Activation Of GTP-binding Protein Research Articles

Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells.

Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.

CAMP Stimulates Human Immunodeficiency Virus (HIV-1)from Latently Infected Cells of Monocyte-Macrophage Lineage: Synergism with TNF-α

The present experiments were designed to study whether GTP binding protein activation and the resulting cAMP plays any role in HIV replication. The results showed that cholera toxin (CT) enhanced HIV replication dose dependently in myelo-monocytic cell lines latently infected with HIV-1, U1, and J22HL-60. Three- to 4-fold enhancement of virus production was observed in U1 cells and 4- to 11-fold enhancement in J22HL-60 cells 4 days after treatment with 100 ng/ml of CT. The increment of intracellular cAMP accumulation was parallel with HIV augmentation by CT in both cells. Even at the low concentration 0.1 ng/ml, TNF enhanced virus production to about an 80-fold higher level than the untreated U1 control cella as described previously (11). However, a synergistic effect (80- to 238-fold enhancement) was observed, when TNF-α and CT were added together to U1 cells. Similar synergism was seen in J22HL-60 cells. HIV antigen positive cells and gp 120 expression were also increased to a similar degree. Phosphodiesterase inhibitor IBMX had no effect on HIV production alone, but potentiated HIV induction by CT and TNF. Adenylate cyclase activator, forskolin (FK), at 100 μM also significantly augmented HIV production (>4-fold) and potentiated TNF induction in J22HL-60 and U1 cells. On the other hand, CT did not show any effect on HIV replication as well as TNF induction in HIV-1-infected T cell line. Northern blot experiment confirmed that this enhancement was mediated through the activation of HIV transcription. These data suggest that cAMP augments HIV replication and potentiates TNF induction in a particular monocyte-macrophage system.

NMR structure of a receptor-bound G-protein peptide.

Heterotrimeric GTP-binding proteins (G proteins) regulate cellular activity by coupling to hormone or sensory receptors. Stimulated receptors catalyse the release of GDP from G protein alpha-subunits and GTP bound to the empty alpha-subunits provides signals that control effectors such as adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Three cytoplasmic loops of the activated receptor are thought to interact with three sites on the heterotrimeric G protein to provide high-affinity interaction and catalyse G-protein activation. The carboxyl terminus of the alpha-subunit is particularly important for interaction with the receptor. Here we study the structure of part of the active interface between the photon receptor rhodopsin and the G protein transducin, or Gt, using nuclear magnetic resonance. An 11-amino-acid peptide from the C terminus of the alpha-subunit of Gt (alpha t (340-350)) binds to rhodopsin and mimics the G protein in stabilizing its active form, metarhodopsin II. The peptide alpha t (340-350) binds to both excited and unexcited rhodopsin and conformational differences between the two bound forms suggest a mechanism for activation of G proteins by agonist-stimulated receptors. Insight into receptor-catalysed GDP release will have broad application because the GTP/GDP exchange and the intrinsic GTPase activity of GTP-binding proteins constitute a widespread regulatory mechanism.

Relationship between alcohol consumption and the activity of GTP-binding regulatory proteins in human erythrocyte membranes

Activity of stimulatory GTP-binding regulatory protein (Gs) in human erythrocyte membranes was assessed by activation of adenylate cyclase in S49 murine lymphoma variant cells to elucidate a relationship to alcohol consumption. In apparently healthy subjects, alcohol consumption < 50 g ethanol per week did not alter the Gs activity, but it was significantly higher ( 14.3%, P < 0.05) in moderate drinkers (50–150 g/week) than non-drinkers. Then, the Gs activity declined with a further increase in alcohol consumption (150–550 g/week). Those subjects with drinking levels of > 50 g/week also showed significant increases in other alcohol-related markers, Na +, K +-ATPase and γ-glutamyltransferase. The Gs activity was significantly low in alcoholics (a 34.9% reduction). No such reduction was noted in patients with other diseases. The results indicate that the Gs activity in erythrocyte membranes is an alcohol-related marker in humans. The variation of Gs activity is distinctive from those of other alcohol-related markers.

Activation of protein kinases by insulin and non-hydrolyzable GTP analogs in permeabilized 3T3-L1 adipocytes.

