Physiological effects of Na+/Ca2+ exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes

Martin K. Slodzinski1,3 and Mordecai P. Blaustein1,2,3

Departments of 1 Physiology and 2 Medicine and the 3 Center for Vascular Biology and Hypertension, University of Maryland School of Medicine, Baltimore, Maryland 21201

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Antisense oligodeoxynucleotides (AS-oligos) targeted to the Na+/Ca2+ exchanger (NCX) inhibit NCX-mediated Ca2+ influx in mesenteric artery (MA) myocytes [Am. J. Physiol. 269 (Cell Physiol. 38): C1340-C1345, 1995]. Here, we show AS-oligo knockdown of NCX-mediated Ca2+ efflux. In initial experiments, the cytosolic free Ca2+ concentration ([Ca2+]cyt) was raised, and sarcoplasmic reticulum (SR) Ca2+ sequestration was blocked with caffeine and cyclopiazonic acid; the extracellular Na+-dependent (NCX) component of Ca2+ efflux was then selectively inhibited in AS-oligo-treated cells but not in controls (no oligos or nonsense oligos). In contrast, the La3+-sensitive (plasmalemma Ca2+ pump) component of Ca2+ efflux was unaffected in AS-oligo-treated cells. Knockdown of NCX activity was reversed by incubating AS-oligo-treated cells in normal media for 5 days. Transient [Ca2+]cyt elevations evoked by serotonin (5-HT) at 15-min intervals in AS-oligo-treated cells were indistinguishable from those in controls. When cells were stimulated every 3 min, however, the peak amplitudes of the second and third responses were larger, and [Ca2+]cyt returned to baseline more slowly, in AS-oligo-treated cells than in controls. Peak 5-HT-evoked responses in the controls, but not AS-oligo-treated cells, were augmented more than twofold in Na+-free media. This implies that NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness. The repetitive stimulation data suggest that the NCX may be important during tonic activation of arterial myocytes.

serotonin; lanthanum; caffeine; cyclopiazonic acid

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CYTOSOLIC CALCIUM is a ubiquitous signal transduction element. In vascular smooth muscle (VSM), agonist- and depolarization-triggered vasoconstrictions are initiated by an increase in the cytosolic free Ca2+ concentration ([Ca2+]cyt) (20, 28). This "activator Ca2+" is mobilized from Ca2+ stores in the sarcoplasmic reticulum (SR) and/or enters the cells from the extracellular fluid (ECF) (20, 28). Conversely, reduction of [Ca2+]cyt promotes vasodilation (20, 28). Ca2+ is removed from the contractile apparatus by resequestration into the SR by the SR Ca2+ (SERCA) pump and/or extrusion to the ECF (17, 20, 28). Ca2+ extrusion is mediated by the plasma membrane (PM) Ca2+ (PMCA) pump (17) and the Na+/Ca2+ exchanger (NCX) (3, 19, 28). The relative roles of the PMCA pump and NCX in VSM are controver- sial. Some investigators have suggested that the NCX in VSM is "latent" (26), or that it plays only a minor role in VSM Ca2+ homeostasis (18, 27). In contrast, others have proposed that the NCX plays a key role in modulating SR Ca2+ and, thus, vascular reactivity (1, 3, 19).

One method to measure the influence of a particular ion transporter is to study the physiology of the cell or organ of interest under normal conditions and after selective block of the transporter. Unfortunately, no sufficiently selective antagonists of the NCX have yet been identified. Removal of extracellular Na+ can inhibit NCX-mediated Ca2+ efflux, but this manipulation also affects the cytosolic Na+ concentration ([Na+]cyt) and alters other Na+-dependent processes such as Na+/H+ exchange (see Ref. 25). Moreover, this reduction in the Na+ electrochemical gradient may promote Ca2+ entry via the NCX. Amiloride analogs (e.g., 3,4-dichlorobenzamil) and a recently described isothiourea derivative (compound 7943) are not sufficiently selective inhibitors of NCX (8, 25, 30). The exchanger inhibitory peptide (XIP), which must be injected into cells, targets the NCX putative calmodulin binding domain on the large cytoplasmic loop of the exchanger (13), but XIP also affects other calmodulin-dependent systems including, for example, the PMCA pump (5). A specific inhibitor of NCX would be especially helpful for resolving questions about NCX function in VSM under physiological conditions.

