Multiple Calcium Pathways Induce the Expression of SNAP-25Protein in Chromaffin Cells

Esther Garcı´a-Palomero, Carmen Montiel, Carlos J. Herrero, Antonio G. Garcı´a,*Rocio M. Alvarez, Francisco M. Arnalich, †Jaime Renart, ‡Hernan Lara, and

*Ana M. Cardenas

Instituto de Farmacologı´a Teofilo Hernando, Departamento de Farmacologı´a, Facultad de Medicina, Universidad Auto´noma deMadrid; †Instituto de Investigaciones Biome´dicas “Alberto Sols,” CSIC-UAM, Madrid, Spain;*Laboratorio de Farmacologı´a,

Escuela de Medicina, Universidad de Valparaı´so, Valparaı´so; and‡Laboratorio de Neuroquı´mica, Facultad de CienciasQuımicas y Farmaceu´ticas, Universidad de Chile, Santiago, Chile

Abstract: Incubation of bovine adrenal chromaffin cells inhigh K1 (38 mM) during 24–48 h enhanced 2.5 to five timesthe expression of SNAP-25 protein and mRNA, respec-tively. This increase was reduced 86% by furnidipine (anL-type Ca21 channel blocker) but was unaffected by eitherv-conotoxin GVIA (an N-type Ca21 channel blocker) orv-agatoxin IVA (a P/Q-type Ca21 channel blocker). Com-bined blockade of N and P/Q channels with v-conotoxinMVIIC did, however, block by 76% the protein expression.The inhibitory effects of furnidipine were partially reversedwhen the external Ca21 concentration was raised from 1.6to 5 mM. These findings, together with the fact that nicotinicreceptor activation or Ca21 release from internal stores alsoenhanced SNAP-25 protein expression, suggest that anincrement of cytosolic Ca21 concentration ([Ca21]i), ratherthan its source or Ca21 entry pathway, is the critical signalto induce the protein expression. The greater coupling be-tween L-type Ca21 channels and protein expression mightbe due to two facts: (a) L channels contributed 50% to theglobal [Ca21]i rise induced by 38 mM K1 in indo-1-loadedchromaffin cells and (b) L channels undergo less inactiva-tion than N or P/Q channels on sustained stimulation ofthese cells. Key Words: Chromaffin cells—Calciumchannels—SNAP-25.J. Neurochem. 74, 1049–1058 (2000).

The presence of L, N, and P/Q subtypes of high-threshold-activated, voltage-dependent Ca21 channels inneurons (Nowycky et al., 1985; Llina´s et al., 1989;Olivera et al., 1994) poses the interesting question oftheir specialization to control different cell functions. Inneurons, N- and P/Q-type channels are predominantlyfound along the length of apical dendrites; these channelsare found also in axon terminals forming synapses ondendrites (Westenbroek et al., 1992), where they controlthe release of various neurotransmitters (Olivera et al.,1994; Wheeler et al., 1994; Garcı´a et al., 1996). How-ever, L-type channels, predominantly located in the cellbody and proximal dendrites (Ahlijanian et al., 1990;

Westenbroek et al., 1990), have been associated withnonexocytotic functions such as the regulation of geneexpression and enzyme activity in cortical and hip-pocampal neurons (Murphy et al., 1991; Bading et al.,1993; Elliot et al., 1995; Westenbroek et al., 1995; Deis-seroth et al., 1998).

Because of this asymmetric distribution of Ca21 chan-nel subtypes in neurons (soma, dendrites, or axon termi-nals), it is easy to understand such type of specialization.However, in spherical bovine adrenal medullary chro-maffin cells that, like neurons, also express L, N, and P/Qsubtypes of channels (Gandı´a et al., 1993; Albillos et al.,1996; Lomax et al., 1997), it is harder to assign specificfunctions to each channel type only on the basis of adifferent regional distribution. In spite of this limitation,attempts have been made to assign special functions toeach channel subtype, as far as its role in mediating theK1-evoked release of catecholamine is concerned. Thus,Ca21 entering through L- and P/Q-type channels triggersexocytosis, whereas Ca21 entering through N-type chan-nels does not (Lo´pez et al., 1994b). Furthermore, usingvarious Ca21 gradients we provided evidence favoringthe idea that in bovine chromaffin cells, P/Q channels arecloser to secretory vesicles than L channels (Lara et al.,1998).

Considering these data, it seems that bovine chromaf-fin cells are an archetypical model to study whether theelevation of the cytosolic Ca21 concentration ([Ca21]i)induced by Ca21 entering through one or the other Ca21

Received August 3, 1999; revised manuscript received November 5,1999; accepted November 5, 1999.

Address correspondence and reprint requests to Dr. C. Montiel atDepartamento de Farmacologı´a, Facultad de Medicina, UniversidadAutonoma de Madrid, Arzobispo, Morcillo, 4, 28029 Madrid, Spain.E-mail: carmen.montiel@uam.es

Abbreviations used:[Ca21]i, cytosolic Ca21 concentration; [Ca21]o,external Ca21 concentration; DMPP, dimethylphenylpiperazinium;SNAP-25, synaptosomal-associated protein of 25 kDa.

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channel subtype, or on the release of Ca21 from intra-cellular stores, is important to regulate functions unre-lated with the immediate exocytotic event. To explorethis question we studied the expression of synaptosomal-associated protein of 25 kDa (SNAP-25), one of thecritical proteins of the SNAP–receptor complex(SNARE). This protein forms part of the exocytoticmachinery in neurons (Blasi et al., 1993; Binz et al.,1994) and in bovine chromaffin cells (Gutie´rrez et al.,1995, 1997; Foran et al., 1996; Kannan et al., 1996;Lawrence et al., 1997), and its expression is enhanced bythe rise of [Ca21]i on depolarization of hippocampalexplants and PC12 cells (Sepu´lveda et al., 1998). Wehave therefore measured the expression of this proteinafter exposing the chromaffin cells to depolarizing con-centrations of K1 to activate the different subtypes ofCa21 channels; also, the protein levels were determinedon nicotinic receptor activation of the cells with dimeth-ylphenylpiperazinium (DMPP) or with caffeine, whichinduces the release of Ca21 from intracellular stores. Allthese maneuvers produce the rise of [Ca21]i in bovinechromaffin cells, a signal for gene expression in manycell types (Bito et al., 1997; Finkbeiner and Greenberg,1997; Ginty, 1997; Hardingham et al., 1997). Dissectionof the various Ca21 entry pathways has been achievedthrough the use of selective blockers of the differentsubtypes of voltage-dependent Ca21 channels.

