Glucocorticoids Prolong Ca2+ Transients in Hippocampal-Derived H19-7 Neurons by Repressing the Plasma Membrane Ca2+-ATPase-1
Aditi Bhargava,
Robert S. Mathias,
James A. McCormick,
Mary F. Dallman and
David Pearce
Departments of Medicine, Cellular and Molecular Pharmacology (A.B., J.A.M., D.P.), Physiology (A.B., M.F.D.), and Pediatrics (R.S.M.), University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Dr. David Pearce, Department of Medicine, Box 0532, 513 Parnassus Avenue, University of California, San Francisco, California 94143. E-mail: pearced{at}medicine.ucsf.edu.
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ABSTRACT
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Calcium ions (Ca2+) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca2+-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca2+ levels ([Ca2+]i) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca2+]i that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca2+ transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca2+ transients by down-regulating Ca2+ extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca2+]i, is likely to compromise neuronal function and viability.
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INTRODUCTION
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THE HIPPOCAMPUS, a brain region critical to learning and memory acquisition, is highly sensitive to the effects of glucocorticoids (GC). GC effects in hippocampal neurons are mediated by two nuclear receptors, a high affinity type I or mineralocorticoid receptor (MR) and a lower affinity type II or glucocorticoid receptor (GR) (1), which exert coordinated effects on gene transcription (2). Although acute rises in GCs are neuroprotective, long-term elevations, as seen in chronic stress, result in hippocampal atrophy and neurodegeneration (3). Hippocampal neurons are also vulnerable to stressful conditions such as seizures, ischemia, and hypoglycemia (4, 5) and at the same time exhibit remarkable plasticity and show dendritic remodeling (6), synaptic turnover, and neurogenesis (7). Although alterations in the intracellular Ca2+ concentration are known to play a crucial role in mediating GC effects on the plasticity, excitability, and viability of hippocampal neurons (8, 9), the mechanism underlying these effects remains unclear.
The intracellular Ca2+ concentration ([Ca2+]i) is determined by the balance among entry through Ca2+ channels (10), release from intracellular stores (11), and buffering and efflux processes. GC regulation of evoked Ca2+ transients has focused primarily on increased entry through ligand- and voltage-gated calcium channels (12, 13); however, several lines of circumstantial evidence suggest that GCs might reduce calcium extrusion as well (14, 15, 16).
In rat, four isoforms of plasma membrane Ca2+-adenosine triphosphatase-1 (PMCA) exist (17), which are expressed in a developmentally regulated manner (18). PMCA1 and -4 are ubiquitously expressed whereas PMCA2 and -3 exhibit tissue-specific expression, with prominent expression in the brain, heart, skeletal muscle, and kidney. Each isoform has been shown to have four to six alternative splice variants, with variants a and b exhibiting developmental switch and differential calcium-calmodulin affinities (19, 20). The PMCAs are energy-dependent Ca2+ pumps that extrude calcium (21, 22) and contribute to maintenance of the low resting level of cytosolic Ca2+.
Recently, we identified PMCA1 as a GC-repressed gene in rat hippocampus (14). In adrenalectomized animals, PMCA1 was repressed by injections of high, but not low, doses of the GC, corticosterone (B), suggesting a GR-mediated effect. Moreover, in response to stress there was an inverse correlation between B level and PMCA1 mRNA in adrenal-intact animals. Repression of PMCA1 was also observed in cultured conditionally differentiated H19-7 hippocampal neurons, but only when the cells were in the differentiated state. With these observations in mind, we were interested in directly examining whether modulation of PMCA1 was one mechanism by which glucocorticoids regulate [Ca]i. We therefore used H19-7 cells as a model system in which PMCA1 levels could be manipulated in the presence and absence of GCs, whereas calcium transients were monitored.
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RESULTS
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Expression Pattern of PMCAs and Glutamate Receptor Subunits in Cultured Neuronal Cells
We first examined the expression pattern of PMCA isoforms in H19-7 cells. H19-7 cells are conditionally differentiated neuronal cells derived from rat embryonic d 17 (E17) hippocampus (23). In the rat, four PMCA isoforms exist, which are expressed in the hippocampus in a developmentally regulated manner (18). As shown in Fig. 1A
, RT-PCR demonstrated that isoforms 1 and 4 were expressed, whereas isoforms 2 and 3 were undetectable in H19-7 cells. This is consistent with the in vivo expression pattern of PMCA isoforms; PMCA1 and -4 are expressed throughout development, whereas PMCA2 and -3 are expressed after E18 (24).

