Abbreviations used : ACh, acetylcholine ; VAChT, vesicular acetylcholine transporter.
Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation
Version of Record online: 18 JAN 2002
Journal of Neurochemistry
Volume 72, Issue 1, pages 166–173, January 1999
How to Cite
Issa, A. M., Gauthier, S. and Collier, B. (1999), Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation. Journal of Neurochemistry, 72: 166–173. doi: 10.1046/j.1471-4159.1999.0720166.x
- Issue online: 18 JAN 2002
- Version of Record online: 18 JAN 2002
- Okadaic acid;
- Calyculin A;
- Acetylcholine compartmentation;
- Acetylcholine release;
- Synaptic vesicle heterogeneity
Abstract : The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P3 fraction, but cytoplasmic ACh contained in the S3 fraction was not significantly affected. The P3 fraction is known to be heterogeneous ; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K+-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [3H]ACh from [3H]choline, the ratio of [3H]ACh in the S3 to P3 fractions was not much changed by exposure of tissue to calyculin A or okadaic acid ; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.
Acetylcholine (ACh) is synthesized in the cytoplasm of cholinergic nerve terminals, after which it is transported into synaptic vesicles by the action of a vesicular ACh transporter, VAChT (see reviews by Parsons et al., 1993 ; Eiden, 1998). Vesicles containing ACh are mobilized to sites of transmitter release in response to stimuli that initiate synaptic transmission (reviewed by Prior and Tian, 1995 ; Langley and Grant, 1997). Releasable ACh appears to be compartmentalized such that a part is more readily releasable than the rest (Birks and MacIntosh, 1961 ; Zimmermann and Denston, 1977 ; Zimmermann and Whittaker, 1977 ; Whittaker, 1984 ; Agoston et al., 1985 ; Collier et al., 1993), and there is considerable biochemical support for the notion of cholinergic synaptic vesicle heterogeneity (Barker et al., 1972 ; von Schwarzenfeld, 1979 ; Zimmermann, 1979 ; Giompres et al., 1981 ; Ágoston et al., 1986 ; Prior and Tian, 1995). However, which of the fractions that can be isolated biochemically relate to transmitter pools identified functionally remains uncertain.
The mechanisms that regulate ACh transport activity and vesicle mobilization also are not fully understood. Some evidence suggests that the level of phosphorylation activity in the nerve terminal might play a role in VAChT activity (Barbosa et al., 1997), and considerable support exists for the idea that phosphorylation regulates vesicle mobilization (Sihra et al., 1989 ; Greengard et al., 1993 ; Betz and Henkel, 1994 ; Südhof, 1995). These evidences suggest that inhibition of dephosphorylation might have consequences for ACh compartmentation, and this is the focus of the present study.
Calyculin A and okadaic acid have been shown to inhibit specifically and potently the serine/threonine phosphatases 1 and 2A (Ishihara et al., 1989 ; Cohen et al., 1990 ; Suganuma et al., 1990). Previously, we have reported that ACh synthesis was inhibited and that endogenous transmitter content was reduced in hippocampal slices incubated in the presence of calyculin A and okadaic acid, but that spontaneous ACh release was not affected (Issa et al., 1996). These findings suggested that phosphorylation-dephosphorylation processes are involved in the regulation of ACh synthesis and possibly in compartmentation of the transmitter. The objective of the present experiments was to investigate this latter notion further by exploring whether these agents alter the subcellular distribution of ACh and whether such tests can clarify the relationship between transmitter release and ACh stores. We have reported some of these results in abstract form (Issa et al., 1995).
MATERIALS AND METHODS
ACh chloride was obtained from Hoffmann-La Roche (Basel, Switzerland), and butyronitrile was from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). Amberlite CG-400 (chloride form), tetraphenylboron (sodium salt), physostigmine sulfate, choline kinase (EC 22.214.171.124 ; ATP : choline phosphotransferase, from Saccharomyces cerevisiae), acetylcholinesterase (EC 126.96.36.199 ; ACh hydrolase, type V-S), HEPES, EGTA, and choline chloride were all purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [γ-32P]ATP (10 Ci/mmol), [3H]choline chloride (86.6 Ci/mmol), and [3H]ACh iodide (100 mCi/mmol) were from New England Nuclear (Boston, MA, U.S.A.). Calyculin A and okadaic acid were purchased from GibcoBRL Life Technologies (Burlington, Ontario, Canada) and Research Biochemicals International (Natick, MA, U.S.A.), respectively ; these lyophilysates were dissolved in 10% dimethyl sulfoxide and diluted to a final solvent concentration of 0.001% before use. Fisher Scientific (Montréal, Québec, Canada) supplied all other chemicals and reagents.