Recently, we described antisense oligodeoxynucleotide (AS-oligo) knockdown of NCX in primary cultured arterial myocytes (24). In those experiments, AS-oligo-treated VSM cells proliferated normally, maintained normal morphology, and responded appropriately to physiological agonists when compared with "control" cells grown either without oligos, or with scrambled (NS) oligos. [NS-oligos had the same base composition as AS-oligos, but the bases were mismatched.] [Ca2+]cyt was only slightly elevated in quiescent VSM cells in which NCX was knocked down with AS-oligos. In striking contrast to control cells or those treated with NS-oligos, however, AS-oligo-treated cells did not exhibit an increase in [Ca2+]cyt when external Na+ was removed to promote Ca2+ influx via NCX, even after exposure to ouabain (to raise [Na+]cyt). Yet, serotonin (5-HT) was able to evoke a rise in [Ca2+]cyt in AS-oligo-treated cells as well as in control groups. In controls, however, but not in AS-oligo-treated cells, the transient rise in [Ca2+]cyt in response to 5-HT (applied in Ca2+-free media) was augmented after treatment of the cells with ouabain. This implies that the NCX plays a role in ouabain-induced augmentation of SR Ca2+ stores. Taken together, these earlier results demonstrate that AS-oligos targeted to the NCX mRNA can be used to knock down NCX function selectively in arterial myocytes.

To distinguish between the roles of the PMCA pump and the NCX in Ca2+ extrusion, independent inhibition of the PMCA pump and the NCX is required. Eosin can inhibit the PMCA pump, but eosin acts on the cytoplasmic face (9) and also affects other P-type ATPases such as the Na+ pump and the SERCA pump (9). Vanadate inhibits the PMCA pump and other ATPases and may also affect NCX (4). In contrast, low concentrations of La3+ (0.03-0.25 mM) appear to inhibit the PMCA pump selectively in VSM (7, 22). These low La3+ concentrations do not affect the extracellular Na+-dependent (NCX-mediated) component of Ca2+ removal from the cytosol of primary cultured rat VSM cells.

In this report, we address the aforementioned controversy about the physiological role of NCX in VSM. These two tools, AS-oligos and low-dose La3+, were used to identify the mechanisms involved in [Ca2+]cyt recovery after evoked elevation of [Ca2+]cyt.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Primary cell culture. Primary cultured arterial myocytes were prepared from rat mesenteric arteries (MA). The arteries were aseptically excised and placed in Hanks' solution containing a 1% dilution of penicillin/streptomycin solution (Sigma, St. Louis, MO). After removal of connective tissue, the blood vessels were incubated for 35 min at 37°C in Hanks' solution with collagenase (2 mg/ml). The adventitia was then separated from the arterial media; the latter was incubated overnight in DMEM containing 10% FCS (95% air-5% CO2, 37°C). The next day the vessels were placed in Hanks' balanced salt solution containing collagenase (2 mg/ml) and elastase (0.5 mg/ml) and incubated for 40 min (95% air-5% CO2, 37°C). The dissociated cells were triturated with a Pasteur pipette and resuspended in DMEM containing heat-inactivated 10% FCS and the appropriate oligos (the FCS was heated 55°C for 4 h to minimize exonuclease activity; Ref. 21). The suspended cells were plated onto glass coverslips on 35-mm culture plates. The medium was changed three times weekly.

