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NEUROPHARMACOLOGY

Nifedipine-Mediated Mobilization of Intracellular Calcium Stores Increases Spontaneous Neurotransmitter Release at Neonatal Rat Motor Nerve Terminals

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Celular y Molecular and Instituto de Fisiología, Biología Molecular y Neurociencias-Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Received March 11, 2003; accepted April 16, 2003.

	Abstract

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The modulation of spontaneous release of acetylcholine by specific Ca²⁺ channel blockers was studied at neonatal rat neuromuscular junction. During early postnatal periods (0–4 days), blockers of N- and P/Q-type Ca²⁺ channels did not affect miniature endplate potential (MEPP) frequency. Unexpectedly, treatment with the L-type Ca²⁺ channel antagonist nifedipine, although not when treated with isradipine, nitrendipine, or calciseptine, resulted in strong increase in MEPP frequency. The potentiation effect of nifedipine was dose-dependent with a 56-fold maximum effect with 15 µM. The effect decreased during the first two postnatal weeks and disappeared by the third. The effect of nifedipine was not dependent on extracellular Ca²⁺ and was not altered by the presence of other Ca²⁺ channel blockers. In contrast, it was abolished by depleting intracellular Ca²⁺ stores with 2 µM thapsigargin and was partially inhibited by 10 µM ryanodine. In conclusion, we report a new ryanodine receptor-mediated effect of nifedipine on neonatal neuromuscular junction that may indicate the developmental expression of a specific receptor channel that interacts with intracellular Ca²⁺ stores. This effect of nifedipine should also be considered when using this drug as either a therapeutic or a research tool.

Different types of VDCCs support neurotransmitter release at many synapses (Uchitel, 1997). At mature mammalian neuromuscular junctions (NMJs), evoked release is mediated by VDCCs of the P/Q-type (Uchitel et al., 1992; Protti and Uchitel, 1993; Katz et al., 1997), whereas spontaneous release is partially inhibited by N- and L-type VDCCs blockers (Losavio and Muchnik, 1997; Protti et al., 1991). At neonatal rat phrenic-diaphragm neuromuscular junctions (NMJs), evoked transmitter release is mediated by N- and P/Q-type VDCCs (Rosato Siri and Uchitel, 1999), but is inhibited through mechanisms that involve L-type VDCCs (Sugiura and Ko, 1997). Developmental changes observed in the role of VDCCs on evoked transmitter release (Rosato Siri and Uchitel, 1999) prompted us to search for similar changes on the regulation of spontaneous release. For this purpose, we have studied the modulation of the frequency of spontaneous release by specific VDCCs blockers during neonatal period.

We found that neither N-nor P/Q-type VDCCs blockers affect spontaneous release. In contrast, the L-type VDCC blocker nifedipine induces strong increase of spontaneous release mediated by a novel effect on intracellular Ca²⁺ stores.

	Materials and Methods

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Animals and Nerve Muscle Preparation. Experiments were carried out on phrenic-diaphragm preparation of 0- to 22-day-old Sprague-Dawley rats. At all stages, animals were cared for in accordance with national guidelines for the humane treatment of laboratory animals, which are as protective as those of the National Institutes of Health. Rats were anesthetized with ether and immediately exsanguinated. The muscle with its nerve supply was excised and dissected on a Sylgard-coated Petri dish containing normal mammalian solution consisting of 137 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 12 mM NaHCO₃, 1 mM Na₂HPO₄, and 11 mM glucose, and continuously bubbled with 95% O₂, 5% CO₂, pH 7.2 to 7.3.

Electrophysiological Recordings. The preparation was transferred to a recording chamber (volume 1 ml) to which different solutions and drugs were applied. Experiments were performed at room temperature. Miniature endplate potentials (MEPPs) were recorded intracellularly with conventional glass microelectrodes filled with 3 M KCl (20–30-M resistance). Spontaneous activity in each fiber was recorded for at least 2 min. Records were rejected when over 5-mV change of membrane potential was observed during acquisition.

