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1. Intracellular recording of membrane potential was made from the separated longitudinal muscle of the guinea-pig terminal ileum in physiological salt solution.

2. When acetylcholine was washed from the tissue following a brief application the membrane repolarized and then hyperpolarized (`after-hyperpolarization') beyond the level existing before the application of acetylcholine.

3. No after-hyperpolarization was observed following acetylcholine in potassium-free solution, in sodium-deficient (17 mM) solution, or in the presence of ouabain (1.7 x 10^-6 M). Repolarization under these conditions was delayed, especially after the membrane potential reached -20 to -30 mV, and was generally incomplete.

4. The after-hyperpolarization was significantly (P < 0.01) greater when acetylcholine was applied in chloride-deficient (13 mM) solution.

5. It was incidentally observed that the membrane potential in the presence of acetylcholine was more positive in potassium-free solution (significance P < 0.025), unchanged in chloride-deficient solution (P > 0.4), and much more negative in sodium-deficient (17 mM) solution (P << 0.001), confirming previous results using carbachol.

6. When a 2 min application of 1.4 x 10^-6 M carbachol was made, the membrane potential 15-20 sec after beginning its application was not affected by ouabain (10^-5 M), but showed a significantly (P < 0.005) greater positive shift subsequently, so that the potential after 120 sec in carbachol was significantly (P < 0.025) more positive in the presence of ouabain. After 45 sec in 5.5 x 10^-5 M carbachol the membrane potential was also significantly (P < 0.005) more positive in the presence of ouabain (10^-5 M).

7. Calculations based on hypotheses concerning the movements of sodium and potassium showed that the positive shift of the membrane potential in the presence of carbachol when sodium pumping was arrested, could be quantitatively explained by a decline in the sodium and potassium gradients across the membrane. It appeared that the electrogenic fraction of the sodium pumped was small in the presence of carbachol.

8. It was concluded that the application of acetylcholine or carbachol (> 10^-6 M) to this smooth muscle disturbs the sodium and potassium gradients across the membrane. These disturbances are in a direction which stimulates electrogenic sodium pumping. Some limitation of depolarization results, and the increased electrogenic extrusion of sodium is responsible for the after-hyperpolarization which follows the application of acetylcholine.