Polycations as prostaglandin synthesis inducers. Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(L-lysine) and other synthetic polycations

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.     

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts.

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of 3H-arachidonic acid and 3H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE1 analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradykinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of 3H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis

Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells.     

Beclobrinic acid--a new hypolipidemic agent--inhibits in vitro human platelet activation by blocking prostaglandin synthesis

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis

Thrombin and certain prostaglandins are both capable of stimulating the proliferation of cultured cells. Since thrombin stimulates the release and metabolism of arachidonic acid, the precursor of prostaglandins, we examined the relationship between this release and metabolism and the stimulation of cell division in cultured fibroblasts. We also examined the role of prostaglandin synthesis in thrombin-stimulated phosphatidylinositol synthesis. The data in this report demonstrate that the release and metabolism of arachidonic acid are not necessary for thrombin-stimulated cell division. The presence of a low concentration of chymotrypsin prevented thrombin-stimulated arachidonic acid release and metabolism without affecting the stimulation of cell division. Furthermore, thrombin-stimulated cell division occurred in the presence of indomethacin concentrations that prevented cyclooxygenase-mediated metabolism of arachidonic acid. The following experiments showed that thrombin-stimulated phosphatidylinositol synthesis was brought about by a cyclooxygenase-mediated metabolite(s) of arachidonic acid. Indomethacin inhibited the cyclooxygenase-mediated metabolism of arachidonic acid without affecting the thrombin-stimulated release of arachidonic acid. Indomethacin also inhibited thrombin-stimulated phosphatidylinositol synthesis. The dose dependence of this inhibition paralleled the inhibition by indomethacin of cyclooxygenase-mediated metabolism of arachidonic acid. In addition, prostaglandin F2 alpha stimulated phosphatidylinositol synthesis in the presence of indomethacin concentrations which prevented thrombin-stimulated phosphatidylinositol synthesis.     

Hydrocortisone inhibition of the bradykinin activation of human synovial fibroblasts.

Human synovial fibroblasts in culture respond to bradykinin with a 20-fold increment in intracellular cyclic AMP concentrations, however bradykinin does not directly activate adenylate cyclase activity in a particulate fraction derived from these cells. Bradykinin evokes a release of labeled arachidonic acid and prostaglandins E and F from synovial fibroblasts pre-labeled with 3H-arachidonic acid. Hydrocortisone inhibits the bradykinin induced increment in cyclic AMP and the release of arachidonic acid and prostaglandins E and F from synovial fibroblasts. Indomethacin, which also inhibits the cyclic AMP response to bradykinin, has no effect on the release of arachidonic acid from synovial fibroblasts. Indomethacin does, however, inhibit the quantity of prostaglandins released into the medium. These studies support the hypothesis that bradykinin does not activate human synovial fibroblast adenylate cyclase, but presumably activates a phospholipase whose products in turn result in the synthesis of prostaglandins. These and other investigations also suggest that a product(s) of the prostaglandin pathway causes the increment in cyclic AMP.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3: Inhibition by cytochalasin B

Cytochalasin B inhibits the production of prostaglandins by serum-, thrombin-, and bradykinin-stimulated MC5-5 cells. The serum-stimulated release of arachidonic acid from cellular phospholipids also is inhibited. Cytochalasin B does not affect the cells' prostaglandin synthetase activity when exogenous arachidonic acid is present. Deacylation of phospholipids may be the step affected by cytochalasin B possibly as a result of disruption of microfilament organization. Colchicine and vinblastine, two drugs that can disrupt microtubule organization, do not inhibit prostaglandin production by cells.     

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of ³H-arachidonic acid and ³H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE₁ analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradkinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of ³H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Stimulation of histamine release and arachidonic acid metabolism in rat peritoneal mast cells by thapsigargin, a non-TPA-type tumor promoter

Thapsigargin, a non-TPA-type tumor promoter, releases histamine and stimulates arachidonic acid metabolism in rat peritoneal mast cells. In order to clarify the relationship between the histamine-releasing activity and the arachidonic acid metabolism-stimulating activity of thapsigargin in mast cells, the effects of cyclooxygenase inhibitors, indomethacin and ibuprofen, a lipoxygenase inhibitor, AA861, and dual inhibitors for cyclooxygenase and lipoxygenase, nordihydroguaiaretic acid and BW755C, on histamine release and arachidonic acid metabolism were examined. High-performance liquid chromatography analysis revealed that the peritoneal mast cells preferentially produce prostaglandin D2 by thapsigargin treatment. These inhibitors suppressed thapsigargin-induced prostaglandin D2 production in a dose-dependent manner, but failed to inhibit histamine release, suggesting that the mechanisms for stimulation of histamine release by thapsigargin is not dependent on increased arachidonic acid metabolism. Time-course experiments of histamine release and the release of radioactivity from [3H]arachidonic acid-labeled mast cells also provide evidence for a difference in mechanism.     

