Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene.

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.

Haematopoietic colony stimulating factors CSF-1 and GM-CSF increase phosphatidylinositol 3-kinase activity in murine bone marrow-derived macrophages.

The activity of phosphatidylinositol (PI) 3-kinase was examined in murine bone marrow-derived macrophages (BMM) stimulated with the haematopoietic growth factors colony stimulating factor-1 (CSF-1) and granulocyte/macrophage-CSF (GM-CSF). PI 3-kinase was immunoprecipitated from cell lysates using anti-phosphotyrosine antibody or an antibody directed against the 85K subunit of PI 3-kinase, and the activity assayed by the phosphorylation of PI in the presence of [gamma 32P]-ATP. The results demonstrate that CSF-1 increases the activity of PI 3-kinase, as compared to the non-stimulated control, in murine macrophages. Maximum activity was seen after 10 min of stimulation with CSF-1 at 3000-5000 U/ml. The dose-response of CSF-1 is consistent with other biochemical effects of CSF-1 seen in the BMM. GM-CSF also stimulated PI 3-kinase activity although to a lesser extent than CSF-1, correlating well with their degree of mitogenic activity on the BMM. Non-mitogenic macrophage activating agents, such as the phorbol myristate acetate, lipopolysaccharide, concanavalin A and formyl-methionyl-leucyl-phenylalanine, did not significantly increase the PI 3-kinase activity. Furthermore, CSF-1 failed to stimulate PI 3-kinase activity in resident peritoneal macrophages, a population of macrophages with poor proliferative capacity. These results suggest that the PI 3-kinase activity may be involved in the haemopoietic growth factor signalling pathways regulating macrophage growth.

Influence of growth rate and nutrient limitation on monobactam production and peptidoglycan synthesis in Pseudomonas aeruginosa.

The effects of growth rate and nutrient limitation on monobactam production, peptidoglycan content and mean cell length in Pseudomonas aeruginosa was studied using continuous culture techniques. All three parameters increased progressively with growth rate, a greater response being shown under carbon limitation compared to that occurring under nitrogen limiting conditions. Interestingly, monobactum production mirrored peptidoglycan synthesis. In addition, the monobactam exhibited a broad range of antibacterial activity and bound preferentially to PBP 1A in the producing organism. Moreover, addition of the monobactam to a growing culture inhibited cell wall synthesis. These results are discussed in relation to the control and regulation of peptidoglycan synthesis.

Thrombin stimulates the production of phosphatidylinositol 3,4-bisphosphate in human erythroleukemia cells.

The human erythroleukemic cell line, HEL, which has numerous platelet markers, shows enhanced inositol phosphate production in response to thrombin. We investigated the production of phosphoinositides in HEL cells and showed that thrombin stimulates the turnover of several phosphoinositides including the synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Phosphatidylinositol 3-monophosphate is also produced in HEL cells and its synthesis is not stimulated by thrombin. Pretreatment of HEL cells with the stable prostacyclin analog iloprost inhibits the thrombin-induced increase in the production of PtdIns(3,4)P2. 3-Phosphorylated phosphoinositides have been implicated in signal transduction and regulation of cell proliferation in other cells and may be involved in signal transduction in HEL cells.

The production of phosphatidylinositol trisphosphate is stimulated by thrombin in human platelets.

Untreated human platelets labeled to equilibrium with 32Pi contained undetectable levels of 3-phosphorylated phosphoinositides. Stimulation of platelets with thrombin for 5 min resulted in an enormous increase in the amount of phosphatidylinositol 3,4-bisphosphate (Nolan and Lapetina, J. Biol. Chem. 265, 2441-2445; 1990). We now report that the levels of phosphatidylinositol 3,4,5-trisphosphate are greatly elevated within 90 s of treatment of platelets with thrombin. Phosphatidylinositol 3,4,5-phosphate might have an important role in platelet aggregation.

Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.

Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.

Thrombin stimulates the production of a novel polyphosphoinositide in human platelets.

