On the mechanism of M-current inhibition by muscarinic m1 receptors in DNA-transfected rodent neuroblastoma x glioma cells.
1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108-15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)-dependent K+ conductance, producing an outward current, and it inhibits a voltage-dependent K+ conductance (the M conductance), thus diminishing the M-type voltage-dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo-1 using whole-cell patch-clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/- 3.2% (n = 23). Following pressure-application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G-protein was tested by adding 0.5 mM GTP-gamma-S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half-times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml-1 pertussis toxin or on adding up to 10 mM GDP-beta-S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh-induced outward current and/or intracellular Ca2+ transient but did not block ACh-induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach-induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Robbins
J
J
Department of Pharmacology, University College London.
Marsh
S J
SJ
Brown
D A
DA
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000502:physiology; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D005910:Glioma / Q000201:enzymology / Q000378:metabolism*; D006822:Hybrid Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007293:Inosine Triphosphate / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects*; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004273:DNA, Neoplasm; D015221:Potassium Channels; D011976:Receptors, Muscarinic; D007293:Inosine Triphosphate; D009569:Nitric Oxide; D010741:Phospholipases A; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D001920:Bradykinin; D002118:Calcium