The confluence-dependent interaction of cytosolic phospholipase A2-alpha with annexin A1 regulates endothelial cell prostaglandin E2 generation

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Differential effect of diacylglycerols and phorbol ester on arachidonic acid metabolism in bone marrow-derived macrophages

Murine bone marrow-derived macrophages were induced to prostaglandin synthesis by activators of protein kinase C, the phorbolester TPA and the diacylglycerols dioctanoylglycerol (diC8) and diolein (diC18:1). As short term stimulation of prostaglandin synthesis is mainly dependent on the availability of free arachidonic acid, the modulation of arachidonic acid liberation and reacylation was investigated. DiC8 inhibited the reacylating enzyme lysophosphatide acyltransferase in the in vitro assay, but there was no evidence for an inhibitory effect of TPA or diacylglycerols on the activity of the lysophosphatide acyltransferase in whole cells. The release of arachidonic acid from prelabelled cells was stimulated by TPA and the diacylglycerols even in the presence of an inhibitor of reacylation, indicating an activation of phospholipase A2. An activation of phospholipase A2 was measured in membranes derived from TPA-stimulated macrophages. These data indicate that the enhanced pool of free arachidonic acid, which drives prostaglandin synthesis, is primarily due to a stimulation of the liberation of arachidonic acid from membrane phospholipids.     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3: Effect of oxidative phosphorylation inhibitors

Oligomycin, antimycin, and 2,4-dinitrophenol, compounds that are known to inhibit oxidative phosphorylation by different mechanisms, inhibit the production of prostaglandins by serum-stimulated MC5-5 cells. The prostaglandin production that is stimulated by thrombin and bradykinin is inhibited by 2,4-dinitrophenol. Prostaglandin synthesis by MC5-5 cells from exogenously-supplied arachidonic acid, however, is not affected by 2,4-dinitrophenol. Antimycin and 2,4-dinitrophenol also inhibit the serum-stimulated release of arachidonic acid from the cellular lipids, suggesting that it is the expression of phospholipase activity, a prerequisite for synthesis of prostaglandins by MC5-5 cells, that is dependent on oxidative phosphorylation.     

Hydrocortisone inhibition of the bradykinin activation of human synovial fibroblasts

Human synovial fibroblasts in culture respond to bradykinin with a 20-fold increment in intracellular cyclic AMP concentrations, however bradykinin does not directly activate adenylate cyclase activity in a particulate fraction derived from these cells. Bradykinin evokes a release of labeled arachidonic acid and prostaglandins E and F from synovial fibroblasts pre-labeled with ³H-arachidonic acid. Hydrocortisone inhibits the bradykinin induced increment in cyclic AMP and the release of arachidonic acid and prostaglandins E and F from synovial fibroblasts. Indomethacin, which also inhibits the cyclic AMP response to bradykinin, has no effect on the release of arachidonic acid from synovial fibroblasts. Indomethacin does, however, inhibit the quantity of prostaglandins released into the medium. These studies support the hypothesis that bradykinin does not activate human synovial fibroblast adenylate cyclase, but presumably activates a phospholipase whose products in turn result in the synthesis of prostaglandins. These and other investigations also suggest that a product(s) of the prostaglandin pathway causes the increment in cyclic AMP.     

Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Platelet-derived growth factor stimulates phospholipase C-gamma 1, extracellular signal-regulated kinase, and arachidonic acid release in rat myometrial cells: contribution to cyclic 3',5'-adenosine monophosphate production and effect on cell proliferation

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-beta receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-gamma 1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI(2)). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA(2) inhibitor, and in the absence of Ca(2+). U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI(2) biosynthesis. PDGF-BB also increased cell proliferation and [(3)H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.