VSM cell culture purity was determined immediately after physiological experiments. Cells were fixed in 95% ice-cold ethanol. A nuclear stain, 4,6-diamidino-2-phenylindole (DAPI), was used to label all cells. Myocytes were then identified by immunochemical methods with mouse anti-rat smooth muscle alpha -actin antibodies (Boehringer Mannheim); FITC-conjugated goat anti-mouse antibodies were used as the secondary stain (Jackson ImmunoResearch Laboratories, West Grove, PA).

AS-oligos. A tandem pair of chimeric phosphorothioated AS-oligos was designed to target the region upstream (5') to, and encompassing the ATG start codon (15) of the NCX1 mRNA (from base -26 to base -10, and from base -9 to base +6) (24). These chimeric phosphorothioated oligos have natural deoxynucleotides flanked by four phosphorothioate modified deoxynucleic acids at the 3'- and 5'-ends (5'-TGAGACTTCCAATTGTT-3' and 5'-AAGCATGTT<UNL>GT<B>A</B></UNL>CAA-3', where bold letters refer to phosphorothioated bases and underlined region corresponds to the location of the start codon). Chimeric phosphorothioated oligos are stable (exonuclease resistant) and are transported into cells better than unmodified oligos (6, 14, 31).

A second set of oligos, with the same base composition, but with scrambled (nonsense, NS-oligos) sequence (5'-AGTACCTTCTATGAGT-3' and 5'-CAGATATATCAGATG-3'), was used to control for nonspecific or toxic effects of the oligos. To ensure that the antisense knockdown of extracellular Na+-dependent Ca2+ efflux effect was dependent on sequence and not on secondary structure, a sense (S-oligos) sequence (5'-AACAATTGGAAGTCTCA-3' and 5'-TTGTACAACATGCTT-3') of complementary base pair composition to our antisense probe served as an additional control in some experiments. All probes were compared with known sequences in GenBank and European Molecular Biology Laboratories using the "Wisconsin Package" sequence analysis program; no significant homologies to other sequences were found.

Control cells were grown without oligos. In parallel, cells were grown in media containing the AS-oligo pair, the S-oligo pair, or the NS-oligo pair. The final concentration of the oligos was 0.5 µM from the time of initial plating until imaging 7-10 days later.

[Ca2+]cyt determination using fura 2. Details of the imaging methods are published (3). Cells were incubated (30 min, 20-22°C, 5% CO2-95% O2) in culture media containing 4.0 µM fura 2-AM, the membrane-permeable acetoxymethyl ester of fura 2. The coverslips were transferred to a tissue chamber mounted on a Nikon Diaphot microscope stage. The cells were superfused with standard physiological salt solution (PSS) for 30 min at 32-34°C to wash away extracellular dye and to allow time for cellular esterases to hydrolyze the fura 2-AM.

The imaging system was designed around a Nikon Diaphot microscope optimized for ultraviolet transmission. Fura 2 fluorescence (510-nm light emission; 380- and 360-nm excitation) and background fluorescence were imaged using a Nikon UV-Fluor objective (×40, numerical aperture 1.3). Fluorescent images were obtained using a micro-channel plate image intensifier (Amperex XX1381, Opelco, Washington, DC) coupled, by fiber optics, to a Pulnix charge-coupled device video camera (Stanford Photonics, Stanford, CA).

Images were acquired, background subtracted, and transformed to Ca2+ images using the MetaFluor imaging system (Universal Imaging, West Chester, PA). Video frames were digitized at a resolution of 512 horizontal × 480 vertical pixels and 8 bits using a Matrox LC imaging board. To improve the signal-to-noise ratio, 16 consecutive video frames were averaged. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (10); 360 nm is the fura 2 isosbestic point. Ca2+ images were acquired at a rate of one averaged image every 3 s to one every 60 s depending on the experimental protocol. The [Ca2+]cyt measurements described in RESULTS were limited to small (~2 µm2) peripheral cytosolic "areas of interest" so as to avoid the nucleus and dense perinuclear organelles. Each image contained 6-12 VSM cells per field; one area of interest was studied in each cell.

