Taro Imaizumi

Bradykinin-induced intracellular Ca2+ elevation in neuroblastoma × glioma hybrid NG108-15 cells; Relationship to the action of inositol phospholipids metabolites

The effect of bradykinin on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator quin 2. Bradykinin induced two phases of change in [Ca2+]i. Bradykinin induced a spike phase of [Ca2+]i increase which was detectable within 15 s and decayed to near-basal concentration in 3 min and then a prolonged plateau phase of [Ca2+]i increase which continued for 15 min. The bradykinin-induced spike phase was not diminished by decreasing extracellular Ca2+ concentration ([Ca2+]o) to 1 microM. On the contrary, the plateau phase was dependent on [Ca2+]o and inhibited by Ca2+ blockers, verapamil (50 microM), nifedipine (1 microM). The iontophoretic injection of inositol-trisphosphate (IP3) into the single cell induced the increase of [Ca2+]i, which was independent of [Ca2+]o. These results indicate that the bradykinin-induced spike phase is mediated by the release of intracellular Ca2+ stores induced by IP3, while the plateau phase is mediated by influx of extracellular Ca2+ probably through voltage-sensitive Ca2+ channels.

Effects of phosphorylation of inhibitory GTP‐binding protein by cyclic AMP‐dependent protein kinase on its ADP‐ribosylation by pertussis toxin, islet‐activating protein

Pretreatment of rat cardiac myocytes with the β‐adrenergic agonist, db‐cAMP or forskolin decreased ADP‐ribosylation of 40–41 kDa protein by islet‐activating protein (IAP) in cell membranes. Addition of activated cyclic AMP‐dependent protein kinase (protein kinase A) catalytic subunit and MgCl2 also decreased ADP‐ribosylation of 40–41 kDa protein by IAP in cell membranes. The α‐ and β‐subunits of partially purified inhibitory GTP‐binding protein (Gi) were both phosphorylated by protein kinase A. The amounts of phosphate incorporated into the subunits of Gi were 0.34 and 0.18 mol/mol protein. These show that phosphorylation of Gi by protein kinase A results in a decrease in its ADP‐ribosylation by IAP.

1-oleoyl-2-acetyl-glycerol and phorbol diester stimulate Ca2+ influx through Ca2+ channels in neuroblastoma × glioma hybrid NG108-15 cells

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator, quin 2. OAG and TPA induced an increase in [Ca2+]i from 100 +/- 19 to 187 +/- 24 nM and 192 +/- 15 nM, respectively, within 15 min. The increase in [Ca2+]i induced by activators of protein kinase C was dependent on the extracellular Ca2+ concentration [Ca2+]o) and was inhibited by the Ca2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca2+]i increase is mediated by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels.

Heterologous desensitization of bradykinin-induced phosphatidylinositol response and Ca2+ mobilization by neurotensin in NG108-15 cells

The heterologous desensitization of the bradykinin (BK)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) by neurotensin was studied in neuroblastoma x glioma hybrid NG108-15 cells. The addition of neurotensin to the cells resulted in an increase in [Ca2+]i and an increase in the formation of inositol phosphates in Ca2+-free medium. Pretreatment of the cells with neurotensin resulted in 43% decrease in the BK-induced increase of [Ca2+]i. The increase in [Ca2+]i induced by ionomycin, which causes Ca2+ release from the intracellular pool, was not decreased by pretreatment with neurotensin. This indicates that the inhibitory effect of neurotensin on the BK-induced increase of [Ca2+]i was not due to depletion of the intracellular Ca2+ pool. Pretreatment with neurotensin also caused a 47% decrease in the BK-induced formation of inositol trisphosphates (IP3). This decrease was not due to depletion of phosphatidylinositol bisphosphates. Neurotensin did not inhibit [3H]BK binding to cell membranes. These results show that neurotensin desensitizes the BK responses of NG108-15 cells, heterologously, perhaps by changes in phospholipase C and/or guanine nucleotide-binding protein (G-protein).

Phosphorylation of Gi protein by cyclic AMP-dependent protein kinase inhibits its dissociation into α-subunits and βγ-subunits by Mg2+ and GTPγS

Abstract Pretreatment of partially purified inhibitory GTP-binding protein (G i , 41 kDa) with activated cyclic AMP-dependent protein kinase (PKA) decreases its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). We examined whether this decrease was associated with dissociation of the trimer of αβγ-subunits of G i protein into α-subunits and βγ-subunits. Results showed that phosphorylation of the G i protein by PKA impaired its dissociation into α-subunits and βγ-subunits by 50 mM Mg 2+ and 100 μM GTPγS. The results suggested that phosphorylation of the G i protein by PKA possibly caused a conformational change of the trimer G i protein.

