Guanosine Triphosphate-binding Protein Research Articles

Recombinant Interleukin-8 Induces Changes in Cytosolic Ca2+ in Human Neutrophils

Activation of polymorphonuclear leukocytes (PMNL) by most soluble stimulants is associated with a marked increase in cytosolic free Ca2+ ([Ca2+]i). Interleukin-8 (IL-8), a monocyte-derived neutrophil chemotactic factor and potent neutrophil-activating cytokine, effectively enhanced the resting free [Ca2+]i within human PMNL in a dose-dependent manner (maximal effect with 100 ng/mL). The increase in [Ca2+]i was substantially (55%) inhibited in the absence of extracellular Ca2+. Thus, the increase was due to extra- and intracellular cooperative mobilization of Ca2+, as supported by the reduced effect of IL-8 on [Ca2+]i after quenching with Mn2+. Granulocyte-macrophage colony-stimulating factor and interferon-gamma failed to induce a change in [Ca2+]i, suggesting that they may operate through different signal pathways. Pretreatment with Bordetella pertussis toxin largely inhibited the IL-8-induced change in [Ca2+]i. Thus, IL-8-induced cooperative mobilization of intra- and extracellular Ca2+ leads to a net Ca2+ influx into the cytoplasm through a process mediated by a guanosine triphosphate-binding protein.

Affinity cross-linked δ-opioid receptor in NG108-15 cells is low molecular weight (25 kDa) and coupled to GTP-binding proteins

The affinity cross-linking of the δ-opioid receptor in neuroblastoma X glioma NG108-15 cells was undertaken using (3-[ 125Iiodotyrosyl 27)human-β-endorphin ([ 125I]β-endorphin) and disuccinimidyl suberate (DSS)or bis(sulfosuccinimidyl) suberatc (BS3) in order to estimate molecular size. Following sodium dodccyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective δ-opioid agonist, [D-Pen 2,D-Pen 5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-lhio)triphosphate (GTPγS) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound β-endorphin (3.6 kDa) into consideration, suggest that the δ-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).

A GDP Dissociation Inhibitor That Serves as a GTPase Inhibitor for the Ras-Like Protein CDC42Hs

Members of the family of Ras-related guanosine triphosphate (GTP) binding proteins appear to take part in the regulation of a number of biological processes, including cell growth and differentiation. Three different classes of proteins that regulate the GTP binding and GTP hydrolytic activities of the Ras family members have been identified. These different regulatory proteins inhibit guanosine diphosphate (GDP) dissociation (designated as GDIs), stimulate GDP dissociation and GDP-GTP exchange (designated as GDSs), or stimulate GTP hydrolysis (designated as GAPs). In the case of the Ras-like protein CDC42Hs, which is the human homolog of a Saccharomyces cerevisiae cell division cycle protein, the GDI protein also inhibited both the intrinsic and GAP-stimulated hydrolysis of GTP. These findings establish an additional role for the GDI protein--namely, as a guanosine triphosphatase (GTPase) inhibitory protein for a Ras-like GTP binding protein.

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11.

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.

Facilitatory effects of diltiazem on dopamine release in the central nervous system. Focus on interactions with D2 autoreceptors and guanosine triphosphate binding proteins.

The purpose of the present study was to investigate the effects of a Ca antagonist (diltiazem) on dopamine release in the central nervous system. Rat striatal slices prelabeled with [3H]dopamine (DA) and superfused with Krebs-Ringer solution were stimulated electrically at a frequency of 1 Hz. Exposure to diltiazem (3.3 x 10(-7) to 1 x 10(-5) mol/L) significantly increased both the basal and stimulation-evoked [3H]DA release in a dose-dependent manner. Exogenously applied unlabeled DA inhibited the stimulation-evoked [3H]DA release. Diltiazem significantly antagonized the capacity of the unlabeled DA to inhibit stimulation-evoked [3H]DA release. The blockade of D2 receptors by a preferential D2 antagonist, sulpiride, reduced the facilitatory effect of diltiazem on stimulation-evoked [3H]DA release. Pretreatment with pertussis toxin, which interferes with the coupling of the inhibitory guanosine triphosphate-binding proteins to adenylate cyclase, significantly diminished the effects of diltiazem on stimulation-evoked [3H]DA release. These results show that diltiazem increased DA release in rat striatum, at least partially by interactions with the D2 autoreceptors and pertussis toxin-sensitive guanosine triphosphate-binding proteins.

Modulation of norepinephrine release by galanin in rat medulla oblongata.

