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Muscarinic modulation of calcium current in neurones from the interatrial septum of bull‐frog heart.
Author(s) -
Tse A,
Clark R B,
Giles W R
Publication year - 1990
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1990.sp018164
Subject(s) - muscarinic acetylcholine receptor , oxotremorine , acetylcholine , medicine , endocrinology , chemistry , gtp' , muscarine , biophysics , voltage clamp , membrane potential , biology , receptor , biochemistry , enzyme
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.