EXPERIMENTAL PROCEDURES

Cell culture and treatmentsBovine adrenal chromaffin cells were isolated following

standard methods (Livett, 1984) with some modifications(Moro et al., 1990). Cells were suspended in Dulbecco’s mod-ified Eagle’s medium supplemented with 10% fetal calf serum,50 IU/ml penicillin, and 50mg/ml streptomycin. Cells (1.83 107) were plated on plastic 10-cm-diameter culture plates ata density of 13 106/ml or on 25-mm-diameter glass coverslipsat a density of 2.53 104 cells/ml, for the immunoblot andcytosolic Ca21 experiments, respectively. Cells were kept in awater-saturated incubator at 37°C, in a 5% CO2/95% air atmo-sphere. Treatments with high K1 for SNAP-25 expressionexperiments were carried out in cells after 1 day in culture. Toobtain a final concentration of 38 mM KCl, 2 ml of an osmot-ically balanced solution with a high concentration of K1 (170mM KCl, 0.9 mM CaCl2, 1.3 mM MgCl2, and 10 mM HEPES,pH 7.4) was added to 8 ml of Dulbecco’s modified Eagle’smedium. In all experiments, except when indicated, the finalconcentration of external Ca21 used was 1.6 mM. Control cellswere manipulated as experimental cells, but they were incu-bated in a medium in which high KCl has been replaced byNaCl. For the cytosolic Ca21 experiments, cells after 2–3 daysin culture were used.

Isolation of RNA and northern blot analysisTotal RNA was extracted from chromaffin cells using a

guanidinium-thiocyanate method (Chomczynski and Sacchi,1987). To assess that equal amounts of the two RNA sampleswere loaded onto the gel, the fluorescence intensities of 28Sand 18S ribosomal RNAs were monitored. Northern blot anal-ysis was performed as described previously (Lara et al., 1990)by hybridization with an 881-bp complementary probe synthe-

sized from a mouse SNAP-25 cDNA cloned in theEcoRI siteof pBluescript (Oyler et al., 1989). The RNA probe was ob-tained from 500 ng ofSalI-linearized plasmid using a Promegatranscription kit with T7 polymerase and 100mCi of[a-32P]CTP (New England Nuclear–DuPont, Claremont, CA,U.S.A.). Hybridization was done with 23 106 cpm/10 ml ofthe antisense probe (109 cpm/mg). Membranes were washed,air-dried, and exposed to Kodak Biomax-MS film for 3 days.Autoradiograms were analyzed by densitometry using an Ac-tion Scanner II (Epson) to digitize the image. Results areexpressed as the ratio of SNAP-25 signals in 38 mM K1-treatedcells compared with those in control 38 mM Na1-treated cells.

Immunoblot analysisEqual amounts of cell lysate proteins (typically 70mg of

total proteins) were separated by sodium dodecyl sulfate–poly-acrylamide gel electrophoresis in Mini-Protean electrophoresiscells (Bio-Rad) using the Laemmli buffer system (Laemmli,1970). Proteins were electrophoretically transferred to nitrocel-lulose membranes by standard procedures. Membranes wereincubated with a monoclonal anti-SNAP-25 antibody (Stern-berger Monoclonals) diluted in phosphate-buffered saline (1:1,000) for 1 h at room temperature. After washing, the blotswere incubated for 1 h with a secondary antibody labeled withhorseradish peroxidase, and the immunoblot was developedwith 4-chloro-1-naphthol as substrate. To compare changes inthe expression of SNAP-25, the blots were scanned, and thedensity and area of each band were determined with the NIHImage Version 1.6 program. Results are expressed as mean6 SEM values of the relative increase of SNAP-25 expressionin depolarized cells in relation to control cells (considered as 1).When the effects of the different Ca21 channel blockers onSNAP-25 expression induced by high K1 were considered, thedata are represented as percentages of expression induced bythe depolarizing stimulus in the presence of the blocker, com-pared with the K1-induced expression (defined as 100%).

Cytosolic calcium measurementsChromaffin cells plated on coverslips were incubated with

10mg/ml indo-1 acetoxymethyl ester (Molecular Probes) for 30min at 37°C. Changes in [Ca21]i evoked by different stimuliwere measured as described previously (Grynkiewicz et al.,1985; Sepu´lveda et al., 1998), using a fluorescence invertedmicroscope (Diaphot-200; Nikon Corp.).

RESULTS

High K 1 enhances mRNA and protein levels ofSNAP-25

To examine whether a depolarizing stimulus, whichraises [Ca21]i by opening voltage-dependent Ca21 chan-nels, also elevates SNAP-25 mRNA levels, bovine chro-maffin cells were incubated with 38 mM K1 for 24 h. Inall experiments referred to here, control cells were ma-nipulated like experimental cells, but elevating the NaClinstead of KCl level. Northern blot analysis revealed thepresence of mRNA species of 2.2 kb (Fig. 1A), corre-sponding to SNAP-25 mRNA (Oyler et al., 1989). Therewas a fivefold increase of SNAP-25 mRNA levels inK1-treated cells, compared with control cells of the sameculture. This mRNA level rise was correlated with anincrease of SNAP-25 protein expression. Thus, whenchromaffin cells were incubated with 38 mM KCl for48 h, protein expression increased 2.56 0.2-fold (n5 28

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batches of cells) compared with parallel control cells(Fig. 1B). Figure 1C shows an original immunoblot ofSNAP-25 expression in control and K1-treated cells.

Effects of voltage-dependent Ca21 channel blockerson [Ca21]i transients induced by briefdepolarizations

It was important to be sure that a slight depolarization,such as that produced by 38 mM K1, was able to openthe three Ca21 channel subtypes present in chromaffincells; therefore, the [Ca21]i rise induced by such stimulusin the absence or presence of different Ca21 channelblockers was studied in single indo-1-loaded chromaffincells. To obtain reproducible [Ca21]i signals induced byrepetitive 38 mM K1 pulses, brief stimulation periods(15 s) were applied every 15 min. After two or three

initial K1 pulses, successive depolarizations gave repro-ducible [Ca21]i signals (.1 mM). Addition of v-cono-toxin MVIIC (1 mM), a blocker of non–L-type Ca21

channels (N and P/Q subtypes), 15 min before and duringthe next K1 pulse, halved the [Ca21]i signal. Subsequentaddition of the dihydropyridine furnidipine (3mM), aselective blocker of L-type Ca21 channels, combinedwith v-conotoxin MVIIC practically abolished the K1-induced response. A typical record of the [Ca21]ichanges obtained with this experimental protocol in asingle cell is shown in Fig. 2A. Figure 2B shows aver-aged results from different cells obtained assaying eachCa21 channel blocker in separate cells.v-ConotoxinMVIIC blocked the [Ca21]i signal by 436 3%, furni-

