Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells

Abstract

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

Keywords: G-proteins; mAchRs; methacholine; nAchRs; nicotine.

Figure 1.

Inhibition of nAChRs currents by prepuff of muscarinic agonists. (a) Inhibition of nAChRs currents by a prepuff of MCh. Left panel shows nicotine (Nic)-induced currents in a SCG neuron via perforated whole-cell recording at –70 mV. The inward currents were evoked by Nic (100 µM) before applying MCh (left trace), during applying MCh (middle trace) and after wash of MCh (right trace). All traces were from the same neuron. Right panel shows the statistics of the experiments in both SCG neurons and adrenal chromaffin cells. The inhibition of nAChRs current by a 10 s prepuff of 1 mM MCh was 77 ± 4% in SCG neurons (n = 19), and 74 ± 3% in adrenal chromaffin cells (n = 11). (b) Dose-dependence of MCh inhibition of nAChRs currents. Left panel, the inhibition of 100 µM Nic-induced currents at –70 mV were 20% and 80% for 10 s-prepuff of 0.1 µM MCh (left traces, neuron 1) and 1 mM MCh (right traces, neuron 2), respectively. Right panel, the dose curves of MCh versus currents induced by Nic (100 μM) (n = 12), and of Nic versus nAChRs currents (n = 9). The EC50 and IC50 of maximum effects were 69 μM and 4 μM for nicotine and MCh, respectively.

Figure 2.

Time course of the ‘MiI’ on inhibition of nAChRs currents at room temperature in SCG neurons. (a) The inhibition of 100 µM Nic-induced currents was strongly dependent on prepuff-time of MCh (1 mM) solution. In addition to the MCh-prepuff solution, MCh (1 mM) was also included in the Nic-puffing solutions, except the currents of ‘control’ and ‘recover’. Note that the ‘control’ current is nearly overlapped by the current of 0 s prepuff. MCh was washed out using standard external solution during and after the 150 s break. All traces in this panel were from one neuron. (b) Time course of the MCh effect in (a). The data points of the prepuff effect were well described by an exponential function with time constant of 1.3 s. Prepuff for 6 s was sufficient to get maximum MiI of 81 ± 1% (n = 8). (c) The recovery of nAChRs inhibition induced by MCh (1 mM) was strongly dependent on wash time. All traces in this panel were from another neuron. (d) Statistics of the wash experiments in (c). At 22°C, the data points were well fitted by a two exponential function with time constants of 10.0 s and 88.7 s, respectively (n = 7).

Figure 3.

Temperature sensitivity of the M-ihhibition. (a) In the absence of prepuff, MCh or ACh could inhibit nAChRs current only at 36°C but not 22°C. Left panels, Nic (1 mM), ACh (1 mM) and MCh (1 mM) + Nic (1 mM) induced currents at 22°C and 36°C, respectively. Right panel, the statistics of the experiments using protocol of left panel. Without prepuff at 36°C, the inhibition of nAChRs current was 33 ± 3% by 1 mM ACh (n = 5), and 34 ± 4% (n = 6) by 1 mM MCh. Note, there was no inhibition of nAChRs by MCh or ACh at 22°C. (b) The MiI of nAChRs was strongly dependent on temperature and prepuff time. Left two panels show currents induced by 100 µM-Nic at 22°C (neuron 1) and 36°C (neuron 2), respectively. MCh (1 mM) was prepuffed with MCh-prepuff-solution. MCh (1 mM) was also included in the Nic (100 µM)-puffing solution for the traces marked with the prepuff times (0 s, 0.5 s, etc.), except where only ‘Nic’ and ‘wash’ are marked. Right panel shows the statistics. The time constants of MiI versus prepuff time were 1.32 s, 0.58 s and 0.27 s, corresponding to 22°C (n = 11), 30°C (n = 8) and 36°C (n = 8), respectively.

Figure 4.

G-Proteins are involved in MiI of nAChRs currents in adrenal chromaffin cells. (a) Intracellular GTP-γ-S inhibited nAChRs currents. Left panel, rundown of whole-cell currents induced by Nic (100 μM). Middle panel, intracellular whole-cell dialysis (Rs = 11 MΩ, Cm = 14 pF) of GTP-γ-S (100 µM) for 8 min inhibited 72% of nAChRs (cell 2). Right panel, statistics of GTP-γ-S experiments. The GTP-γ-S inhibition of nAChRs is significant (p < 0.01). (b) GDP-ß-S removed MiI of nAChRs. Left panel shows that 5 µM MCh inhibited 35% of nAChRs under perforated whole-cell mode at –70 mV (cell 3). Middle panel, intracellular whole-cell dialysis (average Rs = 12, Cm = 12 pF) of GDP-ß-S (1 mM) for 8 min removed 5 µM MCh-inhibition to 10% (cell 4). Right panel, statistics of GDP-ß-S experiments. The effect of GDP-β-S on the MiI was significant (p < 0.01). (c) Effects of pertussis toxin (PTX) on MiI. PTX removed partial MiI of nAChRs under perforated whole-cell mode at –70 mV. Left two panels show that, after PTX treatment (150 ng ml⁻¹ for overnight), MCh (5 µM) inhibition was reduced from 35% (left, cell 1) to 16% (middle, cell 2). Right panel, statistics of PTX experiments. On average, PTX removed MCh (5 µM) inhibition of nAChRs significantly from 34 ± 2% to 20 ± 3% (p < 0.01). (d) Effects of BAPTA on MiI. Left three panels show that, after intracellular dialysis of 20 mM BAPTA for 10 min, 5 µM MCh-inhibition was reduced from 34% (left first panel) to 21% (left second panel). The MiI was further reduced to 12% by combined PTX and intracellular dialysis of 20 mM BAPTA (left third pane). Right panel, statistics of BAPTA experiments. On average, BAPTA and BAPTA + PTX-treatment reduced 5 µM MCh-inhibition of nAChRs significantly from to 34 ± 2% to 21 ± 3% (p < 0.01) and 12 ± 2% (p < 0.01), respectively.

