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Previous Article | Next Article
The Journal of Neuroscience, May 1, 2001, 21(9):3135-3143
Mobilization of Calcium from Intracellular Stores, Potentiation of Neurotransmitter-Induced Calcium Transients, and Capacitative Calcium Entry by 4-Aminopyridine
Maurizio Grimaldi1, Marco Atzori2, Pulak Ray1, and Daniel L. Alkon3 1 Laboratory of Adaptive Systems, National Institute for Neurological Disorders and Stroke and 2 National Institute of Deafness and other Communicative Disorders, National Institutes of Health, Bethesda, Maryland 20892, and 3 Blanchette Rockefeller Neurosciences Institute, Rockville, Maryland 20850
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
In this study we analyzed the effect of 4-aminopyridine (4-AP) on free cytosolic calcium concentration ([Ca2+]i) in basal conditions, after stimulation with neurotransmitters, and during capacitative calcium entry.
Using fura-2 ratiometric calcium imaging, we found that 4-AP increased [Ca2+]i in type I astrocytes, neurons, and in skeletal muscle cells. The [Ca2+]i elevation induced by 4-AP was concentration-dependent and consisted of two phases: the first was dependent on intracellular calcium mobilization, and the second was dependent on extracellular calcium influx. 4-AP also increased the second messenger inositol trisphosphate in both neurons and astrocytes.
In astrocytes, 4-AP treatment potentiated the sustained phase of the [Ca2+]i elevation induced by ATP and bradykinin. In addition, capacitative calcium entry was potentiated severalfold by 4-AP, in astrocytes and muscle cells but not in neurons. These effects of 4-AP were completely and promptly reversible. 4-AP blocked voltage-sensitive K+ currents in astrocytes. However, voltage-sensitive K+ channel blockers inhibiting these currents did not affect agonist-induced calcium transients or capacitative calcium entry, indicating that 4-AP effects on [Ca2+]i were not caused by the blockade of voltage-gated K+ channels.
We conclude that 4-AP is able to affect calcium homeostasis at multiple levels, from increasing basal [Ca2+]i to potentiating capacitative calcium entry. The potentiation of capacitative calcium entry in astrocytes or muscle cells may explain some of the therapeutic activities of 4-AP as a neurotransmission enhancer.
Key words: neuron; astrocytes; muscle cell; capacitative calcium entry; intracellular calcium stores; voltage-sensitive K+ potassium channels
INTRODUCTION
4-aminopyridine (4-AP) and related molecules have been widely recognized for their ability to block voltage-sensitive K+ channels (Aronson, 1992
). 4-AP has also been used as a therapeutic agent in many neurological and neuromuscular junction disorders. A number of reports address the beneficial role of 4-AP and related compounds in multiple sclerosis (Schwid et al., 1997
There are, however, a number of reports addressing a possible direct effect of 4-AP on calcium homeostatic mechanisms (Glover, 1981
MATERIALS AND METHODS
Preparation of primary cultures of cortical type I astrocytes. Embryonic type I astrocyte cultures were obtained from embryonic day 17 rat fetuses, according to a published protocol, with slight modifications (Grimaldi et al., 1994
Primary cultures of cortical neurons. Neuronal cultures were prepared as described above, with some modifications (Grimaldi and Cavallaro, 1999
L6 rat skeletal muscle cell line. L6 cells were purchased from the American Tissue Culture Collection (Rockville, MD). On arrival, cells were cultured, expanded, and frozen. Cell aliquots were thawed and used between passage 1 and 5. Cells were maintained in DMEM with 10% fetal bovine serum (HyClone, Logan, UT) and Pen/Strep (Life Technologies).