The animals used in these experiments were male Sprague-Dawley rats (weighing 175-250 g) obtained from Charles River Breeding Laboratories (St. Constant, Québec, Canada). All experimental procedures were in accordance with the guidelines of the Canadian Council on Animal Care and of the Medical Research Council of Canada ; they were approved by the local university Animal Care Committee.
Preparation and incubation of hippocampal slices
Brain slices were prepared as previously described (Issa et al., 1996). In brief, rats were decapitated, and their brains were rapidly removed into ice-cold Krebs buffer (composed of 120 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSo4· 7H2O, 9.9 mM dextrose, and 25 mM NaHCO3) equilibrated with 5% CO2 in 95% O2 to maintain a pH of 7.4. Hippocampi were dissected from the surrounding tissue on ice, and 0.3-mm-thick slices were prepared using a McIlwain tissue chopper.
The slices were preincubated in Krebs buffer for 45 min at 37°C, after which they were incubated in either the absence (controls) or the presence of calyculin A (50 nM) or okadaic acid (50 nM). Choline chloride (10 μM, except during exposure to [3H]choline when it was 1 μM) was always included in the incubation medium. When ACh release was measured, the medium contained 15 μM physostigmine hemisulfate. For measurement of basal ACh release, slices were incubated for 20 min in normal Krebs medium, and when evoked ACh release was measured, slices were subsequently resuspended in a 50 mM K+ Krebs medium (in which the NaCl concentration was reduced by an equivalent amount) and incubated for a further 10 min. In the experiments that measured [3H]ACh, slices were incubated in the absence (controls) or presence of calyculin A or okadaic acid, as described above, and then with [3H]choline having a specific activity of 550 dpm/pmol for the experiment that measured [3H]ACh in the cytoplasmic (S3) and occluded (P3) fractions and 1,100 dpm/pmol for the experiment that measured [3H]ACh in the nerve terminal-bound subfractions of P3 for 10 min in the absence (controls) or continued presence of calyculin A or okadaic acid.
Preparation of subcellular fractions
The method described by Říčný and Collier (1986) was used to prepare the different subcellular fractions from rat hippocampal slices. Following incubation, the slices were separated from the medium, washed with 0.9% NaCl, and homogenized using 8 up-and-down strokes in a Teflon-glass vessel (0.15-0.23 mm wall clearance) with sucrose (0.32 M) containing HEPES (5 mM ; pH 7.4) and EDTA (1 mM) in the presence or absence of physostigmine hemisulfate (15 μM) depending on the experimental objective described below. The homogenate was centrifuged at 1,000 g at 4°C for 10 min. The supernatant (S1) produced was centrifuged at 10,000 g for 15 min at 4°C to obtain the P2 pellet. This P2 pellet (containing synaptosomes) was either used to obtain nerve terminal occluded (P3) and cytoplasmic (S3) pools by lysis and further centrifugation or layered following lysis onto a discontinuous sucrose gradient to obtain the nerve terminal-bound fractions. In the first instance, physostigmine hemisulfate (15 μM) was included in the solutions used for initial homogenization and lysing. The P2 synaptosomal fraction was lysed in HEPES (5 mM) and EGTA (1 mM) and centrifuged at 100,000 g for 60 min in a Beckman T-40 rotor at 4°C to yield the S3 and P3 fractions, which were then extracted with trichloroacetic acid and processed for ACh or [3H]ACh assay.
For experiments that measured ACh or [3H]ACh in nerve terminal-bound subfractions of P3, physostigmine was not present in any of the solutions. The lysed P2 pellet was layered onto a discontinuous sucrose gradient that consisted of 0.4, 0.8, and 1.2 M sucrose layers and centrifuged in a swinging bucket rotor at 53,000 g for 2 h at 4°C. Fractions were then recovered from the 0.4 (referred to as the D fraction), 0.8 (referred to as the E-F fraction), and 1.2 M (referred to as the G-H fraction) layers and subsequently extracted with trichloroacetic acid and processed for ACh or [3H]ACh assay. This procedure is modified from the method developed by Whittaker et al. (1964), and the nomenclature of the fractions used here is that originally used by these authors.
Quantification of ACh and [3H]ACh
The methods described previously (Issa et al., 1996) were used to extract and assay ACh and [3H]ACh. In brief, medium was separated from the tissue and used directly for the extraction of ACh and assay of released ACh ; the S3 fractions were also used directly. For the P3 and nerve terminal-bound subcellular fractions, the fractions were extracted with trichloroacetic acid, and the ACh or [3H]ACh was recovered by removal of the acid by ether. To recover the ACh or [3H]ACh, all samples were subsequently subjected to an extraction procedure using the method of Fonnum (1969) as described previously by Welner and Collier (1984) and Issa et al. (1996). ACh was assayed using the method of Goldberg and McCaman (1973). [3H]ACh was separated from [3H]choline and other metabolites and quantified as described by Issa et al. (1996).
