Hydrolysis-Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin-Permeabilized PC12 Cells

Abstract: The effect of the hydrolysis-resistant GTP analogs, guanosine 5'- O -(3-thiotriphosphate) (GTPγS) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca²⁺ (10 μ M ) was not affected by GTP7S or GMPPNP, secretion in the absence of Ca²⁺ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTPγS or GMPPNP was approximately 80% of that induced by 10 μ M Ca²⁺. Half-maximum stimulation was induced by 30 μ M GTPγS or GMPPNP. Both Ca²⁺-stimulated and GTPγS-stimulated secretion were ATP dependent and inhibited by N -ethylmaleimide. The GTPγS-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca²⁺ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12- O -tetradecanoyl-phorbol 13-acetate caused a 50% increase in both Ca²⁺-stimulated and GTP7S-stimulated secretion. Cholera and pertussis toxins did not affect Ca²⁺-stimulated or GTPγS-stim-ulated NE secretion. Guanosine 5'- O -(2-thiodiphosphate) (GDPβS) and GTP inhibited GTPγS-stimulated secretion but not Ca²⁺-stimulated secretion. The inability of GDPβS to inhibit Ca²⁺-stimulated secretion indicates that the process affected by GTPγS is not an essential step in the Ca²⁺-stimulated pathway.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

Characterization of the Functional Activity of Dopamine Ligands at Human Recombinant Dopamine D4 Receptors

Abstract: The human D₄ dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [³⁵S]GTPγS binding assay. By measuring D₄ receptor stimulation of [³⁵S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D₄ receptor, dopamine, quinpirole, and dp -2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [³⁵S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [³⁵S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D₄ receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [³H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.     

Stimulation of Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [³⁵S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [³⁵S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [³⁵S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [³⁵S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G_i/G_o, but not to G_s, and support the possibility that activation of G_i/G_o mediates the stimulatory effect on adenylyl cyclase activity.     

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1 . Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C₁₈ fatty acid. The KlLAC1 protein preferred long-chain (C₁₈) fatty acids to very-long-chain (C₂₆) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Phospholipase D activity is required for actin stress fiber formation in fibroblasts

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha-actinin.     

GTPγS antagonizes the mastoparan-induced in vitro activity of PIP2-phospholipase C from symbiotic root nodules of Phaseolus vulgaris

Phospholipase C (PLC) has been suggested to have a role in signal perception by Nod factors (NFs) in legume root hair cells. For instance, mastoparan, a well-described agonist of heterotrimeric G protein, induces nodulin expression after NFs treatment or Rhizobium inoculation. Furthermore, it has been recently demonstrated that mastoparan also mimics calcium oscillations induced by NFs, suggesting that PLC could play a key role during the nodulation process. In this study, we elucidate a biochemical relationship between PLC and heterotrimeric G proteins during NFs signaling in legumes. In particular, the effect of NFs on in vitro PLC activity from nodule membrane fractions in the presence of guanosine 5'-[γ-thio]triphosphate (GTPγS) and mastoparan was assayed. Our results indicate that for phosphatidylinositol 4,5 bisphosphate (PIP₂)-PLC, there is a specific activity of 20–27 nmol mg⁻¹ min⁻¹ in membrane fractions of nodules 18–20 days after inoculation with Rhizobium tropici . Interestingly, in the presence of 5 μ M mastoparan, PIP₂-PLC activity was almost double the basal level. In contrast, PIP₂-PLC activity was downregulated by 1–10 μ M GTPγS. Also, PLC activity was decreased by up to 64% in the presence of increasing concentrations of NFs (10⁻⁸ to 10⁻⁵  M ). NFs are critical signaling molecules in rhizobia/legume symbiosis that can activate many of the plant's early responses during nodule development. Calcium spiking, kinases, PLC activity and possibly G proteins appear to be components downstream of the NFs perception pathway. Our results suggest the occurrence of a dual signaling pathway that could involve both G proteins and PLC in Phaseolus vulgaris during the development of root nodules.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

Ceramide Induces Apoptosis in Cultured Mesencephalic Neurons

Abstract: The death of dopaminergic and other neurons in primary cultures of the mesencephalon could be induced by treatment with ceramide, as in lymphocytes where it mediates activation by the cytokines tumor necrosis factor-α and interleukin-1β of a novel sphingomyelin-dependent signaling pathway leading to apoptosis. The morphological hallmarks of this form of cell death—bleb formation, cell body shrinkage, nuclear chromatin condensation, and fragmentation—were observed in degenerating neurons. Internucleosomal DNA degradation could also be evidenced by gel electrophoresis. The C₂ and C₆ analogues as well as native ceramide, administered in a dodecane suspension, had a similar effect, whereas the closely related C₂-dihydroceramide, which lacks the 4–5 trans double bond in the sphingosine chain, failed to induce apoptosis. Neuronal death could be delayed by serum factors, dibutyryl cyclic AMP, and the protein synthesis inhibitor cycloheximide.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.     

Uptake and utilization of n-octacosane and n-nonacosane by Arthrobacter nicotianae KCC B35

Arthrobacter nicotianae KCC B35 isolated from blue-green mats densely covering oil sediments along the Arabian Gulf coast grew well on C₁₀ to C₄₀ n -alkanes as sole sources of carbon and energy. Growth on C₂₀ to C₄₀ alkanes was even better than on C₁₀ to C₁₈ alkanes. Biomass samples incubated for 6 h with n -octacosane (C₂₈) or n -nonacosane (C₂₉) accumulated these compounds as the predominant constituent alkanes of the cell hydrocarbon fractions. The even chain hexadecane C₁₆ and the odd chain pentadecane C₁₅ were the second dominant constituent alkanes in C₂₈ and C₂₉ incubated cells, respectively. n -Hexadecane-incubated cells accumulated in their lipids higher proportions of C₁₆-fatty acids than control cells not incubated with hydrocarbons. On the other hand, C₂₈ and C₂₉-incubated cells did not contain any fatty acids with the equivalent chain lengths, but the fatty acid patterns of the cell lipids suggest that there should have been mid-chain oxidation of these very long chain alkanes. This activity qualifies A. nicotianae KCC B35 to be used in cocktails for bioremediating environments polluted with heavy oil sediments.     