MEPP frequency was evaluated in a saline solution composed of 137 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM CaCl₂, 1 mM MgSO₄, and 11 mM glucose (recording solution), and continuously bubbled with O₂, pH 7.2 to 7.3. Raising the Ca²⁺ concentration to at least twice its normal level increased the stability of the recording (Redfern, 1970; Dennis et al., 1981). In this article, MEPP frequency was expressed as number of MEPPs per minute. Normalized MEPP frequency expressed in Figs. 2 to 4 and in Table 1 was obtained from the mean MEPP frequency of each treatment divided by the mean MEPP frequency of their own control (in the absence of any drug).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Developmental changes of basal MEPP frequency and nifedipine increased MEPP frequency. A, plot shows the relationship between age and MEPP frequency. Each point represents the mean ± S.E.M. of data pooled from at least two muscles (at least 12 endplates/muscle) from animal's different ages. The least-squares fit was obtained by nonlinear regression according to a two parameters sigmoidal function (r = 0.98, a = 38.27, b = 1.89, x0 = 16.12) (see Materials and Methods). B, time course plot of nifedipine effect on MEPP frequency during development (0–22-day-old rats). Nifedipine effect has a negative correlation with age (r = –0.68, p < 0.0001). The strongest potentiation effect of nifedipine was found at postnatal day 6 (151.58 ± 21.31; n = 32, 2), and it decreased as development proceeded, up to disappearance at postnatal day 22 (1.33 ± 0.15; n = 22, 2). Each point represents mean ± S.E.M. of nifedipine-treated muscles at indicated ages.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Increased MEPP frequency by nifedipine involves ryanodine-sensitive calcium stores. A, nifedipine and Bay-K effects were evaluated in zero Ca2+ solution. Nifedipine strongly increases MEPPs frequency in zero calcium solution from 0.68 ± 0.08 (n = 17, 2) to 25.09 ± 7.21 (n = 19, 2). In contrast, Bay-K potentiation is very weak but significant, and MEEPs frequency increases from 1.0 ± 0.14/min (n = 22, 3) to 1.63 ± 0.33/min (n = 28, 3). *, p < 0.05 compared with control. **, p < 0.0001 compared with control. B, nifedipine effect was blocked by depletion of intracellular calcium stores. Muscles were incubated in zero Ca2+ solution in the presence of 2 µM thapsigargin + 2 mM caffeine for 1 h and then bathed in the recording solution (10 mM Ca2+) in presence of nifedipine + thapsigargin (b) or thapsigargin only (a). Thapsigargin + nifedipine-treated muscles did not show higher MEEP frequency (2.62 ± 0.68 MEPPs/min; n = 38, 4) than the one for thapsigargin-treated muscles (6.50 ± 1.92 MEPPs/min; n = 29, 2). Indeed, muscles treated with nifedipine + thapsigargin showed lower MEPP frequency than that of muscles treated with thapsigargin only. If thapsigargin is omitted (c), the effect of nifedipine is present and MEPP frequency increases significantly (80.26 ± 10.39 MEPPs/min; n = 24, 2). **, p < 0.0001 compared with thapsigargin-treated muscles. ##, p < 0.0001 compared with thapsigargin + nifedipine-treated muscles. C, ryanodine blocked nifedipine effect. Nifedipine increases significantly MEPP frequency (22.12 ± 3.45-fold; n = 35, 3). This increase was partially reverted by addition of 10 µM ryanodine (3.63 ± 0.84; n = 38, 3). Ryanodine alone slightly but significantly decreases MEPP frequency (0.88 ± 0.17-fold; n = 23, 2). *, p < 0.05 compared with control fibers. **, p < 0.0001 compared with control fibers. ##, p < 0.0001 compared with nifedipine + ryanodine-treated fibers.

To study MEPP frequency in a nominal zero Ca²⁺ solution, MEPPs were recorded in a solution composed of 137 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM EGTA, 2 mM MgSO₄, and 11 mM glucose.