Galanin, a 29-amino acid peptide, is widely distributed in both the central and peripheral nervous systems and is colocalized with catecholamines, although its physiological significance remains to be elucidated. In the present study we investigated the regulatory mechanisms of galanin on norepinephrine release in rat medulla oblongata. In slices of medulla oblongata of Sprague-Dawley rats, galanin inhibited the stimulation-evoked [3H]norepinephrine release in a concentration-dependent manner (fractional release ratio during electrical stimulation: control 0.937 +/- 0.043, mean +/- SEM, n = 6; galanin 1 x 10(-7) M 0.501 +/- 0.037, n = 6, p less than 0.05; and galanin 1 x 10(-6) M 0.299 +/- 0.018 n = 6, p less than 0.05). Galanin potentiated inhibition of [3H]norepinephrine release by the alpha 2-agonists (UK 14,304 and clonidine). The blockade of alpha 2-adrenergic receptors by RX 781094 diminished the inhibition of norepinephrine release by galanin. Pretreatment of pertussis toxin, which interferes with the coupling of inhibitory guanosine triphosphate-binding proteins to adenylate cyclase, significantly attenuated the suppressive effects of galanin on norepinephrine release. In slices of medulla oblongata obtained from spontaneously hypertensive rats (SHR), the inhibitory effect of galanin on norepinephrine release was significantly less than in those from age-matched Wistar-Kyoto rats. These results show that galanin might inhibit the stimulation-evoked norepinephrine release in rat medulla oblongata, at least partially mediated by alpha 2-adrenergic receptors and the pertussis toxin-sensitive guanosine triphosphate-binding proteins. Moreover, less suppression of norepinephrine release by galanin in SHR suggests that galanin might be involved in the regulation of central sympathetic nervous activity in hypertension.

Phospholipids regulate growth and function of MDCK cells in hormonally defined serum free medium.

The effects of the simple phospholipids phosphatidic acid (PA) and lysophosphatidic acid (LPA) on the growth and function of Madin Darby Canine Kidney (MDCK) cells has been studied. We observed that PA and LPA not only stimulated the growth of MDCK cells (at 20 microM), but also stimulated the growth of normal rabbit kidney cells in serum free medium (albeit at a lower dosage of 5 microM). Evidence was obtained that PA interacts synergistically with insulin so as to elicit a growth stimulatory effect. Recently, extracellular PA and LPA were proposed to stimulate mitogenesis in several types of animal cells by binding to particular sites on the plasma membrane which are coupled to signaling mechanisms such as adenylate cyclase via a pertussis toxin sensitive, inhibitory guanosine triphosphate binding protein (Gi protein) (15). However, even when the pertussis toxin dosage was increased to 50 ng/ml, LPA still had a dramatic growth stimulatory effect on MDCK cells. In the absence of LPA pertussis toxin was slightly growth stimulatory to MDCK cells. Phospholipids such as PA and LPA have been observed to prevent prostaglandin-induced increases in adenylate cyclase activity in other cell types via their effects on such a pertussis toxin sensitive Gi protein. If PA and LPA act on MDCK cells in this manner, then these phospholipids may possibly prevent the effect of PGE1 on the growth of normal MDCK cells. However PGE1 was still growth stimulatory to normal MDCK cells. The effects of PA on PGE1 independent variants of MDCK cells, which have elevated intracellular cyclic AMP levels (22), were also examined. In the presence of PA, PGE1 remained growth inhibitory, rather than growth stimulatory to the PGE1 independent cells. However, the PA dosage required to elicit an optimal growth response (5 microM) was dramatically reduced, as compared with normal MDCK cells (20 microM). This altered dosage requirement could be explained by the elevated intracellular cyclic AMP levels in the PGE1 independent variants. Like PGE1 and 8-bromocyclic AMP, PA and LPA also significantly increased the initial rate of Rb+ uptake by confluent monolayers of MDCK cells. The increase in the initial rate of Rb+ uptake could be explained by an increase in the ouabain-sensitive component of Rb+ uptake. An increase in the initial rate of ouabain-insensitive Rb+ uptake was also observed in LPA treated MDCK cell cultures.

