The fade of the response to acetylcholine at the rabbit isolated sino‐atrial node.

Abstract

SUMMARY1. The effect of acetylcholine (ACh) on the frequency of spontaneous action potentials, recorded with glass microelectrodes from small preparations of the sinoatrial node of the rabbit, has been investigated.2. On application of ACh there was a rapid increase in cycle length (the interval between successive action potentials) but then, despite the continued presence of the ACh, cycle length decreased once again; i.e. the response to ACh 'faded' in the presence of ACh. This fade of the chronotropic response to ACh has been characterized.3. To observe fade it was found to be important to use a fast flow bath in order to increase the ACh concentration at the preparation abruptly. In other words, the response to ACh was sensitive to the rate of increase in the ACh concentration at the tissue. This may explain why in one study of the sino-atrial node of the guinea-pig the authors failed to observe fade.4. The concentration dependence of fade was studied in two ways: either as the fade in the response with different doses of ACh, or as the decrease in the response to a fixed test dose applied after a variable conditioning dose. Both methods indicated that fade increased with concentration over the range of roughly 10' to 1O' MACh.5. The onset of fade, i.e. the decline in cycle length during an exposure to ACh, was monophasic with low doses of ACh and biphasic (with fast and slow phases) with higher doses. The mean half-times of the fast and slow phases of fade were 10 + 2 s and 240+ 30 s (mean+S.E.M.; n = 5). However, in some preparations, rather than a smooth decrease in cycle length, there were discontinuities and cycle length could decrease in a stepwise fashion or even transiently alternate between two distinct states.6. Time was required for recovery from fade because a response to a test dose applied soon after a conditioning dose was depressed. Recovery was studied by applying test doses at different times after conditioning doses. Recovery occurred with a half-time of 40 + 8 s (mean + S.D.; n = 8) after a short exposure (30 s) to ACh, but was slower (half-time, 250 + 20 s; n = 6) after a longer exposure (5 min).7. Immediately after washing off ACh, cycle length decreased below its original value, although it then returned to this value over several minutes. This 'rebound' of cycle length may be the 'mirror image' of the fade during the exposure to ACh. M. R. BOYETT AND A. ROBERTSRebound was greater and more prolonged after higher concentrations of ACh or longer exposures to ACh.8. This characterization of fade is important because the vagal nerves to the sinoatrial node are tonically active and the chronotropic response to vagal stimulation may be markedly affected by the phenomenon of fade.

On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea‐pig atrial cells.

“…Figure 2B is a semilogarithmic plot of the fade of 'K, ACh during the exposure and it shows that the decay can be fitted by two exponentials. The time constant of the fast phase of desensitization in this example was 1-4 s and in sixteen cells was 1-58 + 014 s. The amplitude of the fast phase of desensitization in different cells was variable and perhaps, in part, depended on how abruptly ACh was applied to the cell (Boyett & Roberts, 1987). If there is mixing of the control and ACh-containing solutions, the ACh concentration next to the cell will increase gradually rather than in a step-like manner.…”

“…This supports the conclusion that at least two processes are responsible for desensitization. In the rabbit sino-atrial node, recovery of the chronotropic response after a 30 s exposure to ACh, during which only the fast phase of desensitization develops, is fast with a time constant of 58 s, whereas recovery after a 5 min exposure, during which the slow phase of desensitization also develops, is slow with a time constant of 362 s (Boyett & Roberts, 1987). The time constants of the recovery of IK, ACh (52 and 222 s) and the chronotropic response (58 and 362 s) from the fast and slow phases of desensitization are therefore similar and this further supports the hypothesis that fade of IK, ACh is responsible for fade of the chronotropic response.…”

“…Throughout this study 10 AM ACh was used, because desensitization is marked with high concentrations of ACh (Carmeliet & Mubagwa, 1986;Boyett & Roberts, 1987;Kurachi, Nakajima & Sugimoto, 1987). Furthermore, 10 /M ACh has been shown to activate IK, ACh near maximally (Carmeliet & Mubagwa, 1986).…”

“…Martin, Levy & Matsuda, 1982;Martin, 1983;Boyett & Roberts, 1987;Boyett, Kirby, Orchard & Roberts, 1988). Because the vagal nerves to the heart are tonically active in the intact animal, desensitization is likely to occur under normal physiological conditions; desensitization is therefore important.…”

Role of internalization of M2muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+current in heart

“…5E; this shows that, after a 20-min exposure to CCh, recycling occurred over several hours. We have found that recovery from desensitization takes longer, the longer the exposure to a muscarinic agonist (2,38); this is also true of receptor internalization. Figure 5F shows M 2 -receptor labeling in groups of control cells, cells incubated without CCh for 24 h (labeling unchanged), cells exposed to 10 M CCh for 2 h (labeling greatly reduced as expected), and cells exposed to 10 M CCh for 2 h; CCh was then washed off for either 12 or 24 h. Figure 5F shows that, after a 2-h exposure to CCh, recycling was not complete even after 24 h; therefore, recycling after a 2-h exposure to CCh (Fig.…”