Total inositol phosphate accumulation assay. Inositol phosphate (InsPt) accumulation was assayed in astrocytes and neurons as previously described (Grimaldi and Cavallaro, 1999
Single cell [Ca2+]i measurements. Astrocytes, L6, or neurons were seeded on glass coverslips (Assistent, Germany). Before each experiment, the cells were washed once in KRB and loaded with 2 µM fura-2 AM (Molecular Probes, Eugene, OR) for 22 min at room temperature, to minimize probe compartimentalization (Roe et al., 1990
Image pairs obtained every 2 sec by exciting the preparations at 340 and 380 nm were used to obtain ratio images. Excitation wavelengths were changed using a filter wheel (Metaltek), and the emission wavelength was set to 510 nm. Captured images were processed with a Matrox-LC acquisition board and analyzed by using the software MetaFluor (Universal Imaging, West Chester, PA). Regions of interest were obtained by delimiting the profile of the cells and averaging the fluorescence intensity within the delimited area. Intensity values were converted to [Ca2+]i using different methods for neurons, muscle cells, and astrocytes. Ratio values were calibrated to [Ca2+]i for neurons and muscle cells obtaining Fmax and Rmax and Fmin and Rmin by exposing the cells to 10 µM ionomycin in presence of 10 mM calcium. After the maximal signal was obtained, cells were perfused with calcium-free KRB containing 10 mM EGTA. [Ca2+]i in neurons and muscle cells was then calculated using the equation developed by Grynkiewicz et al. (1985)
Patch-clamp recording. Astrocytes seeded on glass coverslips were washed in KRB, placed in a perfusion chamber, and then perfused continuously. Glass pipettes were pulled (2-3 M
) and filled with a solution containing (in mM): 130 K+Gluconate, 10 HEPES, 5 BAPTA, 2 ATP, 0.3 GTP, and 1.0 MgCl2. Cells were voltage-clamped in the whole-cell configuration using an Axopatch-1D amplifier driven by a personal computer running the pClamp acquisition software (Axon Instruments, Foster City, CA). Data were analyzed off-line using the same software.
Fast and transient currents were measured at the peak, whereas steady-state currents were measured at the end of the voltage pulse, at a delay
150 msec. In a second set of experiments, voltage changes before and after drug application were measured in the current-clamp configuration, after waiting several minutes for membrane voltage stabilization and after adjusting the resting voltage to
75 mV with a steady current injection. A fixed current pulse inducing a voltage step of ~5 mV was delivered every 8 sec to continuously monitor the cell input resistance. Only cells with stable holding currents before drug application were considered in the analysis.
Materials. All materials were purchased from Sigma (St. Louis, MO), unless otherwise specified in the text.
Use of laboratory animals. Adequate measures were taken to minimize unnecessary pain and discomfort to the animal and to minimize animal use, according to National Institutes of Health regulations on animal handling and care (Guide for the Care and use of Laboratory Animals; National Academy Press, 1996). Pregnant animals were killed by exposure to CO2.
Statistical analyses. Experiments were performed at least three times using different cell preparations. For [Ca2+]i measurements, digital images were converted to analog data and imported to a spreadsheet. Numeric values, representing the [Ca2+]i determined every 2 sec, were averaged, and the SE was calculated. Data are displayed as averages ± SE. When statistical validation was required, data were analyzed by ANOVA followed by a t test and shown as a bar inset in the corresponding figure. Differences were considered statistically significant when the p
0.05.
RESULTS
Effect of 4-AP on [Ca2+]i
4-AP moderately increased [Ca2+]i in cortical type I astrocytes (Figs. 1, 2A), cortical neurons (Fig. 3A), and L6 cells (see Fig. 7B, inset). In astrocytes, increasing concentrations of 4-AP between 1 and 20 mM caused a linear elevation of [Ca2+]i, with an apparent plateau at 20 mM (Fig. 1, inset). The calculated EC50 was between 5 and 10 mM of the drug (Fig. 1, inset). The elevation of [Ca2+]i induced by 4-AP had a slow onset and reached a steady-state level that was maintained as long as the drug was applied (Figs. 1, 2A). After washing, calcium concentration returned promptly to baseline values (Figs. 1, 2A). The 4-AP-induced [Ca2+]i elevation did not show desensitization (Fig. 1). Similar results were found in neurons (Fig. 3A) and in muscle cells (see Fig. 8B, inset).