Solutions. PSS contained (in mM) 140 NaCl, 5.9 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES, titrated to pH 7.4 with NaOH. For Ca2+-free PSS, CaCl2 was replaced by 1.8 mM MgCl2 (total = 3.2 mM), and 0.05 mM EGTA was added. In Na+-free PSS, the NaCl and NaHCO3 were isosmotically replaced by N-methyl-D-glucamine (NMDG), and the pH was adjusted with HCl. In the La3+ PSS, phosphate and bicarbonate salts were replaced by chloride salts, and EGTA was omitted. LaCl3 was added to solutions immediately before use to minimize the formation of La2(CO3)3. Stock solution containing fura 2-AM (1 mM) was prepared in DMSO. Cyclopiazonic acid (CPA, 5 µM), an inhibitor of the SERCA pump, and caffeine (10 mM) were added to the bathing solution to increase [Ca2+]cyt in certain experiments.

Materials. Chimeric phosphorothioated oligos were synthesized by Oligos Etc. (Wilsonville, OR). FCS was obtained from Hyclone (Logan, UT). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). NMDG, DAPI, 5-HT, elastase, Hanks' solution, LaCl3, CPA, caffeine, verapamil, and ouabain were purchased from Sigma. DMEM was obtained from GIBCO BRL (Grand Island, NY). Collagenase was obtained from Worthington Biochemical (Freehold, NJ). All other reagents were analytical grade or the highest purity available.

Statistical analysis and data presentation. Numerical data are presented as means ± SE of a total of n single cells from at least three independent experiments. Where appropriate, ANOVA was used to calculate the significance of the differences of the means. As noted in RESULTS and the legends to Figs. 1-6, fluorescence data (reported as [Ca2+]cyt) correspond to values from 1) a representative single cell, 2) the averaged values for 6-12 cells in a single field on one coverslip, or 3) the means of values from a number of cells on each of several coverslips from different experiments. In graphs of [Ca2+]cyt vs. time, the data points were connected, and the curves were smoothed using a Fourier transform with discontinuities at the times of solution changes. Microcal Origin software (Northhampton, MA) was used for calculations, graphics, and curve smoothing.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Do AS-oligos affect the La3+-sensitive reduction of [Ca2+]cyt? Previously, we described AS-oligo knockdown of the cytosolic Na+-dependent increase of [Ca2+]cyt in Na+-free media (24); this rise in [Ca2+]cyt is a manifestation of NCX-mediated Ca2+ influx. To determine the role of NCX in removal of Ca2+ from the cytosol, however, we must distinguish between Ca2+ extrusion via the PMCA and the NCX. Previous studies indicate that the PMCA pump (7, 22) and NCX (24) can be independently inhibited, by La3+ and AS-oligos, respectively, in MA myocytes.

Figure 1A illustrates the reversible inhibitory effect of La3+ on the removal of Ca2+ from the cytosol in representative single cells. Initially, cells were superfused with Na+- and Ca2+-free medium to block Ca2+ entry and inhibit extracellular extracellular Na+-dependent Ca2+ efflux via NCX. This medium also contained 0.25 mM LaCl3 to inhibit the PMCA pump and 10 µM verapamil to block La3+ entry (22). Application of CPA and caffeine increased [Ca2+]cyt in these cells by mobilizing SR Ca2+ and blocking resequestration. The elevated [Ca2+]cyt was sustained because the major mechanisms involved in removing Ca2+ from the cytosol were then inhibited. When the La3+ was subsequently removed from the perfusion medium, [Ca2+]cyt rapidly declined as a result of reactivation of the PMCA pump (22). This effect, about an 8- to 10-fold increase in the rate of [Ca2+]cyt decline, was observed in AS-oligo-treated cells as well as in controls and NS-oligo-treated cells (Fig. 1B). Apparently, neither of these oligos adversely affected the activity of the PMCA pump; indeed, the slightly faster recovery rate in AS-oligo-treated cells (Fig. 1B) could have been the result of upregulation of the PMCA pump.