The sequential actions of phosphoinositide 4-kinase and 5-kinase and hydrolysis of phosphatidylinositol (PtdIns) 4,5-P2 are stimulated during platelet activation. Recently, a phosphoinositide 3-kinase has been implicated in signal transduction in several cell types. Stimulation of PtdIns(3,4)P2 synthesis has been shown in polyoma middle T-transformed and platelet-derived growth factor-stimulated cells, and this novel lipid has been implicated in signal transduction and regulation of cell proliferation. We demonstrate the formation of PtdIns(3,4)P2 in human platelets and show that the synthesis of this lipid (and of PtdIns(4,5)P2) is stimulated during activation of platelets by thrombin. This indicates the presence of phosphoinositide 3-kinase activity in platelets. We postulate that PtdIns(3,4)P2 is involved in signal transduction in platelets and discuss the possibility that this novel lipid is a substrate for phospholipase C.

Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells.

The alpha-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three alpha-adrenergic receptor subtypes have been cloned: the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors in cells lacking endogenous alpha-adrenergic receptors. Both alpha 2-C10 and alpha 2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. alpha 2-C10 inhibits adenylyl cyclase more efficiently than alpha 2-C4. Effects of the alpha 2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the alpha 1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. alpha 1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the alpha-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.

The effect of iloprost on the ADP-ribosylation of Gs alpha (the alpha-subunit of Gs).

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

Endogenous ADP-ribosylation in human platelets.

We have studied ADP-ribosyltransferase activity in platelet cytosol and electropermeabilized platelets. Cytosolic activity causes ADP-ribosylation or of a 37 kDa protein that is activated by increasing the concentration of potassium phosphate. ADP-ribosylation is inhibited by thiol reagents, an effect partially reversed by cholera toxin. In electropermeabilized platelets incubated with [alpha-32P]NAD, the 37 kDa protein is also ADP-ribosylated as are other proteins and albumin. Under these conditions, ADP-ribosylation is partially inhibited by nicotinamide. This experimental design could be used to determine the effect of cell agonists on endogenous ADP-ribosylation of proteins.

Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets.

The phosphatase-induced hydrolysis of [3H]inositol 1,4-bisphosphate [Ins(1,4)P2)] and [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was studied in platelet subcellular fractions. The activity that hydrolyses Ins(1,4)P2 is cytosolic, whereas the activity that hydrolyses Ins(1,4,5)P3 is present in both particulate and cytosolic fractions. The cytosolic Ins(1,4)P2 phosphatase hydrolyses the 1-phosphate of Ins(1,4)P2, whereas the cytosolic and membrane-bound Ins(1,4,5)P3 phosphatases hydrolyse the 5-phosphate of Ins(1,4,5)P3. In the presence of ATP, it is possible to observe a cytosolic Ins(1,4,5)P3 3-kinase that phosphorylates Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate. Apparent Km values for the particulate and the cytosolic Ins(1,4,5)P3 phosphatases are 100 microM and 40 microM respectively. A large proportion of the membrane-associated Ins(1,4,5)P3 phosphatase can be extracted with 1 M-NaCl, and the Mr of this enzyme, as determined by hydrodynamic studies, is 49,000, whereas that of the cytosolic enzyme is 59,000. The Km values for the cytosolic Ins(1,4)P2 phosphatase is 40 microM; this enzyme has an Mr of 49,000. The highest specific activity of the Ins(1,4,5)P3 phosphatase is present in a highly purified plasma-membrane fraction.

Role of arachidonic acid metabolism in the mitogenic response of BALB/c 3T3 fibroblasts to epidermal growth factor.

We have investigated the involvement of arachidonic acid release and metabolism in the mitogenic response, i.e., [3H]thymidine incorporation, to epidermal growth factor (EGF) in BALB/c 3T3 cells. EGF induces release of arachidonate and prostaglandin (PG) formation after its addition to BALB/c 3T3 cells at the same concentrations that stimulate mitogenesis. Further, EGF-stimulated mitogenesis is blocked by inhibitors of arachidonate metabolism including indomethacin, eicosatetraynoic acid, and dexamethasone, whereas the addition of major arachidonate products in BALB/c 3T3 cells, PGE2, PGF2 alpha, and their intermediates PGG2 and PGH2, stimulate mitogenesis in synergism with EGF. The addition of PGs to BALB/c 3T3 cells also overcame indomethacin- and eicosatetraynoic acid-inhibited responses to EGF. Indomethacin must be added with EGF in order to block arachidonate metabolism and subsequent mitogenesis. These results suggest that the release of arachidonic acid and its subsequent metabolism is an apparent early requirement for the initiation of cell cycle traversal by EGF.