FIG. 2. Effect of different voltage-dependent Ca21 channelblockers on the [Ca21]i rise induced by brief high K1 pulses insingle indo-1-loaded chromaffin cells. To generate [Ca21]i sig-nals, a depolarizing solution containing 38 mM KCl was appliedfor 15 s at 15-min intervals. A: Original record of the [Ca21]iincrements obtained on successive depolarization pulses (blacksquares at the bottom of the panel), in the absence or presenceof v-conotoxin MVIIC (Ctx-MVIIC; 1 mM ). The first [Ca21]i riserepresents the control response, when a stable [Ca21]i peakevoked by the K1 pulse was obtained. At this moment, Ctx-MVIIC alone or combined with furnidipine (3 mM ) was added forthe intervals indicated by the horizontal lines at the bottom of thepanel. B: Inhibitory effect of furnidipine (FURNI; 3 mM ), Ctx-MVIIC (1 mM ), v-conotoxin GVIA (Ctx-GVIA; 1 mM ), and FURNI(3 mM ) plus v-agatoxin IVA (Aga-IVA; 1 mM ) on the [Ca21]i riseevoked by K1 pulses with protocols similar to those described inA. Data, expressed as percent inhibition of the control response,are mean 6 SEM (bars) values obtained in the number of cellsshown in parentheses.

FIG. 1. Effect of a high K1 concentration on SNAP-25 mRNAand protein levels in bovine chromaffin cells. Cells were incu-bated with 38 mM K1 for 24 or 48 h, respectively, for mRNA andprotein determinations. A: A typical autoradiogram (of three) ofan original northern blot. Total RNAs (25 mg) obtained fromcontrol (C) or depolarized (K1) cells were loaded in the first andsecond lane, respectively. The position of the synthetic poly(A)1

RNA markers (2.4 and 1.3 kb) is indicated on the right side of theautoradiogram. B: Relative increment of SNAP-25 protein ex-pression obtained in cells treated with K1 for 48 h comparedwith control cells. ***p , 0.001. Data are mean 6 SEM (bars)values obtained in 28 different batches of cells. C: An originalimmunoblot of SNAP-25 expression obtained from control cells(C) and cells treated with high K1 for 48 h (K1).

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dipine by 516 5%, andv-conotoxin GVIA (1mM), aselective N-type Ca21 channel blocker, by 286 5%.Simultaneous blockade of L- and P/Q-type Ca21 chan-nels with furnidipine plusv-agatoxin IVA (1 mM) re-duced the [Ca21]i signal induced by high K1 by 676 5%. This relative contribution of each Ca21 channelsubtype to the [Ca21]i increase induced by 38 mM K1

differs from previous data concerning the relative densi-ties of Ca21 channel subtypes in bovine chromaffin cells.Thus, using45Ca21 uptake in cells stimulated with 70mM K1 (Villarroya et al., 1997) or depolarizing pulsesfrom 280 mV to 0 mV in voltage-clamped cells (Al-billos et al., 1996), we came to the conclusion that Lchannels account for 20%, N channels for 30%, and P/Qchannels for 50% of the total population of Ca21 chan-nels present in bovine chromaffin cells. In neurons andchromaffin cells, L-type channels are activated at morenegative potentials than non–L-type channels (Carboneet al., 1990; Albillos et al., 1994); also, in chromaffincells N and P/Q subtypes are more prone than the Lsubtype to inactivation at depolarizing voltages (Villar-roya et al., 1999). Thus, the milder depolarization used inthis study (38 mM K1) might condition different gradesof activation/inactivation of the various channel sub-types; in spite of their lower density, L channels had agreater contribution to the [Ca21]i rise under the presentexperimental conditions.

Effects of furnidipine and v-conotoxin MVIIC onexpression of SNAP-25 protein

These experiments were designed to test how Ca21

ions entering through L- or non–L-type Ca21 channelsaffected the increase of SNAP-25 protein expressioninduced by high K1. Cells were incubated with furnidip-ine (3mM) 10 min before and during the entire depolar-ization period (38 mM K1, 48 h). The same was done inthe presence ofv-conotoxin MVIIC (1 mM) or with acombination of furnidipine plusv-conotoxin MVIIC.

Results obtained in different batches of cells areshown in Fig. 3A. In these batches, the relative enhance-ment of the protein expression level induced by high K1,compared with control nondepolarized cells, was 2.96 0.4-fold; this value was defined as 100%. Furnidipineinhibited protein expression by 866 5%. A similar resultwas obtained usingv-conotoxin MVIIC, which caused78 6 5% blockade, or a cocktail of 1mM v-conotoxinGVIA plus 1 mM v-agatoxin IVA, which caused 806 6% blockade (experiment not shown). The incubationof cells with a combination of furnidipine plusv-cono-toxin MVIIC completely prevented the increase of pro-tein expression. One original immunoblot of the effectsof furnidipine and/orv-conotoxin MVIIC on SNAP-25expression, induced by high K1, is shown in Fig. 3B.Note the sharp increase of the band density correspond-ing to K1-treated cells compared with control and itsattenuation on treatment with the various Ca21 channelblockers.

Effects of v-conotoxin GVIA and v-agatoxin IVAon expression of SNAP-25 protein

Simultaneous blockade of the two non–L-type Ca21

channels (N and P/Q) withv-conotoxin MVIIC drasti-cally reduced the expression of SNAP-25 induced bydepolarization. It was of interest to assess the contribu-tion of each of the two non–L channel subtypes to theregulation of protein expression. Thus, experiments sim-ilar to those described above were performed usingv-conotoxin GVIA (1mM) andv-agatoxin IVA (1mM)to block N- or P/Q-type Ca21 channels separately. Inthese batches of cells, 38 mM K1 increased the proteinexpression 1.96 0.1- (n5 4) and 2.06 0.2-fold (n5 3),respectively, in experiments carried out in the presenceof v-conotoxin GVIA andv-agatoxin IVA. Results onthe effects ofv-toxins are shown in Fig. 4; the selective

FIG. 3. Inhibitory effects of furnidipine (FURNI) and v-conotoxinMVIIC (Ctx-MVIIC) on SNAP-25 protein expression induced byhigh K1. Cells were incubated with 38 mM K1 for 48 h in theabsence or presence of furnidipine (3 mM ) or Ctx-MVIIC (1 mM ).In three batches of cells FURNI and Ctx-MVIIC were addedtogether. Blockers were present since 10–20 min before andduring the entire depolarization period (48 h). A: Mean 6 SEM(bars) protein expression values obtained in cells depolarizedwith K1 in the absence or presence of Ca21 channel blockers.The number of experiments done is in parentheses. Values areexpressed as percentages of protein expression induced by highK1 in the absence of blockers (defined as 100%). ***p , 0.001compared with K1 values in the absence of blockers. B: Arepresentative immunoblot of SNAP-25 expression in nondepo-larized (control) and in depolarized chromaffin cells (K1), incu-bated in the absence or the presence of FURNI or Ctx-MVIIC.