Cyclic AMP-dependent protein kinase interferes with GTP gamma S stimulated IP3 formation in differentiated HL-60 cell membranes.

The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA (0.1 mg/ml) in the presence of MgATP for 15 min. at 37 degrees C decreased GTP gamma S-stimulated inositol trisphosphate (IP3) formation by about 30%, but had no influence on Ca2+-stimulated IP3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation.

Effects of lithium ion on ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin, islet-activating protein

Studies were made on the effects of Li+ on ADP ribosylation of inhibitory GTP-binding (Gi) protein by islet-activating protein (IAP), pertussis toxin. The ADP ribosylation of 40-41 kDa proteins of the membranes of rat cardiac ventricular cells by IAP was reduced by the addition of a nonhydrolyzable analog of guanine nucleotide, GTP gamma S, indicating that these proteins included Gi protein. The addition of LiCl (0.5-10 mM) to the membrane fractions of the cells attenuated the ADP ribosylation of the Gi protein of the cell membranes by IAP dose-dependently. The effects of LiCl were reversible. Of the monovalent ions tested, Li+ inhibited the ADP ribosylation of the protein by IAP most strongly. The effects of LiCl (2 mM) were observed even in the presence of 150 mM KCl. Moreover, LiCl decreased the ADP ribosylation of purified Gi protein by IAP. These results support that Gi proteins are one of the targets for the therapeutic effects of lithium.

CHANGES OF ADP‐RIBOSYLATION OF GTP‐BINDING PROTEIN BY PERTUSSIS TOXIN IN HUMAN PLATELETS DURING LONG‐TERM TREATMENT OF MANIC DEPRESSION WITH LITHIUM CARBONATE

1. The study was done to determine whether long‐term treatment with lithium carbonate affected the guanosine triphosphate (GTP)‐binding protein in platelet membranes from patients with manic depression.

Effects of lithium ion on the inhibitory GTP-binding protein and its coupling response

Addition of lithium ion to the inhibitory GTP-binding (Gi) protein resulted in a decrease of its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). The possibility that this decrease was due to dissociation of the Gi protein trimer was examined. Results showed that lithium ions had no appreciable effect on either the Gi protein trimer or its dissociation into its three subunits induced by Mg2+ and GTP gamma S. Next, the effect of lithium ions on Gi protein-mediated adenylate cyclase inhibition and alpha 2-adrenoceptor in human platelet membranes was examined. Lithium ion was found to impair adenylate cyclase inhibition of alpha 2-adrenoceptor stimulation of forskolin-stimulated enzyme activities. The monovalent ion also abolished guanine nucleotide modulation (GTP shift) of agonist binding, while it had no remarkable effects on antagonist binding in alpha 2-adrenoceptor of human platelet membranes. These results suggested that lithium ion caused functional change of the Gi protein without remarkable change of its dissociation, causing modulation in a coupling between alpha 2-adrenoceptor and Gi protein.

The H-L subgroup of guinea-pig cardiac M2 receptors (M2β) regulates inositol phosphate formation

In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgroups, M2 alpha and M2 beta, with different affinities for agonists and that the M2 alpha subgroup is coupled with inhibition of adenylate cyclase. We now studied which subgroup was responsible for the formation of inositol mono- (IP), bis- (IP2), tris- (IP3) and tetrakis- (IP4) phosphates in guinea pig heart. Carbachol (1 mM) significantly stimulated the formation of all four IPs in [3H]myoinositol-preloaded slices of guinea-pig ventricles. Acetylcholine (1 mM) also stimulated the formation of IP2, IP3 and IP4. However, oxotremorine (1 mM) only slightly stimulated the formation of IP2, and pilocarpine did not stimulate the formation of any IP. The pED50 values of carbachol for IP2 and IP3 formation were 3.76 and 4.23, respectively, which coincided with the pKd values of the low-affinity agonist binding site (L site) measured by competition of carbachol with [3H]quinuclidinyl benzilate [( 3H]QNB) binding while the pKd value for inhibition of adenylate cyclase coincided with the pKd value of the high-affinity agonist binding site (H site). Treatment of animals with pertussis toxin decreased the formation of IP2 and IP3 by carbachol to 66 and 54%, respectively, but resulted in complete inhibition of adenylate cyclase. These results suggested that muscarinic stimulation of the formation of IPs was manifested through a different receptor subgroup (M2 beta) and GTP binding protein different from those for inhibition of adenylate cyclase.