Exocytotic Release of [ 3 H]-Acetylcholine by Ouabain Involves Intracellular Ca 2+ Stores in Rat Brain Cortical Slices

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Cellular and Molecular Neurobiology [cemn] pp971-cemn-472180 November 3, 2003 17:36 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 23, No. 6, December 2003 ( C© 2003)

Exocytotic Release of [3H]-Acetylcholineby Ouabain Involves Intracellular Ca2+ Storesin Rat Brain Cortical Slices

Rosangela S. Lomeo,1 Renato S. Gomez,2 Marco Antonio M. Prado,1

Marco Aurelio Romano-Silva,1 Andre R. Massensini,3 and Marcus V. Gomez1,4

Received December 18, 2002; accepted April 7, 2003

SUMMARY

1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat braincortical slices was investigated.

2. The ouabain-induced release of [3H]ACh was calcium-independent and not blockedby EGTA.

3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect sug-gesting the involvement of intracellular calcium stores.

4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhib-ited by 73% the ouabain-induced release of [3H]ACh, suggesting exocytotic release of theneurotransmitter.

5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices.

6. Confocal microscopy and calcium imaging showed that ouabain increased the levelsof [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma.

7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellularstores that can increase [3H]ACh exocytosis from rat brain cortical slices.

KEY WORDS: ouabain; acetylcholine release; exocytotic; intracellular calcium.

INTRODUCTION

Ouabain, the ligand of Na+,K+-ATPase is a steroid derivative (Kawamura et al., 2001)that unlike other steroids hormones, binds specifically to an integral plasma mem-brane protein. Despite extensive research physiological role of the ouabain/Na,K-ATPase complex remains controversial (Aizman et al., 2001). However, ouabain isfound in mammalian tissues (Kawamura et al., 1999; Schoner, 2000).

1 Departamento de Farmacologia, ICB, UFMG, Minas Gerais, Belo Horizonte, MG, Brazil.2 Departamento de Cirurgia, Faculdade Medicina, UFMG, Minas Gerais, Belo Horizonte, MG, Brazil.3 Departamento de Fisiologia e Biofısica, ICB, UFMG, Minas Gerais, Belo Horizonte, MG, Brazil.4 To whom correspondence should be addressed at Departamento de Farmacologia-ICB-UFMG,

Av. Antonio Carlos 6627-Pampulha, 31270-901, Belo Horizonte, MG, Brazil; e-mail: [email protected].

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0272-4340/03/1200-0917/0 C© 2003 Plenum Publishing Corporation

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918 Lomeo, Gomez, Prado, Romano-Silva, Massensini, and Gomez

Ouabain causes the release of acetylcholine (ACh) from brain tissues indepen-dent on the presence or absence of extracellular Ca2+ (Adam-Vizi and Ligeti, 1984;Gomez et al., 1975; Meyer and Cooper, 1981; Vyas and Marchbanks, 1981). Calciumchannels blockers do not seem to affect the ouabain-induced release of ACh fromrat brain cortical slices (Adam-Vizi et al., 1991; Casali et al., 1995); however, theyinhibited the ouabain-evoked release of ACh from the myenteric plexus longitudi-nal muscles of guinea pig ileum (Gomez et al., 1996). The influx (Pocock, 1983) orefflux of Ca2+ was not affected by ouabain even when ACh release was substantiallyincreased (Adam-Vizi and Ligeti, 1986). Thus the process of calcium transport ap-pears not to be involved in the ouabain-induced release of ACh in the central nervoussystem (CNS).

The possible participation of internal Ca2+ stores in the ouabain-induced releaseof ACh was suggested by Vizi and Ligeti (1984), although ouabain can increase AChrelease from synaptosomes without the increase in [Ca2+]i or even Na+ content(Satoh and Nakasato, 1992). Therefore, the mechanism underlying the release ofACh by ouabain is still controversial.

Ionized calcium is a key carrier of information in cells (Berridge, 1998). It hasbeen previously shown that ouabain induces a moderate and rapid increase in cy-tosolic calcium in rat adrenal glomerulosa cells that is dependent of voltage-activatedcalcium channels (Yingst et al., 1999). Recently, it was shown a new mechanism forouabain signaling via plasma membrane receptors that underlie a novel role forouabain as a physiological inducer of [Ca2+]i oscillations involved in transcriptionalregulation in mammalian cells (Aizman et al., 2001). Ouabain inhibits the Na+ pumpcreating conditions that lead to the accumulation of Na+. This accumulation mayinduce an increase in [Ca2+]i and a subsequent release of neurotransmitters.