Stimulation of arachidonic acid metabolism by adherence of alveolar macrophages to a plastic substrate. Modulation by fetal bovine serum.

In previous studies on arachidonic acid (AA) metabolism by pulmonary macrophages in vitro, we observed that the presence of serum in the culture medium influenced the profile of AA metabolites released. To further characterize this phenomenon, rat alveolar macrophages were placed in plastic tissue culture dishes and allowed to adhere in the presence or absence of 7.5% fetal bovine serum (FBS) for 1 h. Adherent cells were then maintained in medium (equilibration) with or without FBS for 3.5 h before stimulation with the calcium ionophore A23187. The release of thromboxane B2 (TXB2) (the stable metabolite of TXA2) and leukotriene B4 (LTB4) during culture was measured by radioimmunoassay and reverse-phase high pressure liquid chromatography, respectively, at the end of each culture step. Cell adhesion to the plastic substrate in FBS-free medium induced an intense stimulation of AA metabolism, with the release of both TXB2 and LTB4. Adhesion and the accompanying TXB2 release appear to be mediated by trypsin-sensitive components since trypsin-pretreated macrophages showed a dramatic reduction in both adherence and TXB2 synthesis. The presence of FBS during the attachment phase of culture reduced both adhesion and release of TXB2 and LTB4 by more than 50%. On the other hand, addition of FBS to cells that had completed adhesion in serum-free medium stimulated release of both metabolites. When challenged with calcium ionophore after 4.5 h of culture, macrophages that had adhered in FBS-free medium released a much smaller amount of TXB2 than did macrophages that had been cultured in the presence of FBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of prostacyclin synthesis in cultured bovine aortic endothelial cells by vitamin K1.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation, is the predominant metabolite of arachidonic acid (AA) in endothelial cells derived from large blood vessels. Vitamin K1 (1-100 microM) inhibited the release of PGI2 and prostaglandin E2 (PGE2) by bovine aortic endothelial cells in culture, as measured by radioimmunoassay of 6-keto-prostaglandin F1 alpha (6-keto-PGE1 alpha) and PGE2. The conversion of exogenous AA to PGI2 was not affected by vitamin K1 as measured by radioimmunoassay and high performance liquid chromatography of radiolabeled AA metabolites. Similarly, vitamin K1 did not affect the conversion of prostaglandin H2 (PGH2) by in vitro enzyme systems. However, vitamin K1 inhibited the calcium ionophore A23187-induced release of [3H]AA from membrane phospholipids of bovine aortic endothelial cells. Inhibition of [3H]AA release from other cells of vascular origin was also observed after exposure to vitamin K1, but this effect was not observed in cells of non-vascular origin, including platelets. Therefore, vitamin K1 modulates the release of AA in vascular cells and thus inhibits the capacity of blood vessels to synthesize PGI2.

Regulation of prostaglandin production by osteoblast-rich calvarial cells.

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were all shown to stimulate PGE2 and 6-keto-PGF1 alpha (the hydration product of PGI2) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE2 and PGI2 (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occurring at approximately 10(-7) M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments in vivo which have indicated that the compounds stimulated PGI2 production. Finally, since the osteoblast is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early times EGF stimulated PGE2 release, however, the predominant effect of the growth factor was an inhibition of both PGE2 and PGI2 production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the osteoblast and that any increase in PG production by these cells may be in response to vascular agents.

Conversion of triostins to quinomycins by protoplasts of Streptomyces echinatus.