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Figure 1. PMCA1 and PMCA4 Are Expressed in H19-7 Cells and Are GC Regulated
A, RT-PCR was used to amplify PMCA isoforms 14 from H19-7 cell RNA. Isoforms 1 and 4 (lanes 1 and 4) were present, whereas 2 and 3 (lanes 2 and 3) were undetectable. B, Effect of B treatment on PMCA1 and -4 immunoreactive protein in H19-7 cells. Cell lysates from control and 3-h B-treated cells were prepared. Two micrograms of total protein were separated by SDS-PAGE, and proteins were transferred to nylon membranes. Blots were probed with PMCA1 (NR1-1) antibody, then stripped and reprobed with PMCA4 antibody (JA9), as described in Materials and Methods. Densitometry data were analyzed using NIH Image. Error bars represent ± SEM (n = 35). A t test was used to determine significance. *, P = 0.029; #, P = 0.002.
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Recently it has been shown that in vivo both B and stress repress PMCA1 mRNA expression in rat hippocampus (14). Moreover, B treatment of differentiated, but not undifferentiated, H19-7 hippocampal cells resulted in the repression of PMCA1 message, as assessed by Northern blot analysis. We therefore examined the effect of B on PMCA1 and PMCA4 protein levels. As shown in Fig. 1B
, 3 h of B treatment resulted in a 1.8- to 2.0-fold (or
46%) reduction of PMCA1 protein level (P = 0.029, by t test) and a 2.5-fold or approximately 57% (P = 0.002) reduction of PMCA4 protein (Fig. 1B
), consistent with previously identified effects on gene expression (14).
We further characterized the H19-7 cells with respect to expression pattern of some of the key subunits of glutamate receptors by RT-PCR. These data are summarized in Table 1
. The expression patterns of these subunits are in agreement with those seen in vivo in the brain (25).
Measurements of [Ca2+]i in Single Neuronal Cells
To determine whether B-induced repression of PMCA1 and -4 was associated with a physiologically significant effect on [Ca2+]i, the effect of B on agonist-induced [Ca2+]i was determined. We first tried the more traditional neurotransmitters such as bradykinin, N-methyl-D-aspartate, glutamate, and the synthetic glutamate analog kainic acid to evoke Ca2+ transients in H19-7 cells in view of the above results. Surprisingly, these traditional neurotransmitters failed to evoke Ca2+ transients in 9598% of H19-7 cells. KCl was able to evoke Ca2+ transients in H19-7 cells; however, the response was not robust. It has been demonstrated that even though the glutamate receptor subunits are expressed as early as stage E14, mere expression of these subunits does not result in a functional receptor. It is only toward the end of embryonic development that neurons begin to respond to N-methyl-D-aspartate or potassium depolarization (26). Thus, it appears that although H19-7 cells express the glutamate receptor subunits, the resulting receptors are probably not functional. They appear largely to reflect the dynamics of the developing embryonic brain, and although they can be conditionally differentiated and express neuronal markers, they are functionally immature.
Thrombin, a potent inducer of Ca2+ transients in most cell types, was recently shown to induce intracellular Ca2+ spikes in primary hippocampal neurons (27) and in vitro in a neuroblastoma cell line (28). Receptors for thrombin are present throughout the central nervous system, including the hippocampus, and endogenous thrombin appears to protect hippocampal CA1 neurons against ischemic insults and mobilizes Ca2+ in Fura-2-loaded CA1 neurons (27). Other studies indicate that thrombin is implicated in a wide variety of processes that include changes in gene expression in the central nervous system, response to injury, long-term potentiation, neuronal plasticity, and dendritic remodeling (29, 30). Thus, thrombin appears to be a physiological regulator of calcium in hippocampal neurons. Importantly for the present purposes, both undifferentiated and differentiated H19-7 cells respond to thrombin with a rapid increase in [Ca2+]i (Fig. 2
). However, in undifferentiated, vehicle-treated cells, the agonist-evoked Ca2+ transient returned rapidly to baseline (Fig. 2A
). Consistent with the lack of repression of PMCA1, treatment with B had no effect on the duration of evoked Ca2+ transients (Fig. 2B
). In differentiated vehicle-treated cells, stimulated transients returned rapidly to baseline (Fig. 2C
). In contrast, B treatment of differentiated cells markedly prolonged the duration of agonist-evoked Ca2+ transients, which remained elevated for at least 510 min (compare Fig. 2
, C and D). In addition, the amplitude of the peak was consistently reduced by B treatment in differentiated cells. Thus, B treatment both repressed PMCA1 expression and had a profound effect on evoked calcium transients in differentiated, but not undifferentiated, H19-7 cells. Treatment with B for periods shorter than 2 h (15, 30, or 60 min) did not result in repression of PMCA1 or prolongation of agonist-evoked Ca2+ transients (data not shown), consistent with the idea that transcriptional repression of PMCA1 is required for this effect.

