ATP-inhibition of M current in frog sympathetic neurons involves phospholipase C but not Ins P(3), Ca(2+), PKC, or Ras.
Suppression of the voltage-activated, noninactivating K(+) conductance (M conductance; g(M)) by muscarinic agonists, P(2Y) agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/(11) class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) but not by the inactive isomer, U73343. The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) failed to affect ATP-induced g(M) suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that "lipid-signaling" mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g(M) by ATP in amphibian sympathetic neurons.
Stemkowski
Patrick L
PL
Department of Pharmacology and University Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Tse
Frederick W
FW
Peuckmann
Vera
V
Ford
Christopher P
CP
Colmers
William F
WF
Smith
Peter A
PA
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D046508:Culture Techniques; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D009433:Neural Inhibition / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D011760:Pyrrolidinones / Q000494:pharmacology; D011892:Rana catesbeiana; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D013564:Sympathetic Nervous System / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*; D018631:ras Proteins / Q000502:physiology
D000470:Alkaloids; D053119:Benzophenanthridines; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D004963:Estrenes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010617:Phenanthridines; D011760:Pyrrolidinones; D018160:Receptors, Cytoplasmic and Nuclear; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; C016299:chelerythrine; D011493:Protein Kinase C; D010738:Type C Phospholipases; D018631:ras Proteins; D002118:Calcium