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interruption of Ca21 entry through N or P/Q channels didnot modify SNAP-25 expression induced by depolariza-tion. This contrasts with the sharp reduction in proteinexpression when both channel types were blocked byv-conotoxin MVIIC (Fig. 3A) or with a cocktail of 1mMv-conotoxin GVIA plus 1mM v-agatoxin IVA.

Role of extracellular Ca21 on SNAP-25 expressionPrevious data from our group have demonstrated that

raising the external Ca21 concentration ([Ca21]o) en-hanced the [Ca21]i signal generated by depolarization ofchromaffin cells (Montiel et al., 1984; Lo´pez et al.,1994a; Michelena et al., 1995; Lara et al., 1998). There-fore, we performed new experiments to study how theincrease of [Ca21]o affected the SNAP-25 expressioninduced by depolarization in the absence (control) orpresence of different Ca21 channel blockers.

The SNAP-25 expression induced by depolarizationwas enhanced 4.06 0.5-fold (n5 4) in 5 mM [Ca21]o.Such an increase was significantly higher (p , 0.05)than that obtained in 1.6 mM [Ca21]o. Furthermore, theblockade of SNAP-25 expression by furnidipine in low[Ca21]o was partially reversed in 5 mM [Ca21]o. This

result is shown in Fig. 5, where the inhibition by furni-dipine reached 55.66 6% (n5 4), a value significantlylower (p , 0.01) than that obtained in 1.6 mM [Ca21]o(compare Figs. 3 and 5). Also, simultaneous blockade ofL- and N-type Ca21 channels (with furnidipine plusv-conotoxin GVIA) in high [Ca21]o did not preventcompletely the activation of protein expression inducedby depolarization (Fig. 5). We have not performed ex-periments withv-conotoxin MVIIC in high [Ca21]o be-cause the binding of this toxin to its receptor has beendescribed as being markedly reduced by high concentra-tions of divalent cations (Albillos et al., 1996; Lara et al.,1998). It is noteworthy that although the global [Ca21]isignal generated by 38 mM K1 in 5 mM [Ca21]o nearlyduplicated the response in 1.6 mM [Ca21]o, the contri-bution of each Ca21 channel subtype to the global[Ca21]i signal induced by depolarization was similar inboth experimental situations (data not shown).

SNAP-25 expression as a function of thedepolarizing interval

These experiments were designed to determine theminimal duration of the depolarizing stimulus required totrigger SNAP-25 expression. Cells were incubated with38 mM K1 for different times (10, 30, or 60 min or 24 h).At the end of each depolarizing period, Ca21 entry wasimmediately stopped by addition of EGTA (3 mM) to theincubation medium. Ten minutes later, EGTA waswashed out, and the external solution was changed to afresh normal medium. Then, cells were maintained for anadditional interval for a total, in all cases, of 48 h afterinitiation of K1 treatment. A significant increase in pro-

FIG. 5. High-calcium medium partially reversed the blockade byfurnidipine of the SNAP-25 expression induced by 38 mM K1.Cells were depolarized in 5 mM [Ca21]o, in the absence (control)or in the presence of furnidipine (FURNI; 3 mM ) or FURNI plusv-conotoxin GVIA (Ctx-GVIA; 1 mM ). Protein expression in thepresence of the blockers was expressed as a percentage of thecontrol expression induced by high K1 (defined as 100%). Dataare mean 6 SEM (bars) values. The number of experiments doneis in parentheses. ***p , 0.001 compared with K1 values in theabsence of blockers.

FIG. 4. Effects of v-conotoxin GVIA (Ctx-GVIA) and v-agatoxinIVA (Aga-IVA) on SNAP-25 protein expression induced by highK1. Cells were depolarized with 38 mM K1 for 48 h in theabsence or presence of Ctx-GVIA (1 mM ) or Aga-IVA (1 mM ).Each v-toxin was assayed in different cell batches. Toxins werepresent 10–20 min before and during the entire depolarizationperiod. A: Toxin effect expressed as a percentage of the proteinexpression induced by high K1 in the absence of the blocker(defined as 100%). Data are mean 6 SEM (bars) values. Thenumber of experiments done is in parentheses. B: Two repre-sentative immunoblots of SNAP-25 expression in nondepolar-ized [control (C)] and in depolarized chromaffin cells (K1) incu-bated in the absence or the presence of Ctx-GVIA or Aga-IVA.

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tein expression was observed already after a 10-mindepolarization (1.496 0.16-fold, n 5 4; p , 0.05,compared with paired untreated cells). Similar increaseswere obtained in cells preincubated for 30 or 60 min inhigh K1 (1.606 0.08- and 1.586 0.16-fold, respective-ly). However, a 24-h depolarization caused an additionalincrease (2.326 0.21-fold, n5 4) that was statisticallydifferent from the expression values obtained using 10–60-min depolarizing periods (Fig. 6A) but not from thevalue obtained on 48 h of high K1 treatment (Fig. 1B).In Fig. 6B, a representative immunoblot of SNAP-25expression is shown. Observe the different SNAP-25expression levels after different periods of K1 exposure.In this experiment, control cells were treated with highNaCl for 30 min (there were no statistical differencesamong results of expression in control cells incubatedwith NaCl for different intervals; data not shown).

Time course of [Ca21]i changes on sustaineddepolarization of indo-1-loaded chromaffin cells

The activation of SNAP-25 expression was alreadyvisible after depolarizing challenges as short as 10 min.It was therefore interesting to study the time course of[Ca21]i changes along a prolonged depolarizing stimu-

lus, in either the absence or the presence of various Ca21

channel blockers. Figure 7A shows the time course of[Ca21]i rises in cells depolarized with 38 mM K1 during60 min. During the first 10 min, a transient initial Ca21

spike (1,237 nM) was followed by a plateau (200 nM)that lasted the 60-min exposure to K1. In the presence ofv-conotoxin MVIIC (1 mM), a similar transient [Ca21]irise followed by a plateau phase was observed. However,the peak amplitude and the area of the [Ca21]i transientresponse during the first 10 min of depolarization werelower (54 and 65%, respectively) than those obtained inthe absence of toxin. Another interesting differencefound was that the plateau phase only lasted 20 min,declining slowly to reach the basal [Ca21]i (Fig. 7B). Inthe presence of furnidipine plusv-conotoxin GVIA,either the peak or the area of the [Ca21]i signal inducedby K1 during the first 10 min was smaller (27 and 40%of those obtained in cells depolarized in the absence ofblockers; Fig. 7C). Finally, in the presence of furnidipineplus v-agatoxin IVA, the peak and the [Ca21]i areaduring the first 10 min were 33 and 45% of those ob-tained in depolarized cells in the absence of blockers(Fig. 7D).