In the present work we demonstrate that ouabain induces an exocytotic ex-tracellular calcium-independent release of ACh that was blocked by inhibitors ofryanodine and inositol 1,4,5-triphosphate (InP3) receptors in rat brain cortical slices.Moreover ouabain was also able to increase [Ca2+]i in cholinergic SN56 cells.

MATERIALS AND METHODS

Chemicals

Ouabain, tetrodotoxin, Percoll, EGTA, BAPTA-AM, cadmium, tetracaine,dantrolene, fluo 3-AM, and paraoxon were obtained from Sigma Chemical Co. (St.Louis, MO) and [methyl-3H]choline, 60–90 Ci/mmol from New England Nuclear(Boston, MA). The effective (−)-enantiomer of vesamicol was a gift of Dr S. M.Parsons (University of California at Santa Barbara, CA) and in this paper the termvesamicol refers to the (−)-enantiomer. All other chemicals and reagents were ofanalytical grade.

Release of [3H]-ACh From Rat Brain Cortical Slices

All procedures were approved by the local ethics committee. Slices of cerebralcortex (0.5 mm) were obtained from Wistar rats (180–200 g) of either sex using a

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Exocytotic Release of [3H]-Acetylcholine by Ouabain 919

McIlwain Tissue Slicer (Brinckman Instruments, Inc.). The release of [3H]-ACh intothe incubated fluid (Krebs-Trizma medium) was performed as previously described(Casali et al., 1995). The slices were previously incubated for 15 min in the presenceor absence tetrodotoxin, vesamicol, dantrolene, tetracaine, EGTA, or BAPTA-AMat the indicated concentrations followed by stimulation in the presence of ouabain(100µM) for 30 min or otherwise stated. In the experiments with EGTA or BAPTA-AM calcium was removed from the incubation medium.

Cell Culture

SN-56 cells were a gift from Prof Bruce Wainer (Department of Pathology,Emory University, School of Medicine). The cells were maintained in Dulbecco’smodified Eagle’s medium, 10% of fetal bovine serum (Gibco Life Technologies),2 mM L-glutamine and 1% penicillin/streptomycin in 50-mL culture bottles in a 5%C02 atmosphere at 37◦C. Cells were differentiated in the same medium above butlacking fetal bovine serum and supplemented with 1 mM dibutyryl-cyclic AMP forat least 3 days.

Measurements of [Ca2+]i on Cholinergic Cells SN56 Cells

Experiments were performed at room temperature (20–25◦C). SN-56 cells wereplaced on coverslips and incubated in HEPES-buffered salt solution (HBSS)(124 mM NaCl, 1.2 mM MgCl2, 10 mM glucose, and 25 mM HEPES) for 60 min,in the presence of the alpha-methyl-ester form (AM) of FLUO-3 (10 µM) to allowpenetration of the dye into cells. Cells were mounted on the perfusing chamber,placed on the microscope stage and continuously superfused by a gravity drivenapparatus with HBSS in the presence or absence of 2 mM CaCl2.

Imaging was performed with a Bio-Rad MRC1024 laser scanning confocal sys-tem running the software Lasersharp 3.0 coupled to a Zeiss microscope (Axiovert100) with a water immersion 40X, 1.2 NA objective. A Krypton/argon laser was usedto excite the preparation through 488 nm and emitted light selected with band passfilter 522/35 as previously described (Kushmerick et al., 1999). Image acquisitions intime series were performed using the Biorad Lasersharp Timecourse software, v.3.2.Both images and numerical data were used in the processing and analyses procedures.

Statistical

Statistical analysis was performed by analysis of variance using the Student–Newman–Kuels test (Steel and Torrie, 1960) with a significance of P < 0.05.

RESULTS

Ouabain caused a dose-dependent increase on [3H]ACh release (Fig. 1(A)).Thus, at the concentrations of 10, 50, and 100µM, ouabain increased the [3H]AChrelease during 30 min of incubation by approximately 1.5-, 2.1-, and 2.2-fold,

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Fig. 1. The effect of concentration (A) and incubation time in the ouabain-induced release of[3H]ACh in rat brain cortical slices. Brain slices were incubated in Krebs-Trizma medium for 30 min(A) or for 5, 10, 15, 30, or 60 min (B) in the absence (control) or at the indicated concentrations ofouabain of 0.1, 1.0, 10.0, 50.0, or 100 µM (A) or 100 µM (B). The values represent mean ± SEM forduplicates of at least three experiments. For other details, see Methods.

