Neurons translate electrical activity into chemical information via Ca2+ signaling. On depolarization of a neuronal cell, Ca2+ enters through voltage-gated Ca2+ channels. There are several subtypes of Ca2+ channels that open at different voltages and have different characteristics and functions. The L-type channel (Cav) is a high-voltage-activated channel that can be blocked with high specificity by dihydropyridines such as nitrendipine (NTP) and is found predominantly in the cell bodies and proximal dendrites of neurons.1 The L-type channel has been shown to be important in the transfer of extracellular signals to the cell nucleus. The movement of Ca2+ through L-type Ca2+ channels in certain neuronal cells is specifically linked to the binding of Ca2+ to calmodulin (CaM) and the rapid translocation of CaM to the cell nucleus.2 This process leads to the activation of the transcription factor, cyclic adenosine monophosphate response element-binding protein (CREB), through phosphorylation by CaM-sensitive kinases and results in the stimulation of new gene expression.3 This series of events links Ca2+ entry to nuclear signaling.
We have previously shown in SH-SY5Y cells, a human neuroblastoma cell line, that about one-third of the depolarization-induced Ca2+ transient is initiated by Ca2+ entry through the L-type channel.4 The entry of Ca2+ through the L-type channel is reversibly inhibited by halothane5 and isoflurane (ISO) (this study) at clinically relevant concentrations. Furthermore, it has been shown that the administration of 1.2% ISO and N2O to rats results in a significant decrease in CREB phosphorylation (P-CREB) in the rat hippocampus when compared with that of control.6 Others have shown that [Ca2+]cyt elevation in SH-SY5Y cells results in the formation of P-CREB.7 These considerations lead us to question whether ISO exposure of SH-SY5Y cells would result in inhibition of P-CREB formation and whether that inhibition is linked to Ca2+/CaM interaction and translocation.
Cell Culture and Treatment
Undifferentiated human SH-SY5Y neuroblastoma cells were maintained in 75-cm2 flasks for 14–20 days in RPMI 1640 medium that contained 12% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in a 37°C humidified incubator consisting of 5% CO2. All cell culture related reagents were purchased from Invitrogen, Carlsbad, CA. The cells were centrifuged at 1900g for 5 min after washing with Dulbecco’s phosphate buffered saline. The cells were rinsed once with incubation buffer (IBG) containing (in mM) 140 NaCl, 5 KCl, 5 NaHCO3, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. The cell pellet was resuspended in IBG buffer and approximately 1.25 × 106 cells added to each microcentrifuge tube. The cell suspension was incubated in IBG at room temperature for 10 min. After incubation without or with 10 μM NTP (5 min), 100 nM ω-conotoxin GVIa (CTX) (5 min), or 0.2–0.8 mM ISO (10 min) at 37°C, 1.5 mM CaCl2 was added to the cell suspension. Three minutes later, the reaction was initiated with the addition of KCl (100 mM, 30 s). The reaction was stopped by placing the cell suspension on ice.
For Western blot and enzyme-linked immunosorbant assay (ELISA), after KCl stimulation, the reaction was stopped by dropping the reaction tubes containing the cell suspension into liquid nitrogen.
Measurement of L-type Ca2+ Currents
Ca2+ currents were measured in SH-SY5Y cells as previously described.5 A cell-attached single-channel patch was used to measure the ISO effect. The bath solution contained (in mM) 120 KAspartate, 25 KCl, 2 MgCl2, 0.5 CaCl2, 2 EGTA, 2 HEPES, and 1 μM tetrodotoxin at pH 7.4. The pipette solution contained (in mM) 2 CaCl2, 110 sucrose, 23 TEACl, 2 EGTA, 2 HEPES, and 100 μM picrotoxinin, 0.1 μM CTX, 1 μM tetrodotoxin at pH 7.4. Calcium currents were recorded in response to 150 ms test pulse to −10 mV applied every 4 s from a −60 mV holding potential. Wash (bath solution) and ISO solution (in bath solution) were applied directly on top of the cell through a Millimanifold (MLF-4 from ALA Scientific, NY; dead space <50 μL) at a 2.8 μL/s flow rate. Data were filtered at 2 kHz and acquired at 20 kHz. Current traces in the figure were filtered at 500 Hz. Data analysis was done using Clampfit 9.2 software (Molecular Devices, Sunnyvale, CA). Anesthetic application and measurement were as previously described.5
Fluorescence Measurement of Ca2+ Transients
SH-SY5Y cells in confluent culture were loaded with 5 μM fura-2 for 30 min at 37°C as previously described.4 After washing and suspending, cells were equilibrated for 5 min before the addition of 1.5 mM CaCl2; after an additional 5 min, 100 mM KCl was added to the reaction mixture. Fluorescence ratios were monitored at 510 nm after excitation at 340 and 380 nm. The cytosolic Ca2+ concentration was determined from the fluorescence ratios as previously described.4
Isoflurane Addition and Measurement
An aliquot of pure ISO or an aliquot of a saturated solution of ISO (13.4 mM) was added to a suspension of cells with a Hamilton syringe as previously described to achieve the desired concentration. For cells attached on coverslips, each coverslip was mounted in a sealed chamber with 1 mL of liquid. An aliquot of saturated solution of ISO was injected into the sealed chamber and incubated with shaking for 10 min. The concentration of the ISO in the buffer solution was measured by gas chromatography as previously described.4
CREB phosphorylation was measured by both the Western blot and ELISA method.