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Figure 2. The Effect of B Treatment on Thrombin-Evoked [Ca2+]i in H19-7 Cells
Cells were cultured and loaded with Fura-2/AM as described in Materials and Methods. A, Undifferentiated (UnD), vehicle-treated H19-7 cells; B, undifferentiated, B-treated cells; C, vehicle-treated, differentiated (Dif) H19-7 cells; D, 10-7 M B-treated, differentiated (Dif) H19-7 cells. Measurements were made from at least five to eight independent cells per condition. One representative tracing from each condition is shown.
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Exogenous Expression of PMCA1 Prevents B-Mediated Prolongation of Ca2+ Transients
It was reasoned that if repression of PMCA1 were necessary for the B-induced prolongation in Ca2+ transients, then heterologous expression of PMCA1 from a non-B-regulated promoter would prevent the B-induced prolongation in Ca2+ transients. To address this issue directly, a PMCA1 expression vector driven by the cytomegalovirus promoter was transiently transfected into H19-7 cells along with a green fluorescent protein (GFP) expression vector to identify transfected cells. Control cells transfected with the cytomegalovirus-driven empty vector and GFP continued to show B-mediated prolongation in agonist evoked Ca2+ transients (Fig. 3
, A and B). However, in striking contrast, neuronal cells overexpressing exogenous PMCA1 were resistant to the B-induced prolongation of Ca2+ transients (Fig. 3
, C and D). Heterologous overexpression of PMCA1 did not have any effect on basal cytosolic levels of calcium, consistent with previous observations in other cell types (31, 32). It was also determined by immunocytochemistry that the heterologously overexpressed PMCA1 was targeted appropriately to the plasma membrane and was not retained in the endoplasmic reticulum, whereas the endogenously expressed PMCA1 was undetectable in control cells [data not shown and Brini et al. (32)]. These data demonstrate that PMCA1 overexpression prevents the B-induced prolongation in Ca2+ transients, consistent with the idea that repression of PMCA1 is necessary for this effect.

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Figure 3. Exogenous Expression of PMCA1 Rescues B-Mediated Prolongation of [Ca2+]i in H19-7 Cells
A, GFP- plus vector-transfected, vehicle-treated control cells; B, GFP- plus vector-transfected, 10-7 M B-treated cells; C, GFP- plus PMCA1-transfected, vehicle-treated cells; D, GFP- plus PMCA1-transfected, 10-7 M B-treated cells. Measurements were made from at least 810 independent cells/condition.
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RNA Interference Technique (RNAi) against PMCA1 Results in Prolongation of Intracellular Calcium Transients
To determine whether down-regulation of PMCA1 by itself caused prolongation of agonist-induced Ca2+ transients, RNAi was used to selectively decrease PMCA1 levels. RNAi is a highly specific and effective method for generating phenotypic knockouts in a variety of animal and cell culture models, including Caenorhabditis elegans (33, 34), Drosophila (35), and mouse (36) tissues and cultured cells (37, 38), although it has not been effective in all cell types. To our knowledge this is the first report showing that RNAi works in cultured mammalian cells to produce a specific downstream effect of physiological significance. We first established that transfection of PMCA1-specific RNAi resulted in a reduction of PMCA1 protein expression relative to that in cells transfected with control RNAi (Fig. 4A
). Expression of PMCA4 remained unaffected by either RNAi treatment (Fig. 4A
).