Cytosolic [Ca21]i rise and SNAP-25 expressioninduced by caffeine and DMPP

We have also studied the expression of SNAP-25 byraising [Ca21]i with caffeine, which induces the releaseof Ca21 from internal stores, and the nicotinic receptoragonist DMPP, which activatesa3b4 and a7 neuronalnicotinic acetylcholine receptors in chromaffin cells(Lopez et al., 1998). Figure 8 shows two traces of the[Ca21]i signals induced by caffeine and DMPP. Treat-ment of indo-1-loaded cells with caffeine (10 mM) for 30min produced a [Ca21]i peak with an amplitude around350 nM. The area under the curve (first 10 min ofstimulation) was 27% of that obtained in high K1-stim-ulated cells. The response to caffeine declined slowlyand reached basal levels in 15 min (Fig. 8A). DMPP (20mM) evoked a [Ca21]i signal whose peak amplitudereached a value close to that obtained in high K1-treatedcells; however, the [Ca21]i signal induced by DMPP wastransient (lasting only 13 min) and was not followed bya plateau phase as with K1 (Fig. 8B).

Figure 9A shows mean6 SEM values of SNAP-25expression induced by caffeine and DMPP. Expressionof SNAP-25 in chromaffin cells incubated for 48 h withcaffeine (10 mM) or DMPP (20mM) was increased 1.46 0.1- (p , 0.05) and 2.66 0.2-fold (p , 0.005),respectively, compared with parallel control cells. Whenthe expression induced by DMPP was compared withthat produced by 38 mM K1 in cells from the sameculture day, no significant differences were found be-tween these two stimuli. However, the protein levelinduced by caffeine was significantly lower than thatevoked by high K1 in cells belonging to the same cul-ture. Figure 9B shows two typical immunoblots obtainedin two different batches of cells treated with caffeine orDMPP for 48 h. For the sake of comparison, two samples

FIG. 6. Time course of expression of SNAP-25 protein. Cellswere depolarized with 38 mM K1 for 10, 30, or 60 min or 24 h asdescribed in Results. Protein expression was determined 48 hafter addition of K1 began. A: Mean 6 SEM (bars) values ofprotein expression induced by each depolarization period ob-tained from the number of experiments shown in parentheses. *p, 0.05, **p , 0.01, ***p , 0.001 compared with control, non-depolarized cells; †p , 0.05, ††p , 0.01 compared with 24 h ofK1 treatment. B: A typical immunoblot under these experimentalconditions. Control (C) cells were treated with NaCl for 30 min.

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from parallel cells, untreated (control) or treated with 38mM K1 for 48 h, were also assayed.

DISCUSSION

Exposure of bovine chromaffin cells to a high K1

medium for different intervals led to an increase ofSNAP-25 protein expression. This is a Ca21-dependentphenomenon because blockade of Ca21 entry during celldepolarization suppressed SNAP-25 expression. Regula-tion of gene transcription and protein expression trig-gered by enhanced Ca21 entry and the subsequent in-crease of [Ca21]i is well established in neurons (Murphyet al., 1991; Bading et al., 1993; Bito et al., 1997; Ginty,1997; Hardingham et al., 1997) and in PC12 cells (Me-nezes et al., 1996; Sepu´lveda et al., 1998). However, it isunclear whether such regulation by Ca21 is selectivelycoupled to a given Ca21 entry pathway and/or a Ca21

source, i.e., extracellular Ca21 entry versus Ca21 mobi-lization from intracellular stores.

As far as the Ca21 entry pathway is concerned, wefound that using a mild depolarization stimulus (38 mMK1 in 1.6 mM [Ca21]o), the selective blockade of L-typeCa21 channels suppressed SNAP-25 expression, whereasthe separate blockade of N- or P/Q-type channels did not.A priori, this observation might suggest that L-type Ca21

channels are tightly coupled to the protein expression,whereas N- or P/Q-type channels are not. However, thefact that simultaneous blockade of N- plus P/Q-typechannels markedly reduced the protein expression to an

FIG. 7. Increase in [Ca21]i levelsgenerated by prolonged exposureof cells to high K1. Indo-1-loadedchromaffin cells were stimulatedwith 38 mM K1 for 60 min, (A) inthe absence of Ca21 channelblockers or in the presence of (B)1 mM v-conotoxin MVIIC (Ctx-MVIIC), (C) 3 mM furnidipine plus1 mM v-conotoxin GVIA (Ctx-GVIA), or (D) 3 mM furnidipine plus1 mM v-agatoxin IVA (Aga-IVA).Blockers were added 10 min be-fore and during all the K1 stimu-lation period. Traces are aver-aged from three different records.

FIG. 8. Cytosolic [Ca21]i rise generated by prolonged stimula-tion with (A) caffeine (10 mM, 30 min) or (B) DMPP (20 mM, 30min) added to single indo-1-loaded chromaffin cells. Traces areaverages of four different records.

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1055CALCIUM PATHWAYS AND SNAP-25 EXPRESSION

extent similar to that obtained on blocking of L-typechannels (78% vs. 86%) casts doubts to this hypothesis.

Rather than a specialized Ca21 entry route, our resultssuggest that Ca21-activated SNAP-25 expression inchromaffin cells seems to be related more to quantitativeaspects of the [Ca21]i signals generated on depolariza-tion. Thus, a threshold value of [Ca21]i seems to be thecritical signal to trigger SNAP-25 expression; under thisvalue there is no activation of protein synthesis. Thisview is supported by two findings: (a) the need of block-ing .45–50% of the Ca21 entry, either with furnidipine(L-type Ca21 channels blockade) or withv-conotoxinMVIIC (N- plus P/Q-type channels blockade), to inhibitthe protein expression; and (b) the absence of an effecton the SNAP-25 expression of the separate blockade ofN channels withv-conotoxin GVIA or P/Q channelswith v-agatoxin IVA. These last maneuvers did notprevent the [Ca21]i signal from reaching the criticalvalue necessary to activate the protein expression. Incontrast, the threshold [Ca21]i required to induce theprotein expression was not reached when both non–L-type Ca21 channels were simultaneously inhibited withv-conotoxin MVIIC or when Ca21 influx through L-typeCa21 channels was interrupted with furnidipine. That the

effect of v-conotoxin MVIIC on SNAP-25 expressionwas the result of a specific blockade of non–L-type Ca21

channels, and not the consequence of a cumulative effecton L-type channels, could be proved by the fact thatv-conotoxin GVIA plusv-agatoxin IVA, added simul-taneously to block non–L-type Ca21 channels, also pro-duced the same effect as that observed withv-conotoxinMVIIC.

