Routes of acetylcholine leakage from cytosolic and vesicular compartments of rat motor nerve terminals

Neuroscience Letters, 135 (1992) 5-9

5

© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 08323

Routes of acetylcholine leakage from cytosolic and vesicular compartments of rat motor nerve terminals Dean O. Smith Department of Physiology, University of Wisconsin, Madison, WI 53706 (U.S.A.) (Received 3 May 1991; Revised version received 5 September 1991; Accepted 9 October 1991)

Key words: Acetylcholine; Motor nerve; Cytosolic; Vesicular; AH5183; Leakage; Vesamicol Acetylcholine el'flux at the rat neuromuscular junction was assayed following blockage of ACh transport into synaptic vesicles by 2-(4-phenylpiperidino)cyclohexanol (AH5183). [2H4]Choline was used as a labeled precursor. AH5183 completely blocked ACh efflux from the cytosolic compartment but had comparatively less effect on release from the unlabeled vesicular pool. Tissue [2H4]ACh levels increased after AH5183 addition due to cytosolic ACh retention. Thus, ACh in the non-vesicular pool (calculated to be 34% of the total ACh) may efflux solely via the AH5183-sensitive ACh transporter inserted into the terminal membrane. ACh released from the vesicular fraction was about 100-fold more than could be accounted for by miniature end-plate potentials; possible causes of this overestimate are discussed.

It has been well established that acetylcholine (ACh) is distributed into a cytosolic and a vesticular compartment. ACh in the cytosolic pool is incorporated into the synaptic vesicles via a transporter located in the vesicle membrane [1]. This transporter requires a proton gradient for its operation [1] and is specifically blocked by the tertiary nitrogen-containing compound 2-(4-phenylpiperidino)cyclohexanol, AH5183 (also known as vesamicol) [2, 15]. During release of vesicular ACh, the transporter becomes part of the nerve-terminal membrane as the vesicle fuses into the plasma membrane. In this configuration, transport activity will move ACh from the cytosolic compartment into the extracellular space. This has been suggested as a pathway for non-quantal ACh release from cytosolic stores [5, 7, 18]. Alternative pathways have not been ruled out, though, for blockage of the transporter by AH5183 has not been found to block non-quantal ACh release entirely [5, 7, 18]; previous studies had obtained only incomplete (e.g. 50%) blockage (e.g. ref. 7). While investigating the effects of AH5183 on ACh efflux from rat motor nerve terminals, we discovered that leakage in the absence of stimulation could be blocked completely by the drug. This fortuitous result indicated that the transporter may be the sole pathway for nonquantal release at the neuromuscular junction (cf. ref. 8). Correspondence: D.O. Smith, Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, U.S.A.

It also allowed us to estimate the sizes of the cytosolic and the vesicular ACh pools. All experiments were conducted using the extensor digitorum longus (EDL) preparation from Fischer 344 rats. After anesthetizing the animals with chloral hydrate (2.8 mmol/kg, i.p.), EDL musles with approximately 1 cm of the associated peroneal nerve were dissected from both legs. Average (+ standard error) wet weights of the dissected samples were 0.146 (+ 0.002) g. The tissue was then pinned down at resting length in a small chamber containing 4 ml of saline solution containing the following constituents (mM): NaC1 124, KC1 5.1, MgCI 2 1.3, KH2PO4 1.22, NaHCO3 25.5, CaCI2 2, and glucose 10.2; the pH was 7.4. Fresh saline, aerated with 95% 02-5% CO2 and maintained at 37°C, circulated over the tissue at a rate of 10 ml/min; oxygen saturation was maintained at greater than 80% (608 mmHg). Electrophysiologic measurements of miniature endplate potential (m.e.p.p.) rates and amplitudes were obtained from 8 different recording sites, each in a different animal, using standard intracellular recording procedures [19]. ACh and choline were assayed using gas chromatography/mass spectrometry (GCMS) and a [2H4] label. The tissue was first incubated for 60 min in saline containing [2H4]choline (10 ¢tM). The nerve was stimulated (1 Hz) during the last 10 min to accelerate loading of choline into the tissue and ACh synthesis. It was then placed in fresh choline-free saline for 4 min. Eserine (50/IM) was

