Capsaicin modulates acetylcholine release at the myoneural junction

Abstract

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.

Keywords: Acetylcholine release; Capsaicin; Capsaicin (PubChem CID: 1548943); Endocytosis; Exocytosis; Motor nerve terminal; Phosphatidylinositol-4,5-bisphosphate (PubChem CID: 5311358GTPL2387); TRPV1; capsazepine (PubChem CID: 2733484.

Figure 1. Mouse NMJ expresses TRPV1

A. Western blot showing TRPV1 in lysates of mouse diaphragm and HEK293 cells transfected with TRPV1. TRPV1 expression is undetectable in wild type HEK293 cells. B. Representative western blot (n = 3 independent experiments) for TRPV1 in wild type and TRPV1−/− diaphragms. GAPDH was used as loading control.

Figure 2. CAP (CAP) inhibits stimulus evoked twitch tension of hemidiaphragm nerve muscle preparations and capsazepine (CPZ) antagonizes the effects of CAP

A. Time course of stimulus evoked twitch tension (%) of control, + CAP (1 µM) and + CPZ (10 µM) + CAP treated hemidiaphragm nerve muscle preparations. B. Representative twitch tensions of control, + CAP and + CPZ + CAP (1 µM) preparations at 0 min (before any treatment) and at 250 min. after treatment with CAP alone or + CPZ + CAP. The data are from 8–12 nerve-muscle preparations obtained from 12 mice. ** represents statistical significance for P<0.001.

Figure 3. CAP concentration-response curve in vitro

A. IC50 of CAP determined by measuring the concentration-dependent inhibitory effects of CAP on nerve stimulus evoked twitch tensions of diaphragm NMP in vitro. Data collected at 90 min. period following the addition of different concentrations CAP were shown. No more than two concentrations of CAP were studied in one experiment.

Figure 4. CAP inhibits stimulus evoked neurotransmitter release and capsazepine (CPZ; 10 µM) antagonizes the effects of CAP

A. Representative traces of 1 Hz stimulus evoked end plate currents (EPC) of control (a and c), + CAP (b), + CPZ (d), and + CPZ + CAP (e) treated nerve muscle preparations (n = 4–6, 16–22) at the end of 120 min. after the application of either vehicle alone or the drug. CPZ was pretreated and was present throughout the experiments (d and e). B. EPC amplitudes ± S.E.M of control (a and c), + CAP (b), + CPZ (d), and + CPZ + CAP (e) treated nerve muscle preparations (n = 4–6, 16–22). C, D and E. Represent the spontaneous miniature end plate current (mEPC) amplitude, frequency and quantal content of control, + CAP, + CPZ + CAP, and + CPZ treated preparations. Numbers in parentheses represent the numbers of nerve muscle preparations and fibers respectively obtained from six mice for each condition. (Quantal content = ratio of EPC amplitude to mEPC amplitude). ** represents statistical significance for P<0.001.

Figure 5. CAP inhibits EPC of wild type but not TRPV1^−/−

A. Representative EPC (top) and mEPC (bottom) traces for control and CAP treated diaphragm from TRPV1^−/− mice. B. Bar graph represent the mean EPC ± S.E.M. for the diaphragm obtained from wild type and TRPV1^−/− animals (n = 6–8 animals, 18–24 endplates). C. EPC amplitudes for control and CAP-treated wild type and TRPV1^−/− diaphragm at 120 min. vehicle or CAP application. ** represents statistical significance for P<0.001.

Figure 6. CAP pretreatment alters the subcellular disposition of AP-2 in Neuro 2a cells

Confocal micrographs represent the immunostaining for AP-2 µ1 in control and CAP treated wild type Neuro 2a cells and TRPV1 expressing Neuro 2a cells.

Figure 7. Wortmannin (WMN; 20 µM) inhibits stimulus evoked neurotransmitter release

A. Representative traces of 1 Hz stimulus evoked end plate currents (EPC) of control and WMN treated nerve muscle preparations (n = 4–6 muscles and 16–22 neuromuscular junctions) at the end of 120 min. after the application of either vehicle alone or WMN (120 min. treatment). B. EPC amplitudes ± S.E.M of control and WMN treated NMP (n = 3–5, 12–26). C, D and E. Represent the spontaneous miniature end plate current (mEPC) amplitude, frequency, and quantal content of control and WMN treated preparations. Numbers in parentheses represent the numbers of nerve muscle preparations and fibers respectively. ** represents statistical significance for P<0.001.

Figure 8. WMN but not CAP inhibits stimulus evoked neurotransmitter release in TRPV1^−/− NMP

A. Mean amplitudes (nA) in response to 1 Hz stimulus evoked end plate currents (EPC) of control, CAP (1 µM) or WMN (20 µM) treated NMP (n = 4–6 muscles and 14–20 neuromuscular junction, from 6 mice) at the end of 120 min. after the application of either vehicle alone (first bar) or CAP or WMN (second and third bars respectively). ** represents statistical significance for P<0.001.

Figure 9. Model describing the effect of CAP or WMN on PIP2 dependent exo-endocytic mechanism

PIP2 regulates exocytosis by facilitating SNARE complex formation and synaptic vesicle docking, fusion and release. PIP2 also participates in the formation and nucleation of endocytic machinery. The mechanism of how PIP2 regulation of endocytosis couples to exocytosis still remains unclear. CAP activates TRPV1 and causes decrease in PIP2 via Ca²⁺-PLC dependent mechanisms. WMN inhibits PI Kinase and decreases PIP2 levels. The reduction in PIP2 level down-regulates endocytosis as well as exocytosis. + sign denotes activation. ? Denotes unknown mechanism by which endocytosis and exocytosis couple with each other. Upward and downward arrows represent increase and decrease respectively.