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Fig. 1.   Effects of antisense oligonucleotides (AS-oligos) and La3+ on rate of decline of cytosolic Ca2+ concentration ([Ca2+]cyt) after mobilization of Ca2+ from sarcoplasmic reticulum (SR). A: representative original [Ca2+]cyt data from single cells cultured for 9 days in control medium (no oligos) or in medium containing AS-oligos or nonsense oligonucleotides (NS-oligos). Each curve corresponds to a single representative cell that was superfused in Na+- and Ca2+-free medium to inhibit Ca2+ entry and extracellular Na+-dependent Ca2+ efflux [via Na+/Ca2+ exchange (NCX)]. La3+ (0.25 mM) was included to inhibit plasma membrane Ca2+ (PMCA) pump and, as indicated, intracellular Ca2+ stores were mobilized with 10 µM cyclopiazonic acid (CPA) and 5 mM caffeine (CAF) to raise [Ca2+]cyt; 10 µM verapamil was also present, to block La3+ entry (22, and see text). At time indicated, La3+ was washed out to reactivate PMCA pump. B: pooled data from 3 experiments similar to and including the one in A. Ordinate corresponds to rate of [Ca2+]cyt decline during 30 s preceding La3+ washout (crosshatched bars) and 30 s immediately after initiating La3+ washout (solid bars). Rate constant for washout of chamber was 0.3 s-1 (22), ~10 times faster than rates of [Ca2+]cyt decline measured here. In all 3 groups of cells, increase in rate of [Ca2+]cyt decline after La3+ washout was significantly greater (ANOVA, P < 0.001) than rate before washout.

Do AS-oligos affect the extracellular Na+-dependent reduction of [Ca2+]cyt? The effects of AS-oligos on the extracellular Na+-dependent (NCX-mediated) Ca2+ efflux in MA myocytes are shown in Fig. 2. In these experiments [Ca2+]cyt was again elevated by CPA plus caffeine in Na+- and Ca2+-free medium containing 0.25 mM La3+ and 10 µM verapamil. In this case, however, external Na+ was reintroduced to reactivate NCX-mediated Ca2+ extrusion while the PMCA pump was still inhibited by the continued presence of La3+. Under these circumstances, the rate of [Ca2+]cyt decline was accelerated by ~10- to 16-fold in control cells and in those treated with S- or NS-oligos, but not in myocytes treated with AS-oligos, in which the rate increased only 1.7-fold (Fig. 2, A and B). Thus Ca2+ extrusion by NCX was markedly reduced in the AS-oligo-treated cells. The AS-oligos did not, however, significantly affect the ability of the SR to store Ca2+, or the ability of CPA and caffeine to mobilize the stored Ca2+. Furthermore, the extracellular Na+-dependent decline in [Ca2+]cyt, presumably a manifestation of NCX activity (22), in the control, NS-oligo, and S-oligo groups was apparently unaffected by 0.25 mM La3+.


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Fig. 2.   Effects of AS-oligos and Na+ on rate of decline of [Ca2+]cyt after mobilization of Ca2+ from SR. A: representative original [Ca2+]cyt data from single cell cultured for 7 days in control medium (no oligos) or in medium with AS-, NS-, or sense (S)-oligos. Cells were superfused in Na+- and Ca2+-free medium to inhibit Ca2+ entry and extracellular Na+-dependent Ca2+ efflux (via NCX). La3+ (0.25 mM) was included to inhibit PMCA pump and, as indicated, intracellular Ca2+ stores were mobilized with CPA + caffeine to raise [Ca2+]cyt; 10 µM verapamil was also present. At time indicated, Na+ was added back to medium to reactivate NCX. B: pooled data from 3 similar experiments (including one in A). Ordinate corresponds to rate of [Ca2+]cyt decline during 30 s before adding back Na+ (crosshatched bars) and 30 s immediately after adding back Na+ (solid bars). Rate of [Ca2+]cyt decline accelerated markedly (ANOVA, P < 0.001) in control cells and in those treated with NS- or S-oligos when external Na+ was added back. Rate accelerated only minimally (and insignificantly, P > 0.05) when Na+ was added back to AS-oligo-treated cells.

