Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes

Abstract

1. Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (F(in)) and inhibited K+ currents gated by cAMP (I(K,cAMP)) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs. 2. Inhibition of I(K,cAMP) was about 100 times more sensitive to ACh than F(in) activation; the half-maximal effective concentrations (EC50) were 6.6 ± 0.4 and 784 ± 4 nM, respectively. 3. Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide blocked the two effects with very different potencies. F(in) was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 ± 0.07 nM, whilst the IC50 for I(K,cAMP) inhibition was 5.9 ± 0.2 μM. 4. Oxotremorine, a muscarinic agonist, preferentially stimulated I(K,cAMP) inhibition (EC50 = 15.8 ± 1.4 μM), whilst F(in) was only weakly activated. In contrast, oxotremorine inhibited F(in) generated by ACh with an IC50 of 2.3 ± 0.7 μM. 5. F(in) elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling. 6. Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in I(K,cAMP) inhibition. 7. In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype and the other the M3 subtype. These receptors are coupled to distinct membrane mechanisms leading to independent regulation of two membrane conductances.