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Fig. 2. Effect of ouabain and tityustoxin in the release of [3H]ACh in rat brain cortical slices. The brainslices were preincubated in Krebs-Trizma medium for 15 min in the absence (control) or presence oftetrodotoxin (1.0µM) and then stimulated for 30 min in the presence of ouabain (100µM) or tityustoxin(0.5 µM). The values represent mean ± SEM for duplicates of three experiments. For other details,see Methods. ∗P < 0.05 when compared with the control value. ∗∗P > 0.05 by comparing with thecontrol value.

respectively. The ouabain-induced release of the neurotransmitter was a functionof the incubation time with a maximum effect at 30 min of incubation (Fig. 1(B)).Only at 5-min incubation time, the ouabain-induced release of [3H]ACh was notdifferent from the control value (P > 0.05).

Tetrodotoxin, an Na+ channel blocker, had no effect on the ouabain-inducedrelease of [3H]ACh but it was capable to inhibit the scorpion tityustoxin-inducedrelease of the neurotransmitter (Fig. 2). Tityustoxin is an α-type toxin that binds tosite 3 of the sodium channel causing depolarization and increasing the release of[3H]ACh. Thus tetrodotoxin-sensitive Na+ channels are not involved in the ouabainaction.

EGTA 0.5 mM and Cd2+100 µM had no effect on the ouabain-induced releaseof [3H]ACh (data not shown), while BAPTA-AM (50 µM) inhibited almost totallythe ouabain-induced release of neurotransmitter (Fig. 3), suggesting the involvementof intracellular calcium stores in the ouabain-induced release of [3H]ACh from braincortical slices. To identify these calcium stores, we studied the effects of tetracaine anddantrolene, blockers of InP3 and ryanodine receptors, on the ouabain-induced releaseof [3H]ACh. Figure 4 shows that dantrolene (100 µM) and tetracaine (500 µM)inhibited by 57 and 66%, respectively, the ouabain-induced release of [3H]ACh frombrain cortical slices (P < 0.05).

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Fig. 3. Effect of BAPTA-AM in the ouabain-induced release of [3H]ACh in brain cortical slices. Thebrain slices were preincubated in Krebs-Trizma medium for 15 min in the absence (control) or presenceor BAPTA-AM (50 µM) and then stimulated for 30 min with ouabain (100 µM). The values representmean±SEM for duplicates of three experiments. For other details, see Methods. ∗P < 0.05 by comparingwith the control value; ∗∗P > 0.05 by comparing with the control value.

To test the possibility that ouabain causes exocytotic release of [3H]ACh, we usedvesamicol, a drug that blocks acetylcholine transport to synaptic vesicles. Figure 5shows that the ouabain-induced release of [3H]ACh was inhibited in 73% by vesam-icol (P < 0.05), suggesting that the great majority of the ouabain-induced release of[3H]ACh comes from synaptic vesicles.

By using confocal microscopy, we tested the effect of ouabain on [Ca2+]i , usingthe cholinergic cells SN 56. We observed that ouabain caused a great increase in[Ca2+]i in these cells (Fig. 6(A)). The effect was a function of time and the maximumincrease of [Ca2+]i in the cells was reached at 20 min. Note that the increase in[Ca2+]i was concentrated in the cell soma (Fig. 6(B)). The ouabain-induced increaseon [Ca2+]i from SN56 cells was dependent of extracellular Ca2+ ions and in theirabsence it was not observed (data not shown).

DISCUSSION

The ouabain-elicited release of ACh from CNS is calcium-independent (Adam-Vizi and Ligeti, 1984; Casali et al., 1995; Gomez, et al., 1975; Vizi et al., 1972). Despitethese data it was shown that verapamil (500 µM) inhibited the ouabain-inducedrelease of ACh in synaptosomes (Satoh and Nakasato, 1992). A reason for this dis-crepancy is that at the above concentration verapamil also inhibits sodium channels(Hille, 1992; Triggle, 1981).

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Fig. 4. The effect of tetracaine and dantrolene in the ouabain-induced release of [3H]ACh inrat brain cortical slices. The brain slices were preincubated in Krebs-Trizma medium for 15 minin the absence (control) or presence of tetracaine (500 µM) or dantrolene (100 µM) and themstimulated for 30 min with ouabain (100µM). The values represent mean± SEM for duplicates ofthree experiments. For other details, see Methods. ∗P < 0.05 by comparing with the control value;∗∗P > 0.05 by comparing with the control value.

Tetrodotoxin, a blocker of Na+ channels, had no effect in the ouabain-inducedrelease of ACh in brain slices as previously shown (Gomez et al., 1975). Thus, byinhibiting the Na+ pump, ouabain creates a condition that increases intracellularNa+ leading to an increase in [Ca2+]i and the subsequent release of [3H]ACh inbrain cortical slices.