Protein content was measured by the assay of Lowry et al. (1951) using bovine serum albumin as the standard. An aliquot of the P2 synaptosomal preparation was used for protein determination, and the results were expressed per milligram of this synaptosomal protein.
Determination of radioactivity
Radioactivity was determined by liquid scintillation spectrometry (LKB). Five and 10 ml of Optiphase HiSafe II scintillation cocktail was used to measure 3H and 32P, respectively. Counting efficiency was 30-39% for 3H and ~99% for 32P.
Results are expressed as mean ± SEM values of the number of experiments indicated. All data were analyzed by one-way ANOVA with post hoc Newman-Keuls analysis.
Effects of calyculin A and okadaic acid on subcellular distribution of ACh
Initial experiments tested whether exposure of hippocampal tissue to calyculin A or okadaic acid altered the subcellular distribution of ACh. Hippocampal slices were incubated (20 min) in the absence (controls) or presence of calyculin A (50 nM) or okadaic acid (50 nM) and homogenized, and subcellular fractions were prepared. The synaptosomal pellet P2 was lysed in the presence of physostigmine and centrifuged to yield the soluble S3 fraction, containing cytoplasmic ACh, and the pellet P3, containing occluded ACh.
Endogenous ACh was significantly depleted in the P3 fraction of tissue exposed to calyculin A (p < 0.05) or okadaic acid (p < 0.05) (Fig. 1B). ACh in the S3 fraction was not significantly different from that in the control in the presence of calyculin A or okadaic acid (p > 0.98) (Fig. 1A). This depletion of nerve terminal ACh is less than what was predicted from the results with tissue ACh content of hippocampal slices (see Issa et al., 1996) ; it is unclear why this is so.
The P3 fraction is known to be heterogeneous, and therefore we explored whether the phosphatase inhibitors altered the subcellular distribution of ACh in fractions prepared from P3. To obtain the nerve terminal-bound subcellular fractions, the synaptosomal P2 pellet was lysed without physostigmine, layered onto discontinuous sucrose gradients, and centrifuged at high speed in a swinging bucket rotor. ACh content of the D (0.4 M sucrose), E-F (0.8 M sucrose), and G-H (1.2 M sucrose) fractions was assayed. The ACh level of the D fraction was reduced significantly (p < 0.05) when prepared from tissue exposed to calyculin A or okadaic acid, but the other nerve terminal-bound pools were not so affected (p > 0.8 ; Fig. 2).
Effects of calyculin A and okadaic acid on K+-evoked ACh release from hippocampal slices
We tested whether the reduction in ACh content of the vesicular nerve terminal fractions (P3 and the vesiclebound D fraction) was associated or not with a decrease in K+-evoked ACh release. For this, hippocampal slices were incubated (20 min) in normal Krebs buffer to assay basal ACh release and then resuspended and incubated in 50 mM K+ Krebs buffer to measure the evoked ACh release (both Krebs buffers containing 15 μM physostigmine to preserve the released ACh) in the absence (controls) or presence of calyculin A (50 nM) or okadaic acid (50 nM), and the amount of endogenous ACh released into the medium was measured. Consistent with our previous results (Issa et al., 1996), spontaneous release was not significantly affected (p > 0.9) by either of the phosphatase inhibitors. However, the amount of endogenous ACh released from K+-depolarized slices was significantly (p < 0.01) decreased in the presence of calyculin A and okadaic acid (Fig. 3). The decrease in evoked release (K+-stimulated - basal) from the tissues treated with the phosphatase inhibitors was ~60%, rather more than depletion of total nerve terminal ACh (see Fig. 1) but similar to the proportional depletion of the D fraction (see Fig. 2).
Effects of calyculin A and okadaic acid on synthesis and incorporation of [3H]ACh from [3H]choline into subcellular stores
These experiments were designed to test whether inhibition of phosphatase activity alters the ability of newly synthesized ACh to be taken up into synaptic vesicles. For this test, hippocampal slices were incubated as described above (20 min) in the absence (controls) or presence of calyculin A (50 nM) or okadaic acid (50 nM) and then with [3H]choline for 10 min in the continued absence (controls) or presence of these agents. Slices were subsequently homogenized, and the P2 pellet was lysed in either the presence of physostigmine (for the preparation of S3 and P3 fractions) or in its absence (for the gradient centrifugation preparation of the nerve terminal-bound fractions) and assayed for both total ACh and [3H]ACh.