Protoplasts of Streptomyces echinatus have been used to investigate the biosynthesis of echinomycin (quinomycin A). It has been shown that this organism has the capacity to convert a series of triostins to the corresponding quinomycins by a mechanism involving methylation. Evidence is presented which suggests that triostin A is the natural precursor of echinomycin. Conversion of tetra-N-demethyl analogues of triostin A to corresponding analogues of echinomycin was not detected.

Pericardial release of prostacyclin induced by bradykinin and angiotensin II: effects on coronary blood flow in the dog.

Angiotensin II and bradykinin are potent releasers of prostanoids, and it has been suggested that these prostanoids may modulate vascular tone in a number of organs. We previously showed that the pericardium is an important site of prostacyclin biosynthesis, and in this study we have investigated whether or not prostacyclin released into the pericardial fluid influences coronary blood flow. Angiotensin II and bradykinin were infused intra-aortically into anaesthetized dogs, and coronary blood flow in the circumflex artery was measured with electromagnetic probes. Krebs solution irrigating the pericardial surfaces was monitored for prostanoid release using a cascade of bioassay tissues as previously described. Angiotensin II infusions (25 ng/kg/min) increased arterial blood pressure, decreased coronary blood flow, and increased the release of a prostacyclin-like substance into the pericardial irrigating fluid. Inclusion of indomethacin (1 microgram/ml) in the pericardial irrigating fluid abolished angiotensin II-induced release of the prostacyclin-like substance from the heart, did not affect resting coronary flow, but potentiated the coronary vasoconstrictor response to intra-arterial angiotensin II. Bradykinin infusions (0.2 microgram/kg/min) also released the prostacyclin-like substance into the pericardial fluid, and caused a transient decrease in arterial pressure and increase in coronary blood flow. Inclusion of captopril (1 microgram/ml) in the irrigating fluid increased slightly the release of the prostacyclin-like substance, but did not alter the increase in coronary blood flow produced by bradykinin. Moreover, when prostacyclin release was abolished by pericardial indomethacin, the coronary vasodilator response to bradykinin was not altered. A large intravenous dose of indomethacin (5 mg/kg) increased the coronary vasoconstrictor response to angiotensin II, but, again, did not alter the vasodilator response to bradykinin.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin produced by the pericardium and its influence on coronary vascular tone.

To determine the influence of pericardial fluid prostacyclin on coronary blood flow, the latter was measured in the circumflex artery of anesthetized dogs. Intraaortic infusions of angiotensin II (25 ng . kg-1 . min-1) reduced blood flow and released prostacyclin into pericardial fluid. Epicardial and pericardial superfusion with indomethacin (1 micrograms/ml) abolished prostacyclin release and significantly increased the coronary vasoconstrictor effect of angiotensin II; this treatment did not appear to affect vascular synthesis of prostacyclin. Pericardial prostacyclin may modulate the coronary vasoconstrictor effect of angiotensin, but its general role as a regulator of coronary vascular resistance is probably limited. A more important effect of pericardial prostacyclin may be exerted on the large coronary vessels in the epicardial surface. Release of prostacyclin into pericardial fluid represents a potential mechanism for opposing coronary vasospasm, especially if platelet activation is found to be a contributory factor in vasotonic angina pectoris.

Prostanoids in platelet-vascular interactions.

Prostacyclin, the labile prostanoid product of arachidonic acid metabolism in vascular endothelium, is the most potent known inhibitor of platelet aggregation and is highly effective in relaxing vascular smooth muscle. Its production is probably critically important in the maintenance of an intact vasculature. Although there is some evidence that prostacyclin circulates as a hormone, it is probably most important as a locally active agent in preventing thrombosis and maintaining patent vessels. Several factors can influence prostacyclin production, the most important of which probably act locally at sites of vessel wall injury. The most promising therapeutic approaches toward using prostacyclin's beneficial effects in vascular disease may lie in the use of drugs aimed at increasing prostacyclin production. Among these are thromboxane synthesis inhibitors, which act by diverting prostaglandin endoperoxides through the prostacyclin synthetase pathway, and lipoxygenase inhibitors, which might act chiefly by preventing formation of metabolites capable of inhibiting prostacyclin synthetase.