There are studies indicating that ouabain can increase the levels of intrasyn-aptosomal-free Ca2+ in the presence of extracellular calcium (Nachshen, 1985a,b).The increase in [Ca2+]i as the mechanism by which ouabain induces the release ofACh was refuted by Satoh and Nakasato (1992). We here describe that in the cholin-ergic cells SN56, ouabain increased [Ca2+]i and this increase was concentrated in thecell soma. However this effect in these cells was calcium-dependent. The calciumdependency may reflect tissue differences as was observed in the myenteric plexusand in sympathetic ganglion where the ouabain-induced release of ACh is calcium-dependent and in the former is inhibited by Ca2+ channel blockers (Collier et al.,1993; Gomez et al., 1996). The inhibition of the ouabain-evoked release of [3H]AChby BAPTA-AM, a chelator of intracellular Ca2+, and also by inhibitors of intracel-lular calcium stores InP3 and ryanodine indicates the involvement of [Ca2+]i in the

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Fig. 5. The effect of vesamicol in the ouabain-induced release of [3H]ACh in rat braincortical slices. The brain slices were preincubated in Krebs-Trizma medium for 15 min inthe absence (control) or presence of vesamicol 1.0 µM and then stimulated for 30 min withouabain (100 µM). The values represent mean ± SEM for duplicates of three experiments.For other details, see Methods. ∗P < 0.05 by comparing with the ouabain value.

ouabain-induced release of [3H]ACh in brain cortical slices. Ouabain can selectivelymodulate Na+ and Ca2+ concentration in sarcoplasmic reticulum and can therebyregulate Ca2+ signalling (Blaustein et al., 2002). Ca2+ signalling producing cytoso-lic calcium fluctuations via ryanodine and InP3 receptors triggers cytoplasmic Ca2+

signals important for the synaptic transmission and plasticity (Verkhratsky, 2002).It has long been known that the release of neurotransmitters requires the pres-

ence of extracellular Ca2+. According to the calcium hypothesis, the synaptic releaseof neurotransmitters occurs through an exocytotic process triggered by Ca2+ inflowassociated with presynaptic depolarization (Katz and Miledi, 1967). However, evi-dence showing transmitter release in the absence of external Ca2+ has been reported(Adam-Vizi, 1992). Notwithstanding all contradictory data, it is possible that underspecific circumstances the neurotransmitter release could be entirely independent ofextracellular Ca2+ and triggered by the efflux of Ca2+ from internal stores.

Regarding the neurotransmitter release in Ca2+-free medium, most of the liter-ature suggests that under these conditions neurotransmitter release occurs throughthe efflux of transmitters from cytoplasmic sources. Indeed in the neuromuscularjunction, ouabain caused a calcium-dependent nonquantal release of ACh not inhib-ited by vesamicol. This release was explained by an effect of the drug blocking thevesicle recycling (Zemkova et al., 1990). However our present data with vesamicol, acompound known to inhibit the ACh transporter of cholinergic vesicles (Ricny andCollier, 1984), shows that most of the release of [3H]ACh evoked by ouabain frombrain cortical slices occurs from a compartment that uses the vesicular ACh trans-porter to accumulate ACh. The results here presented are distinct from previous

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Fig. 6. Removal of extracellular Ca2+ abolished ouabain-induced increase in [Ca2+]i of SN56 cells.(A). Changes in the fluorescence of SN56 cells were determined using the calcium indicator Fluo-3as described in Methods. Cells were perfused with 200 µM ouabain in the presence (•) or absence(©) of CaCl2. Curves are mean± SEM for three experiments in which the number of individual cellswere at least 5 per experiment. Traces a and b are time points where the representative images in Bwere taken. (B). Differential interference contrast (DIC) image of typical SN56 cells differentiated withdibutyryl-cyclic AMP for 3 days. Confocal fluorescence images of differentiated SN56 cells from anotherculture labeled with fluo-3 before (a) and 30 min after addition of 200 µM of ouabain (b). Images arerepresentative of all experiments.

data showing that the extracellular Ca2+-independent release does not require thevesicular ACh pool (Adam-Vizi, 1992; Adam-Vizi et al., 1991) but are in agreementwith data showing the independency of extracellular Ca2+ for the exocytotic releaseof neurotransmitters (Gomez et al., 1999; Westerink et al., 1989).

In conclusion we suggest that in cholinergic neurons, the ouabain-induced in-crease in [Na+]i alters intracellular calcium homeostasis increasing [Ca2+]i and causes

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a calcium-independent exocytotic release of ACh that is inhibited by blockers ofintracellular calcium stores.

ACKNOWLEDGMENTS

We thank Adriane A. Pereira and Andreia A. Guimaraes for technical assistanceand L. A. De Marco for reading and suggestion in this paper. Supported by Pronex,PADCT, CNPq, and Fapemig.

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Exocytotic Release of [ 3 H]-Acetylcholine by Ouabain Involves Intracellular Ca 2+ Stores in Rat Brain Cortical Slices

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