present throughout the procedure. At the conclusion of each experiment, the tissue was rinsed 3 times in a Buchner funnel over a vacuum and was then transferred to a tared tube containing ice-cold formic acid (15%, I N) in acetone and internal standards. The bathing medium was also collected into tared chilled extraction tubes containing 1.6 ml dichloromethane plus internal standards ([~H9]ACh and [2H9]choline). Labeled and unlabeled (endogenous) choline and ACh were then assayed using techniques identical to those described in Smith and Weiler [21]. Measurements of labeled choline or ACh will also include any label which had not been rinsed out of the extracellular space. The calculated amounts of choline and ACh distributed in this space (27/~1) represent less than about 1% of the tissue levels [20]. Therefore, this contribution has been ignored throughout the analysis. During incubation in [2H4]choline (60 min), 2H4 was incorporated into [2H4]ACh that then distributed into the cytosolic and the vesicular compartments. The relative amounts in the two compartments were determined by measuring the fractional amount of [2H4]ACh ([2H4]ACh/total ACh) released during 4 min under resting or stimulated (1 Hz) conditions. The average values were 0.100 (+0.051) and 0.085 (+0.009), respectively (n=6); the corresponding fractional amount of [2H4]choline in the tissue was 0.098 (+0.022). The differences between these values are not statistically significant. Thus, the fractional amounts of [2H4]ACh in the pools for resting and evoked release are similar following the 60-min incubation period (cf. ref. 20). To block ACh transport, AH5183 (0.1-1 ~M) was added to the saline during the incubation in [ZH4]choline. As shown in Fig. 1, addition of AH5183 to the bath completely blocked [2H4]ACh efflux in the absence of stimulation. There was, however, continuing leakage of unlabeled (endogenous) ACh, although at a slightly reduced rate. This unlabeled ACh was most likely incorporated into vesicular sources before the experiment began since AH5183 had blocked [2H4]ACh loading into vesicles. Indeed, the addition of AH5183 had little effect on spontaneous release of vesicular ACh (m.e,p.p.s.) (cf. ref. 14). In the absence of AH5183, the m.e.p.p, rate and amplitude were 1.64 (+0.39) per second and 0.58 (+0.05) mV, respectively; the corresponding values obtained from the same recording sites following 20-rain incubation in 1-/IM AH5183 were 1.56 (+0.28) per second and 0.51 (+ 0.12) inV. By inference, all ACh leakage through the AH5183-sensitive transport pathway is, conversely, derived from cytosolic pools and not some other less-defined pathway [7]. Tissue levels of [2H4]ACh, though, increased nearly two-fold after addition of AH5183, as shown in Fig. 2.

Since endogenous ACh levels did not change appreciably, elevated tissue [2H4]ACh may be attributed to retention of labeled ACh in the terminal following blockage of the leakage pathway by the drug. Estimates of the fraction of ACh in the cytosolic compartment can be calculated from the data summarized in Figs. 1 and 2. Leakage specifically from the cytosolic pool equals the total ACh efflux in the absence of AH5183 (6.2+0.6 pmol) minus the vesicular efflux (3.8+0.3 pmol in the presence of 1/.tM AH5183), this is 2.4+0.7 pmol. Since there is equal distribution of [2H4]ACh into both the vesicular and the cytosolic pools in the absence of AH5183, the fractional amount of [2H4]ACh in the total leakage from the cytosolic pool ([2H4]ACh leakage/total ACh leakage = 0.17_+0.02) should be the same as the fractional amount of [2H4]ACh in the cytosolic ACh pool. Therefore, total cytosolic ACh content can be obtained by dividing the tissue [2H4]ACh levels in the presence of AH5183 (9.4+0.8 pmol) by its fractional amount (0.17); this value is 55.3_+7.6 pmol. Furthermore, this cytosolic ACh represents 34% (+ 5%) of the total tissue ACh ( 165.0 + 9.5 pmol in the absence of AH5183). In this preparation, AH5173 was particularly effective in blocking ACh efflux from the cytosolic compartment. Similar results were obtained using the mouse diaphragm by Edwards et al. [7]. In non-stimulated tissue, they observed a complete inhibition of membrane hyperpolarization ('H effect') upon exposure to I>tubocurarine when 1 ~M AH5183 was present. Likewise 'spontaneous' ACh leakage under resting conditions is reduced in the presence of AH5183 in other tissues, including rat striatum [18] and mouse brain [5], although the reductions were somewhat less than 100% as seen in the EDL. In general, though, these results all indicate that ACh efflux from the cytosolic compartment is via the AH5183-sensitive ACh transporter, which is probably incorporated into the plasma membrane during vesicle exocytosis. Indeed, this appears to be the sole pathway at the neuromuscular junction [8]. The results further indicate that the ACh within the cytosolic compartment represents 34% of the total ACh. This value is about midway between indirect estimates, ranging from 10 to 73%, obtained from Torpedo [22, 23], sympathetic ganglion [3], and motor nerve terminals of frog and rat [10, 13]. Variability between preparations is to be expected, however. Using a two-compartment model that assumes negligible exchange between the vesicular and the cytosolic ACh compartments under resting conditions, Weiler et al. [23] also estimated the relative size (22%) of the cytosolic ACh pool in Torpedoterminals. Under this assumption, [2Ha]ACh, which is thus present only in the cytoso-