Drugs were applied to the recording chamber solution and incubated for 1 h. When using dihydropyridines (DHPs), experiments were performed with extreme care to minimize exposure of drug solutions to light. Moreover, stability of nifedipine during the experiment was tested by measuring UV absorbance at 280, 310, and 360 nm before and after the experiment by a UV-visible ChemStation (Hewlett Packard, Palo Alto, CA). Nifedipine photoxidization was achieved by 4-h exposure to strong visible light (Majeed et al., 1987) and checked by measuring UV absorbance.

In Fig. 2A, data were fitted to a two-parameter sigmoidal function of the following form.

Statistics. Values were expressed as mean ± S.E.M. (n = number of muscle fibers, number of muscles studied). The statistical significance (p values in table and figure legends) was evaluated by the nonparametric Mann-Whitney U test. Null hypothesis was rejected when p < 0.05.

Toxins and Chemicals. Salts and reagents used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO). The synthetic polypeptide -conotoxin GVIA (-CgTx GVIA), ryanodine, calciseptine, and thapsigargin, were purchased from Alomone Labs (Jerusalem, Israel). The synthetic polypeptide -Agatoxin IVA (-Aga IVA) was purchased from Peptides Institute, Inc. (Osaka, Japan). Nifedipine, nitrendipine, Bay-K, and isradipine were purchased from Sigma/RBI (Natick, MA). Caffeine was purchased from local pharmacies. Stock solutions of ryanodine, -Aga IVA, -CgTx GVIA, calciseptine, and caffeine were dissolved in distilled water. Stock solutions of thapsigargin, nifedipine, isradipine, and nitrendipine were dissolved in dimethyl sulfoxide. When using these drugs, final concentration of dimethyl sulfoxide in the bath medium never exceeded 0.2%. Control experiments were performed by incubating muscles with final concentrations of drugs' vehicle.

	Results

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Nifedipine Enhances Spontaneous Release. The effect of different VDCC blockers on the rate of MEPPs was studied in neonatal (0–4-day) rat NMJ, a developmental stage where multiple Ca²⁺ channels mediate evoked transmitter release (Rosato Siri and Uchitel, 1999). MEPP frequency from 350 muscle fibers studied in 29 untreated neonatal muscles was 2.61 ± 0.19/min. This observation agrees with previous reports on this preparation (Diamond and Miledi, 1962).

Spontaneous release is not as dependent on extracellular Ca²⁺ as evoked release (Elmqvist and Feldman, 1965). Therefore, VDCC blockers were expected to exert a small effect on MEPP frequency. In fact, 1-h incubation with N- or P/Q-type VDCCs blockers did not change the rate of spontaneous transmitter release. MEPP frequency after incubation with 5 µM -CgTx GVIA was 2.49 ± 0.6 (n = 59, 4) and 3.10 ± 0.58/min (n = 35, 3) after incubation with 100 nM -Aga IVA (Table 1). However, treatment with 10 µM of the L-type VDCC agonist Bay-K or the antagonist nifedipine (Fig. 1) produced a strong increase in the rate of spontaneous release. After treatment with these drugs, MEPP frequency increased 13- and 50-fold, respectively (Table 1). The reversibility of nifedipine was tested after washing the preparation with drug-free saline solution for 1 h. Reversal was almost complete because MEPP frequency was reduced more than 10 times (17.39 ± 3/min; n = 31, 3)

View larger version (32K): [in this window] [in a new window]   Fig. 1. Nifedipine increases MEPP frequency. Representative traces of 2-min recording of control and after 1-h incubation with 10 µM nifedipine. Apparent synchronization of MEPPs' occurrence is a consequence of the recording method that uses MEPP as the trigger.

Surprisingly, the strong potentiation effect of nifedipine contrasted with the lack of effect on MEPP frequency of other L-type VDCC antagonists such as the DHPs isradipine and nitrendipine or the peptide calciseptine (Table 1).