Our finding of the greater coupling between L-typeCa21 channels and SNAP-25 expression might be theresult of two facts: (a) These channels contribute by 50%to the global [Ca21]i rise induced by 38 mM K1, and (b)L-type Ca21 channels suffer less inactivation than non–L-type channels, as we have recently demonstrated onprolonged depolarization of bovine chromaffin cells(Villarroya et al., 1999). Thus, it is understandable thatunder the present experimental conditions (cell depolar-ization lasting minutes to hours), Ca21 entry throughnoninactivating L-type Ca21 channels might provide abetter signal for gene expression than Ca21 entry throughinactivating N- or P/Q-type channels.

According to our hypothesis, the only requirement totrigger the Ca21-dependent SNAP-25 expression inchromaffin cells is that the Ca21 influx through a givenCa21 channel provides a sufficient threshold [Ca21]isignal. If this is true, then the blockade of protein ex-pression observed in our experiments, when L-type chan-nels were blocked in 1.6 mM [Ca21]o, should be reversedby a higher [Ca21]o and the consequent increase of Ca21

influx through the other two channel subtypes (N andP/Q). In fact, this is what happened when cells weretreated with 38 mM K1 in 5 mM [Ca21]o; under theseexperimental conditions the blockade of protein expres-sion by furnidipine in low [Ca21]o was partially reversed(compare Figs. 3 and 5). Because the [Ca21]i signal andprotein expression evoked by 38 mM K1 in high [Ca21]oalmost doubled those obtained in low [Ca21]o, this lastmaneuver allows, in the presence of furnidipine, a largerCa21 influx through non–L-type channels. Another in-teresting finding was that the Ca21 entry through P/Q-type channels (on blockade of L- and N-channels) wasstill able to activate the protein expression in high[Ca21]o (see Fig. 5). That the Ca21 entry pathway orCa21 source does not condition the Ca21-dependentSNAP-25 expression is further supported by the caffeineexperiments; just by promoting Ca21 release from intra-cellular Ca21 stores, caffeine was able to enhanceSNAP-25 expression.

Because the SNAP-25 expression evoked by depolar-ization of chromaffin cells seems to be a Ca21-activatedprocess, we tried to correlate the amplitude and durationof the [Ca21]i signal generated by a prolonged depolar-izing stimulus with the time course of the protein expres-sion levels. Our results show that 38 mM K1, applied for1 h, evoked a biphasic [Ca21]i response that consisted ofan initial vigorous peak that subsequently declined over10 min to a sustained low plateau during the entirestimulation period. The time course of SNAP-25 expres-sion revealed two peaks according with the duration of

FIG. 9. Expression of SNAP-25 protein in cells stimulated withcaffeine (CAFF) or DMPP. Effects of CAFF (10 mM, 48 h) orDMPP (20 mM, 48 h) on protein expression were assayed inseparate batches of cells. In parallel with each of these stimuli,expression induced by 38 mM K1 was also determined. A: Mean6 SEM (bars) relative values obtained in high K1-, DMPP-, andCAFF-treated cells compared with parallel nontreated cells (con-trol). *p , 0.05, ***p , 0.001 compared with control. B: Twotypical immunoblots of the CAFF or DMPP effects. C, control.

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1056 E. GARCI´A-PALOMERO ET AL.

the depolarizing stimulus: The first expression peak wasseen after depolarization periods of 10–60 min, whereasthe second peak was observed on longer K1 exposureperiods (24–48 h). Because the first protein peak wasalready obtained with a depolarization period of 10 min,most probably it corresponds with the activation of asignaling pathway that would require a high threshold of[Ca21]i; this level could be reached during the transient[Ca21]i phase induced by depolarization. The secondpeak of SNAP-25 expression would be the result of theactivation of a second signaling pathway by lower andprolonged [Ca21]i levels, at.1 h, which could be pro-vided by the plateau phase of the late [Ca21]i response.In fact, a similar observation has been described in Blymphocytes, where two phases (transient and plateau) inthe [Ca21]i response induced by chicken egg lysozymeantigen were able to control, differentially, the activationof several Ca21-sensitive proinflammatory transcrip-tional regulators (Dolmetsch et al., 1997). However, wecannot exclude that the second peak of SNAP-25 expres-sion observed on prolonged depolarization of chromaffincells may be the result of the inhibition of physiologicalmechanisms, i.e., dephosphorylation of transcription fac-tors, that down-regulate gene transcription, as proposedafter prolonged periods of synaptic input in neurons(Bito et al., 1997).

In this context, we believe that the regulation by Ca21 ofSNAP-25 expression must surely take place at the transcrip-tion level because the SNAP-25 mRNA level was alsoincreased on depolarization. However, the transcriptionalpathway that, when activated by the Ca21 entry, conveysthe signal to the nucleus to stimulate the SNAP-25 genetranscription in depolarized chromaffin cells remains to beelucidated. Furthermore, our results suggest that the in-crease of SNAP-25 protein levels by Ca21 could be phys-iologically relevant because a selective agonist for nicotinicreceptors, DMPP, which mimics the effect of endogenouslyreleased acetylcholine on splanchnic nerve stimulation inthe intact adrenal gland, also augmented SNAP-25 expres-sion. As SNAP-25 is a critical protein of the exocytoticmachinery in bovine chromaffin cells (Gutie´rrez et al.,1995, 1997; Foran et al., 1996; Lawrence et al., 1997), itsinduction during cell activation might have important im-plications for the regulation of the rate and extent of exo-cytosis. This result adds a new member to the list ofproteins implicated in catecholamine release that are up-regulated on activation of secretion in PC12 and chromaffincells. These proteins include not only the granule-secretedproteins chromogranin A (Tang et al., 1996) and proen-kephalin A (Stachowiak et al., 1990), but also the catechol-amine biosynthetic enzymes such as tyrosine hydroxylase,phenylethanolamine-N-methyltransferase, and dopamine-b-hydroxylase (Kilbourne et al., 1992; Hiremagalur et al.,1993; Nankova et al., 1993).

In conclusion, the results of this study demonstratethat on depolarization of chromaffin cells, Ca21 entrythrough different voltage-dependent Ca21 channel sub-types was able to regulate the expression of SNAP-25protein. In these cells, the amount of Ca21 entering the

cell at a given time, rather than a specialized Ca21

channel subtype or Ca21 source, seems to be the criticalsignal to trigger the expression of SNAP-25. This is thefirst time that Ca21 channels of the P/Q and N subtypeshave been involved in the regulation of gene transcrip-tion. At this point, an interesting question is whether theincrease of SNAP-25 protein content following depolar-ization of chromaffin cells modifies the late functionalefficacy of the secretory machinery.