The ELISA immunoassay kit was purchased from Biosource International, Inc. (Camarillo, CA). The anti-CREB antibody was from Cell Signaling Technology (Danvers, MA). The anti-P-CREB (Ser133) antibody was from Cell Signaling Technology and Chemicon (Temecula, CA). The anti-CaM antibody was from ABR (Golden, CO).
Immunoprecipitation and Western Blotting
The frozen SH-SY5Y cell suspension was thawed at room temperature for 20 min and sonicated three times for 10 s on ice. Cell lysates were then incubated on ice with 2 μL/sample of undiluted monoclonal anti-P-CREB antibody for 2 h. Immune complexes were collected on protein G-Sepharose (Sigma, St. Louis, MO) minicolumns and washed four times with cold phosphate buffered saline (PBS) by centrifugation and then eluted with denatured solution containing 0.6 M Tris/HCl, 1% SDS, 10% sucrose, 0.5% β-mercaptoethanol, and 0.5% bromophenol blue. The mixture was loaded on to the 10% SDS-PAGE gel for electrophoresis and then transferred to a polyvinylidene fluoride membrane (Amersham Biosciences, Bucks, UK). Nonspecific binding sites on the membrane were blocked with 5% skimmed milk. The membrane was then probed first with anti-P-CREB (Ser133) antibody on the immunoblots and then with secondary antibody, which was conjugated with horseradish peroxidase and developed with Opti-4CN Kit (BioRad, Hercules, CA) for colorimetric detection.
P-CREB Quantification by ELISA
Quantification of CREB phosphorylation was performed by a noncompetitive ELISA on soluble fractions obtained from cell lysates. The lysates were loaded onto the wells of the microtiter strips, which were coated with monoclonal antibody specific for CREB. During the 2-h incubation at room temperature, the P-CREB antigen from the lysate was bound to the immobilized antibody. After washing four times with buffer, the detection antibody specific for P-CREB at serine-133 was added. After 1-h incubation followed by four washings, the horseradish peroxidase-labeled anti-rabbit IgG (anti-rabbit-IgG-HRP) was added. Thirty minutes later, the complex was washed with substrate solution, and then the stabilized chromogen was added. After a 30-min incubation, the stop solution (2 M sulfuric acid) was added, and the absorbance at 450 nm was read by a BioRad microplate reader.
The SH-SY5Y cells were grown on coverslips placed in 12-well plates. After treatment as described above, they were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 for 45 min. The cells were exposed to 3% horse serum in PBS/Tris buffered saline followed by incubation with anti-P-CREB (Cell Signaling Technology) or anti-CaM (ABR) primary antibody (1:100), for 1 h at 37°C, respectively. Cells were then incubated with an Alexa Fluor 568 goat anti-mouse IgG as the secondary antibody (Invitrogen) (1:500) for 1 h at room temperature. After washing with PBS/Tris buffered saline, the stained cells were mounted on glass slides with mounting medium (Polysciences, Inc., Warrington, PA). P-CREB and CaM localization were examined by fluorescence imaging. All images were taken with a high resolution digital B/W CCD camera (ORCA-ER, Hamamatsu Photonics K.K., Hamamatsu City, Japan) connected to a CARV confocal module (Atto Instruments, Rockville, MD). A Zeiss Axiovert S100 (inverted type) microscope with a Plan-Apo 63× objective (Carl Zeiss Jena GmbH, Jena, Germany) was used.