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Figure 4. RNAi Against PMCA1 Results in Selective Degradation of PMCA1 Protein and Prolonged Evoked Ca2+ Transients
Cells were transfected with dsRNA as described in Materials and Methods. A, PMCA1 protein is selectively reduced in cells that received RNAi against PMCA1 compared with cells treated with control dsRNA. The same blot was stripped and probed with antibody against PMCA4, whose expression remains unchanged with treatment. Densitometry data were obtained from above blots, and the mean average density was calculated using NIH Image. Error bars represent ± SEM (n = 35). A t test was used to calculate P values. *, P = 0.04. B, Selective removal of PMCA1 by RNAi results in prolongation of [Ca2+]i in the absence of GCs. B, H19-7 cells were transfected with dsRNA against PMCA1 (left tracing) or control dsRNA (right tracing). Measurements were made from at least five to eight independent cells per condition, with similar results.
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Next, the effect of RNAi on agonist-stimulated [Ca2+]i was determined. As shown in Fig. 4B
, H19-7 cells transfected with RNAi against PMCA1 consistently exhibited prolonged evoked Ca2+ transients, although not to the same extent as with B treatment. Importantly, transfection of several nonspecific species of RNAi had no effect on Ca2+ transients (Fig. 4B
and data not shown). Hence, we conclude that selective repression of PMCA1 protein levels is sufficient to prolong Ca2+ transients in the absence of GCs.
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DISCUSSION
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These results provide direct support for the idea that regulation of PMCA1 levels is an important mechanism by which GCs modulate evoked Ca2+ transients in neurons and possibly other cell types. The B-induced prolongation in Ca2+ transients was completely suppressed by heterologous expression of PMCA1, on the one hand, and was partially mimicked by specific inhibition of PMCA1 with RNAi, on the other. In support of the relevance of our findings to the intact adult animal, Ca2+ transients in neurons from hippocampal explants treated with high doses of B (sufficient to activate GR) are prolonged, whereas those treated with low doses of B (sufficient to activate the higher affinity MR, but not GR) are foreshortened (39).
Heterologous overexpression of PMCA1 in differentiated cultured cells was able to overcome the repressive effects of B treatment. Similar to the effect of B, heterologous expression of PMCA1 did not affect basal calcium levels, consistent with previous findings in other cell types overexpressing PMCA1 that do not show altered basal [Ca2+]i (31, 32). Interestingly, differentiated H19-7 cells overexpressing PMCA1 were resistant to B-induced prolongation of evoked Ca2+ transients, suggesting that repression of PMCA1 is necessary to decrease the rate of cytosolic Ca2+ extrusion. However, the caveat exists that this overexpression may rescue or compensate for the influence other candidate proteins may exert on recovery, including the endoplasmic Ca2+-adenosine triphosphatases (SERCAs), other PMCA isoforms, or the Na+/Ca2+ exchanger when cells are challenged with B. Overexpression of PMCA1 does not seem to dramatically alter the amplitude of the [Ca2+]i response (after agonist stimulation) and is not qualitatively different from that seen in untreated cells. Notably, this also suggests that B-mediated decreases in efflux of Ca2+ are not due to diminished ATP, because isolated overexpression of PMCA1 (whose activity is ATP dependent) rescues the inhibitory effects of B.
The other calcium extrusion candidate, the Na+/Ca2+ exchanger, has been shown to be present mainly in the dendrites in neurons and in presynaptic nerve terminals (40). In addition, the Na+/Ca2+ exchanger and mitochondria play a minor role during [Ca2+]i recovery from [Ca2+]i elevations that do not exceed 50 nM (41, 42). In our study, [Ca2+]i measurements were made from the cell body and not the dendrites; hence, the role played by the Na+/Ca2+ exchanger in the extrusion phase may not be very significant. Nonetheless, its role in [Ca2+]i extrusion in H19-7 neurons will be of interest to determine.
Interestingly, we found that B treatment does not inhibit PMCA1 expression in undifferentiated cells, nor does it alter agonist-stimulated Ca2+ transients. This argues that repression of PMCA1 by B is state and context dependent; however, repression of PMCA1 appears to be both necessary and sufficient for prolongation of evoked [Ca2+]i transients. Differentiated H19-7 cells show inhibition of PMCA1 upon B treatment, and the duration of agonist-induced [Ca2+]i is also increased. In addition, B treatment reduced the amplitude of the peak in differentiated cells. The reduction in peak height could be due to decreased release from the endoplasmic reticulum or more rapid uptake into the intracellular stores upon agonist treatment. It is conceivable that B treatment alters the expression of other components of the system (such as SERCAs) that influence intracellular levels of calcium. Shorter periods of treatment with B did not repress PMCA1 mRNA, nor did it result in prolongation of agonist-evoked Ca2+ transients consistent with a transcriptional mechanism.