As illustrated in Figs. 1B and 2B, the rates of recovery from the elevated [Ca2+]cyt in control and NS-oligo-treated cells were faster after Na+ replacement (Fig. 2B) than after La3+ washout (Fig. 1B). With the assumption that La3+ dissociation from the PMCA pump molecules was not rate limiting, these data suggest that the NCX in these MA myocytes may have been able to extrude Ca2+ a little faster than the PMCA pump under the conditions of these experiments.

Can the effects of AS-oligos be reversed? An important control for the specific antagonism by AS-oligos is reversibility. Figure 3 shows data from cells that were treated with either AS-oligos or no oligos (controls) for 7 days. Then, on day 7, some of the cells that had been treated with DMEM plus 10% FCS containing AS-oligos were returned to normal (oligo-free) DMEM plus 10% FCS medium (the "recovery" group). The three groups were continued in culture for an additional 5 days. Extracellular Na+-dependent reduction of [Ca2+]cyt was then measured, using the protocol illustrated by the experiment in Fig. 2A. External Na+ greatly accelerated the decline in [Ca2+]cyt in recovery cells (by 8-fold) as well as in the controls (by 15-fold), but not in the cells incubated with AS-oligos for all 12 days (Fig. 3). The slower recovery rates of the control cells in these experiments, compared with those of Fig. 2B, may be attributed to the older age of the cultures in the experiments of Fig. 3.


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Fig. 3.   Recovery of NCX activity after treatment with AS-oligos. Cells were divided into 3 groups: 1 group (control) was continuously incubated in culture medium without oligos for 12 days; the other 2 groups were incubated with AS-oligos for 7 days, then 1 of the groups (recovery) was returned to oligo-free medium for next 5 days of culture, whereas the other group (antisense) was incubated with AS-oligos for all 12 days. Protocol for testing NCX activity (extracellular Na+-dependent decline in [Ca2+]cyt) was identical to that shown in experiment of Fig. 2A. Results shown here are pooled data from 3 independent experiments. "Antisense" group exhibited no detectable NCX activity, whereas both "control" and "recovery" groups showed large (ANOVA, P < 0.001) extracellular Na+-dependent declines in [Ca2+]cyt.

Little recovery was observed in AS-oligo-treated cells that were subsequently incubated without AS-oligos for only 1 or 2 days (data not shown). Thus the knockdown of NCX function by AS-oligos appears to be reversible in MA myocytes, but only after more than 2 days of recovery without the oligos.

Does NCX knockdown affect 5-HT-evoked Ca2+ transients? To determine the role that NCX plays in the response to physiological agonists, control cells and AS- or NS-oligo-treated cells were stimulated with brief (30 s) applications of 10 µM 5-HT (a maximal effective dose). The Ca2+ transients in AS-oligo-treated cells evoked by this single, brief exposure to 5-HT were indistinguishable from those in control cells (Fig. 4A and see Table 1). Furthermore, the Ca2+ transients in controls (including NS-oligo-treated cells) and AS-oligo-treated cells were negligibly affected if external Ca2+ was removed 30 s before the 5-HT was introduced (Fig. 5; and see Table 2, no La3+). Thus these transients can be attributed primarily to Ca2+ mobilization from the SR, and not to Ca2+ entry from the extracellular fluid (2).


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Fig. 4.   Ca2+ transients evoked by repetitive applications of serotonin (5-HT) in primary cultured mesenteric artery myocytes. Representative original [Ca2+]cyt data from single cells cultured for 9 days without oligos (control) or with AS- or NS-oligos. 5-HT (10 µM) was superfused for 30 s at 10-min intervals (A) and at 3-min intervals (B). Pooled results from several such experiments are presented in Tables 1 and 3, respectively.

