Stimulation of the 23-Kd protein cAMP dependent phosphorylation by inositol 1,4,5 trisphosphate in human platelet membrane vesicles

The effect of inositol 1,4,5 trisphosphate (IP3) has been investigated on the cAMP-induced phosphorylation of the 23-Kd protein involved in platelet calcium fluxes by isolated membrane vesicles. The studies were conducted using the catalytic subunit of the cAMP-dependent protein kinase (C. Sub.). A dose-dependent stimulation of the 23 Kd protein phosphorylation induced by C. Sub. was initiated by IP3 with a half-maximal effect of 0.5 microM. The maximal effect was observed after 1-2 min. The effect was detected in the absence of Ca2+ and in the presence of phosphatase inhibitors. These results can suggest that the 23 Kd is an associated protein to the IP3 receptor in human platelets.     

Interferon-gamma binds to high and low affinity receptor components on murine macrophages

Interferon-gamma (IFN-gamma), a glycoprotein secreted by antigen-or mitogen-activated lymphocytes, modulates the activities of lymphocytes and macrophages. For mouse macrophages, murine IFN-gamma (MuIFN-gamma) stimulates several functions, including phagocytosis, tumoricidal activity, and the increased expression of Ia and H-2 antigens of the major histocompatibility complex. Recent reports have suggested that IFN-gamma specifically binds to cell surface receptors corresponding to a single class of binding sites with a Kd of 10(-8) to 10(-10) M. We present evidence that, on the murine macrophage cell line WEHI-3, MuIFN-gamma specifically binds to two classes of binding sites with Kd of 9.1 X 10(-11) M (500 sites/cell) and 2.7 X 10(-9) M (4400 sites/cell). The higher affinity sites most likely represent the physiologically relevant receptors that mediate at least some of the actions of MuIFN-gamma.     

Identification of platelet-activating factor receptors in P388D1 murine macrophages

Platelet-activating factor (PAF) binding and metabolism by eight murine and human cell lines was analyzed. Only the murine P388D1 macrophage line had specific, high affinity PAF binding sites. PAF binding reached saturation within 10 min at room temperature and was irreversible. Minimal PAF metabolism was observed at the time binding saturation was achieved. Scatchard analysis of PAF binding revealed a single class of PAF receptors (7872 +/- 1310/cell) which had a dissociation constant of 0.08 +/- 0.01 nM (mean +/- SEM, eta = 6). The dissociation constant was confirmed independently by quantifying the kinetics of initial specific PAF binding. PAF binding was stereospecific, required an sn-2 acetyl substituent, and was inhibited by structurally diverse PAF antagonists including kadsurenone, BN 52021, triazolam, and CV3988. The fact that the receptors are functionally active was shown by the observation that 1 to 100 pM PAF increased free intracellular calcium in P388D1 cells in a dose-related manner. These studies demonstrate that P388D1 macrophages have functional PAF receptors whose affinity and structural specificities are similar to PAF receptors in other cells. The availability of a stable cell line that binds but does not metabolize PAF will greatly facilitate studies of the PAF receptor.     

Involvement of protein kinase C in platelet-activating factor-stimulated diacylglycerol accumulation in murine peritoneal macrophages