In explants from CA3/CA1 regions of the hippocampus, treatment with low doses of B result in small ionic conductance and transmitter responses (43) and decreased Ca2+ conductance (44). The evoked Ca2+ transients return faster to baseline under conditions of low B administration, whereas neuronal cells treated with high B show a decrease in the clearance rate of [Ca2+]i (39). Large [Ca2+]i transients are associated with long-term potentiation modulating synaptic plasticity associated with learning and memory, whereas smaller magnitudes of [Ca2+]i transients are associated with long-term depression (45, 46). Qualitatively, our observations in conditionally differentiated cultured hippocampal neurons are similar to those seen in dissociated hippocampal cells under conditions of high B treatment. Thus, our findings in H19-7 cells appear to reflect in vivo regulation of [Ca2+]i transients by high levels of GC, with the caveat that H19-7 cells express only low levels of MR, and hence, the effects of low B concentrations mediated by MR could not be studied.
It is striking that repression of PMCA1 is not only necessary but is also sufficient to produce prolongation of stimulated Ca2+ transients in differentiated H19-7 cells. However, neither PMCA1 repression nor overexpression had a detectable effect on basal Ca2+ levels in H19-7 cells, similar to previous observations in a variety of other neuronal and nonneuronal cell types (31, 32, 39, 47, 48). These observations are consistent with the idea that under resting conditions, pump levels are in excess, and turnover number is substrate limited even when PMCA levels are repressed (47). In contrast, under conditions of elevated [Ca2+]i, as seen during evoked transients, Ca2+ extrusion becomes limited when PMCA1 is repressed.
Although the effects of B and RNAi on evoked transients were similar, it is noteworthy that the [Ca2+]i elevation during the later phase (after 35 min) was substantially greater in the B-treated cells. In view of the complete suppression of the B effect by heterologous PMCA1 (Fig. 3
) and the lack of increase in peak [Ca2+]i, this difference suggests that B treatment alters the expression of other genes involved in the removal of cytoplasmic Ca2+ (for example, PMCA4 or SERCAs); however, late effects on Ca2+ channels could also be implicated. In support of a role for PMCA4, this PMCA isoform was also repressed by B treatment in differentiated H19-7 cells, whereas its levels remained unchanged in neurons treated with RNAi against PMCA1. It remains to be established whether selectively inhibiting PMCA4 by RNAi also results in prolongation of Ca2+ transients in H19-7 cells.
During fetal/embryonic development, hippocampal neurons are in a dynamic state, establishing new synaptic contacts and undergoing a plethora of active remodeling (49). At this stage of development, the onus of maintaining [Ca2+]i by actively extruding elevated levels of Ca2+ falls upon PMCA1 and -4, whereas tissue-specific PMCA isoforms 2 and 3 do not begin to be expressed until later during embryonic development (24). Thus, any modulations or oscillations in [Ca2+]i required by the neurons will be achieved by regulating the activity and/or levels of PMCA1 and -4. H19-7 cells derived from E17 hippocampal neurons express both PMCA1 and 4, and both isoforms are regulated by B in differentiated cells. Thus, H19-7 cells provide an attractive model system to study dynamic changes taking place in the developing embryonic hippocampus.
Although the transient high levels of GCs associated with circadian peaks or brief stress promote adaptive behavioral responses, persistently elevated levels in conjunction with chronic stress or Cushings syndrome contribute to depression, posttraumatic stress disorder, and dementia (4, 50). At the cellular level, acute GC-induced elevations in [Ca2+]i result in changes in synaptic plasticity and neuronal excitability, whereas chronic elevations lead to remodeling of neurons, enhanced apoptosis (51), and, ultimately, neurodegeneration (3). Delineating the role of GC regulation of PMCA1 in neuronal Ca2+ homeostasis provides a mechanistic basis for these GC-induced effects.
