guanosine-diphosphate and inositol-1-3-4-5-tetrakisphosphate

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

Topics: Animals; Calcium; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Inositol Phosphates; Mice; Proteins; ras GTPase-Activating Proteins; ras Proteins; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Thionucleotides; Tumor Cells, Cultured

Previous studies have reported that the inwardly rectifying K+ conductance (GKir) in macrophages is modulated by intracellular perfusion with inositol 1,4,5-triphosphate (InsP3), inositol 1,3,4,5-tetrakisphosphate (InsP4), or GTP analogues and by exposing cells to macrophage-specific colony-stimulating factor (CSF) I. This study uses both conventional whole cell and amphotericin B perforated patch studies to investigate GKir modulation in thioglycollate-elicited mouse peritoneal macrophages (MO). Under whole cell recording conditions with 150 mM Cl- in the patch pipette, GKir decreased within 25 min. The GKir decrease was slowed by exchanging glutamate for Cl- as the major anion in the pipette or by adding guanosine 5'-O-(2-thiodiphosphate) (50 nM) or ATP (0.5 mM) to the pipette. Addition of InsP3 or InsP4 to the pipette had no effect on the magnitude of GKir or its rate of decrease but activated an outward current in the voltage range of +60 to +120 mV in 57% of the cells studied. Thus in murine MO GKir may be modulated by G proteins but is unaffected by inositol phosphate metabolites, which have been reported to enhance GKir in phorbol 12-myristate 13-acetate (PMA)-differentiated HL-60 cells. In contrast to whole cell studies, perforated patch recordings of murine MO GKir were stable for > 1 h. Perforated patch studies demonstrated that murine MO also differ from PMA-differentiated HL-60 cells in that CSF I had no effect on GKir. Additionally, arachidonic acid, PMA, and H2O2, agents implicated in macrophage activation, did not modulate GKir. We conclude that GKir regulation in murine MO differs from that reported in PMA-differentiated HL-60 cells and that although our data suggest that GKir is modulated by G protein(s), they differ from the G proteins involved in MO responses to CSF I and the other agents tested.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Female; Guanosine Diphosphate; Humans; Hydrogen Peroxide; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Potassium Channels; Protein Kinase C; Thionucleotides

In mouse peritoneal macrophages, alpha 1-adrenoceptor stimulation evokes a Ca(2+)-dependent K+ current [Io(Adr)][Hara et al. (1991) Pflügers Arch 419:371-379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP3) and a GTP-binding protein (G protein) in Io(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of InsP3 (5-50 microM) evoked transient outward currents [Io(InsP3)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5'-[3-thio]triphosphate (GTP[gamma S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10-90 s. Io(InsP3) and the GTP[gamma S]-induced current [Io(GTP[gamma S])] were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K+ concentration. Fura-2 measurements revealed that Io(InsP3) and Io(GTP[gamma S]) result from a rise in intracellular free Ca2+ concentration ([Ca2+]i). Removal of extracellular Ca2+ did not abolish Io(InsP3) and Io(GTP[gamma S]). Both were blocked by bath-applied charybdotoxin. Intracellular D-myo-inositol 1,3,4,5-tetrakisphosphate (InsP4, 50 microM) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP3(2,4,5), 20 microM] elicited an outward current at -40 mV. Io(InsP3) was completely blocked by prior dialysis with the InsP3 receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5'-[2-thiol] diphosphate (GDP[beta S], 2 mM) or heparin (5 mg/ml) together with GTP[gamma S] in the patch pipette solution completely blocked Io(GTP[gamma S]). These results indicate that intracellular injection of InsP3 or GTP[gamma S] mimic Io(Adr).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Egtazic Acid; Electric Conductivity; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Heparin; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Macrophages; Mice; Peritoneal Cavity; Potassium; Potassium Channels; Receptors, Adrenergic, alpha; Thionucleotides