                              
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Table 1.   Effects of AS-oligo knockdown of Na+/Ca2+ exchange on 5-HT-evoked [Ca2+]cyt transients with 30-s 5-HT application at 15-min intervals (see Fig. 4A)


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Fig. 5.   Effects of AS-oligos on 5-HT-evoked Ca2+ transients in Ca2+-free media in absence and presence of 0.125 mM La3+. A: representative original [Ca2+]cyt data from single cells cultured for 9 days in medium without oligos (control) or with AS- or NS-oligos. Ca2+ transients were evoked with 10 µM 5-HT in Ca2+-free media to prevent Ca2+ entry. During second exposure to 5-HT, 0.125 mM La3+ was added to inhibit PMCA pump; at this La3+ concentration, no verapamil was needed to inhibit La3+ entry (22). B: expanded time scale (abscissa) to show first-order exponential curve fits (heavy lines) to decline of [Ca2+]cyt during first 30 s after peaks of Ca2+ transients in absence (left) and presence (right) of 0.125 mM La3+. C: time constants (tau ) for decline of [Ca2+]cyt during first 30 s after peak Ca2+ transients; time constants were measured from curve fits such as those illustrated in B. Bar graph shows pooled data from 3 independent experiments similar to (and including) one illustrated in A and B. In absence of La3+, time constants for three groups of cells were not significantly different from one another (see Table 2 and text). In presence of 0.125 mM La3+, mean time constant for AS-oligo-treated cells was significantly larger (ANOVA, P < 0.01) than time constants for other 2 groups of cells.

                              
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Table 2.   Effects of AS-oligo knockdown of Na+/Ca2+ exchange on 5-HT-evoked [Ca2+]cyt transient in absence and presence of La3+ (see Fig. 5A)

Does NCX knockdown affect responses to repetitive applications of 5-HT? Even when 5-HT was repetitively applied at 15-min intervals, the amplitudes of the three consecutive [Ca2+]cyt peaks were relatively constant and did not differ significantly in cells from each of the three groups (controls, AS-oligos, and NS-oligos; Fig. 4A and Table 1). These results demonstrate that the responses to 5-HT were reproducible and that there were no substantial differences among the three groups when the cells were given sufficient time to recover.

When 5-HT was repetitively applied at much shorter intervals, however (e.g., every 3 min, as in Fig. 4B and Table 3), the peak amplitudes of the Ca2+ transients declined progressively (peak 1 > peak 2 > peak 3) in control cells and in those treated with NS-oligos. Explanations for this decline include the possibility that the 5-HT receptor/intracellular signaling (inositol trisphosphate) pathway was temporarily downregulated or that the SR did not have sufficient time to refill between exposures to 5-HT at this higher rate of stimulation.

                              
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Table 3.   Effect of AS-oligo knock down of Na+/Ca2+ exchange on 5-HT-evoked [Ca2+]cyt transients with 30-s 5-HT application at 3-min intervals (see Fig. 4B)

The responses of the AS-oligo-treated cells differed from those of the controls and NS-oligo-treated cells activated at 3-min intervals. One difference was a slower recovery to baseline [Ca2+]cyt of the AS-oligo-treated cells; for example, 2.9 min after each exposure to 5-HT (i.e., just before the next stimulus), [Ca2+]cyt remained higher in the AS-oligo-treated cells than in the controls (Fig. 4B and Table 3, prestimulation). Although these data were obtained in standard PSS, comparable results were obtained when external Ca2+ was temporarily removed during each application of 5-HT (data not shown). These data indicate that the NCX plays a role in the rapid removal of Ca2+ from the cytosol and recovery of [Ca2+]cyt after Ca2+ transients in MA myocytes.

A second difference between the AS-oligo-treated cells and controls in these experiments concerns the amplitudes of the Ca2+ transients. There was a relatively smaller decline in the peak amplitudes of the second and third 5-HT-evoked Ca2+ transients in AS-oligo-treated cells (~10 and 25% decline, compared with the initial response) than in the controls and NS-oligo-treated cells (~35 and 50%, respectively; Figs. 4B and Table 3). This, too, seems consistent with inhibition of NCX-mediated Ca2+ extrusion during recovery after Ca2+ transients; under these circumstances, the SR Ca2+ stores may have been able to refill more rapidly than normal because of the slower extrusion of Ca2+ from the cytosol.