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MATERIALS AND METHODS
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Cell Culture
H19-7 cells of hippocampal origin were grown on poly-L-lysine-coated dishes (or coverslips), in 1% DMEM at 33 C at 5% CO2 as described previously (14). H19-7 cells are transformed with a temperature-sensitive mutant of simian virus 40 T antigen and are conditionally differentiated (23). For differentiation, cells were incubated at 39 C at 5% CO2 in DMEM and N2 supplements, and the process was accelerated by adding 10 ng/ml basic fibroblast growth factor as previously described (23). One day before hormone treatment, cells were provided with medium containing charcoal-stripped fetal bovine serum. Differentiated cells were then treated with 10-7 M B for 0, 30, 60, and 120 min. RNA or cell extracts were prepared as previously described (14).
Western Blot Analysis
H19-7 cells were differentiated and treated with B as described above. Three hours after B treatment, cells were washed twice with ice-cold PBS, and 30 µl lysis buffer were added to the cells. Proteins were electroblotted onto a nylon membrane and immunoprobed as previously described (52). Blots were incubated in appropriate secondary antibody, washed in PBS-Tween 20, and developed using an ECL Plus kit (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the suppliers specifications.
Preparation of RNAi and Cell Transfection
Double-stranded RNA was prepared by synthesizing sense and antisense RNA in vitro using the Riboprobe kit (Promega Corp., Madison, WI) and plasmid pCDNA3.1/rPMCA1 linearized with appropriate restriction enzymes, as described previously (33). For RNAi experiments, at least 15 µg PMCA1 double-stranded RNA (dsRNA) or equivalent pmol amounts of nonspecific dsRNA (pBluescript) were used per well.
For transfection studies, cells were seeded on coverslips at a density of 5 x 104 cells/well in a six-well dish. Sixteen hours later, PMCA1 expression plasmid (or vector) and GFP were transfected using the Lipofectamine method (BRL, Gaithersburg, MD) according to the suppliers specifications. Twenty-four hours after transfection, cells were differentiated using the conditions described above. DNA-transfected cells were then treated with either vehicle (ethanol) or 10-7 M B for various times (15 min to 3 h). [Ca2+]i measurements were performed as described below. After RNAi transfection, cells were differentiated, and Ca2+ measurements were performed without B treatment.
Microscopy and [Ca2+]i Measurements
Cells were incubated in loading buffer containing 5 mM Fura-2/AM for 60 min. Transfected cells were identified by their GFP expression. The coverslips were mounted on a temperature-controlled chamber at 31 C in assay medium (53). Fura-2 fluorescence was measured using a Nikon epifluorescence inverted microscope (Melville, NY) fitted with a rotating holder for excitation filters (340 and 380 nm) as previously described (53). Signals were digitized using a Labmaster interface board (Scientific Solutions, Solon, OH) and were recorded in an IBM style computer using the UMANS software package (Chester Regen, Bio-Rad Laboratories, Inc., Hercules, CA). [Ca2+]i was calculated as previously described (54). Agonist (thrombin, 1.5 U/ml) was added to evoke intracellular calcium transients. Signal from three to five cells per condition per experiment were collected. Each experimental condition was repeated at least three times on different days.
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ACKNOWLEDGMENTS
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Dr. Penniston and Mrs. Filoteo (Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN) are acknowledged for providing the antibodies against PMCA1 (NR1-1) and 4 (JA-9). The authors thank Drs. R. Edwards and D. Julius for their helpful comments on the manuscript.
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FOOTNOTES
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This work was supported by NIH Grants DK-54376 and DK-51151 (to D.P.) and DK-28172 (to M.F.D.).
Abbreviations: ATPase, Adenosine triphosphatase; B, corticosterone; [Ca2+]i, intracellular Ca2+; dsRNA, double-stranded RNA; E17, embryonic d 17; GC, glucocorticoid; GFP, green fluorescent protein; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PMCA1, plasma membrane Ca2+-adenosine triphosphatase-1; RNAi, RNA interference technique; SERCA, sarco/endoplasmic Ca2+-ATPase.
Received for publication November 19, 2001.
Accepted for publication February 26, 2002.