The molecular events that lead from the interaction of insulin with its receptor to the activation of protein serine/threonine kinases are still unknown. In this study, we have examined the role of GTP-binding proteins in this signaling pathway using differentiated 3T3-L1 adipocytes permeabilized with alpha-toxin from Staphylococcus aureus. Addition of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) or insulin to such permeabilized cells markedly increases protein kinase activities in cell lysates using the microtubule-associated protein-2 kinase substrate peptide KRELVE-PLTPSGEAPNQALLR, which contains the threonine 669 phosphorylation site on the epidermal growth factor receptor. Similar stimulations of protein kinase activity by these agents are observed using the peptide KRRRLASLAA, which is selectively phosphorylated by ribosomal protein S6 kinases. The effects of insulin and GTP gamma S are not additive. Importantly, the GTP-binding protein antagonist GDP beta S (guanosine 5'-O-(2-thiodiphosphate)) inhibits the activation of the protein kinase activities by insulin in permeabilized 3T3-L1 adipocytes. These data are consistent with the hypothesis that activation of Ras or other GTP-binding proteins is a key element of the signaling mechanism whereby insulin receptor tyrosine kinase activates the microtubule-associated protein-2 kinase cascade.

公開シンポジウム「不整脈研究の最前線」3)細胞機能修飾の新しい局面 GTP結合蛋白質の活性化

公開シンポジウム「不整脈研究の最前線」3)細胞機能修飾の新しい局面 GTP結合蛋白質の活性化

Tumour suppressors and the regulation of GTP-binding protein activity

GTP-binding proteins regulate a wide variety of intracellular signalling pathways in eukaryotic cells. The Ras GTP-binding proteins have received a great deal of attention since they were found to be modified by amino acid substitutions in a large number of cancers. It is now clear that Ras plays an essential role in regulating normal cell growth and differentiation, although how this is achieved biochemically is not known. The cellular concentration of Ras bound to GTP appears to be the limiting factor for signalling, and, not suprisingly, it is tightly controlled by both positive and negative regulators. There is now convincing evidence that the loss of one of these negative regulators of Ras, neurofibromin, can contribute to the development of malignancy; thus, neurofibromin behaves as a tumour suppressor gene product.

Aluminofluoride activates hyperpolarization- and stretch-activated cationic channels in single smooth muscle cells.

Aluminofluoride (AF) has a variety of biological actions such as activation of GTP binding proteins and inhibition of phosphatases. In the present study, the effects of AF on hyper-polarization- and stretch-activated cationic channels (HA-SACs) were investigated in isolated gastric smooth muscle cells from the toad, Bufo marinus, using the patch-clamp technique. In cell-attached patches extracellular application of AF (20 mM KF plus 20 microM AlCl3) reversibly increased HA-SAC activity without changing its voltage sensitivity. The single channel current amplitude of HA-SACs was not affected during this procedure. The mechanism of AF-induced activation of HA-SACs remains unclear. However, this activation may play a role in contraction of smooth muscle induced by AF.

The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC8) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC8 to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca2+ concentration or with an increase of intracellular Ca2+ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca2+-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A2 (PLA2) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low μmolar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA2-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA2 activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.

Modulation of beta-adrenergic responses of chloride and calcium currents by external cations in guinea-pig ventricular cells.

1. The catecholamine-induced Cl- current and the Ca2+ current were recorded in the single ventricular cells of guinea-pig hearts, using the whole-cell patch clamp technique combined with internal perfusion. Dependence of the beta-adrenergic responses on external monovalent cations was investigated. The Cl- current was recognized by measuring the reversal potential of the agonist-induced current. 2. The amplitude of the Cl- current, activated by 1 microM adrenaline or 0.01-0.1 microM isoprenaline, was decreased when the external Na+ concentration ([Na+]o) was reduced by replacement with Tris+. The conductance of the catecholamine-induced Cl- current was proportional to the logarithm of the [Na+]o over a range of 15-140 mM. When the conductance was plotted against the concentration of Tris+, a dose-dependent inhibition of the Cl- response by Tris+ was suggested with a half-maximum concentration of 95 mM. 3. The inhibitory effect of the Na+ substitute TEA+ on the Cl- current was not affected by either increasing the buffer for the internal Ca2+ (10 mM BAPTA) or for the pH (50 mM HEPES). 4. In the relationship between agonist concentration and the Cl- conductance, the half-maximum concentration (K1/2) of isoprenaline was 0.013 microM in the control Na+ solution, and was shifted to 0.07, 0.08, 0.1 and 0.3 microM in the Li+, Cs+, TEA+ and Tris+ external solutions, respectively. The maximum slope conductance was not significantly affected, except for a slight depression on the Tris+ solution. When the current was induced by adrenaline, qualitatively the same finding was obtained; K1/2 was 0.15 and 3.2 microM in the Na+ and Tris+ solutions, respectively. 5. As a substitute for the external Na+, sucrose seemed to be inert. The activation of the inward Cl- current was conserved in the 300 mM sucrose solution ([Cl-]o = 8 mM) with a K1/2 value of 0.015 microM isoprenaline. 6. The Cl- current, when activated by either an external application of forskolin (0.2-10 microM) or an internal perfusion of cyclic AMP (100-500 microM), was not affected by replacing external Na+ with other cations. Activation of the Cl- current by 0.2-5 microM histamine was also insensitive to a substitution of Na+. These findings indicate that the inhibition by the Na+ substitute is at a point before the activation of GTP-binding protein. 7. The effects of Na+ substitution were not affected by varying the Na+ concentration (0-115 mM) in the internal solution, excluding an involvement of a change in the [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Pertussis toxin-insensitive G protein mediates carbachol activation of phospholipase D in rat pheochromocytoma PC12 cells.

