G-PROTEIN DIFFUSION DURING MUSCARINIC ACTIVATION

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We have studied the muscarinic response of rat lacrymal gland cells to acetylcholine with the tight-seal whole-cell technique. To measure the liberation of calcium from internal stores in that cell we monitored the calcium dependent K and Cl currents resulting from application of acetylcholine. Intracellular inclusion of IP2 liberated calcium. To show that polyphosphoinositide (PIP2) hydrolysis is a step in muscarinic activation we added neomycin, which binds to PIP2 and blocked activation by acetylcholine. The response to agonist diminished over several minutes after initiation of whole-cell dialysis. We found that the response was stable for some minutes, then decreased exponentially. The delay and time constant of the washout was directly proportional to the size of the pathway for diffusion and inversely proportional to the cell volume. It appeared that some soluble intracellular factor, needed after receptor activation, was needed for liberation of intracellular calcium. Attempts to constantly replenish PIP2 by inclusion of CTP, ATP, and inositol did not halt washout. Intracellular inclusion of ATP, GTP, cAMP, cGMP, together or alone, did not stop washout. We observed washout in one cell, gently removed the pipette after no further response to acetylcholine was seen, and sealed to the same cell a new pipette containing IP3. A rapid and sustained response was seen upon initiation of the whole cell mode. Intracellular inclusion of GTP-S potentiated the muscarinic response and slowed washout. We conclude that the washout is due to the loss of a diffusible factor which acts after muscarinic receptor activation and before polyphosphoinositol release. We suspect the action of the factor to involve a G protein.