We have reported previously that treatment with these phosphatase inhibitors reduces ACh synthesis (Issa et al., 1996) ; thus, the assessment of any consequence on ACh sequestration in the present tests rested on a comparison of the ratio of [3H]ACh in the S3 fraction to that in the P3 fraction and analysis of the specific radioactivity of ACh in the fractions. The amount of [3H]ACh in the S3 fraction was significantly (p < 0.01) reduced by calyculin A and okadaic acid. [3H]ACh content of the P3 fraction was also significantly (p < 0.01) decreased in the presence of calyculin A and okadaic acid (Fig. 4A). Total ACh was significantly (p < 0.001) decreased only in the P3 fraction in the presence of calyculin A and okadaic acid ; that of the S3 fraction was not significantly different from the control value (p > 0.6 ; Fig. 4B). The total [3H]ACh accumulated by the P3 fraction was 12% in the controls and 9% in the drug-treated tissues, suggestive of, at best, a modest effect of the phosphatase inhibitors on ACh sequestration. It would be expected that if ACh uptake were affected considerably by inhibition of phosphatase activity, this would be reflected in a reduction in specific activity of the P3 fraction greater than that of the S3 fraction. The measures of specific activity of ACh in the two fractions did not reflect an effect of the phosphatase inhibitors on ACh uptake : The decrease in specific activity of ACh in the P3 fraction (~30%) caused by calyculin A or okadaic acid was not more than that of the S3 fraction (~40% ; Fig. 4C).
To determine if the phosphatase inhibitors had a particular effect on the uptake of newly synthesized transmitter by any subfraction of the P3 fraction, the experiment above was repeated with analysis of the ACh in fractions D, E-F, and G-H. The [3H]ACh level of the D fraction was significantly (p < 0.01) reduced in tissue that had been exposed to calyculin A or okadaic acid (Fig. 5A). Incorporation of [3H]choline into [3H]ACh in the G-H fraction was also reduced significantly (p < 0.05), but this was less marked than that of the D fraction. [3H]ACh content of the E-F fraction was somewhat reduced in the presence of these drugs, but this was not statistically significant (p > 0.4). Total ACh of the D fraction was also significantly (p < 0.05) reduced in the presence of these drugs, but calyculin A or okadaic acid had no significant effect on the ACh content of the E-F (p > 0.7) or G-H (p > 0.9) fraction (Fig. 5B). Analysis of the specific activity of ACh in the different fractions (Fig. 5C) shows an inhibitory effect of both drugs on fractions D (p < 0.01) and G-H (p < 0.05) but not fraction E-F (p > 0.25). Thus, the decrease in ACh specific activity of the affected fractions (~30%) was not more than the decrease in S3 specific activity (~40% ; see Fig. 4C).
The first objective of the present study was to examine the effects of the phosphatase inhibitors calyculin A and okadaic acid on the release and subcellular distribution of ACh in the rat hippocampal formation. Calyculin A and okadaic acid selectively decreased the ACh content of the P3 fraction but did not affect the transmitter content of the cytosolic S3 compartment.
Further subfractionation of the vesicular fraction revealed that these agents selectively reduced the ACh content of a fraction of monodispersed synaptic vesicles (the D fraction) ; other nerve terminal-bound pools (fraction E-F or G-H) were not so affected in the presence of calyculin A or okadaic acid.
The amount of transmitter released usually depends on the tissue content, and a reduced ACh store of a vesicular fraction would be expected to yield reduced exocytotic ACh release if that store contributes to release. Thus, we explored whether the observed selective depletion of ACh from the vesicular fraction by calyculin A and okadaic acid was accompanied by a reduction in K+-evoked ACh release. Both calyculin A and okadaic acid reduced K+-evoked ACh release, a finding that is consistent with the results of Betz and Henkel (1994) and Vickroy et al. (1995). Okadaic acid has also been shown to inhibit K+-evoked release of other neurotransmitters (Verhage et al., 1995), suggesting that phosphorylation-dephosphorylation reactions are important contributors to release mechanisms of different transmitters. The present observation that decreased ACh release was accompanied by a reduced amount of vesicle-bound ACh when nonvesicle ACh was not depleted is compatible with the concept that vesicles are the source of ACh released from cholinergic nerve terminals (see reviews by Zimmermann, 1979 ; Ceccarelli and Hurlbut, 1980 ; van der Kloot, 1988 ; van der Kloot and Molgó, 1994 ; Prior and Tian, 1995), a matter that remains somewhat contentious (see Dunant and Israël, 1998).