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Figure 1. Effect of 4-AP on [Ca2+]i. Near-confluent cultured type I astrocytes were loaded with fura-2. Cells were perfused with increasing 4-AP concentrations applied at the times indicated by the top broken line arrows and washed at the times indicated by the solid line arrows below the trace. Increasing concentrations of 4-AP caused proportional [Ca2+]i elevation that reached an apparent plateau at 20 mM 4-AP, as shown in the inset. Removal of 4-AP caused a prompt return to baseline [Ca2+]i. The EC50 of 4-AP was ~10 mM.
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Figure 2. Characterization of 4-AP-induced calcium response in type I astrocytes. A, Treatment with 10 mM 4-AP caused a slow-onset long-lasting [Ca2+]i elevation, which was promptly reversed by washout. B, When extracellular calcium was removed, 4-AP was still able to increase [Ca2+]i. However, the shape of the response was modified to a transient peak with a quick return to baseline. C, The effect of 4-AP was tested in the absence of extracellular calcium and after depletion of intracellular calcium stores with thapsigargin (Thap). D, A 10 mM concentration of 4-AP was applied to astrocytes in calcium-free KRB. Subsequently calcium was reintroduced, and [Ca2+]i was monitored. Perfusion of the testing substances is indicated by the horizontal bars.
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Figure 3. Characterization of 4-AP-induced [Ca2+]i rise in cultured cortical neurons. A, A 10 mM concentration of 4-AP was applied to cortical neurons plated on glass coverslips. The compound caused a slow-onset long-lasting [Ca2+]i elevation. The [Ca2+]i elevation was promptly reversed by washing out the compound. B, When extracellular calcium was removed, 4-AP (10 mM) was still able to increase [Ca2+]i. However, the response was transient, and [Ca2+]i returned promptly to baseline values. C, ICS were depleted in the absence of extracellular calcium with thapsigargin and CCE after reintroduction of calcium was measured. D, ICS were depleted with 4-AP in calcium-free medium. The neurons were subsequently exposed to thapsigargin, and 60 sec later calcium was reintroduced in the extracellular solution to elicit CCE. Perfusion of the testing substances is indicated by the horizontal bars.
We analyzed the role of ICS and extracellular calcium in 4-AP-induced [Ca2+]i elevation in astrocytes and in neurons. In the absence of extracellular calcium, 4-AP was still able to elevate [Ca2+]i in both astrocytes and neurons (Figs. 2B, 3B). However, the prolonged sustained phase of [Ca2+]i elevation was absent (Figs. 2B, 3B). Muscle cells displayed a similar behavior (see Fig. 8B, inset). These data suggest that this property of 4-AP is general rather than cell type-specific. In the absence of extracellular calcium, treatment with thapsigargin prevented 4-AP-induced calcium mobilization in astrocytes and neurons (Fig. 2C). The latter evidence suggests that 4-AP-induced calcium elevation is caused by release of calcium from thapsigargin-sensitive ICS.