Nuclear Protein Preparation
After treatment as described above, the cells, at 0°C–4°C in the presence of fresh 1× protease inhibitor cocktail (PIERCE #78410, Rockford, IL) and 1 mM dithiothreitol (Sigma), were centrifuged at 1900g for 10 min and washed once with 5× pellet volume of Dulbecco’s phosphate buffered saline. The cell pellets were rapidly resuspended in 5× pellet volume of hypotonic buffer containing 10 mM HEPES, pH 7.9 at 4°C, 1.5 mM MgCl2, and 10 mM KCl, and then centrifuged at 1900g for 5 min. The pelleted cells were resuspended in 3× pellet volume of hypotonic buffer on ice for 10 min. The suspension was homogenized with 10 up-down strokes using a plastic pestle. The nuclei were collected by centrifugation at 3300g for 15 min. The nuclei were then resuspended in one-half pellet volume of low-salt buffer containing 20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, and 0.2 mM EDTA, pH 7.9 at 4°C. An additional one-half pellet volume of low-salt buffer but containing 1.6 mM KCl was added in a dropwise fashion. The nuclei were extracted for 30 min with continuous gentle stirring. The extract was centrifuged at 17,500g for 40 min, and the supernatant was saved. Protein concentration of the supernatant was determined by the Lowry method.
CaM Detection by Western Blotting
The nuclear extract was mixed with denaturing solution containing 0.6 M Tris/HCl, 1% SDS, 10% sucrose, 0.5% β-mercaptoethanol, and 0.5% bromophenol blue. After boiling for 5 min and a brief centrifugation, the supernatant was loaded onto a 4%–20% SDS-PAGE premade gel (Invitrogen) for electrophoresis and then transferred to a polyvinylidene fluoride membrane pretreated with 100% methanol. Nonspecific binding sites on the membrane were blocked with 5% skimmed milk. The membrane was probed with anti-CaM (Invitrogen) antibody that recognized both Ca2+-free and Ca2+-bound CaM in the One-Step Western Blot Kit (Columbia Bio LLC, Elmhurst, NY) and developed with SuperSignal West Pico Chemiluminescent Substrate kit (PIERCE, Rockford, IL) for detecting horseradish peroxidase on immunoblots. The exposed and developed film was scanned and bands quantified by using NIH ImageJ.
Figure 1A demonstrates the ISO-mediated inhibition of Ca2+ currents through single L-type Ca2+ channels in SH-SY5Y cells. The averaged currents and individual traces clearly demonstrate the inhibition with ISO and the remarkable burst of channel activity when ISO is washed away.
P-CREB has been shown to be related to Ca2+ entry through L-type channels during depolarization. We have demonstrated that depolarization with 100 mM KCl induced an increase in P-CREB (Figs. 1B and C) and that exposure of cells to NTP inhibited P-CREB (Figs. 1B and C). Figure 2A demonstrates the increase in cell Ca2+ on depolarization with 100 mM KCl. On addition of 100 mM KCl, the cytosolic Ca2+ transient increases rapidly. We have shown previously that the transient is almost totally dependent on Ca2+ entry through L- and N-type Ca2+ channels.4 The formation of P-CREB has been shown to be quite rapid in other systems and it occurs rapidly in SH-SY5Y cells, with peak phosphorylation achieved at 30 s (Figs. 2B and 3A). Despite the increase in P-CREB, no evident change occurred in the amount of CREB as measured by Western blot (Figs. 3A and B).
The exposure of cells to ISO before and during depolarization resulted in a concentration-dependent inhibition of KCl-evoked P-CREB (Fig. 4). Figure 5 demonstrates a similar finding using immunocytochemistry and an antibody to P-CREB. KCl-induced depolarization causes a marked increase in P-CREB fluorescence and exposure to ISO inhibits that increase. Exposure of cells to Bay K 8644, an L-type Ca2+ channel agonist, even in the presence of ISO, leads to an increase in P-CREB fluorescence relative to ISO alone. This result suggests that the ISO inhibition of P-CREB fluorescence is related to the inhibition of the entry of Ca2+ through the L-type channel because Bay K 8644 can partially overcome ISO inhibition as has been shown in other systems.8
Others have shown that P-CREB is linked to the entry of Ca2+, its binding to CaM, and the translocation of the complex to the cell nucleus. In SH-SY5Y cells, a similar process apparently occurs. After exposure to KCl, cells were rapidly fixed and permeabilized and exposed to CaM antibody followed by exposure to an Alexa-Fluor 568 goat anti-mouse IgG secondary antibody. Figure 6A demonstrates the marked increase in CaM fluorescence after KCl depolarization. The CaM fluorescence is essentially absent when KCl depolarization occurs in the presence of the L-type Ca2+ channel antagonist NTP. Exposure to CTX, the N-type Ca2+ channel blocker, during KCl depolarization does not prevent the CaM fluorescence from appearing. We have previously shown in SH-SY5Y cells that 70% of Ca2+ entry on depolarization occurs through N-type channels. These results suggest that Ca2+ entering through the L-type Ca2+ channel and not the N-type Ca2+ channel activates CaM and allows its translocation to the nucleus as others have found.9
We then examined the effect of increasing concentrations of ISO on CaM fluorescence. As can be seen in Figure 6B, after KCl depolarization, there was a decrease in CaM fluorescence with increasing concentrations of ISO. We questioned whether the effect of ISO related to inhibition of Ca2+ entry through the L-type channel or some other downstream process such as Ca2+ binding to CaM or translocation of Ca2+/CaM to the nucleus. To test these possibilities, we exposed cells to Bay K 8644 and ISO before KCl stimulation. As can be seen in Figure 6B, Bay K 8644 exposure attenuated the inhibition of Ca2+/CaM fluorescence by 0.8 mM ISO.