The low molecular weight guanosine triphosphate-binding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes

The Rab genes have recently been cloned and sequenced in mammals, and their products represent good candidates for low molecular weight guanosine triphosphate-binding proteins involved in the regulation of intracellular transport of vesicles in higher eukaryotes. Remarkably, each of the Rab proteins appears to be associated with a distinct step of either the exocytic or endocytic pathway. In particular, Rab6p has been localized to the outermost Golgi cisternae in normal rat kidney cells, where its function remains unclear. In this work, we have carried out a series of immunocytochemical analyses of the subcellular distribution of Rab6p in a polarized cell, the electrocyte of Torpedo marmorata, to elucidate the molecular mechanisms involved in the sorting and targeting of synaptic proteins. We report that, aside from its Golgi localization, the bulk of Rab6p associates with clusters of post-Golgi vesicles, primarily those located at the cytoplasmic face of the innervated membrane of the electrocyte. Consequently, Rab6p presents a polarized distribution in this cell. Furthermore, we show that this distribution is dependent upon the integrity of the microtubule network of the electrocyte. These data are coherent with the notion that Rab6p is involved in the regulation of membrane traffic from the trans-Golgi network to the innervated plasma membrane, delivering, by way of a microtubule-based organelle transport mechanism, synaptic proteins to their appropriate locations.

Calcitonin gene-related peptide in noradrenergic transmission in rat hypothalamus.

In the present study, we examined the regulatory mechanisms of calcitonin gene-related peptide on norepinephrine release in rat hypothalamus. Calcitonin gene-related peptide inhibited the stimulation-evoked norepinephrine release from hypothalamic slices of Sprague-Dawley rats in a dose-dependent manner, although the peptide did not affect basal release of norepinephrine. The blockade of the alpha 2-adrenergic receptors by RX 781094 failed to modulate the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. Pretreatment of slices with islet activating protein, a toxin that interferes with the coupling of the inhibitory receptors to adenylate cyclase, did not affect the suppression of norepinephrine release by calcitonin gene-related peptide. However, Bay K 8644, a dihydropyridine-sensitive calcium channel agonist, significantly reversed the inhibitory effects of calcitonin gene-related peptide on norepinephrine release. These results show that calcitonin gene-related peptide might inhibit norepinephrine release in rat hypothalamus, partially mediated by interactions with dihydropyridine-sensitive Ca2+ channels but not by interactions with presynaptic alpha 2-adrenergic receptors and inhibitory guanosine triphosphate binding proteins. Furthermore, the finding suggests the possible involvement of calcitonin gene-related peptide in the regulation of sympathetic nervous activity in the central nervous system.

G proteins: implications for psychiatry.

There is mounting evidence that a family of guanosine triphosphate binding proteins (G proteins) play an obligatory role in the transduction of a vast array of extracellular, receptor-detected signals across cell membranes to intracellular effectors. The author reviews the literature dealing with G protein coupling to second messenger generation, the role of G proteins in regulating both the convergence and divergence of neurotransmitter action in the CNS, and the involvement of G proteins in a variety of clinical conditions, with an emphasis on psychiatric conditions and their treatments. G proteins from the basis of signal integration in the CNS, endowing the neuron with a large degree of functional diversity. Abnormalities in the function and/or expression of G proteins have been implicated in a variety of pathophysiologic states, and a number of currently available psychotropic drugs affect G proteins. The understanding of the mechanisms by which G proteins modulate neuronal activity may be one of the keys to understanding the functioning and the complexities of the nervous system. Given their widespread, critical roles in the regulation of neuronal function, it seems likely that G proteins are involved in the pathophysiology of various major psychiatric illnesses. The development of novel, site-specific drugs with primary G protein targets remains an exciting prospect for the future.

Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells.

Activation of B lymphocytes by engagement of their immunoglobulin M antigen receptors results in phosphorylation of a number of proteins on tyrosine residues. One such protein is p95vav, the product of the vav proto-oncogene. Tyrosine phosphorylation of p95vav occurred within seconds of immunoglobulin M cross-linking and was independent of other events induced during stimulation of B cells, such as protein kinase C activation, guanosine triphosphate-binding protein signaling, and calcium mobilization. Moreover, engagement of antigen receptors induced the rapid (approximately 5 seconds) and transient (approximately 60 seconds) association of p95vav with a 70-kilodalton tyrosine-phosphorylated protein, Vap-1, an interaction mediated by the Src homology 2 domain of p95vav. These results suggest that the vav proto-oncogene participates in the signaling processes that mediate the antigen-induced activation of B lymphocytes.

Subcellular Distribution of the Rap1A Protein in Human Neutrophils: Colocalization and Cotranslocation With Cytochrome b559

AbstractRaplA, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that RaplA is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocal-ized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the RaplA was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule-associated Rap1A was translocated to the plasma membrane in PMA-stimu-lated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total RaplA labeling was associated with the phagolysoso-mal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of RaplA with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide-generating system. 1992 by The American Society of Hematology

Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti- cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule- associated Rap1A was translocated to the plasma membrane in PMA- stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide- generating system.

