Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum

Abstract

1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5. We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.

Figure 1

Effect of drugs on the stimulation-induced release of [³H]-acetylcholine. The two-stimulation (S₂/S₁) paradigm was used. Columns from the left to the right: control; carbachol 1 μM; carbachol 10 μM; carbachol 100 μM; atropine 1 μM; nifedipine 1 μM; nifedipine 1 μM with carbachol 1 μM; nifedipine 1 μM with carbachol 10 μM; haloperidol 1 μM; haloperidol 10 μM; domperidone 1 μM; and ω-conotoxin MVIIC 0.1 μM. Ordinate: S₂/S₁ ratio. Data are means (±s.e.mean) of measurements on 3–26 slices. ^*=significantly different (P<0.05) from control.

Figure 2

Stimulated release of [³H]-acetylcholine from slices pretreated with toxins. Columns from the left to the right: control; ω-agatoxin IVA (AGA; 300 nM for 30 min), ω-conotoxin GVIA (CTX; 100 nM for 30 min); both toxins (CTX+AGA). Ordinate: Stimulated release of radioactivity expressed as per cent of total radioactivity present in the slice. Data are means (±s.e.mean) of measurements on 6–40 slices. ^*=significantly different (P<0.01) from control.

Figure 3

Effect of carbachol on the stimulated release of [³H]-acetylcholine from slices pretreated with ω-agatoxin IVA (AGA) and ω-conotoxin GVIA (CTX). The first stimulation (S₁) was on superfused slices which had been preincubated for 30 min with 300 nM AGA or 100 nM CTX. The second stimulation (S₂) was on the same slices in the same medium without any further addition (columns denoted as AGA or CTX), or with the addition of 10 μM carbachol (columns denoted as AGA+car or CTX+car). Ordinate: S₂/S₁ ratio. Data are means (±s.e.mean) of measurements on six slices. ^*=significantly different from corresponding control without carbachol (P<0.01). #=significantly different (P<0.01) from AGA+CAR.

Figure 4

Effects of ω-conotoxin GVIA and ω-agatoxin IVA on the stimulated release of [³H]-acetylcholine from slices pretreated with paraoxon. Columns from the left to the right: slices pretreated with paraoxon (50 μM for 30 min; PX); slices pretreated with paraoxon and ω-agatoxin IVA (300 nM for 30 min; PX+AGA); slices pretreated with paraoxon and ω-conotoxin GVIA (100 nM for 30 min; PX+CTX); Ordinate: Stimulated release of radioactivity, expressed as per cent of total radioactivity in the slices. Data are means (±s.e.mean) of measurements on 11–23 slices. ^*=significantly different from PX (P<0.05).

Figure 5

Effects of 1 μM atropine (atr) on the stimulated release of [³H]-acetylcholine from slices preincubated with paraoxon (PX) alone, or with paraoxon in combination with ω-agatoxin IVA (AGA) or ω-conotoxin GVIA (CTX). Preincubations lasted 30 min and the applied concentrations were 50 μM for paraoxon, 100 nM for CTX, and 300 nM for AGA. Ordinate: S₂/S₁ ratios. Data are means (±s.e.mean) of measurements on 5–6 slices. The 6.1 fold increase in the release induced by atropine from slices pretreated with PX+AGA was significantly higher (^*P<0.05) than the 4.2 fold and 2.8 fold increases from slices pretreated with PX or PX+CTX, respectively.