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells.

GTP-binding proteins have been shown to serve as second messengers in the transduction of hormone signals across animal cell plasma membranes. We present here three lines of evidence to demonstrate that GTP-binding proteins are also involved in the elicitation of the defense response of cultured soybean cells. First, the antigen-binding fragment (Fab) of an antibody that specifically recognizes GTP-binding proteins in plants and animals was delivered into soybean cells using a non-destructive biotin-mediated delivery technique developed previously. Internalization of this Fab enhanced up to 10-fold the rapid oxidative burst induced by elicitor molecules, whereas internalization of its heat-denatured counterpart or unrelated proteins had no effect. Because the antibody recognizes a protein of molecular mass approximately 45 kDa in soybean cell membranes that is protected from ADP-ribosylation by GTP gamma S (guanosine 5'-O-(thiotriphosphate), we propose the 45-kDa GTP-binding protein is responsible for these effects. Second, mastoparan, a specific activator of GTP-binding proteins, was shown to induce the defense-related oxidative burst in the absence of elicitor stimulation, thus mimicking an activated receptor as it is thought to do in mammalian systems. Finally, but admittedly less convincing, the A subunit of cholera toxin, an activator of certain stimulatory GTP-binding proteins (Gs), was found to weakly enhance the conventional elicitor-induced oxidative burst. Taken together, these data argue for the involvement of GTP-binding proteins in elicitor signal transduction in soybean cells.

Inhibitory effects of substance P analogs on the activation of GTP-binding regulatory proteins by receptors

Inhibitory effects of substance P analogs on the activation of GTP-binding regulatory proteins by receptors

Mechanism of prostaglandin E 2-induced arachidonic acid release in osteoblast-like cells: Independence from phosphoinositide hydrolysis

We previously reported that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in the coupling of prostaglandin E 2 (PGE 2) receptor to phospholipase C in osteoblast-like MC3T3-E1 cells (1). In the present study, we analyzed the mechanism of PGE 2-induced arachidonic acid (AA) release in MC3T3-E1 cells. PGE 2 stimulated the release of AA and the formation of inositol trisphosphate (IP 3) dose dependently in the range between 1 nM and 10 μM. The effect of PGE 2 on AA release (ED 50 was 80 nM) was more potent than that on IP 3 formation (ED 50 was 0.8 μM). Quinacrine, a phospholipase A 2 inhibitor, suppressed the PGE 2-induced AA release but had little effect on the IP 3 formation. NaF, a GTP-binding protein activator, mimicked PGE 2 by stimulating the AA release. The AA release stimulated by a combination of PGE 2 and NaF was not additive. PTX had little effect on the PGE 2-induced AA release. These results strongly suggest that the AA release and the phosphoinositide hydrolysis are separately stimulated by PGE 2 in osteoblast-like cells, and the PGE 2-induced AA release is mediated by PTX-insensitive GTP-binding protein.

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca 2+-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca 2+ concentration. ACh also suppressed the axoplasmic transport in Ca 2+-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca 2+ ions.

Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells.

Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF2 precipitation. In 0.5 mM external Ca2+, NaF inhibited PTH release and lowered the cAMP content by 50-70% of the effects attained with 3.0 mM Ca2+. The NaF-induced increase of [Ca2+]i was considerably smaller than that obtained with rise of external Ca2+. It seems likely that NaF activates the inhibitory G1-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca2+]i induced by NaF is due to a direct effect of a G-protein on Ca2+ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca2+ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca2+]i and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca(2+)-inhibited PTH release and Ca2+ triggered increase of [Ca2+]i. Consistent with observations of down regulation of the parathyroid Ca2+ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.

Membrane-bound conformation of mastoparan-X, a G-protein-activating peptide

Mastoparan-X, a tetradecapeptide from wasp venom, has been proposed to cause secretion from various kinds of cells by the direct activation of GTP-binding regulatory proteins (G proteins) that couple to phospholipase C. The mechanism of the activation has been shown to be very similar to that of G-protein-coupled receptors in vitro, and the interaction with membranes seems to be very important for the activation of G proteins that are membrane-bound [Higashijima, T., Uzu, S., Nakajima, T., & Ross, E. M. (1988) J. Biol. Chem. 263, 6491-6494]. We report here the precise vesicle-bound conformation of mastoparan-X in the presence of perdeuterated phospholipid vesicles, determined by two-dimensional 1H-NMR analyses of transferred nuclear Overhauser effects, combined with distance geometry and molecular dynamics calculations. Of 14 amino acid residues, the C-terminal 12 residues take an alpha-helical conformation upon binding to the phospholipid bilayer. The overall structure of the alpha-helix is amphiphilic, with three lysine side chains located on one side and with hydrophobic side chains on the other side. This conformation of mastoparan-X was maintained both in the gel and in the liquid-crystalline phases of the membranes. The conformation described herein will provide a useful basis for understanding conformation-activity relationships of mastoparan analogs as activators of G proteins. These studies will help to design novel potent analogs for the regulation of G proteins and to analyze receptor-G-protein interactions.

Selective regulation of Gi alpha 1 expression and function in PC12 cells by cAMP

Hormone and neutrotransmitter receptor systems regulate both the activity and expression of GTP-binding proteins (G-proteins). However, relatively little is known about the mechanism by which this regulation occurs. One G-protein subtype, Gi alpha 1, is expressed primarily in neuronal cells. Here, we demonstrate the selective regulation of Gi alpha 1 mRNA and protein levels by cAMP. Treatment of PC12 cells with forskolin increases Gi alpha protein levels. Similarly, incubation of PC12 cells with agents that increase intracellular levels of cAMP, including forskolin, dibutyryl-cAMP, and 8-bromo-cAMP, induce a two- to fourfold increase in Gi alpha 1 mRNA levels. Furthermore, the effect of increased intracellular cAMP is specific for Gi alpha 1 mRNA expression; the levels of mRNA encoding other G-protein subtypes remain unaltered. cAMP-stimulated Gi alpha 1 expression occurs within hours of treatment and is sustained for days. Increasing intracellular cAMP by activation of cell surface adenosine receptors also increases Gi alpha 1 mRNA levels. Treatment of PC12 cells with phorbol esters, NGF, or depolarizing concentrations of KCl did not increase Gi alpha 1 mRNA expression, demonstrating that Gi alpha 1 expression is specifically regulated by cAMP. Guanine nucleotide-mediated inhibition of adenylate cyclase activity was measured in order to determine if the change in Gi alpha protein expression was accompanied by a change in G-protein function. Adenylate cyclase activity in PC12 cells treated with an adenosine analog and therefore expressing higher levels of Gi alpha protein is more sensitive to inhibition by guanine nucleotides than in nontreated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative effect of PGD 2 on osteoblast-like cells; Independent activation of pertussis toxin-sensitive GTP-binding protein from PGE 2 or PGF 2α

PGD 2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 μM in osteoblast-like MC3T3-E1 cells. PGD 2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 μM. The formation of inositol trisphosphate (IP 3) induced by PGD 2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE 2 or PGF 2α and both PGE 2 and PGF 2α affected PGD 2-induced IP 3 formation additively. Pertussis toxin (PTX) inhibited both PGD 2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 μg/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD 2-induced DNA synthesis as well as the formation of IP 3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD 2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD 2 was apparently different from that by PGE 2 or PGF 2α. These results indicate that PGD 2 activates PTX-sensitive GTP-binding protein independently from PGE 2 or PGF 2α and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.