Effects of calcium-antagonists on dopamine release in the central nervous system--possible interactions with D2-autoreceptors and guanosine triphosphate-binding proteins.

The effects of calcium antagonists (verapamil and nicardipine) on central dopaminergic activity were investigated in vitro. Rat striatal slices prelabelled with (3H)dopamine and superfused with Krebs-solution were stimulated electrically at a frequency of 1 Hz. Exposure to verapamil (3.3 x 10(-7) - 1 x 10(-5) M) significantly increased both basal and stimulation-evoked (3H)dopamine release in a concentration-dependent manner. Nicardipine produced no changes in stimulation-evoked (3H)dopamine release, although a high concentration of nicardipine slightly increased basal release of (3H)dopamine. Exogenously applied unlabelled dopamine (1 x 10(-7) M) inhibited the stimulation-evoked (3H)dopamine release. Verapamil (1 x 10(-6) M) significantly antagonized the capacity of the unlabelled dopamine to inhibit stimulation-induced (3H)dopamine release. The blockade of D2-receptors by a preferential D2-antagonist, sulpiride, reduced the facilitatory effect of verapamil on stimulation-induced (3H)dopamine release. Pretreatment with pertussis toxin, which interferes with the coupling of the inhibitory guanosine triphosphate-binding proteins to adenylate cyclase, significantly diminished the effects of verapamil on stimulation-induced (3H)dopamine release. The results of the present study show that verapamil (but not nicardipine) increased dopamine release in rat striatum, at least partially via interactions with the D2-dopamine autoreceptors and the pertussis toxin-sensitive guanosine triphosphate-binding proteins. Furthermore, a close interaction between verapamil and the dopamine receptors might partially explain the central effects of verapamil.

Regulation of carbachol- and histamine-induced inositol phospholipid hydrolysis in a human oligodendroglioma.

A stable cell line derived from a human oligodendroglioma (HOG) was used to study the regulation of muscarinic- and histamine receptor-mediated phosphoinositide hydrolysis. Both carbachol and histamine increased inositol monophosphate (InsP) accumulation in a dose- and time-dependent manner in the presence of lithium and the effect of simultaneous addition of carbachol and histamine was additive, implying independent signal transduction pathways. Homologous desensitization of muscarinic, but not histamine receptors, could be demonstrated although neither receptor type appeared to be heterologously desensitized. [3H]InsP accumulation in HOG cells was also stimulated by fluoride, suggesting guanosine triphosphate (GTP)-binding protein involvement, but phosphoinositide (PtdIns) hydrolysis was not sensitive to pertussis toxin. Phorbol ester-activation of protein kinase C (PKC) inhibited both muscarinic and histamine receptor-stimulated InsP release but did not attenuate either the fluoride-induced release of InsP nor beta-adrenergic receptor-mediated stimulation of adenylate cyclase activity. Taken together, we conclude that muscarinic and histamine receptors are differentially regulated through both PKC-dependent and -independent mechanisms, and that feedback inhibition of PtdIns turnover occurs proximal to the GTP binding proteins.

Subcellular Distribution of Low Molecular Weight Guanosine Triphosphate-Binding Proteins in Adipocytes: Colocalization with the Glucose Transporter Glut 4*

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Since low mol wt GTP-binding proteins (LMW-GTP-binding proteins) have been implicated in the regulation of vesicular trafficking, we have analyzed these proteins in adipocytes. Isolated adipocytes were incubated in the absence or presence of insulin before separation of plasma membranes (PM) and low density microsomes (LDM). [alpha-32P]GTP binding to proteins transferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific and distinct subsets of proteins in the PM and LDM; those proteins were more abundant in PM than in LDM. [alpha-32P]GTP binding to these proteins was specific for the guanylnucleotides, since it was competed for by GTP and guanosine 5'-O-(3-thiotriphosphate), but not by ATP or adenosine 5'-O-(3-thiotriphosphate). The LMW-GTP-binding proteins were tightly associated with the membranes, as treatment with 1.5 M KCl did not modify this association. The distribution of the LMW-GTP-binding proteins in the fractions and their affinity for guanylnucleotides were the same in control and insulin-treated adipocytes. When the presence of Gi alpha subunits was looked for with a specific antibody, Gi alpha 1 and Gi alpha 2 were found almost exclusively in PM. By contrast, the same antibody revealed the presence of a 100 kDa band in the LDM. Insulin treatment of adipocytes did not modify the amounts of those G-proteins in PM or LDM fractions, although it promoted the translocation of Glut 4 proteins from LDM to PM. LDM fractions contain a specific subset of vesicles markedly enriched in Glut 4 molecules. When those vesicles were isolated from the total LDM fraction by immunoadsorption on highly specific antibodies to Glut 4 protein, LMW-GTP-binding proteins were found in the immune pellet. Those proteins were absent when immunoprecipitation was performed after solubilization of the vesicles with 1% Triton X-100. Our results strongly suggest that the vesicles containing the Glut 4 protein also contained LMW-GTP-binding proteins and indicate that these GTP-binding proteins could play a role in the exocytosis of the Glut 4-containing vesicles.

