cyclic GMP synthesis. In each case, the EC50 was approximately 100-fold higher and the maximal response was smaller in car1- than wild-type cells. These data indicate that cAR1 normally mediates responses to cAMP. The ligand specificity suggests that the responses seen in car1- mutants are mediated by a second receptor, cAR3. To test this hypothesis, we constructed a cell line containing deletions of both cAR1 and cAR3 genes. As predicted, these lines are totally insensitive to cAMP. We conclude that the functions of the cAR1 and cAR3 receptors are partially redundant and that both interact with the same heterotrimeric G-protein to mediate these and other responses.     

Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.     

Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy

In Dictyostelium discoideum amoebae, cAMP-induced phosphorylation of the surface cAMP receptor is associated with a discrete transition in its electrophoretic mobility. The native and modified forms of the receptor are designated R and D (Mr = 40,000 and 43,000). The relationship of the number of receptors which are modified as a function of the receptors which bind cAMP was investigated. Modification was assessed by determining the amounts of R and D forms in Western blots which detect all receptors whether or not they are exposed on the surface. Cyclic AMP or the analog, adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS), induced a loss of cAMP-binding activity (down-regulation), which was not accompanied by a loss of the receptor protein. About 60% of the receptors do not bind cAMP in the absence of Ca2+ and are unmasked by 10 mM Ca2+. However, the fraction of receptors which are modified in response to cAMP is equal in the absence or presence of Ca2+. (Rp)-cAMPs induces down-regulation (50%) but not modification. Addition of cAMP, following down-regulation by (Rp)-cAMPS, causes all receptors to be modified. cAMP induces both down-regulation (80%) and modification. Modification is more readily reversed than down-regulation: 30 min after removal of cAMP, receptors remain down-regulated (57%) but are found in the R form. All receptors shift to the D form when cAMP is readded to the cells. These results indicate that exposed, as well as cryptic and down-regulated receptors, are modified in response to the cAMP stimulus.     

Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor

Extracellular ATP promotes a wide range of physiological effects in many tissues. Of particular interest is the effect of ATP on leukaemia-derived HL-60 and NB4 cell lines, which are induced to mature to neutrophil-like cells. The differentiation process appears to be mediated by ATP binding to a cell-surface purinergic P2Y receptor, resulting in the stimulation of adenylyl cyclase, elevation of cAMP levels and activation of protein kinase A. In 1997, a novel ATP-selective P2Y receptor, P2Y11, was cloned and shown to be linked to both cAMP and Ca2+ signalling pathways. The pharmacological profile of ATP analogues used by P2Y11 for cAMP production in transfected cells is reviewed in the present paper and shown to be closely similar to the profiles for cAMP production and differentiation of myeloblastic HL-60 cells and promyelocytic NB4 cells, both of which express P2Y11. Additional data are provided showing that HL-60 mature to neutrophil-like cells in response to extracellular ATP, as measured by upregulation of the N-formyl peptide receptor, N-formyl peptide-mediated actin polymerization and superoxide production. It is proposed that P2Y11 is responsible for the ATP-mediated differentiation of these cells lines and that this receptor may play a role in the maturation of granulocytic progenitors in the bone marrow.     

Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin

To characterize the requirements for T cell proliferation, we have studied the response of purified populations of human T cells to varying concentrations of the mitogen phytohemagglutinin (PHA). Concentrations of PHA which induce optimal proliferative responses induce increases in cytosolic free calcium ([Ca2+]i), expression of interleukin 2 (IL 2) receptors, and production of IL 2. As the concentration of PHA is decreased, each of these processes decreases in parallel. At suboptimal concentrations of PHA, the addition of exogenous IL 2 reconstitutes both the proliferative response and the expression of the IL 2 receptor, as measured by immunofluorescence with antibodies directed against the TAC/IL 2 receptor molecule, but without reconstituting the increase in [Ca2+]i. Therefore, the concentration dependence of responses to PHA appears to be secondary to an absence of IL 2 production due to a failure to induce an increase in [Ca2+]i. The addition of the calcium ionophores A23187 and ionomycin or of accessory cells to low concentrations of PHA induces increases in [Ca2+]i and subsequent proliferative responses, suggesting that the two events are linked. The proliferative response can be inhibited by antibodies directed towards IL 2 or the IL 2 receptor, indicating that the proliferative response was at least partially dependent on the production and action of IL 2. This suggests that, although increases in [Ca2+]i are an integral event in the induction of proliferation by PHA, the increase in [Ca2+]i is required for the production but not the action of IL 2. In addition, low concentrations of PHA deliver an additional signal to cells, independent of an increase in [Ca2+]i, which induces IL 2 receptor expression and allows a proliferative response in the presence of exogenous IL 2.     

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.     

Apparent Positive Cooperativity at a Surface cAMP Receptor in Dictyostelium

In the large species of the cellular slime mold Dictyostelium , cell aggregation is regulated by extracellular cAMP. During aggregation, cAMP is released in pulses from cells in the aggregation centers and these rhythmic signals are propagated through the population by a signal relay system. In addition to triggering the relay response, the pulsatile signals also regulate the chemotactic movement of the cells and early cell differentiation. These different cellular responses to exogenous cAMP are thought to be mediated via cAMP receptors, which appear on the cell surface shortly after starvation.
Using a sensitive assay, the equilibrium binding properties of these receptors were analyzed at low cAMP concentrations. As reported earlier, Scatchard plots of cAMP binding to preaggregative amoebae of D. discoideum strain NP187 in the concentration range 2–500 nM were curvilinear suggesting either receptor heterogeneity or negative cooperative interactions. However, at cAMP concentrations below approximately 1.5 nM, the affinity of the receptors was found to decline as a function of decreasing receptor occupancy. This apparent positive cooperativity was observed with binding sites on crude plasma membranes as well as on intact cells, and it occurred at both 0°C and 22°C. Moreover, apparent positive cooperativity was a property of the receptors on all strains of D. discoideum examined and on one strain of D. purpureum . Unlike preaggregative cells, receptors on postaggregative cells often lacked this property.
The lowest concentration of cAMP pulses that can appreciably stimulate membrane differentiation in strain NP187 was found to be 0.15–1.5 nM. Since similar concentrations of exogenous cAMP have been reported to trigger minimal chemotactic and relay responses in D. discoideum , the apparent positive cooperative behavior of the cAMP receptor might function to generate a steep cellular response threshold.     

Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms

Many hormones and neurotransmitters attenuate cyclic AMP (cAMP) accumulation in intact cells by virtue of their ability to inhibit adenylate cyclase activity via the GTP-binding protein denoted as Gi. Nonetheless, a number of physiological findings suggest that attenuation of cAMP production is not sufficient to serve as the only signal for eliciting the diverse physiological effects provoked by these various receptor populations. Additional biochemical and electrophysiological changes are known to occur after occupancy of receptors linked to inhibition of adenylate cyclase, including acceleration of Na+/H+ exchange, activation of K+ conductances, and inhibition of voltage-sensitive Ca2+ channels. This review summarizes the current understanding of how these receptors are coupled to their multiple potential effector mechanisms and offers some speculation about the possible interplay between the biochemical and electrophysiological sequels of receptor occupancy. It is hoped that future studies will establish which constellation of possible signaling mechanisms actually brings about changes in metabolic, secretory, or contractile events in different target cells.     

N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was less apparent after dhcB treatment. To summarize, this work shows that activated N-formyl peptide receptors aggregate and immobilize in the plane of the neutrophil plasma membrane before internalization, a process that is affected, but not significantly reversed, by cytochalasin. The results are consistent with a model where arrested receptors are associated mainly with a cytochalasin-insensitive pool of cytoskeletal elements.     

beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities

The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.     

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.