The effect of nifedipine was further investigated using drugs from different batches and in all cases a strong effect was detected. Furthermore, treatment with nifedipine (10 µM) inactivated by exposure for 4 h to strong visible light (Majeed et al., 1987) did not increase MEPP frequency (3.39 ± 0.97/min; n = 28, 2). Thus, the enhancement of spontaneous transmitter release by nifedipine is most likely attributable to drug effect and not to an anomalous effect or contamination.

Effect of Nifedipine Is Age-Dependent. Diaphragms from 0- to 22-day-old rats were studied before and after incubation with 10 µM nifedipine. MEPPs at a rate of 2 to 3/min were recorded during the first 2 weeks after birth. MEPP frequency increases by postnatal day 15 (12.53 ± 2.22/min; n = 28, 2) and reaches its mature rate at postnatal day 22 (35.61 ± 3.38/min; n = 27, 2) (Fig. 2A). Nifedipine induced enhancement of spontaneous transmitter release at NMJs in newborn but not in adult rats. In our study, the highest potentiation effect of nifedipine was found at postnatal day 6 and it decreased as development proceeded, up to disappearance at postnatal day 22 (Fig. 2B). Although both the increase of basal MEPP frequency and the decrease of nifedipine effect occurred during the same time period, their variation rate was very different, suggesting that they may not be directly linked to each other.

The effect of Bay-K on MEPP frequency was studied in 22-day-old NMJs. Bay-K increases MEPP frequency from an average 40.04 ± 3.83 (n = 26,2) to 116.43 ± 15.51 (n = 25, 2)/min (p < 0.05). Thus, the effect of Bay-K on MEPP frequency was partly maintained at ages where the nifedipine effect has already disappeared (Pancrazio et al., 1989).

Mechanism of Nifedipine Activity. The mechanism underlying the strong potentiation effect of nifedipine on spontaneous release was investigated in 0- to 4-day-old neonatal diaphragm muscle (Rosato Siri and Uchitel, 1999). The potentiation effect of nifedipine was concentration-dependent: 1, 5, 10, and 15 µM nifedipine significantly increased MEPP frequency 3.84 ± 0.85 (n = 41, 3), 32.16 ± 6.5 (n = 45, 3), 49.46 ± 3.89 (n = 101, 6), and 55.36 ± 7 (16, n = 31, 2) fold, respectively (Fig. 3A).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Pharmacological profile of nifedipine effect. A, nifedipine increases MEPPs frequency in dose-dependent manner. The plot shows Hill equation fit to nifedipine effect (nH = 1.74, IC50 = 4.88 µM, maximum effect = 63.30, r = 0.99). B, nifedipine effect (49.46 ± 3.88; n = 101, 6) was not reduced by pretreatment with 1, 3, and 30 µM isradipine or 300 nM calciseptine. Column represents the mean ± S.E.M. of MEPP frequency from pooled data. *, p < 0.05 compared with 10 µM nifedipine. In A and B, 10 µM nifedipine bar is extracted from Table 1.

The finding that both Bay-K and nifedipine increased MEPP frequency prompted us to search for an L-type VDCC agonistic action of nifedipine. To test this hypothesis, nerve muscle preparations were treated with L-type channel blockers that lack any potentiation effect per se before nifedipine. We used the high-affinity DHP isradipine (Scholze et al., 2001) and calciseptine, which interact with the channel at the same site as DHPs, but are membrane-impermeable (Yasuda et al., 1993). Neither calciseptine nor low or high concentrations of isradipine were capable of preventing the strong increase (over 50-fold) of MEPP frequency exerted by nifedipine (Fig. 3B). In the presence of these L-type channel antagonists, nifedipine (10 µM) increased MEPP frequency from control values to 196.9 ± 25.36/min (n = 40, 3 for 3 µM isradipine), 168.83 ± 21.13/min (n = 15, 1 for 30 µM isradipine), and 147.64 ± 21.16/min (n = 25, 2 for 300 nM calciseptine), suggesting that the effect is not attributable to agonistic action of nifedipine on the L-type calcium channel. Furthermore, other VDCCs blockers like -CgTx GVIA and -Aga IVA were also ineffective in preventing the action of nifedipine (data not shown). In contrast, isradipine was able to occlude most of the increase in MEPP frequency induced by Bay-K. Average MEPP frequency in the presence of Bay-K alone was 2.52 ± 0.41 (n = 26, 3) and 6.03 ± 2.19/min (n = 33, 3) in paired diaphragms treated with 3 µM isradipine and Bay-K.

