Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF‐Rβ

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet‐derived growth factor (PDGF)‐BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF‐Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF‐BB. CSMC were enzymatically isolated from Sprague–Dawley rats, and the effect of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, transforming growth factor (TGF), IL‐17A and IL‐2 on CSMC growth and responsiveness to PDGF‐BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid‐induced colitis. Neither CM alone nor cytokines caused proliferation of early‐passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF‐BB. IL‐1β, TNF‐α and IL‐17A, but not other cytokines, increased the effect of PDGF‐BB because of up‐regulation of mRNA and protein for PDGF‐Rβ without change in receptor phosphorylation. PDGF‐BB was identified in adult rat serum (RS) and RS‐induced CSMC proliferation was inhibited by imatinib, suggesting that blood‐derived PDGF‐BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL‐1β and TNF‐α induced the early expression of PDGF‐Rβ, while imatinib blocked subsequent RS‐induced cell proliferation. Thus, pro‐inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF‐Rβ and serum‐derived PDGF‐BB, and control of PDGF‐Rβ expression may be beneficial in chronic intestinal inflammation.     

Demonstration of an antibody against platelet-derived growth factor

An antibody was raised in a rabbit against platelet-derived growth factor (PDGF), purified from human platelets. It recognized specifically ¹²⁵I-labelled PDGF, as demonstrated by gel electrophoretic analysis of precipitated radioactivity. Addition of immunoglobulins from the immune serum to cell cultures together with PDGF inhibited the multiplication-stimulating activity of the growth factor. Further, multiplication-stimulating activity in crude platelet lysate was retained upon passage over a column of Sepharose-bound anti-PDGF; it was recovered after elution of the column with acid. A radioimmunoassay for PDGF was developed, permitting the determination of 5 ng PDGF/ml. Platelet factor 4 and β-thromboglobulin, both being proteins of the platelet α-granule, did not cross-react in the radioimmunoassay for PDGF. The radioimmunoassay was utilized to examine the immunological relationship between PDGF and some other growth factors. The anionic growth factors previously demonstrated in pletelets were found to be antigenically distinct from cationic PDGF. Similar results were obtained with epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, osteosarcomaderived growth factor, a PDGF-like growth factor previously known as a product of cultured human osteosarcoma cells, showed some cross-reactivity in the radioimmunoassay for PDGF. It was concluded that osteosarcoma-derived growth factor (ODGF) has both physical-chemical and immunological properties in common with PDGF.     

Interleukin-1 promotes proliferation of vascular smooth muscle cells in coordination with PDGF or a monocyte derived growth factor

We report that interleukin-1 (IL-1) potentiates the proliferation of vascular smooth muscle cells. Growth of early passage smooth muscle cells was not significantly affected by IL-1 alone. Treatment with IL-1 together with the platelet derived growth factor (PDGF) or another polypeptide growth factor derived from mitogen activated human monocytes (MDGF) resulted in a significant enhancement of cell growth over either PDGF or MDGF alone. DNA synthesis was enhanced only marginally (30-40%) in quiescent cultures treated with an optimal concentration of IL-1 alone. In the presence of 5 units/ml of PDGF or MDGF, IL-1 produced about six- to eightfold higher DNA synthesis than the untreated cultures. Induction of DNA synthesis was linear between 0.1 and 1.0 pM IL-1, dependent on PDGF concentration, and was effectively neutralized by monoclonal antibodies against IL-1 beta. The growth promoting activity of IL-1 was extremely potent producing half-maximum stimulation at a concentration of 0.5 pM. These results suggest that IL-1 may play an important role in the modulation of growth and other activities of vascular smooth muscle cells. These observations are especially important with regard to defining the potential macrophage derived mediators contributing to vascular cell proliferation during inflammation and the pathogenesis of atherosclerosis. It is shown here that elicitation of IL-1 induced growth response requires a coordinated action with another priming growth factor such as PDGF. In this regard, IL-1 mediated proliferation of smooth muscle cells may have analogy with the IL-1 mediated T-cell activation and IL-2 production where concerted actions of antigen/mitogen and IL-1 are required.     