Acknowledgment: This work was supported by grant1991018 from FONDECYT (Chile) to A.C., grant PB97-0047from DGES to C.M., and grant PB94-0150 from DGICYT(Spain) to A.G.G. The cDNA used as a template to synthesizethe SNAP-25 cRNA probe was a gift from Dr. Michael C.Wilson (Scripps Research Institute, La Jolla, CA, U.S.A.). Wewant to thank Mr. Ricardo de Pascual for excellent preparationof cells.

REFERENCES

Ahlijanian M. K., Westenbroek R. E., and Catterall W. A. (1990)Subunit structure and localization of dihydropyridine-sensitivecalcium channels in mammalian brain, spinal cord, and retina.Neuron4, 819–832.

Albillos A., Artalejo A. R., Lopez M. G., Gandı´a L., Garcı´a A. G., andCarbone E. (1994) Calcium channel subtypes in cat chromaffincells.J. Physiol. (Lond.)477,197–213.

Albillos A., Garcıa A. G., Olivera B., and Gandı´a L. (1996) Re-evaluation of the P/Q Ca21 channel components of Ba21 currentsin bovine chromaffin cells superfused with solutions containinglow and high Ba21 concentrations.Pflugers Arch.432, 1030–1038.

Bading H., Ginty D., and Greenberg M. (1993) Regulation of geneexpression in hippocampal neurons by distinct calcium signalingpathways.Science260,181–186.

Binz T., Blasi J., Yamasaki S., Baumeister A., Link E., Sudhof T. C.,Jahn R., and Niemann H. (1994) Proteolysis of SNAP-25 by typesE and A botulinal neurotoxins.J. Biol. Chem.269,1617–1620.

Bito H., Deisseroth K., and Tsien R. W. (1997) Ca21-dependentregulation in neuronal gene expression.Curr. Opin. Neurobiol.7,419–429.

Blasi J., Chapman E. R., Link E., Binz T., Yamasaki S., De Camilli P.,Sudhof T. C., Niemann H., and Jahn R. (1993) Botulinum neuro-toxin A selectively cleaves the synaptic protein SNAP-25.Nature365,160–163.

Carbone E., Sher E., and Clementi F. (1990) Ca21 currents in humanneuroblastoma IMR32 cells: kinetics, permeability and pharma-cology.Pflugers Arch.416,170–179.

Chomczynski P. and Sacchi N. (1987) Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroform ex-traction.Anal. Biochem.162,156–159.

Deisseroth K., Heist E. K., and Tsien R. W. (1998) Translocation ofcalmodulin to the nucleous supports CREB phosphorylation inhippocampal neurons.Nature392,198–202.

Dolmetsch R. E., Lewis R. S., Goodnow C. C., and Healy J. I. (1997)Differential activation of transcription factors induced by Ca21

response amplitude and duration.Nature386,855–858.Elliott E. M., Malouf A. T., and Catterall W. A. (1995) Role of calcium

channel subtypes in calcium transients in hippocampal CA3 neu-rons.J. Neurosci.15, 6433–6444.

Finkbeiner S. and Greenberg M. E. (1997) Spatial features of calcium-regulated gene expression.Bioessays19, 657–660.

Foran P., Lawrence G. W., Shone C. C., Foster K. A., and Dolly J. O.(1996) Botulinum neurotoxin C1 cleaves both syntaxin andSNAP-25 in intact and permeabilized chromaffin cells: correlationwith its blockade of catecholamine release.Biochemistry35,2630–2636.

J. Neurochem., Vol. 74, No. 3, 2000

1057CALCIUM PATHWAYS AND SNAP-25 EXPRESSION

Gandı´a L., Albillos A., and Garcı´a A. G. (1993) Bovine chromaffincells possess FTX-sensitive calcium channels.Biochem. Biophys.Res. Commun.194,671–676.

Garcıa A. G., Albillos A., Gandı´a L., Lopez M. G., Michelena P., andMontiel C. (1996)v-Toxins, calcium channels and neurosecretion, inCellular and Molecular Mechanisms of Toxin Action: Toxins andSignal Transduction(Lazarovici P. and Gutman Y., eds), pp. 155–209. Harwood Academic Publishers, Chur, Switzerland.

Ginty D. D. (1997) Calcium regulation of gene expression: isn’t thatspatial?Neuron18, 183–186.

Grynkiewicz G., Poenie M., and Tsien R. Y. (1985) A new generationof Ca21 indicators with greatly improved fluorescence properties.J. Biol. Chem.260,3440–3450.

Gutierrez L. M., Canaves J. M., Ferrer-Montiel A. V., Reig J. A.,Montal M., and Viniegra S. (1995) A peptide that mimics thecarboxy-terminal domain of SNAP-25 blocks (Ca21)-dependentexocytosis in chromaffin cells.FEBS Lett.372,39–43.

Gutierrez L. M., Viniegra S., Rueda J., Ferrer-Montiel A. V., CanavesJ. M., and Montal M. (1997) A peptide that mimics the C-terminalsequence of SNAP-25 inhibits secretory vesicle docking in chro-maffin cells.J. Biol. Chem.272,2634–2639.

Hardingham G. E., Chawla S., Johnson C. M., and Badin H. (1997)Distinct functions of nuclear and cytoplasmic calcium in thecontrol of gene expression.Nature385,260–265.

Hiremagalur B., Nankova B., Nitahara J., Zeman R., and Sabban E. L.(1993) Nicotine increases expression of tyrosine hydroxylasegene. Involvement of protein kinase A-mediated pathway.J. Biol.Chem.268,23704–23711.

Kannan R., Grant N. J., Aunis D., and Langley K (1996) SNAP-25 isdifferentially expressed by noradrenergic and adrenergic chromaf-fin cells.FEBS Lett.385,159–164.

Kilbourne E. J., Nankova B. B., Lewis E. J., McMahon A., Osaka H.,Sabban D. B., and Sabban E. L. (1992) Regulated expression ofthe tyrosine hydroxylase gene by membrane depolarization. Iden-tification of the responsive elements and possible second messen-gers.J. Biol. Chem.267,7563–7569.

Laemmli U. K. (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4.Nature227,680–685.

Lara B., Gandı´a L., Martınez-Sierra R., Torres A., and Garcı´a A. G.(1998) Q-type Ca21 channels are located closer to secretory sitesthan L-type channels: functional evidence in chromaffin cells.Pflugers. Arch.435,472–478.

Lara H. E., Hill D. F., Katz K. H., and Ojeda S. R. (1990) The geneencoding nerve growth factor is expressed in the immature ratovary: effect of denervation and hormonal treatment.Endocrinol-ogy 126,357–363.

Lawrence G. W., Foran P., Mohammed N., DasGupta B. R., and DollyJ. O. (1997) Importance of two adjacent C-terminal sequences ofSNAP-25 in exocytosis from intact and permeabilized chromaffincells revealed by inhibition with botulinum neurotoxins A and E.Biochemistry36, 3061–3067.