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REFERENCES
|
---|
- Reul JM, van den Bosch FR, de Kloet ER 1987 Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J Endocrinol 115:459467[Abstract]
- Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344[Medline]
- McEwen BS, Angulo J, Cameron H, Chao HM, Daniels D, Gannon MN, Gould E, Mendelson S, Sakai R, Spencer R, Woolley C 1992 Paradoxical effects of adrenal steroids on the brain: protection versus degeneration. Biol Psychiatry 31:177199[Medline]
- Sapolsky RM 2000 Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch Gen Psychiatry 57:925935[Abstract/Free Full Text]
- Mattson MP 2000 Neuroprotective signaling and the aging brain: take away my food and let me run. Brain Res 886:4753[CrossRef][Medline]
- Cameron HA, Gould E 1996 Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy. J Comp Neurol 369:5663[CrossRef][Medline]
- McEwen BS, Sapolsky RM 1995 Stress and cognitive function. Curr Opin Neurobiol 5:205216[CrossRef][Medline]
- Elliott EM, Sapolsky RM 1993 Corticosterone impairs hippocampal neuronal calcium regulation: possible mediating mechanisms. Brain Res 602:8490[CrossRef][Medline]
- Ghosh A, Greenberg ME 1995 Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268:239247[Medline]
- Varadi G, Mori Y, Mikala G, Schwartz A 1995 Molecular determinants of Ca2+ channel function and drug action. Trends Pharmacol Sci 16:4349[CrossRef][Medline]
- Simpson PB, Challiss RA, Nahorski SR 1995 Neuronal Ca2+ stores: activation and function. Trends Neurosci 18:299306[CrossRef][Medline]
- Joels M, de Kloet ER 1989 Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus. Science 245:15021505[Medline]
- Landfield PW, Pitler TA 1984 Prolonged Ca2+-dependent afterhyperpolarizations in hippocampal neurons of aged rats. Science 226:10891092[Medline]
- Bhargava A, Meijer OC, Dallman MF, Pearce D 2000 Plasma membrane calcium pump isoform 1 gene expression is repressed by corticosterone and stress in rat hippocampus. J Neurosci 20:31293138[Abstract/Free Full Text]
- Elliott EM, Sapolsky RM 1992 Corticosterone enhances kainic acid-induced calcium elevation in cultured hippocampal neurons. J Neurochem 59:10331040[Medline]
- Elliott EM, Mattson MP, Vanderklish P, Lynch G, Chang I, Sapolsky RM 1993 Corticosterone exacerbates kainate-induced alterations in hippocampal tau immunoreactivity and spectrin proteolysis in vivo. J Neurochem 61:5767[Medline]
- Shull GE, Greeb J 1988 Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases. J Biol Chem 263:86468657[Abstract/Free Full Text]
- Stahl WL, Eakin TJ, Owens Jr JW, Breininger JF, Filuk PE, Anderson WR 1992 Plasma membrane Ca2+-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization. Brain Res Mol Brain Res 16:223231[Medline]
- Carafoli E, Garcia-Martin E, Guerini D 1996 The plasma membrane calcium pump: recent developments and future perspectives. Experientia 52:10911100[Medline]
- Keeton TP, Burk SE, Shull GE 1993 Alternative splicing of exons encoding the calmodulin-binding domains and C termini of plasma membrane Ca2+-ATPase isoforms 1, 2, 3, and 4. J Biol Chem 268:27402748[Abstract/Free Full Text]
- Carafoli E 1991 Calcium pump of the plasma membrane. Physiol Rev 71:129153[Free Full Text]
- Carafoli E 1992 The Ca2+ pump of the plasma membrane. J Biol Chem 267:21152118[Free Full Text]
- Eves EM, Tucker MS, Roback JD, Downen M, Rosner MR, Wainer BH 1992 Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression. Proc Natl Acad Sci USA 89:43734377[Abstract]
- Strehler EE, Zacharias DA 2001 Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81:2150[Abstract/Free Full Text]
- Goebel DJ, Poosch MS 1999 NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Brain Res Mol Brain Res 69:164170[Medline]
- Barish ME, Mansdorf NB 1991 Development of intracellular calcium responses to depolarization and to kainate and N-methyl-D-aspartate in cultured mouse hippocampal neurons. Brain Res Dev Brain Res 63:5361[Medline]
- Striggow F, Riek M, Breder J, Henrich-Noack P, Reymann KG, Reiser G 2000 The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci USA 97:22642269[Abstract/Free Full Text]