Obligatory action of polypeptide growth factors for the IL-1-mediated prostaglandin E2 production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-1

Our studies show that in connective tissue cells, induction of PGE2 synthesis in response to IL-1 requires costimulation with platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). In cells incubated in medium containing fresh serum, IL-1 induced a dose-dependent synthesis of PGE2. However, when the cells were incubated in medium containing low serum or platelet poor plasma (lacking PDGF), IL-1 alone failed to induce PGE2 synthesis. PGE2 synthesis was restored when platelet poor plasma was supplemented with PDGF. Addition of PDGF or FGF together with IL-1 resulted in a 14- and 66-fold stimulation of PGE2 synthesis, respectively. Stimulation was dependent on the concentration of both IL-1 and the growth factor. PGE2 synthesis was also dependent on the synthesis of new proteins. In cells simultaneously treated with IL-1 and PDGF, PGE2 synthesis was initiated after a lag of 2 to 3 h, proceeded first with a rapid rate for 6 h, and then with a slower rate through 24 h. PGE2 synthesis during the latter, slower phase was greatly enhanced by pretreatment with PDGF, but not by pretreatment with IL-1. PDGF pretreatment also resulted in maintenance of 10- to 12-fold higher cell surface IL-1-binding during this phase. These data provide evidence for potentially novel interactions between PDGF and IL-1 activities, one of which is the modulation of IL-1 receptors by PDGF. Furthermore, these studies suggest that by virtue of their effect on IL-1 activities, PDGF and FGF may play additional roles in connective tissues, including an indirect role in inflammatory processes.     

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.     

Effects of platelet-derived growth factor on human and mouse osteoblastic cells isolated from the trabecular bone surface.

We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by (3H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48-72 hours). In both species, the effect of PDGF (25 ng/ml) was lower than the mitogenic effect of 10% FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH)2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH)2D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.     

Monoclonal antibodies that depress a specific subset of multiple components of the glutathione-induced response ofHydra

Summary Reduced glutathione evokes a feeding response, the tentacle-ball formation inHydra japonica. This response consists of at least 5 components (R1–R5). We raised 6 monoclonal antibodies (mAbs), each of which depressed a specific subset of these components, and we also examined the immunocytochemical localization of antigens with these mAbs at light microscopic level. The 2 mAbs that depressed R2 and R4 bound to the cnidocils of the desmoneme and the stenotele nematocytes; the 3 mAbs that depressed R5 bound to the apical surface adjacent to the cnidocils of the nematocytes; and the 2 mAbs that depressed R1 and R3 bound to the apical spot structures of unidentified cells in the ectoderm.Together with the specificity of the action of the mAbs on the behavioral response, the correspondence between the effects on the response and the structures visualized with these mAbs suggests that these structures include components of the receptor-effector system relevant to chemoreception.     

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.     

Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation

Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of 125I-PDGF to human washed platelets was therefore investigated. It was found 1) to be time-dependent, reaching a plateau at 20 degrees C after 30 min, 2) collagen concentration-dependent, 3) specifically inhibited by unlabeled PDGF, and 4) saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of [32P]phosphatidylinositol 4,5-biphosphate (PIP2) and [32P]phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of [32P]PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that (i) a specific PDGF receptor can be induced by collagen, and (ii) PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.     

Regulation of glycogen synthase activity by growth factors. Relationship between synthase activation and receptor occupancy

Platelet-derived growth factor (PDGF) was found to stimulate the activity of glycogen synthase, an enzyme subjected to regulation by phosphorylation-dephosphorylation reactions. In Swiss mouse 3T3 cells, the time course of enzyme activation by PDGF is very similar to that of epidermal growth factor (EGF) and insulin. A 3-fold maximal stimulation was observed by 30 min, and the enzyme activity ratio returned to basal levels by 100 min. The PDGF effect was maximal at 1 ng/ml (30 pM) and half-maximal stimulation was observed at 0.2 ng/ml (6 pM). Parallel measurements of 125I-PDGF binding indicate that binding was maximal by 10 min and thus should not be rate-limiting for enzyme activation. In addition, presaturation of the receptors with PDGF at 4 degrees C did not expedite subsequent enzyme activation at 37 degrees C. Removal of PDGF in the media after the 4 degrees C pretreatment did not affect the enzyme activation response, indicating that the continued presence of PDGF in the medium is not necessary after receptor binding is saturated. Results of sequential addition of PDGF, EGF, and insulin indicated a refractory period in the response system. This property is evident even when the second addition involved a different growth factor and is independent of the sequence of addition of the factors. There was little additivity in the actions of the three growth factors in effecting enzyme activation and suggests a common intermediate element in the three signalling pathways.     

Chemotactic activity of platelet alpha granule proteins for fibroblasts

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.     

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.     

Ovariectomy increases mitogens and platelet-induced proliferation of arterial smooth muscle

Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGF(AB) and PDGF(BB)), transforming growth factors (TGF-beta(1) and TGF-beta(2)), and vascular endothelial growth factor (VEGF(165)) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGF(BB), but not PDGF(AB), TGF-beta(1), and TGF-beta(2), increased with ovariectomy. VEGF(165) was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGF(BB) in platelets. Higher levels of PDGF(BB) in platelets in synergy with other platelet-derived products could contribute to increased proliferative arterial response to injury after ovariectomy.     

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca]i in human platelets in vitro

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.