Livett B. G. (1984) Adrenal medullary chromaffin cells in vitro.Physiol. Rev.64, 1103–1161.

Llinas R., Sugimori M., Lin J. W., and Cherksey B. (1989) Blockingand isolation of a calcium channel from neurons in mammals andcephalopods utilizing a toxin fraction (FTX) from funnel-webspider poison.Proc. Natl. Acad. Sci. USA86, 1689–1693.

Lomax R. B., Michelena P., Nu´nez L., Garcı´a-Sancho J., Garcı´a A. G.,and Montiel C. (1997) Different contributions of L- and Q-typeCa21 channels to Ca21 signals and secretion in chromaffin cellsubtypes.Am. J. Physiol.272,C476–C484.

Lopez M. G., Albillos A., De la Fuente M. T., Borges R., Gandı´a L.,Carbone E., Garcı´a A. G., and Artalejo A. R. (1994a) LocalizedL-type calcium channels control exocytosis in cat chromaffincells.Pflugers Arch.427,348–354.

Lopez M. G., Villarroya M., Lara B., Martı´nez-Sierra R., Albillos A.,Garcıa A. G., and Gandı´a L. (1994b) Q- and L-type Ca21 channelsdominate the control of secretion in bovine chromaffin cells.FEBSLett. 349,331–337.

Lopez M. G., Montiel C., Herrero C. J., Garcı´a-Palomero E., MayorgasI., Hernandez-Guijo J. M., Villarroya M., Olivares R., Gandı´a L.,McIntosh M., Olivera B. M., and Garcı´a A. G. (1998) Unmasking

the functions of the chromaffin cell alpha7 nicotinic receptor byusing short pulses of acetylcholine and selective blockers.Proc.Natl. Acad. Sci. USA95, 14184–14189.

Menezes A., Zeman R., and Sabban E. (1996) Involvement of intracellularor extracellular calcium in activation of tyrosine hydroxylase geneexpression in PC12 cells.J. Neurochem.67, 2316–2324.

Michelena P., Vega T., Montiel C., Lo´pez M. G., Garcı´a-Perez L. E.,Gandı´a L. and Garcı´a A. G. (1995) Effects of tyramine and calciumon the kinetics of secretion in intact and electroporated chromaffincells superfused at high speed.Pflugers Arch.431,283–296.

Montiel C., Artalejo A. R., and Garcı´a A. G. (1984) Effects of the noveldihydropyridine Bay-K-8644 on adrenomedullary catecholaminerelease evoked by calcium reintroduction.Biochem. Biophys. Res.Commun.120,851–857.

Moro M. A., Lopez M. G., Gandı´a L., Michelena P., and Garcı´a A. G.(1990) Separation and culture of living adrenaline- and noradren-aline-containing cells from bovine adrenal medullae.Anal. Bio-chem.185,243–248.

Murphy T. H., Worley P. F., and Baraban J. M. (1991) L-type voltage-sensitive calcium channels mediate synaptic activation of imme-diate early genes.Neuron7, 625–635.

Nankova B., Devlin D., KvetnˇanskyR., Kopin I. J., and Sabban E. L.(1993) Repeated immobilization stress increases the binding ofc-Fos-like proteins to a rat dopamineb-hydroxylase promoterenhancer sequence.J. Neurochem.61, 776–779.

Nowycky M. C., Fox A. P., and Tsien R. W. (1985) Three types ofneuronal calcium channel with different calcium agonist sensitiv-ity. Nature316,440–443.

Olivera B. M., Miljanich G. P., Ramachandran J., and Adams M. E.(1994) Calcium channel diversity and neurotransmitter release:the omega-conotoxins and omega-agatoxins.Annu. Rev. Biochem.63, 823–867.

Oyler G. A., Higgins G. A., Hart R. A., Battemberg E., Billingsley M.,Bloom F. E., and Wilson M. C. (1989) The identification of a novelsynaptosomal-associated protein, SNAP-25, differentially expressedby neuronal subpopulations.J. Biol. Chem.109,3039–3052.

Sepulveda C. M., Troncoso C. C., Lara H., and Ca´rdenas A. M. (1998)Intracellular calcium and arachidonic acid increase SNAP-25 ex-pression in cultured rat hippocampal explants, but not in culturedrat cerebellar explants.Neurosci. Lett.252,127–130.

Stachowiak M. K., Hong J. S., and Viveros O. H. (1990) Coordinateand differential regulation of phenylethanolamineN-methyltrans-ferase, tyrosine hydroxylase and proenkephalin mRNAs by neuraland hormonal mechanisms in cultured bovine adrenal medullarycells.Brain Res.510,277–288.

Tang K., Wu H., Mahata S. K., Taupenot L., Rozansky D. J., Palmer R. J.,and O’Connor D. T. (1996) Stimulus–transcription coupling in pheo-chromocytoma cells. Promoter region-specific activation of chromo-granin A biosynthesis.J. Biol. Chem.271,28382–28390.

Villarroya M., de la Fuente M. T., Lo´pez M. G., Gandı´a L., and Garcı´aA. G. (1997) Distinct effects of omega-toxins and various groups ofCa21-entry inhibitors on nicotinic acetylcholine receptor and Ca21

channels of chromaffin cells.Eur. J. Pharmacol.320,249–257.Villarroya M., Olivares R., Ruı´z A., Cano-Abad M. F., Pascual R.,

Lomax R., Lopez M. G., Mayorgas I., Gandı´a L., and Garcı´a A. G.(1999) Voltage inactivation of Ca21 entry and secretion associatedwith N- and P/Q-type but not L-type Ca21 channels of bovinechromaffin cells.J. Physiol. (Lond.)516,421–432.

Westenbroek R. E., Ahlijanian M. K., and Catterall W. A. (1990)Clustering of L-type Ca21 channels at the base of major dendritesin hippocampal pyramidal neurons.Nature347,281–284.

Westenbroek R. E., Hell J. W., Warner C., Dubel S. J., Snutch T. P.,and Catterall W. A. (1992) Biochemical properties and subcellulardistribution of an N-type calcium channel alpha 1 subunit.Neuron9, 1099–1115.

Westenbroek R. E., Sakurai T., Elliot E. M., Hell J. W., Starr T. V. B.,Snutch T. P., and Caterall W. A. (1995) Immunochemical identi-fication and subcellular distribution of the alpha 1A subunits ofbrain calcium channels.J. Neurosci.15, 6403–6418.

Wheeler D. B., Randall A., and Tsien R. W. (1994) Roles of N-type andQ-type Ca21 channels in supporting hippocampal synaptic trans-mission.Science264,107–111.

J. Neurochem., Vol. 74, No. 3, 2000

1058 E. GARCI´A-PALOMERO ET AL.