Muscarinic modulation of calcium current in neurones from the interatrial septum of bull-frog heart.
1. The effects of activation of muscarinic receptors on the voltage-dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull-frog (Rana catesbeiana) heart, and were maintained in short-term (1-6 day) tissue culture. ICa was recorded from the cells using whole-cell patch-clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current-voltage relation for ICa (i.e. +10 to +15 mV), whereas at more-negative potentials (i.e. -15 to -25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM-ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1-10 microM), DL-muscarine (0.1-2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide-binding protein (G-protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non-hydrolysable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP-gamma-S produced smaller changes in ICa than in control conditions. Inclusion of another non-hydrolysable analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 0.5-5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G-protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet-activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12-15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP-dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.
Tse
A
A
Department of Medical Physiology, University of Calgary, Canada.
Clark
R B
RB
Giles
W R
WR
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000187:drug effects; D002478:Cells, Cultured; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology; D006346:Heart Septum / Q000294:innervation*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010275:Parasympathetic Nervous System / Q000187:drug effects / Q000502:physiology*; D011892:Rana catesbeiana; D011976:Receptors, Muscarinic / Q000502:physiology*
D015220:Calcium Channels; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D002118:Calcium