Activation of a small GTP-binding protein by nucleoside diphosphate kinase.

Genes that encode nucleoside diphosphate kinases (NDKs) have been implicated as regulators of mammalian tumor metastasis and development in Drosophila melanogaster. However, the cellular pathways through which NDKs function are not known. One potential mechanism of regulation is phosphorylation of guanosine diphosphate (GDP) bound to regulatory guanosine triphosphate (GTP) binding proteins. NDK-catalyzed phosphorylation of bound GDP was investigated for the adenosine diphosphate ribosylation factor (ARF), a 21-kilodalton GTP-binding protein that functions in the protein secretion pathway. Bovine liver NDK, recombinant human NDK, and the protein product of the mouse gene nm23-1, which suppresses the metastatic potential of certain tumor cells, used ARF-GDP as a substrate, thereby allowing rapid and efficient production of activated ARF (ARF-GTP) in the absence of nucleotide exchange. These data are consistent with the proposed function of NDK as an activator of a small GTP-binding protein and provide a mechanism of activation for a regulatory GTP-binding protein that is independent of nucleotide exchange.

Mechanism of inhibitory action of prostaglandins on the growth of human gastric carcinoma cell line KATO III

Prostaglandins (PGs) play important roles in the regulation of various gastric functions. In this study, the effects of various PGs on the growth of the human gastric carcinoma cell line KATO III were investigated. All the PGs tested inhibited KATO III cell growth with a relative potency order of PGE2 > PGE1 > 17S,20-dimethyl-6-oxo PGE1-methyl ester (ornoprostil) > PGF2α. This inhibition was accompanied by an increase of cyclic adenosine monophosphate production. Furthermore, in the presence of guanosine triphosphate, these PGs stimulated adenylate cyclase activity in the plasma membrane of KATO III cells, followed by enhancement of membrane guanosine triphosphatase activity. The relative potencies of these PGs for increasing cyclic adenosine monophosphate levels, activating adenylate cyclase, and enhancing guanosine triphosphatase activity were all comparable to those for inhibiting cell growth. On the other hand, the proliferation of KATO III cells was also inhibited by forskolin as well as dibutyryl cyclic adenosine monophosphate, whereas none of the agents that did not increase cyclic adenosine monophosphate levels had any effect. These results suggest that PGs inhibit KATO III cell growth by stimulating cyclic adenosine monophosphate production via a guanosine triphosphate-dependent process, suggesting the involvement of guanosine triphosphate-binding stimulatory protein, probably coupled to PGE2 receptors, in the action of PGs.

Decreased responsiveness to beta-adrenoceptor agonists in arterial strips from spontaneously hypertensive rats is not associated with alterations in beta-adrenoceptors.

Beta-adrenoceptors in femoral and mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied using radioligand binding assays and were compared with in vitro measurements of beta-adrenoceptor-mediated relaxation. The relaxant responses to noradrenaline via beta-adrenoceptors were significantly decreased in the SHR femoral artery when compared with the WKY femoral artery. However, under the same conditions, arterial relaxant responses to forskolin, an activator of adenylate cyclase, were not significantly different between SHR and WKY rats. Specific binding of 125I-iodocyanopindolol to membranes prepared from femoral arteries of SHR and WKY rats was saturable and of high affinity. Neither the equilibrium dissociation constant of 125I-iodocyanopindolol, nor the maximum number of binding sites were significantly different between SHR and WKY rats. Similar results were obtained in the case of mesenteric arteries from SHR and WKY rats. These results indicate that the decreased responsiveness to beta-adrenoceptor stimulation in SHR arteries is not associated with alterations in beta-adrenoceptors and further support the hypothesis that a reduced function of the stimulatory guanosine triphosphate-binding protein is responsible for the decreased responsiveness to a variety of receptor agonists whose mechanism of action involves adenylate cyclase activation.