It has been accepted that an increased rate in MEPP frequency is associated with a sustained increase in cytosolic Ca²⁺ (Rahamimoff and Alnaes, 1973; Blaustein et al., 1978; Emptage et al., 2001). To determine the source of calcium related to nifedipine effect, the drug was applied to preparations previously incubated in zero Ca²⁺ solution. MEPP frequency in zero Ca²⁺ solution was 0.68 ± 0.08/min (n = 17, 2) and increases went up to 25.09 ± 7.21/min (n = 19, 2) after 1-h incubation in the presence of 10 µM nifedipine, indicating that the effect is not directly dependent on extracellular Ca²⁺ (Fig. 4A). In contrast, the effect of Bay-K was strongly reduced in the absence of extracellular Ca²⁺. The 13-fold increase in MEPP frequency induced by Bay-K in the presence of 2 mM Ca²⁺ was reduce to a 1.63-fold increase in zero Ca²⁺ solution.

Thus, the possibility that nifedipine exerts its effect via Ca²⁺ released from intracellular sources was thereafter investigated. Nerve muscle preparations were incubated in zero Ca²⁺ solution containing 2 µM Ca²⁺-ATPase inhibitor thapsigargin and 2 mM caffeine, to deplete intracellular calcium stores. Afterward, preparations were transferred to the recording solution (10 mM Ca²⁺) with caffeine-free thapsigargin. After this treatment, further addition of nifedipine did not increase MEPP frequency (compare Fig. 4Ba and 4Bb). In contrast, the strong potentiation effect of nifedipine was present in muscles incubated in zero Ca²⁺ solution with caffeine but without thapsigargin, a treatment that avoids depletion of intracellular Ca²⁺ stores (Fig. 4Bc).

Calcium from intracellular stores can be released into the cytoplasm by activation of ryanodine receptor, a phenomenon blocked by the alkaloid ryanodine. Application of 10 µM ryanodine induced a slight but significant 12% decrease in MEPP frequency. In nerve muscle preparations treated with nifedipine and further incubated with 10 µM ryanodine in the presence of nifedipine, MEPP frequency was reduced by 83.57% (n = 38, 3) (Fig. 4C). Thus, the nifedipine-mediated increase of MEPP was strongly antagonized by ryanodine.

	Discussion

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In the present study, we describe a novel effect of nifedipine on spontaneous transmitter release at neonatal rat NMJs. The effect is likely to be the result of an increase in cytosolic nerve terminal Ca²⁺ concentration due to release of Ca²⁺ from the ryanodine-sensitive stores. Several lines of evidence presented in this article indicate that the ryanodine-sensitive Ca²⁺ pool is the main source of nifedipine effect. First, the nifedipine-induced rise in MEPP frequency was observed, although lowered, in the absence of extracellular Ca²⁺. The reduction of nifedipine effect in zero calcium solution might reflect a partial depletion of intracellular calcium stores during incubation. In addition, nifedipine effect was abolished after depleting the intracellular Ca²⁺ storage compartments by incubating the nerve-muscle preparation with nominal zero Ca²⁺ solution containing the Ca²⁺-ATPase inhibitor thapsigargin and caffeine. Furthermore, nifedipine-induced increases on MEPP frequency could be strongly reduced by ryanodine. The small effect of nifedipine still present after treating the nerve terminals with 10 µM ryanodine is consistent with an incomplete locking of the ryanodine channels in the closed configuration (Fill and Copello, 2002). Ryanodine alone only slightly reduced MEPP frequency, suggesting that ryanodine reversal of nifedipine effect was mediated by a specific antagonistic action.