As mentioned in the introductory section, there is evidence of biochemical and morphological heterogeneity of cholinergic synaptic vesicles (Barker et al., 1972 ; von Schwarzenfeld, 1979 ; Zimmermann, 1979 ; Giompres et al., 1981 ; Agoston et al., 1986 ; Prior and Tian, 1995). Recent evidence suggests that this is probably a common feature of various chemical neurotransmitters (Thoidis et al., 1998). Releasable stores of ACh are usually described as behaving as if they are distributed between two compartments : a store that is readily available for release and one that is less so until mobilized to release sites (Birks and MacIntosh, 1961 ; Zimmermann and Denston, 1977 ; Zimmermann and Whittaker, 1977 ; Whittaker, 1984 ; Agoston et al., 1985 ; Collier et al., 1993). It is somewhat unclear whether one of the fractions isolated by subcellular fractionation techniques contains the readily releasable pool of transmitter (see von Schwarzenfeld, 1979 ; Říčný and Collier, 1986). The present results are compatible with the idea that ACh in a fraction (D) of monodispersed synaptic vesicles likely represents the more metabolically active “readily releasable” pool of transmitter : The decreased evoked release of ACh caused by calyculin A or okadaic acid was associated with reductions in the D fraction but not in the E-F or G-H fraction.
There is evidence suggesting that the “releasable” population of synaptic vesicles is capable of docking at special release sites or active zones before undergoing exocytosis, whereas the so-called “reserve” pool of vesicles is constrained by cytoskeletal interactions (Heuser and Reese, 1981 ; Landis et al., 1988 ; Bahler et al., 1990). Several nerve terminal proteins have been shown to be regulated by phosphorylation (reviewed by Benfenati and Valtorta, 1993 ; Greengard et al., 1993), and it seems that phosphorylation of at least some of these proteins is involved in the dynamics of vesicular mobilization and release (for reviews, see Greengard et al., 1993 ; Jahn and Südhof, 1993 ; Augustine et al., 1996 ; Linial, 1997).
Our observations that calyculin A and okadaic acid reduced the ACh content of the P3 vesicular pool, specifically of the D fraction, suggested that these phosphatase inhibitors may decrease K+-evoked ACh release by depleting preformed ACh stores in that particular compartment and preventing their replenishment by mobilization. Alternatively, the present results could indicate that these phosphatase inhibitors block the refilling of vesicular stores with newly synthesized ACh and that this effect is particularly marked for fraction D.
The mammalian VAChT has been isolated, and its structure has been deduced (Erickson et al., 1994, 1996 ; Roghani et al., 1994 ; Schäfer et al., 1994) and found to contain a phosphorylation site (Erickson et al., 1994 ; Usdin et al., 1995). Recently, Barbosa et al. (1997) have suggested that the VAChT is subject to phosphorylation and proposed that ACh uptake characteristics are affected. Thus, it seemed plausible that calyculin A and okadaic acid by acting on the phosphatases 1 and 2A might affect the VAChT and thereby alter storage of ACh into synaptic vesicles. Our test of this involved allowing the tissue to synthesize [3H]ACh from precursor and testing whether that labeled ACh could be recovered in the subcellular fractions prepared. Calyculin A and okadaic acid reduced the synthesis of [3H]ACh, an effect that was anticipated from our earlier study (Issa et al., 1996), and therefore the interpretation of the present experiments rests on whether or not whatever [3H]ACh is synthesized can be taken up by synaptic vesicles. If the phosphatase inhibitors had a major effect on ACh uptake by synaptic vesicles, the expectation was that the ratio of [3H]ACh in S3 to that in P3 would reflect this, as would the specific activity of ACh measured with the fractions prepared.
As analyzed in Results, there was only a small effect of the phosphatase inhibitors on the apparent distribution of [3H]ACh between a cytosolic (S3) and particulate (P3) fraction, and the specific labeling of the P3 subfractions was not affected disproportionately to that of S3. Thus, these experiments suggest that if the VAChT is a substrate for phosphorylation, inhibition of its dephosphorylation has only a modest consequence to its main function, ACh transport, at least under the conditions of the present experiments.
Thus, the main mechanism that is responsible for the depletion of ACh in synaptic vesicles prepared from tissue exposed to phosphatase inhibitors appears not to be inhibition of VAChT, and we suggest that it results from an effect on vesicle mobilization or recycling.
The present work was supported by a grant from the Medical Research Council of Canada to B.C. A.M.I. was the recipient of a doctoral award from Hoechst Marion Roussel Canada.
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