Incubation of murine peritoneal macrophages with platelet-activating factor (PAF; 1-O-alkyl(C16 + C18)-2-acetyl-sn-glycerol-3-phosphorylcholine) results in the rapid accumulation of [3H]inositol phosphates and sn-1,2-diacylglycerol (DAG) and mobilization of intracellular calcium (Prpic, V., Uhing, R. J., Weiel, J. E., Jakoi, L., Gawdi, G., Herman, B., and Adams, D. O. (1988) J. Cell Biol. 107, 363-372). We have further investigated the relationship of phosphoinositide metabolism to accumulation of DAG and the possible involvement of protein kinase C in the accumulation of DAG in response to PAF. DAG accumulation proceeds at a slower rate than the accumulation of either [3H] inositol 1,4,5-trisphosphate or total [3H]inositol phosphates. Accumulation of DAG from additional precursors is suggested from both an estimation of the mass of total inositol phosphates produced and the accumulation of [3H]choline in response in PAF. Down-regulation of protein kinase C by prolonged pretreatment with phorbol ester or inhibition of the enzyme with sphingosine inhibited the PAF-generated accumulation of DAG at 10 min by approximately 80%. Under the same conditions, no inhibition of PAF-stimulated generation of [3H]inositol 1,4,5-trisphosphate was observed. Similar inhibition was observed when 10 microM ionomycin or 0.1 microM phorbol 12-myristate 13-acetate were used to stimulate accumulation of DAG. The results suggest that PAF stimulates the accumulation of DAG from source other than phosphatidylinositol metabolism in peritoneal macrophages and that this occurs subsequent to the activation of protein kinase C.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Intracellular receptors for inositol 1,4,5-trisphosphate in angiotensin II target tissues

Many cells (including angiotensin II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol 1,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol 1,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Sp?t, A., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol 1,4,5-trisphosphate-binding sites are saturable and present in low concentration (104 +/- 48 fmol/mg protein) and exhibit high affinity for inositol 1,4,5-trisphosphate (Kd 1.7 +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol 1,4-bisphosphate (Kd approximately 10(-7) M), inositol 1-phosphate and phytic acid (Kd approximately 10(-4) M), fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately 10(-3) M), with no detectable affinity for inositol 1-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = 17 microM). Furthermore, submicromolar concentrations of inositol 1,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol 1,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.87 +/- 0.31 nM, Bmax = 14.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = 1.66 +/- 0.7 nM, Bmax = 147 +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol 1,4,5-trisphosphate mobilizes Ca2+ in target tissues for angiotensin II and other calcium-dependent hormones.     

The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages

The colony-stimulating factor, CSF-1, stimulates cultured quiescent murine bone marrow-derived macrophages (BMM) to enter DNA synthesis with a lag phase of 10-12 h. The binding, dissociation, internalization, and degradation of 125I-CSF-1 by BMM during the lag phase were investigated. Quiescent BMM express approximately 5 X 10(4) cell surface receptor sites/cell but contain additional cryptic sites (approximately 10(5)/cell) that can appear at the cell surface within 10 min at 37 degrees C. Studies of the binding reaction at both 2 degrees C (Kd less than or equal to 2 X 10(-13) M) and 37 degrees C (Kd approximately 4 X 10(-10) M) are consistent with the existence of a single class of cell surface sites. The disappearance of cell surface 125I-CSF-1 following a 2-37 degrees C temperature shift results from two, competitive, first order processes, internalization and dissociation. Internalization (t1/2 = 1.6 min) is 6 times more frequent than dissociation (t1/2 = 9.6 min). Following internalization, 10-15% of the intracellular CSF-1 is rapidly degraded whereas the remaining 85-90% is slowly degraded by a chloroquin-sensitive first order process (t1/2 greater than 3.5 h). These findings were confirmed and extended by studies of the uptake of 125I-CSF-1 at 37 degrees C. Following addition of 125I-CSF-1, cell surface receptors are rapidly down-regulated (t1/2 approximately 7 min) and their replacement does not commence until 20-60% of pre-existing surface receptor sites have disappeared. Despite receptor replacement, initially from the cryptic pool and later by de novo synthesis and/or receptor recycling (4 molecules/cell/s at steady state), the number of receptors at the cell surface remains low. The process results in the intracellular accumulation of large amounts of 125I-CSF-1 (greater than 10(5) molecules/cell) by BMM. Thus, whereas the kinetics of association, dissociation, and internalization of CSF-1 with BMM and peritoneal exudate macrophages are similar, BMM, which exhibit a higher proliferative response, degrade growth factor 12 times more slowly.     