4-AP increases InsPt production
To test whether the effect of 4-AP on [Ca2+]i was caused by the activation of the phospholipase C (PLC)/InsP3 pathway, we measured InsPt production, an index of PLC activation, in astrocytes (Fig. 4A) and neurons (Fig. 4B) after exposure to 4-AP. In both cell types, after 4-AP treatment a concentration-dependent increase of InsP production was detected (Fig. 4). Sensitivity of neurons and astrocytes to 4-AP was almost identical. Stimulation of the InsPt production caused by 4-AP (10 mM, the EC50 for 4-AP) was not very large, compared with the effectiveness of a less than half-maximal concentration of ATP (Grimaldi et al., 1999
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Figure 4. Effect of 4-AP on InsPt accumulation in astrocytes and neurons. A, 4-AP causes a concentration-dependent elevation of InsPt accumulation in type I astrocytes. Compared with the stimulation obtained with a 10 µM ATP (EC50, 30 µM), 10 mM 4-AP (EC50) was 50% less powerful (inset). B, 4-AP increased InsPt production in cortical neurons. Basal values are indicated by the open triangle and the dashed line. *p
4-AP potentiates CCE in astrocytes and muscle cells
Astrocytes exposed to 10 mM 4-AP in calcium-free buffer released calcium from ICS. The subsequent reintroduction of calcium in the perfusion buffer was followed by elevation of [Ca2+]i, indicating the activation of CCE (Fig. 2D). If the latter finding is compared with CCE evoked by an agonist such as ATP or bradykinin, it is clear that CCE in the presence of 4-AP is potentiated (see Figs. 6A, C). This finding suggested that 4-AP interfered with CCE. To test whether CCE potentiation by 4-AP could modulate agonist-evoked calcium responses, we analyzed calcium transients after exposure to InsP3-linked agonists known to mobilize calcium from ICS. Astrocytes pre-exposed to 4-AP and then challenged with ATP or bradykinin displayed markedly changed calcium transient dynamics. In particular, the amplitude of the sustained phase of the calcium transient, an indicator of CCE, became larger and was significantly prolonged.
In the case of 10 µM ATP, the pattern of [Ca2+]i elevation in control cells was characterized by a spike followed by a lower but prolonged phase of [Ca2+]i elevation (Fig. 5A). When astrocytes were pre-exposed to 10 mM 4-AP and then challenged with 10 µM ATP, the calcium transient induced by ATP lacked the initial spike but had a very high and prolonged plateau phase, which was promptly reversed by removing 4-AP from the perfusion medium (Fig. 5B; statistical validation is displayed in Fig. 5E, values were taken 2 sec before 4-AP removal).
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Figure 5. Effect of 4-AP on neurotransmitter-evoked calcium transients in astrocytes. Astrocytes were challenged with 10 µM ATP (A) or 100 nM bradykinin (C). The calcium response to both agonists was characterized by a fast and sharp [Ca2+]i elevation and a rapid return to a much lower but prolonged [Ca2+]i value. Astrocytes pre-exposed to 10 mM 4-AP and challenged with ATP (B) or bradykinin (D) showed a long-lasting large [Ca2+]i elevation. E and F show statistical analysis of the data extrapolated from the experiments in A and C 2 sec after agonist stimulation, and in B and D 2 sec before 4-AP washout. *p
Bradykinin responses were similarly affected by 4-AP. The typical spike-plateau response to bradykinin in control astrocytes (Fig. 5C) was, after treatment with 10 mM 4-AP, modified to a persistent high [Ca2+]i elevation, which was promptly reversed after washing out the 4-AP (Fig. 5D; statistical validation in Fig. 5F, values extracted 2 sec before 4-AP washout).
To further prove that 4-AP potentiates CCE, we designed a group of experiments in which CCE was measured. Control experiments were conducted to determine [Ca2+]i elevation in response to ICS depletion induced by three different paradigms. First, when ICS were depleted by ATP stimulation in the calcium-free KRB, the reintroduction of calcium induced a very small [Ca2+]i elevation (Fig. 6A). ICS were completely emptied by the ATP pulse, as shown by the lack of response to a second stimulation with ATP (Fig. 6A) or bradykinin (Fig. 6C). When the same experiments were performed in the presence of 10 mM 4-AP, CCE was greatly potentiated, when compared to ATP or 4-AP alone (Fig. 2D). In 4-AP-pretreated astrocytes, ATP-induced emptying of ICS triggered a large CCE after reintroduction of calcium in the extracellular medium. [Ca2+]i rapidly rose to an extremely high plateau, which was maintained as long as 4-AP was present and promptly decreased to baseline, once the compound was washed out (Fig. 6B; see statistical validation in Fig. 6E, values extracted 2 sec before 4-AP washout). A similar CCE potentiation by 4-AP was seen after ICS depletion with bradykinin (Fig. 6D; statistical validation displayed in Fig. 6F, values extracted 2 sec before 4-AP washout). The effect of 4-AP on CCE was concentration-dependent. A 5 mM concentration of 4-AP caused a smaller potentiation than 10 mM 4-AP of CCE induced by ICS depletion with either ATP (control, 173 ± 5 nM; 5 mmM 4-AP, 765 ± 10 nM; 10 mM 4-AP, 1489 ± 75 nM) or bradykinin (control, 182 + 8 nM; 5 mM 4-AP, 488 ± 16 nM; 10 mM 4-AP, 1084 ± 68 nM).
