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 (Fin) and inhibited K+ currents gated by cAMP (IK,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 IK,cAMP was about 100 times more sensitive to ACh than Fin 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-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 +/- 0.07 nM, whilst the IC50 for IK,cAMP inhibition was 5.9 +/- 0.2 microM. 4. Oxotremorine, a muscarinic agonist, preferentially stimulated IK, cAMP inhibition (EC50 = 15.8 +/- 1.4 microM), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 +/- 0.7 microM. 5. Fin 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 IK,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.

Figure 1. Current-voltage and dose-response relationships of follicular responses generated by ACh

A, membrane current-voltage relationships of F_in (•) and I_K,cAMP (▴) elicited by FSH (0.25 μg ml⁻¹), and during I_K,cAMP inhibition (○) by 0.1 μm ACh. Each point indicates the mean (±s.e.m.) from 4-7 follicles (3 frogs) tested in each condition. Reversal potentials for the currents were -21.6 ± 3, -98 ± 2 and -55 ± 9 mV, respectively. B, top traces: follicular F_in elicited by ACh applied consecutively at concentrations of 0, 0.05, 0.1, 0.5, 1 and 10 μm, with 10 min wash intervals in NR solution. The ACh applications started at the point indicated by the arrow and lasted about 80 s. The superimposed traces are from a single epithelium-removed follicle held at -60 mV. Bottom, 10 μm ACh application in a defolliculated oocyte from the same frog. C, top traces: inhibition of I_K,cAMP (generated by FSH) by increasing concentrations of ACh (0, 1, 5, 10, 50 and 100 nm, with 15 min wash intervals). Data from 1 follicle held at -40 mV. In this and subsequent records, the bars indicate the time of drug application, and voltage steps of +20 mV (2 s) were applied periodically to monitor membrane conductance. Bottom, FSH application in a defolliculated oocyte from the same frog. D, dose-response relationships for activation of F_in (•) and inhibition of I_K,cAMP (○) normalized with respect to the maximal response. Means (±s.e.m.) of 12-16 follicles from 5 frogs. Curves are fits to the equation:by the method of non-linear least squares fitting, where EC₅₀ is the half-maximal effective concentration of ACh, n_H is the slope factor (Hill coefficient), A₁ and A₂ are the initial and final normalized I values, respectively, and [ACh] is the concentration of the neurotransmitter.

Figure 2. Dose-response relationships of muscarinic antagonists on follicular responses to ACh

A, effect of 4-DAMP on F_in generated by 800 nm ACh. The traces on the right show 4-DAMP blockade of F_in elicited by ACh; the trace on the left is the control current. Wash periods were of 10 min. Data from a single follicle. B, blocking effect of 1 μm PZP, TPC, MTT or 4-DAMP on 8 nm ACh inhibition of I_K,cAMP generated by 2 μm ADE (open bar below each trace). Superimposed traces in each group correspond to control current (ADE alone; 1), I_K,cAMP inhibition in the presence of ACh and ADE (2), and the same as 2 plus the antagonist which was superfused for 100 s before ACh application (3). Wash intervals were of 15 min. Data from different follicles (2 frogs). C and D, dose-antagonist relationships for the effect of 4-DAMP (squares), MTT (circles), PZP (triangles) and TPC (diamonds) on F_in activation (C) and I_K,cAMP inhibition (D) elicited by ACh at the EC₅₀ concentration. Curves were fitted using the same equation as for Fig. 1D, and each point represents the mean of 4-5 follicles from 2 frogs. I_K,cAMP was generated by 2 μm ADE or 2 μg ml⁻¹ FSH.

Figure 3. Dose-response relationships of muscarinic agonists on follicular responses

A, F_in generated by ACh and oxotremorine in a single follicle. The ACh concentrations were 0.05, 0.1, 0.5, 1, 10 and 100 μm, and the oxotremorine concentrations were 1, 10 and 100 μm. B, inhibition by oxotremorine of I_K,cAMP (elicited by ADE). Oxotremorine (0.5 or 100 μm) was applied at the peak of the K⁺ current. C, preincubation with oxotremorine also blocked I_K,cAMP. The oxotremorine concentrations were 0.1, 1, 10, 100 and 1000 μm. Note that at intermediate concentrations (1-100 μm) I_K,cAMP inhibition was reversed rapidly on washout. D, carbachol (open symbols) and oxotremorine (filled symbols) dose-response relationships for F_in activation (circles) and I_K,cAMP inhibition (triangles). Mean of 9-13 follicles from 5 frogs.

Figure 4. Oxotremorine effect on follicular responses and activation rates of muscarinic responses

A, PZP blockade of oxotemorine-elicited (100 μm) inhibition of I_K,cAMP. Decreasing concentrations of PZP were applied (100, 10, 1 and 0.1 μm), with 15 min wash intervals. Similar results were obtained in 3 other follicles from 2 frogs. B, examples of follicles in which ACh generated both F_in (arrow) and oscillatory Ca²⁺-dependent Cl⁻ currents (top traces), but in which oxotremorine failed to generate any response (middle and bottom traces). The 4 superimposed traces (top) show currents elicited by ACh (0.1, 1, 10 and 100 μm) applied to the same follicle. Similar results were obtained in follicles from 2 other frogs. C, ACh concentration vs. rate of F_in activation (•) or inhibition of I_K,cAMP (○). Rates are expressed as the time necessary to reach 50 % of the maximal response for each concentration. For F_in activation the rate decreased linearly while for I_K,cAMP inhibition it increased following a sigmoidal relationship (3 follicles, 2 frogs).

Figure 5. Oxotremorine effect on F_in elicited by ACh or ATP

A, dose-response relationships for oxotremorine inhibition of F_in elicited by 50 μm ACh (•) or 10 μm ATP (○). Inhibition curves were adjusted as in Fig. 1D. The IC₅₀ for F_in elicited by ACh was 2.3 ± 0.7 μm. Each point represents the mean of 8 follicles from 3 frogs. B and C, sample traces of inhibition produced by oxotremorine on F_in. The trace on the left in each panel is the control current elicited by the agonist alone, and the superimposed traces on the right show the effects of oxotremorine: for F_in activated by ACh the oxotremorine concentrations were 10 nm, 1 and 10 μm, and for F_in activated by ATP the concentrations were 10 and 100 μm. All traces were obtained from a single follicle.

Figure 6. RT-PCR amplification of XlmR transcripts in follicular cells

A, ethidium bromide-stained gel of RT-PCR-amplified products. The left-hand lane contains DNA markers (in bp). +, cDNA from defolliculated oocytes or isolated follicular cells. -, negative controls, RNA without reverse transcriptase. B, Southern blot of the same gel as in A, hybridized with XlmR probe.