Figure 5.

Protein kinases A and C are involved in the MiI. (a) PKC-specific inhibitor, bisindolylmaleimide I (Bis) removes part of the MiI in SCG neurons and adrenal chromaffin cells under perforated whole-cell mode with holding potential –70 mV. Left two panels show 1 mM MCh-induced inhibition of nAChR currents were partially removed by Bis treatment (500 nM, in 37°C for 30 min). Bis reduced the MiI from 74% (left, chromaffin cell 1) to 46% (middle, chromaffin cell 2). Rightmost panel, Bis removed the MiI significantly from 77 ± 6% to 37 ± 9% and 74 ± 6% to 46 ± 5% in SCG neurons and adrenal chromaffin cells, respectively (p < 0.01). The digits in brackets in this and other histograms indicate numbers of cells tested in each condition. (b) Activation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibits partial nAChRs currents. Left two panels, the protocol was similar to (a), except 10s-prepuff of MCh is replaced by 4 min-prepuff of PMA. Without Bis treatment, the nicotine-induced current was reduced 45% compared with that of control (left, SCG neuron 1). Bis removed the PMA-inhibition completely (middle, SCG neuron 2). Rightmost panel, Bis removed the PMA-inhibition significantly from 55 ± 10% to 2 ± 8% and 29 ± 6% to 4 ± 4% in SCG neurons and Adrenal chromaffin cells, respectively (p < 0.01). (c) PKA-specific inhibitor, H-89 removes part of the MiI in SCG neurons and Adrenal chromaffin cells under perforated whole-cell mode with holding potential –70 mV. Left two panels show 1 mM MCh-induced inhibition of nAChR currents were partially removed by H89 treatment (500 nM, in 37°C for 30 min). H89 reduced the MiI from 77% (left, chromaffin cell 3) to 50% (middle, chromaffin cell 4). Rightmost panel, H-89 removed the MiI significantly from 80 ± 5% to 40 ± 9% and 74 ± 6% to 50 ± 8% in SCG neurons and adrenal chromaffin cells, respectively (p < 0.01). (d) Activation of PKA by 8-Bromo-cyclic AMP (8Br-cAMP) inhibits partial nAChRs currents. Without H-89 treatment, the nicotine-induced current was reduced by 40% compared with that of control (left, chromaffin cell 5). H-89 removed partial 8Br-cAMP inhibition (middle, chromaffin cell 6). Rightmost panel, statistically H-89 removed the 8Br-cAMP-inhibition significantly from 47 ± 9% to 5 ± 18% and 40 ± 9% to 10 ± 8% in SCG neurons and adrenal chromaffin cells, respectively (p < 0.01).

Figure 6.

Methacholine inhibits the secretion in SCG neurons and adrenal chromaffin cells. (a) The activation of mAChRs inhibited nAChRs-induced quantal secretion of catecholamines in a SCG neuron. Representative amperometric spikes were induced by Nic (100 μM) in the presence and absence of MCh (1 mM). To reduce the baseline noise, the traces were low pass-filtered to 10 Hz. All traces are from the same neuron. (b) Similar experiments to (a), except the cell was a chromaffin cell under voltage-clamp at –70 mV. The nAChRs currents and quantal secretion were measured by combined patch-clamp and electrochemical amperometry in the presence of Tg (1 μM). Amperometric signals were low-pass filtered at 500 Hz. (c) Statistics of amperometry experiments shown in (a) and (b). On average, in contrast to Nic-induced secretion, MCh inhibited secretion by 16 ± 8% (Nic + MCh without MCh prepuff), 99 ± 1% (MCh prepuff for 10 s), 68 ± 7% (Nic with pre-wash for 10 s) and 2 ± 5% (washout MCh for 3 min) in SCG neurons, respectively (n = 8). In adrenal chromaffin cells, the corresponding inhibitions were 12 ± 6%, 97 ± 1%, 62 ± 5% and 0 ± 10%, respectively (n = 6).