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Figure 6. Effect of 4-AP on CCE triggered by agonist-induced ICS depletion in astrocytes. ICS were depleted with ATP (A) or bradykinin in the absence of extracellular calcium. ICS emptying was controlled with a second ATP (A) or bradykinin (C) stimulation. When calcium was reintroduced, a weak [Ca2+]i elevation was generated because of CCE activation. B, ICS were depleted with 10 µM ATP in the presence of 4-AP. When calcium was reintroduced, [Ca2+]i elevation was very high and lasted until 4-AP was removed from the cells. The same effect was recorded when stores were depleted with bradykinin in the presence of 4-AP (D). E and F show the statistical validation of the data presented in A-D, respectively. Values were extrapolated from the experiments 2 sec before removal of 4-AP. *p value
In a second set of experiments, we analyzed CCE after depletion of ICS with thapsigargin, an irreversible blocker of the smooth endoplasmic reticulum calcium ATPase (SERCA), to exclude that the 4-AP potentiation of CCE observed after ATP- and bradykinin-induced ICS emptying was not caused by interaction with secondary signal-transducing mechanisms activated by the two agonists. Exposure to a maximal concentration of thapsigargin (10 µM) would also allow us assess the role of SERCA blockade in CCE potentiation by 4-AP (Fig. 7A). Thapsigargin, at 10 µM, applied with calcium-free KRB, completely discharged ICS. When calcium was reintroduced in the extracellular environment, a transient [Ca2+]i elevation was triggered, which decreased to a lower steady-state [Ca2+]i (Fig. 7A). When this experiment was conducted in the presence of 4-AP, CCE triggered by thapsigargin was powerfully potentiated (Fig. 7B; statistical validation presented in Fig. 7C, values were extrapolated at the peak of the response). We tested whether a similar phenomenon occurred in muscle cells and neurons. A strong potentiation of CCE induced by 4-AP was also observed in muscle cells (Fig. 8A, B; statistical validation is displayed in Fig. 8C, values were extracted 2 sec before 4-AP washout). On the contrary, potentiation of CCE by 4-AP was not observed in neurons (Fig. 3, compare D, C).
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Figure 7. 4-AP potentiates thapsigargin-induced CCE in astrocytes. A, Calcium stores were depleted with a maximal concentration of thapsigargin (10 µM). B, Thapsigargin exposure in cells pretreated with 4-AP resulted in a large increase of CCE. C, Statistical validation of the data presented in A and B. Peak values were analyzed. *p value
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Figure 8. 4-AP potentiates CCE in L6 cells. A, Application of 10 µM thapsigargin in calcium-free KRB caused an elevation of [Ca2+]i, (see inset to A). Reintroduction of calcium in the extracellular buffer was followed by CCE. The effect of thapsigargin alone it is highlighted in the inset. B, In the presence of 10 mM 4-AP, CCE was increased approximately ninefold. C, Statistical validation of the data extracted from control at peak and from 4-AP-exposed cells 2 sec before 4-AP washout. *p value
4-AP inhibits voltage-dependent K+ currents in astrocytes
Astrocytes were voltage-clamped at a membrane voltage of
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Figure 9. 4-AP inhibits voltage-gated K+ currents in astrocytes. A, Astrocytes were voltage-clamped at a membrane voltage of
Voltage-sensitive K+ channel blockers have no effect on CCE
To assess whether the effects of 4-AP on CCE resulted from blockade of voltage-sensitive K+ channels, we analyzed the effect of specific blockers on CCE. We studied ATP-evoked [Ca2+]i transients in astrocytes pretreated with
-dendrotoxin (DTx), an inhibitor of the fast inactivating K+ current (Ransom and Sontheimer, 1995
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Figure 10. Effect of DTx on CCE in astrocytes. We tested whether voltage-gated K+ channel blocker
Because we found that 10 mM 4-AP caused a slight alkalization of KRB (pH 7.8), we conducted experiments to determinewhether changes in extracellular pH might have been involved in CCE potentiation. Increasing the pH of KRB up to 8.4 was not able to reproduce the effect of 4-AP on CCE (data not shown).