The presence of calcium channels sensitive to DHP blockers at mouse NMJs has been reported previously (Urbano and Uchitel, 1999; Flink and Atchison, 2003); therefore, the effect of nifedipine might be exerted through the activation of these channels. However, nifedipine effect was not blocked by isradipine nor calciseptine, indicating that it is not acting as an L-type VDCC agonist (Triggle, 2003). Nifedipine effect was dose-dependent and saturated at micromolar concentrations, suggesting its action is exerted on a specific saturable type of receptor, which might be linked to ryanodine-sensitive intracellular Ca²⁺ stores.

In contrast with our results, Sugiura and Ko (1997) have reported that nifedipine as well as other L-type Ca²⁺ channel blockers potentiate evoked release at neonatal mouse NMJs. The unique effect of nifedipine on spontaneous release was probably overlooked by these authors because their experiments were performed in the presence of curare. The potentiation exerted by the DHPs, studied by Sugiura and Ko (1997), was blocked by pretreatment with fast but not slow permeant Ca²⁺ chelators, suggesting that DHPs are blocking Ca²⁺ influx via the L type Ca²⁺ channels. Therefore, nifedipine should be considered as a unique compound with two sites of action at neonatal mouse NMJs, one on the DHP receptor at the L-type Ca²⁺ channel and another linked to intracellular Ca²⁺ stores.

A similar unique effect of nifedipine on the frequency of miniature synaptic currents in rat brain supraoptic neurons has been reported recently (Hirasawa and Pittman, 2002). Although the mechanism of action seems to be different from the one we are postulating, it is important evidence of the uniqueness of this DHP compound (Triggle, 2003) and of the fact that this novel effect is not restricted to neonatal NMJs.

Although the agonist Bay-K also increases MEPP frequency, the mechanism of action differs from the one postulated for nifedipine. Bay-K effect is maintained after 3 weeks of development, it is highly dependent on Ca²⁺ extracellular, and is occluded by the DHP antagonist isradipine. Therefore, Bay-K seems to act as expected on the DHP receptors of presynaptic L-type Ca²⁺ channels. However, removal of Ca²⁺ from the extracellular solution or else competition with isradipine, strongly reduced, although it did not abolish the increase in MEPP frequency induced by nifedipine. Thus, as proposed by Pancrazio et al. (1989), Bay-K may exert a dual effect on motor nerve endings, characterized by a primary action on the presynaptic L-type Ca²⁺ channels and by a secondary action similar to that of nifedipine but with substantially lower efficiency.

A cytosolic Ca²⁺ rising effect of DHPs in human fetal skeletal muscle cells has been described recently (Weigl et al., 2000). This effect is a result of Ca²⁺ release from the ryanodine-sensitive pool and is mediated by binding DHPs to their receptor, which shifts the DHPs receptor to a preactivated state that can in turn activate the ryanodine receptor (Weigl et al.,2000). If such a mechanism mediates nifedipine effect on MEPP frequency, then a postsynaptic target could be postulated. Under this hypothesis, nifedipine might exert its action on muscular cells, inducing a retrograde signal that in turn induces a presynaptic increase on spontaneous neurotransmitter release rate.

Although more experiments should be performed to clarify the differences observed between nifedipine and structurally related drugs such as isradipine and nitrendipine, our results suggest that previous reports as DHPs incrementing synaptic transmission should be more carefully interpreted in terms of L-type calcium channels' specificity of DHPs' action

Nifedipine potentiation effect on MEPP frequency was restricted to early stages of development. Coincidental with the findings of Sugiura and Ko (1997), the potentiation effect of L-type Ca²⁺ channel blockers on evoked release also diminished during the first 2 weeks after birth and later on gradually disappeared. During the same period, significant changes in basal MEPP frequency occur, but they are not coincident with the disappearance of nifedipine potentiation. This observation suggests that both processes are not directly related, laying open the possibility that disappearance of the nifedipine potentiation effect reflects modulation of other synaptic properties during development.