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Fig. 1. Effects of AH5183 on ACh efflux from motor nerve terminals. Tissue was pre-incubated for 60 min in saline containing [2H4]choline (10#M) and AH5183 (0-1 #M). This was then replaced by fresh saline, and after 4 rain the saline was collected and assayed for ACh content. Effiux of [2H4]ACh (A), endogenous (unlabeled) ACh (B), and total (labeled plus endogenous) ACh (C) are presented. Average values (+ S.E.M.) of measurements from 4 different animals are shown. Effiux of [2H4]ACb (A) was blocked completely by 0.1 and 1/.tM AH5183; therefore, the S.E.M. was 0. lic fraction, is in isotopic equilibrium with the precursor [2H4]choline pool. Consequently, the cytosolic A C h fraction m u s t equal the ratio o f the fractional a m o u n t o f

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Fig. 2. Effects of AH5183 on motor nerve ACh levels. Tissue was preincubated for 60 min in saline containing [2H4]choline (10 #M) and AH5183 (0-1 #M). This was then replaced by fresh saline, and after 4 min the tissue was assayed for ACh content. Levels of [2H4]ACh (A), endogenous (unlabeled) ACh (B), and total (labeled plus endogenous) ACh (C) are presented. Average values ( + S.E.M.) of measurements from 4 different animals are shown.

labeled A C h to the fractional a m o u n t o f labeled choline precursor. U s i n g data from our study, the estimates range from 0.297 to 0.411 in the absence and the presence (1 # M ) o f A H 5 1 8 3 , respectively. These values are consis-

tent with our more direct estimate and lend support to the study by Weiler et al. [23]. Since all efflux of unlabeled ACh in the presence of AH5183 comes from vesicular sources, the amount of ACh per vesicle may also be estimated from these data. Total efflux from the entire muscle, which contains an average of 3152 end plates [6] was 3.8 pmol/4 min (Fig. 1); this corresponds to 5.0 x 10-18 mol/end plate per second. Under similar resting conditions, m.e.p.p.s that presumably result from the spontaneous release of one vesicle's ACh content are recorded using intracellular microelectrodes at an average rate of 1.64/s. Total ACh per vesicle is thus calculated from these data to be 3.1 × 10 -18 mol or, alternatively, 1.8 x 106 molecules; in a 50nm synaptic vesicle this corresponds to an ACh concentration of 47 mol/liter. This estimate of vesicular ACh content is clearly too high. Indeed, it is 100-fold larger than estimates obtained from iontophoretic applications of ACh by Kuffler and Yoshikami [12]. Therefore, our measurements of ACh efflux or of m.e.p.p, rate require further consideration. The efltux rates are quite close to those reported by many others (e.g. refs. 10, 16, 20, 21). Likewise, the m.e.p.p. rates are about the same as those reported by numerous others (e.g. ref. 9). Trusting the accuracy of the biochemical assay results, we hypothesize several possible explanations for this overestimate of vesicular ACh content. (1) There may be ACh leakage from non-neuronal tissue such as the underlying muscle via an AH5183-insensitive pathway. Measurements of non-neuronal ACh release range from about 15% to 50% of the nerve-terminal levels [17, 21]. Even at the highest estimate, though, efflux from the non-neuronal sources is not sufficient to account for the overestimated vesicular stores. (2) M.e.p.p.s. may result from concurrent release of many vesicles. There is evidence for this possibility [11]. However, this would require the nearly simultaneous release of about 100 vesicles per m.e.p.p., and this too seems unlikely. (3) The most likely explanation is that ACh released spontaneously from most vesicles does not generate a detectable m.e.p.p. This implies that either the ACh is hydrolyzed or is not released sufficiently near ACh receptors to produce an electrical response greater than background noise. Failure to generate a m.e.p.p, due to hydrolysis cannot be the case, though, for inhibition of acetylcholinesterase does not markedly alter m.e.p.p, frequency. Alternatively, it is indeed possible that some vesicular ACh is released at sites remote from a population of ACh receptors (cf. ref. 8). Whether this could account for the large overestimate of vesicle ACh content cannot be assessed, though, until the basic mechanisms of vesicle release have been further clarified.

This work was supported by N I H Grant NS13600. The assistance of Dr. Molly Weiler and Dr. Donald Jenden and his staff for their support while using his GCMS facility and Dr. Stanley Parsons who generously provided the AH5183 are gratefully acknowledged.

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