Heterogenous inositol tetrakisphosphate binding sites in the adrenal cortex

Bovine adrenal cortical microsomes possess high (Kd = 3.68 +/- 1.02 X 10(-9) M) and lower (Kd = 9.20 +/- 1.71 X 10(-8) M) affinity binding sites for inositol 1,3,4,5-tetrakisphosphate. The binding to these sites is rapid, saturable (reaches equilibrium by 15 min at 0 degrees C), and reversible. Competition studies with other inositol phosphate analogs indicate that the high affinity binding sites are clearly distinct from the inositol 1,4,5-trisphosphate receptors which are, however, responsible for a fraction of the lower affinity binding. The characteristics of the inositol 1,3,4,5-tetrakisphosphate binding sites described are compatible with their possible receptor function.     

Inositol trisphosphate and calcium mobilisation in permeabilised enterocytes

Saponin-permeabilised epithelial cells isolated by hyalurodinase incubation from chicken small intestine were used to study 45Ca uptake into intracellular stores. At low (6.7 X 10(-7) M) free Ca2+ concentration most of the Ca2+ appears to be taken up into non-mitochondrial stores, whilst the mitochondria seem to play a major role at high (2 X 10(-5) M) Ca2+ concentration. Addition of inositol trisphosphate (IP3) causes a rapid and reversible release of 45Ca from non-mitochondrial stores, with a half-maximal effect of approximately 1 microM.     

Expression of a mannosyl-fucosyl receptor for endocytosis on cultured primary macrophages and their hybrids

The presence of a pinocytosis receptor, specific for mannose-fucose terminated glycoproteins, has been established on murine resident peritoneal macrophages, thioglycollate-elicited peritoneal macrophages, and macrophages derived from bone-marrow in culture. Macrophagelike cell lines (J-774 and P338.D1), a myelomonocytic cell line (427E), lymphocytes, polymorphonuclear leukocytes, and fibroblasts were negative. Binding and uptake of 125I-mannose-BSA and 125I-beta-glucuronidase, respectively, into thioglycollate-induced peritoneal macrophages is saturable (Kd 4 degrees C = 5.4 X 10(-9) M; Kuptake 37 degrees C = 7 X 10(-7) M) and sugar specific. Macrophage-macrophage (rat X mouse) hybrids prepared by fusing rat alveolar macrophages with J-774-B10 (HAT-sensitive macrophagelike cell line) expresses the mannose-fucose receptor. Karyotypes of the hybrids confirmed a 1:1 fusion of rat and mouse cells. The rat/mouse hybrids express a variety of rat and mouse antigens including Fc receptors. Fibroblast-macrophage hybrids and melanoma-macrophage hybrids were negative for mannose-fucose receptor activity. The expression of the mannose-fucose receptor by macrophages appears to be regulated independently of other macrophage markers.     

Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage

The beta-adrenergic receptor was characterized on BCG-activated rat peritoneal macrophage membranes by radio-ligand binding studies. Saturable binding with [125I]iodocyanopindolol (125I-ICYP) was demonstrated. With Scatchard analysis, rat macrophages demonstrate approximately 1000 receptors per cell with a Kd of 5 X 10(-11) M for 125I-ICYP. Competition curves with (-) and (+) propranolol at concentrations below 10(-6) M confirmed stereospecificity. The potency of various ligands to compete for 125I-ICYP binding sites followed the order: propranolol greater than isoproterenol greater than epinephrine greater than norepinephrine with apparent Kd of 2.0 X 10(-9), 3.9 X 10(-7), 1.0 X 10(-5), and 2.5 X 10(-5) M, respectively. Isoproterenol-stimulated adenylate cyclase activity was two-fold above basal activity. The potential physiologic significance of a beta-adrenergic receptor on rat peritoneal macrophages was suggested by a dose-dependent decrease in phagocytosis of soluble, model immune complexes (aggregated gamma-globulin) by macrophages incubated with metaproterenol. We conclude that the rat macrophage has a beta-adrenergic receptor and that catecholamines may thereby modulate macrophage function.     