A further demonstration of the activation and translocation of Ca2+/CaM was done through cell fractionation and isolation of the nuclear fraction. Cells were exposed briefly to KCl in the presence and absence of NTP or increasing concentrations of ISO. The nuclear fraction was isolated, and immunoblots for CaM were obtained. Figure 7 demonstrates the increased amount of CaM found in the nucleus upon KCl exposure and the NTP- and ISO-evoked inhibition of the KCl-evoked CaM increase, supporting the view that NTP and ISO have an inhibitory effect on Ca2+/CaM translocation.
In this study, we have shown that exposure of SH-SY5Y cells to ISO inhibits the L-type Ca2+ channel current, the appearance of CaM fluorescence, and P-CREB in the cell nucleus. Other investigators have demonstrated that CaM translocation is initiated by the entry of Ca2+ through the L-type voltage-gated Ca2+ channel and the subsequent binding of Ca2+ to CaM.10 We have shown that ISO inhibits the entry of Ca2+ through the L-type channel and that the inhibition of Ca2+ entry is linked to the decreased translocation of CaM and the lower degree of P-CREB. Two pieces of evidence support this contention. First, NTP, the dihydropyridine blocker of the L-type channel, prevents the appearance of CaM fluorescence in the nucleus. CTX, a N-type Ca2+ channel blocker, does not prevent the appearance of CaM fluorescence in the nucleus, suggesting that Ca2+ entering through the L-type channel but not the N-type channel has privileged access to CaM and activates its movement to the nucleus presumably in its Ca2+-bound state. Second, the L-type Ca2+ channel agonist, Bay K 8644, is able to mostly overcome the inhibition of both NTP and ISO. When Bay K 8644 is added to the solution containing ISO, we once again see CaM fluorescence in the nucleus, which is absent when cells are exposed to ISO alone.
In SH-SY5Y cells, we have shown that one-third of the Ca2+ transient induced by KCl depolarization results from the entry of Ca2+ through the L-type channel.4 The Ca2+ that enters through the L-type channel then stimulates the release of Ca2+ from the caffeine-sensitive Ca2+ store. The current evidence does not allow one to distinguish between the activation of CaM by Ca2+ that enters directly through the L-type channel or the Ca2+ that is released from the caffeine-sensitive Ca2+ store. CaM has been shown to be bound to the L-type channel11 but it also has been shown to be a modulator of the ryanodine receptor, which is the conduit that releases Ca2+ from the caffeine-sensitive Ca2+ store.12 Because the L-type channel and the ryanodine receptor are in close proximity and evidence estimates the CaM concentration in the near vicinity of the L-type channel to be 2.5 mM, determining which of these two sources of Ca2+ binds to and activates CaM will require further investigation.11 However, Ca2+ entry through the N-type Ca2+ channel also evokes the release of Ca2+ from the caffeine-sensitive Ca2+ store but has no effect on P-CREB or CaM translocation suggesting a unique role of the L-type channel.
It is also possible that other sites along the path from extracellular Ca2+ to P-CREB might be altered by ISO. The binding of Ca2+ to CaM might be inhibited by ISO. However, that seems unlikely in light of in vitro evidence that at the concentrations of ISO used in this study, the affinity of CaM for Ca2+ is actually increased.13 However, the mechanism of rapid transport of Ca2+/CaM to the nucleus from the plasma membrane is believed to be a facilitated transport and might be a site at which ISO is exerting its inhibitory effect.2 Finally, Ca2+/CaM activation of a kinase in the nucleus might be another ISO-sensitive site. The kinase involved in P-CREB is CaM kinase IV,14 and at present there is no evidence regarding the effect of ISO on this kinase.
CREB activation of new transcription requires phosphorylation of serine-133.15 P-CREB has been directly linked to entry of Ca2+ through the L-type channel and association of that Ca2+ with CaM. P-CREB is stimulated by synaptic activity including those that support long-term potentiation and long-term depression.16 It has further been shown that P-CREB is reduced in the hippocampus of mice that have been anesthetized with ISO and N2O.17 Our demonstration of ISO suppression of Ca2+/CaM translocation and P-CREB in a neuronal model system suggest a link between the effects of ISO on memory and the molecular processes involved. It also suggests that anesthesia with volatile anesthetics might have a considerable effect on gene transcription.
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© 2009 International Anesthesia Research Society
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