The presence of two types of β-cyanoalanine synthase in germinating seeds and their responses to ethylene

Germinating seeds of many species contain two types of β-cyanoalanine synthase (CAS, EC 4.4.1.9) that convert HCN to β-cyanoalanine. One is cytoplasmic CAS (cyt-CAS), which is precipitated by 50 to 60% (NH₄)₂SO₄ and has a pH optimum of 10.5. Cytoplasmic CAS is present at high levels in dry seed and its activity does not increase during imbibition. The activity of cyt-CAS is not affected by exogenously applied ethylene (C₂H₄), except in rice ( Oryza sativa cv. Sasanishiki). The second type of CAS found in seed is mitochondrial CAS (mit-CAS), which is precipitated by 60 to 70% (NH₄)₂SO₄ and has a pH optimum of 9.5. Mitochondrial CAS is present at low levels in dry seed, and its activity increases greatly during imbibition in the seeds of all species tested. Exposure to C₂H₄ stimulated mit-CAS activity in seeds of rice, barley ( Hordeum vulgare cv. Hadakamugi). cucumber ( Cucumis sativus cv. Kagafushinari) and cocklebur ( Xanthium pennsylvanicum ). The increase in the mit-CAS activity in cocklebur in response to C₂H₄ commenced alter a lag period of 2 to 3 h when the duration of soaking was short (16 h), but commenced without a lag period when the seeds were soaked for three months. Application of both chloramphenicol and cycloheximide to the axial and cotyledonary tissues of cocklebur seeds strongly inhibited growth as well as the increase in mit-CAS activity. It is postulated that the mit-CAS is synthesized de novo during imbibition and that its activity is regulated by C₂H₄, CO₂ which also promotes seed germination in some species, was ineffective m stimulating mit-CAS activity in cocklebur seeds.     

Effects of age, supra-ambient oxygen and repeated assays on acetylene reduction and root respiration in pea

An open flow-through gas system was used to investigate the effect of plant age on nitrogenase activity in relation to root respiration (measured as CO₂ release) and supra-ambient O₂ levels in 24- to 51-day-old, nodulated Pisum sativum L. cv. Bodil. The effect of assaying plants repeatedly was also studied. The respiratory efficiency of nitrogenase [mol CO₂ (mol C₂H₄)⁻¹] and the relative decline in nitrogenase (EC 1.7.99.2) activity in response to introduction of C₂H₂ in the gas stream were unaffected by plant age. In contrast, the nitrogenase-linked respiration as a proportion of total root respiration increased with time. Accordingly, the specific respiration linked-to growth and maintenace of the noduled root system decreased with time. C₂H₂ reduction and root respiration were increased by supra-ambient O₂ levels, but the tolerance to high O₂ concentrations seemed to decrease with plant age. Repeated C₂H₂ assays on the same plants decreased their rate of growth and N accumulation: in addition, nitrogenase activity and root respiration were somewhat negatively affected. The results indicate that results from experiments with plants of different ages cannot always be directly compared, and that repeated C₂H₂ assays on the same plants should be applied with caution in physiological work.     

Dimerization of phospholipase d isozymes.

Two mammalian phospholipase D (PLD) isozymes (PLD1 and PLD2) have been reported. In this study, we differentially tagged these isozymes with enhanced green fluorescent protein (EGFP-rPLD1 and EGFP-rPLD2) or Xpress peptide epitope (Xpress-rPLD1 and Xpress-rPLD2) to examine the association between these isozymes. Overexpressed EGFP-rPLD1 coimmunoprecipitated with Xpress-rPLD1 using anti-Xpress antibody. However, the coimmunoprecipitation was independent of the activity of rPLD1. Xpress-rPLD2 also bound to EGFP-rPLD1 although the binding was less efficient than observed with Xpress-rPLD1. The association between rPLD2 and rPLD1 was confirmed by coimmunoprecipitation of EGFP-rPLD2 with Xpress-rPLD1. EGFP-rPLD2 also bound to Xpress-rPLD2 as shown by coimmunoprecipitation. Immunofluorescence staining of COS-7 cells coexpressing EGFP-rPLDs and Xpress-rPLDs showed that the PLD isozymes colocalized in the perinuclear and plasma membrane regions, suggesting that they could associate in a cellular setting. These results suggest that rPLD1 and rPLD2 can exist as homodimers and can form heterodimers.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Influence of positive allosteric modulators on GABA(B) receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA_B receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [³⁵S]GTPγS binding to Gαo and, less markedly, Gαi_1/3 in cortex, whereas Gq and Gs/olf were unaffected. ( R )-baclofen and SKF97581 likewise activated Gαo and Gαi_1/3, expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA_B antagonist, CGP55845A, abolished agonist-induced activation of Gαo and Gαi_1/3 in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [³⁵S]GTPγS binding to Gαo in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Gαi_1/3. Similarly, in human embryonic kidney cells expressing GABA_B(1a+2) or GABA_B(1b+2) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Gαi_1/3, though they increased its potency. To summarise, GABA_B receptors coupled both to Gαo and to Gαi, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi_1/3. It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.     

Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O₂⁻) and nitric oxide (NO^•). Neurotoxicity mediated by NO^• may result from the reaction of NO^• with O₂, leading to formation of peroxynitrite (ONOO⁻). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.