Consistent with our observation that the potentiation effect of nifedipine is restricted to early stages of NMJ development, Losavio and Muchnik (1997) have reported an inhibitory effect on MEPP frequency by L-type calcium channel antagonists, including nifedipine, at adult rat NMJs.

In conclusion, we hereby report a novel effect of the widely used drug nifedipine, mediated by ryanodine-sensitive intracellular calcium storages. This effect of nifedipine should be taken into consideration when using this drug. The exact mechanism responsible for nifedipine effects over intracellular calcium stores remains to be investigated, as well as whether a physiological mechanism involving intracellular calcium stores modulates synaptic transmission at developing NMJs.

	Footnotes

 
This work was supported by the Muscular Dystrophy Association Inc. (Tucson, AZ); Ministerio de Salud, Beca Carrillo Oñativia; Agencia Nacional de Ciencia y Técnica 6220; and National Institutes of Health Grant R03TW01312–02 and UBACYT (Argentina)

DOI: 10.1124/jpet.103.051524.

ABBREVIATIONS: VDCC, voltage-dependent calcium channel; MEPP, miniature endplate potential; DHP, dihydropyridine; NMJ, neuromuscular junction; -CgTx GVIA, -conotoxin GVIA; -Aga IVA, -agatoxin IVA; BAY-K, BAY-K8644, S-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester.

¹ Current address: Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avenida Doctor Arce 37, 28002 Madrid, Spain.

² Current address: Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies, Via Beirut 2-4 (34014), Trieste, Italy.

Address correspondence to: Dr. Osvaldo D. Uchitel, Laboratorio de Fisiología y Biología Molecular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab II 2^de piso, Buenos Aires 1428, Argentina. E-mail: odu{at}fibertel.com.ar

	References

Top Abstract Materials and Methods Results Discussion References

 

Blaustein MP, Ratzlaff RW, and Kendrick NK (1978) The regulation of intracellular calcium in presynaptic nerve terminals. Ann NY Acad Sci 307: 195–212.[Medline]

Castonguay A and Robitaille R (2001) Differential regulation of transmitter release by presynaptic and glial Ca²⁺ internal stores at the neuromuscular synapse. J Neurosci 21: 1911–1922.[Abstract/Free Full Text]

Dennis MJ, Ziskind-Conhaim L, and Harris AJ (1981) Development of neuromuscular junctions in rat embryos. Dev Biol 81: 266–279.[CrossRef][Medline]

Diamond J and Miledi R (1962) A study of foetal and new-born rat muscle fibres. J Physiol (Lond) 162: 393–408.

Elmqvist D and Feldman DS (1965) Calcium dependence of spontaneous acetylcholine release at mammalian motor nerve terminals. J Physiol (Lond) 181: 487–497.[Free Full Text]

Emptage NJ, Reid CA, and Fine A (2001) Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca²⁺ entry and spontaneous transmitter release. Neuron 29: 197–208.[CrossRef][Medline]

Fill M and Copello JA (2002) Ryanodine receptor calcium release channels. Physiol Rev 82: 893–922.[Abstract/Free Full Text]

Flink MT and Atchison WD (2003) Iberiotoxin-induced block of KCa channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals. J Pharmacol Exp Ther 305: 646–652.[Abstract/Free Full Text]

Hirasawa M and Pittman QJ (2003) Nifedipine facilitates neurotransmitter release independently of calcium channels. Proc Natl Acad Sci USA 100: 6139–6144.[Abstract/Free Full Text]

Hubbard JI, Jones SF, and Landau EM (1968) On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals. J Physiol (Lond) 194: 355–380.[Abstract/Free Full Text]

Katz E, Protti DA, Ferro PA, Rosato Siri MD, and Uchitel OD (1997) Effects of Ca²⁺ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction. Br J Pharmacol 121: 1531–1540.[CrossRef][Medline]

Krizaj D, Bao JX, Schmitz Y, Witkovsky P, and Copenhagen DR (1999) Caffeine-sensitive calcium stores regulate synaptic transmission from retinal rod photoreceptors. J Neurosci 19: 7249–7261.[Abstract/Free Full Text]

Llano I, Gonzalez J, Caputo C, Lai FA, Blayney LM, Tan YP, and Marty A (2000) Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients. Nat Neurosci 3: 1256–1265.[CrossRef][Medline]

Losavio A and Muchnik S (1997) Spontaneous acetylcholine release in mammalian neuromuscular junctions. Am J Physiol 273: C1835–C1841.