DISCUSSION
4-AP and its analogs have numerous clinical applications, including treatment of neuromuscular and neurodegenerative disorders and traumatic injuries of the CNS (Li and Zhang, 1994
The data we present show a weak and concentration-dependent increase of total InsPt production in astrocytes and neurons after exposure to 4-AP. We hypothesize that this effect of 4-AP might be ascribed to an increase of PLC activity. Activation of PLC and subsequent production of InsP3 may be responsible for 4-AP-evoked calcium mobilization from ICS.
In addition, 4-AP appears to play a significant role in the regulation of calcium entering the cells after ICS depletion, a phenomenon known as CCE. CCE contributes to the magnitude and duration of agonist-evoked [Ca2+]i transients, to calcium oscillation, and is the principal mechanism through which ICS are refilled (Putney, 1986
We also have demonstrated that 4-AP can potently prolong and increase [Ca2+]i elevations caused by neurotransmitters such as ATP and bradykinin, which are linked to the intracellular messenger InsP3. This latter evidence suggests that the physiology of the response to neurotransmitters can be modified when cells are exposed to 4-AP. This indicates that an interaction in vivo is likely to happen and will result in a longer duration of calcium transients. Because the effect of 4-AP alone on CCE is not so large as when it is triggered by a large calcium mobilization, we believe that other mechanisms must be activated to uncover the potentiation of CCE that we observed. When ICS are depleted either using an agonist able to cause a large production of InsP3, such as ATP or bradykinin, or an agent able to completely discharge ICS, such as thapsigargin, a robust signal is generated that triggers the opening of CRAC. Such a signal has not been definitively identified and characterized. However, in cortical type I astrocytes a soluble factor, probably belonging to the family of the eicosanoid derivatives (Rzigalinski et al., 1999
That 4-AP may interact with other targets cannot be excluded. In particular, ligand-gated calcium channels may participate in the calcium transient evoked by ATP (for review, see Burnashev, 1998
Regardless of the mechanisms underlying CCE potentiation, the prolongation and potentiation of agonist-induced [Ca2+]i elevation may enhance excitation-contraction coupling of the muscle cells with a consequent improvement of neuromuscular function. Moreover, in the CNS such a potentiation of calcium responses may cause astrocytes to change their state of activation and to secrete trophic factors, which could play an important role in repairing mechanisms and in survival of surrounding neurons.
We have also shown that the effects of 4-AP are not attributable to blockade of voltage-sensitive K+ channels. All experiments with different types of compounds affecting voltage-sensitive K+ channels were not able to reproduce the effect of 4-AP on CCE.
In conclusion, we report novel effects of 4-AP, namely mobilization of calcium from ICS, PLC activation, and the potentiation of agonists responses through a large potentiation of CCE. These actions may explain some of the therapeutic effects of 4-AP in disorders in which impairment of neurotransmission is involved. Moreover, changes in calcium homeostasis induced by 4-AP in astrocytes might cause the release of trophic factors that would likely support regrowth of neuronal extensions. Finally, we hypothesize that 4-AP potentiates CCE by interfering with SOC/CRAC channels and may thus be a useful tool to study this channel for which specific agonists and antagonists are not yet developed.
FOOTNOTES Received Nov. 28, 2000; revised Jan. 18, 2001; accepted Jan. 30, 2001.
Correspondence should be addressed to Dr. Maurizio Grimaldi, Department of Neurology, Uniformed Services of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. E-mail: mgrimaldi{at}usuhs.mil.
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