- Yang Y, Akiyama H, Fenton Jr JW, Brewer GJ 1997 Thrombin receptor on rat primary hippocampal neurons: coupled calcium and cAMP responses. Brain Res 761:1118[CrossRef][Medline]
- Mizutani A, Saito H, Matsuki N 1996 Possible involvement of plasmin in long-term potentiation of rat hippocampal slices. Brain Res 739:276281[CrossRef][Medline]
- Weinstein JR, Gold SJ, Cunningham DD, Gall CM 1995 Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 15:29062919[Abstract]
- Brandt PC, Sisken JE, Neve RL, Vanaman TC 1996 Blockade of plasma membrane calcium pumping ATPase isoform I impairs nerve growth factor-induced neurite extension in pheochromocytoma cells. Proc Natl Acad Sci USA 93:1384313848[Abstract/Free Full Text]
- Brini M, Bano D, Manni S, Rizzuto R, Carafoli E 2000 Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca2+ signalling. EMBO J 19:49264935[Abstract/Free Full Text]
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806811[CrossRef][Medline]
- Montgomery MK, Xu S, Fire A 1998 RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 95:1550215507[Abstract/Free Full Text]
- Kennerdell JR, Carthew RW 1998 Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95:10171026[Medline]
- Wianny F, Zernicka-Goetz M 2000 Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2:7075[CrossRef][Medline]
- Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, Hemmings BA, Dixon JE 2000 Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA 97:64996503[Abstract/Free Full Text]
- Hammond SM, Bernstein E, Beach D, Hannon GJ 2000 An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293296[CrossRef][Medline]
- Joels M, Werkman T, Karst H, Juta TAJ, Wadman WJ 1998 Corticosteroids and calcium homeostasis: implication for neuroprotection and neurodegeneration. Amsterdam: Harwood Academic
- Luther PW, Yip RK, Bloch RJ, Ambesi A, Lindenmayer GE, Blaustein MP 1992 Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations. J Neurosci 12:48984904[Abstract]
- Miller RJ 1991 The control of neuronal Ca2+ homeostasis. Prog Neurobiol 37:255285[CrossRef][Medline]
- Werth JL, Usachev YM, Thayer SA 1996 Modulation of calcium efflux from cultured rat dorsal root ganglion neurons. J Neurosci 16:10081015[Abstract]
- Joels M, de Kloet ER 1994 Mineralocorticoid and glucocorticoid receptors in the brain. Implications for ion permeability and transmitter systems. Prog Neurobiol 43:136[CrossRef][Medline]
- Karst H, Wadman WJ, Joels M 1994 Corticosteroid receptor-dependent modulation of calcium currents in rat hippocampal CA1 neurons. Brain Res 649:234242[CrossRef][Medline]
- Cormier RJ, Greenwood AC, Connor JA 2001 Bidirectional synaptic plasticity correlated with the magnitude of dendritic calcium transients above a threshold. J Neurophysiol 85:399406[Abstract/Free Full Text]
- Malenka RC, Kauer JA, Zucker RS, Nicoll RA 1988 Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science 242:8184[Medline]
- Liu BF, Xu X, Fridman R, Muallem S, Kuo TH 1996 Consequences of functional expression of the plasma membrane Ca2+ pump isoform 1a. J Biol Chem 271:55365544[Abstract/Free Full Text]
- Usachev YM, Toutenhoofd SL, Goellner GM, Strehler EE, Thayer SA 2001 Differentiation induces up-regulation of plasma membrane Ca2+-ATPase and concomitant in-crease in Ca2+ efflux in human neuroblastoma cell line IMR-32. J Neurochem 76:17561765[CrossRef][Medline]
- Perry EK, Piggott MA, Court JA, Johnson M, Perry RH 1993 Transmitters in the developing and senescent human brain. Ann NY Acad Sci 695:6972[Abstract]
- McEwen BS 1999 Stress and hippocampal plasticity. Annu Rev Neurosci 22:105122[CrossRef][Medline]
- Roy M, Sapolsky R 1999 Neuronal apoptosis in acute necrotic insults: why is this subject such a mess? Trends Neurosci 22:419422[CrossRef][Medline]
- Filoteo AG, Elwess NL, Enyedi A, Caride A, Aung HH, Penniston JT 1997 Plasma membrane Ca2+ pump in rat brain. Patterns of alternative splices seen by isoform-specific antibodies. J Biol Chem 272:2374123747[Abstract/Free Full Text]
- Huang CL, Takenawa T, Ives HE 1991 Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors. J Biol Chem 266:40454048[Abstract/Free Full Text]
- Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:34403450[Abstract]