Majeed IA, Murray WJ, Newton DW, Othman S, and Al-Turk WA (1987) Spectrophotometric study of the photodecomposition kinetics of nifedipine. J Pharm Pharmacol 39: 1044–1046.[Medline]

Narita K, Akita T, Osanai M, Shirasaki T, Kijima H, and Kuba K (1998) A Ca²⁺-induced Ca²⁺ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals. J Gen Physiol 112: 593–609.[Abstract/Free Full Text]

Neher E (1998) Vesicle pools and Ca²⁺ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20: 389–399.[CrossRef][Medline]

Pancrazio JJ, Viglione MP, and Kim YI (1989) Effects of Bay K 8644 on spontaneous and evoked transmitter release at the mouse neuromuscular junction. Neuroscience 30: 215–221.[CrossRef][Medline]

Protti DA, Szczupak L, Scornik FS, and Uchitel OD (1991) Effect of omega-conotoxin GVIA on neurotransmitter release at the mouse neuromuscular junction. Brain Res 557: 336–339.[CrossRef][Medline]

Protti DA and Uchitel OD (1993) Transmitter release and presynaptic Ca²⁺ currents blocked by the spider toxin omega-Aga-IVA. Neuroreport 5: 333–336.[Medline]

Rahamimoff R and Alnaes E (1973) Inhibitory action of ruthenium red on neuromuscular transmission. Proc Natl Acad Sci USA 70: 3413–3416.

Redfern PA (1970) Neuromuscular transmission in new-born rats. J Physiol (Lond) 209: 701–709.[Abstract/Free Full Text]

Rosato Siri MD and Uchitel OD (1999) Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions. J Physiol (Lond) 514: 533–540.[Abstract/Free Full Text]

Scholze A, Plant TD, Dolphin AC, and Nurnberg B (2001) Functional expression and characterization of a voltage-gated CaV1.3 (1D) calcium channel subunit from an insulin-secreting cell line. Mol Endocrinol 15: 1211–1221.[Abstract/Free Full Text]

Sugiura Y and Ko CP (1997) Novel modulatory effect of L-type calcium channels at newly formed neuromuscular junctions. J Neurosci 17: 1101–1111.[Abstract/Free Full Text]

Triggle DJ (2003) The 1,4-dihydropyridine nucleus: a pharmacophoric template part 1. Actions at ion channels. Mini Rev Med Chem 3: 217–225.

Uchitel OD (1997) Toxins affecting calcium channels in neurons. Toxicon 35: 1161–1191.[Medline]

Uchitel OD, Protti DA, Sanchez V, Cherksey BD, Sugimori M, and Llinas R (1992) P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses. Proc Natl Acad Sci USA 89: 3330–3333.[Abstract/Free Full Text]

Urbano FJ and Uchitel OD (1999) L-Type calcium channels unmasked by cellpermeant Ca²⁺ buffer at mouse motor nerve terminals. Pfluegers Arch 437: 523–528.[CrossRef][Medline]

Weigl LG, Hohenegger M, and Kress HG (2000) Dihydropyridine-induced Ca²⁺ release from ryanodine-sensitive Ca²⁺ pools in human skeletal muscle cells. J Physiol (Lond) 525: 461–469.[Abstract/Free Full Text]

Yasuda O, Morimoto S, Chen Y, Jiang B, Kimura T, Sakakibara S, Koh E, Fukuo K, Kitano S, and Ogihara T (1993) Calciseptine binding to a 1,4-dihydropyridine recognition site of the L-type calcium channel of rat synaptosomal membranes. Biochem Biophys Res Commun 194: 587–594.[CrossRef][Medline]

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