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.     

Dimethyphenylpiperazinium, a nicotinic receptor agonist, downregulates inflammation in monocytes/macrophages through PI3K and PLC chronic activation

Activation of nicotinic acetylcholine receptors (nAChRs) on inflammatory cells induces anti-inflammatory effects. The intracellular mechanisms that regulate this effect are still poorly understood. In neuronal cells, nAChRs are associated with phosphatidylinositol 3-kinase (PI3K). This enzyme, which can activate phospholipase C (PLC), is also present in monocytes. The aim of this study was to assess the role of these proteins in the signaling pathways involved in the anti-inflammatory effect of dimethylphenylpiperazinium (DMPP), a synthetic nAChR agonist, on monocytes and macrophages. The results indicate that PI3K is associated with alpha3, -4, and -5 nAChR subunits in monocytes. The PI3K inhibitors wortmannin and LY294002 abrogated the inhibitory effect of DMPP on LPS-induced TNF release by monocytes. Treatment with DMPP for 24 and 48 h provoked a mild PLC phosphorylation, which was blocked by the nAChR antagonist mecamylamine and reversed by PI3K inhibitors. Treatment of monocytes and alveolar macrophages with DMPP reduced the inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium mobilization induced by platelet-activating factor (PAF), an effect that was reversed by mecamylamine in alveolar macrophages. DMPP did not have any effect on PAF receptor expression. DMPP also inhibited the thapsigargin-provoked calcium release, indicating that the endoplasmic reticulum calcium stores might be depleted by treatment with the nAChR agonist. Taken together, these results suggest that PI3K and PLC activation is involved in the anti-inflammatory effect of DMPP. PLC limited, but constant activation could induce, the depletion of intracellular calcium stores, leading to the anti-inflammatory effect of DMPP.     

Characterization of specific high-affinity receptor for human lymphotoxin

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini.

Previous studies have shown that the dose-response relationship for secretin-stimulated cyclic AMP accumulation is different from that for secretin-stimulated enzyme secretion in the rat exocrine pancreas. Here we show that secretin concentrations of 10(-10) M and higher stimulated a rise in cyclic AMP levels, with maximum effect on cyclic AMP accumulation being achieved already with 10(-8) M-secretin. However, at this concentration of secretin, enzyme secretion rates were approximately half-maximal. Unexpectedly, at concentrations of secretin greater than 10(-8) M there was evidence suggestive of phosphatidylinositol bisphosphate hydrolysis with rapid increases in inositol trisphosphate, cytosolic free calcium and diacylglycerol content of rat pancreatic acini. Furthermore, there was a dose-response relationship among secretin concentration (in the range 10(-8) M-2 X 10(-6) M), increases in inositol trisphosphate and increases in cytosolic free calcium ([Ca2+]i). Contrary to what has been previously believed, these results clearly indicate that in rat pancreatic acini secretin not only stimulates cyclic AMP accumulation but also raises inositol trisphosphate, [Ca2+]i and diacylglycerol. Thus, two second messenger systems may play a role in the regulation of secretin-induced amylase release.     

Calcium modulation of phosphoinositide kinases in transverse tubule vesicles from frog skeletal muscle

Highly purified transverse tubule membranes isolated from frog skeletal muscle phosphorylate phosphatidylinositol to phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate. The two phosphorylation reactions have different calcium requirements. Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate, which takes place in both isolated transverse tubules and sarcoplasmic reticulum membrane, is independent of calcium in a range of concentrations from 10(-9) to 10(-6) M, and is progressively inhibited to 10% of the maximal values by increasing calcium to 10(-4) M or higher (K0.5 = 5 X 10(-6) M). In contrast, phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol (4,5)-bisphosphate, a reaction exclusively present in transverse tubule membranes, is maximal at calcium concentrations higher than 2 X 10(-6) M and decreases to 30% of maximal values at calcium concentrations of 2 X 10(-7) M or lower (K0.5 = 10(-6) M). Unlike frog membranes, transverse tubules from rabbit muscle need exogenous phosphatidylinositol 4-phosphate in order to produce the bisphosphate derivative in the same range of calcium concentrations. Inositol (1,4,5)-trisphosphate has been proposed recently as a chemical messenger in excitation-contraction coupling in skeletal muscle. Calcium regulation of the synthesis of phosphatidylinositol (4,5)-bisphosphate, the membrane-bound precursor of inositol (1,4,5)-trisphosphate, might have physiological implications regarding modulation of excitation-contraction coupling by intracellular calcium levels.     

Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium

Inositol 1,4,5-trisphosphate is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. We have recently identified high levels of specific binding sites for inositol 1,4,5-trisphosphate in brain membranes (Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161) and have now further characterized these sites. In cerebellar membranes, inositol 1,4,5-trisphosphate binding sites are abundant (20 pmol/mg protein) and display high affinity and selectivity for inositol 1,4,5-trisphosphate (KD approximately equal to 40 nM), whereas other inositol phosphates such as inositol 1,3,4,5-tetrakisphosphate (Ki approximately equal to 10 microM) and inositol 1,4-bisphosphate (Ki approximately equal to 10 microM) exhibit much lower affinity for this site. Submicromolar concentrations of calcium strongly inhibit inositol 1,4,5-trisphosphate binding (IC50 approximately equal to 300 nM). A sharp increase in binding occurs at slightly alkaline pH. These results suggest that actions of inositol 1,4,5-trisphosphate are regulated by physiological alterations in intracellular pH and calcium concentrations.     

Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin

Four murine macrophage-like continuous cell lines (P388D1, J774.1, RAW 264.7, and PU5-1.8) and two human cell lines displaying macrophage-monocyte characteristics (HL-60, U-937) have been examined for their ability to degrade both normal and acetylated low-density lipoproteins. All of these cell lines, except PU5-1.8, were demonstrated to have LDL receptors that were induced 2-5-fold by preincubation in lipoprotein-deficient serum. Metabolism of dextran sulfate-LDL complexes by all lines except PU5-1.8 was observed. Three cell lines, P388D1, J774.1 and RAW 264.7, while exhibiting individual differences in their metabolism of acetyl-LDL, all processed acetyl-LDL in a fashion qualitatively analogous to that by murine peritoneal macrophages and human monocytes. Cell lines PU5-1.8, U-937 and HL-60 did not bind or degrade significant quantities of acetyl-LDL. In P388D1 cells, metabolism of acetyl-LDL exhibited time and concentration dependence, was reversibly inhibited by chloroquine, blocked by fucoidan and dextran sulfate, and was calcium independent. Approximately 4 X 10(5) receptors, with an apparent Kd of 3 X 10(-8) M, were present on P388D1 cells. P388D1 cells metabolized 30% as much acetyl-LDL as murine peritoneal macrophages at 37 degrees C and bound 60% as much at 4 degrees C. Chemical measurement demonstrated a 250-fold increase in the cholesteryl ester content of P388D1 cells over 96 h. The accumulation of cholesteryl esters was reversible in the presence of HDL3 and involved continuous hydrolysis and reesterification. These lines represent a convenient resource for examining the metabolism of chemically modified lipoproteins, for isolation of cell mutants, and for isolation of specific lipoprotein receptors.     

Activation of washed chicken thrombocytes by 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (platelet-activating factor)

Washed, [3H]serotonin-labeled chicken thrombocytes aggregated and secreted [3H]serotonin when stimulated in vitro with platelet-activating factor (PAF), collagen and calcium ionophore A23187. The effective dose causing a 25% secretion of [3H]serotonin (ED25) from washed chicken thrombocytes was 10(-8) M for PAF, 5 X 10(-8) M for collagen and 3 X 10(-7) M for A23187. Chicken thrombocyte activation by PAF required Ca2+ and appeared to be mediated through a specific receptor for PAF.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.