Mouse muscle denervation increases expression of an alpha7 nicotinic receptor with unusual pharmacology

Abstract

Neuronal nicotinic alpha7 subunits have been found in chick and rat skeletal muscle during development and denervation. In the present study, reverse transcriptase-polymerase chain reaction was used to detect alpha7 subunit mRNA in denervated mouse muscle. To determine whether the alpha7 subunit forms functional nicotinic acetylcholine receptors (nAChRs) in muscle, choline was used to induce a membrane depolarization because choline has been considered a specific agonist of alpha7-containing (alpha7*) nAChRs. We found, however, that choline (3-10 mM) also weakly activates muscle nAChRs. After inhibiting muscle nAChRs with a specific muscle nAChR inhibitor, alpha-conotoxin GI (alphaCTxGI), choline was used to activate the alpha7* nAChRs on muscle selectively. Four weeks after denervation, rapid application of choline (10 mM) elicited a substantial depolarization in the presence of alphaCTxGI (0.1 microM). This component of the depolarization was never present in denervated muscles obtained from mutant mice lacking the alpha7 subunit (i.e. alpha7-null mice). The depolarization component that is resistant to alphaCTxGI was antagonized by pancuronium (3-10 microM) and by a 4-oxystilbene derivative (F3, 0.1-0.5 microM) at concentrations considered highly specific for alpha7* nAChRs. Another selective alpha7 antagonist, methyllycaconitine (0.05-5 microM), did not strongly inhibit this choline-induced depolarization. Furthermore, the choline-sensitive nAChRs showed little desensitization over 10 s of application with choline (10-30 mM). These results indicate that functional alpha7* nAChRs are significantly present on denervated muscle, and that these receptors display unusual functional and pharmacological characteristics.

Figure 2. Choline and ACh evoke depolarizations mediated by muscle-type nAChRs at the endplate of innervated muscle

Rapid agonist applications were made to fibres from 3 mouse diaphragm muscles. A, trace showing the averaged response to 10 mm choline (Ch; n = 16). The arrowhead indicates agonist application. B, averaged response to 0.3 mm ACh (n = 12) C, concentration-response curves for choline from C57BL/6J mice (n = 4–16), α7+/+ (n = 13–21) and α7-/- mice (n = 9–21). D, concentration-response curve of ACh from C57BL/6J mice (n = 5–12). The peak amplitude of the depolarizations evoked by agonists was calculated and then averaged. E, concentration-response curve demonstrating inhibition by α-conotoxin GI (αCTxGI) of the depolarization evoked by 10 mm choline (n = 3–8). F, concentration-response curve demonstrating inhibition by αCTxGI of the depolarization evoked by 0.3 mm ACh (n = 4–12). These responses were obtained 20 min after treatment with αCTxGI. The peak amplitude of depolarization was normalized as a percentage of the control response observed without αCTxGI. Data values in C-F represent the means ± s.e.m.

Figure 4. Lack of the αCTxGI-resistant response to choline in denervated muscle from α7-null mice

Choline-evoked depolarization was obtained after treatment with 0.1 µmαCTxGI in the denervated soleus muscles. A, traces showing averaged response to 10 mm choline in α7+/+ and α7-/- mice. Stimulation artifacts caused by the agonist applications (arrowheads) have been eliminated. B, peak amplitude of the αCTxGI-resistant depolarization by choline in α7+/+ and α7-/- mice. Data values represent the means ± s.e.m. (n = 17–24, 5–6 muscles, **P < 0.01, determined by unpaired t test).

Figure 6. αCTxGI-resistant depolarization present throughout prolonged application of choline in denervated muscle

Choline-evoked depolarization was obtained after treatment with 0.1 µmαCTxGI in the denervated soleus muscles. A, traces showing typical recordings of the αCTxGI-resistant depolarization evoked by 1 s application of choline (10 mm) in denervated muscles of α7+/+ and α7-/- mice. Horizontal bars below the traces indicate 1 s application of choline. Similar results were obtained in 3 separate muscle fibres from 2 muscles of α7+/+ mice and in 5 separate muscle fibres from 2 muscles of α7-/- mice. B, time course of averaged αCTxGI-resistant responses to prolonged choline application in denervated muscles of C57BL/6 mice. The choline solution (10–30 mm) containing 0.1 µmαCTxGI was continuously applied to the recording area for 10 s via an outflow tube. The horizontal bar in the plot indicates 10 s application of choline. Data values represent the means ± s.e.m. (10 mm choline: ▴, n = 11, 4 muscles; 30 mm choline : •, n = 10, 6 muscles).

Figure 1. Semi-quantified expression level of the α7 nAChR subunit by RT-PCR

Total RNA was extracted from mouse innervated muscle (innerv), 4 week-denervated muscle (denerv), blood and hippocampus (hippoc). RT-PCR products for α7 subunit and β-actin were obtained simultaneously from each sample. A, the α7 mRNA level was normalized as a percentage of the β-actin mRNA level, which was considered to be 100 %. Data values represent the means ± s.e.m. (n = 6–14). A significant difference (*P < 0.05) existed between innervated and denervated muscles as determined by an unpaired t test. B, photo-images in the negative form demonstrating the RT-PCR product for α7 mRNA that yielded the expected band of 509 bp, and the product for β-actin mRNA that showed the expected band of 348 bp.

Figure 3. The αCTxGI-resistant depolarization by choline and ACh in denervated muscle of C57BL/6J mice

All the data were obtained after treatment with αCTxGI in mouse soleus muscles. A, traces showing averaged response to 10 mm choline (Ch) in the presence of 0.1 µmαCTxGI in the innervated (innerv) and denervated (denerv) muscles (n = 10–17, 4–5 muscles). Stimulation artifacts just after the agonist applications (arrowheads) are blanked. B, peak amplitude of 10 mm choline-evoked depolarization in the presence of αCTxGI (0.1–1 µm) in the innervated and denervated states. Data values represent the means ± s.e.m. (n = 10–17). C, traces demonstrating averaged response to 0.3 mm ACh in the presence of 0.1 µmαCTxGI in the innervated and denervated muscles. Stimulation artifacts are blanked (n = 3–6, 3 muscles). D, peak amplitude of 0.3 mm ACh-evoked depolarization in the presence of αCTxGI (0.1–0.5 µm) in the innervated and denervated states. Data values represent the means ± s.e.m. (n = 3–6). *Significantly different response (P < 0.05) to choline or ACh in the presence of 0.1 µmαCTxGI in the denervated muscles from those in the innervated muscle, determined by unpaired t test.

Figure 5. Distinct pharmacological properties of the αCTxGI-resistant depolarization in denervated muscle and the junctional nicotinic response in innervated muscle

Choline-evoked depolarizations were obtained in either the innervated diaphragm muscles without αCTxGI (innerv, ○) or the denervated soleus muscles treated with 0.1 µmαCTxGI (denerv, •) in C57BL/6J mice. The inhibitors (pancuronium, MLA or F3) were applied first, before rapid agonist application was carried out. The concentration-response curves demonstrate the inhibitory effects of pancuronium (A), MLA (B) and F3 (C) on the depolarization induced by 10 mm choline (Ch). Peak amplitude of the depolarization was normalized as a percentage of the control response obtained without these blockers. Data values represent the means ± s.e.m. (A: ○, n = 4-8, 3 muscles; •, n = 7-9, 4 muscles; B: ○, n = 17-18, 3 muscles; •, n = 3-4, 3 muscles; C: ○, n = 3-5, 3 muscles; •, n = 6-8, 3 muscles).