Effects of application of 5-HT on MA myocytes incubated in Na+-free media. If external Na+ was removed at the time that 5-HT was applied, the evoked Ca2+ transients in control and NS-oligo-treated cells were significantly greater than those evoked in these cells in standard PSS (Fig. 6B). In contrast, the removal of external Na+ had little effect on the 5-HT-evoked Ca2+ transients in the AS-oligo-treated cells (Fig. 6, A and B). This can be explained if the reversal of the Na+ electrochemical gradient across the plasma membrane promoted Ca2+ entry via NCX only in the control and NS-oligo-treated cells and thereby augmented the Ca2+ mobilized from the SR stores and directly contributed to the larger Ca2+ transients in these cells (2, 24).


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Fig. 6.   Effects of AS-oligos on 5-HT-evoked Ca2+ transients in Na+-free medium. A: representative original [Ca2+]cyt data from single cells cultured for 9 days in medium without oligos (control) or medium containing AS- or NS-oligos. Na+-free medium was introduced at same time that 10 µM 5-HT was added to medium. 5-HT was removed after 30 s, but superfusion with Na+-free medium was continued for remainder of recording period. B: pooled data from 3 similar, Na+-free experiments (Na-free PSS, right). Peak [Ca2+]cyt both in control cells and in those treated with NS-oligos were significantly greater than peak [Ca2+]cyt in AS-oligo-treated cells (ANOVA, P < 0.001 in both cases). Peak [Ca2+]cyt in control and NS-oligo-treated myocytes during inital 5-HT-evoked responses was also significantly greater (P < 0.001) when cells were bathed in Na+-free medium (right) than in standard PSS (left). The latter data are pooled results from initial 5-HT-evoked transients described in Tables 1 and 3.

Effects of La3+ on 5-HT-evoked Ca2+ transients. To evaluate, further, the effect of AS-oligos on the rate of [Ca2+]cyt recovery from activation by 5-HT, Ca2+ responses evoked in the absence and presence of La3+ were compared. Myocytes were first exposed to 5-HT in Ca2+-free medium to ensure that the evoked increase in [Ca2+]cyt was the result of mobilization of intracellular Ca2+. After recovery from the initial exposure to 5-HT, 0.125 mM La3+ was added to the medium to block the PMCA pump, and 5-HT was reapplied (Fig. 5A); at this La3+ concentration, verapamil is not needed to block La3+ entry (22). The initial 30-s decline of [Ca2+]cyt from the peak of each 5-HT-evoked transient was fitted to a first-order exponential (Fig. 5B). The time constants (tau ) for the decline of [Ca2+]cyt in the absence and presence of La3+ were then compared (Fig. 5, B and C). In the absence of La3+ (i.e., with the PMCA pump functioning), there were no significant differences in the tau  values among the three groups of cells (controls and AS-oligo- and NS-oligo-treated cells), although tau  was slightly higher in these AS-oligo-treated cells (Fig. 5C; and see Table 2). In other experiments, such as those illustrated in Fig. 4, A and B, however, no differences in tau  values were detected (0.18-0.21 min in controls, 0.18-0.25 min in NS-oligo-treated cells, and 0.19-0.26 min in AS-oligo-treated cells). In contrast, when the PMCA pump was inhibited with La3+, the tau  for recovery of AS-oligo-treated cells increased markedly and was ~10-fold larger than that of the control cells and ~3-fold larger than the tau  of the NS-oligo-treated cells. The implication is that during a single exposure to 5-HT, the PMCA pump is able to remove Ca2+ from the cytosol at a rapid rate so that the NCX appears to be superfluous under these conditions. Nevertheless, as noted above, the NCX clearly plays a role in Ca2+ removal from the cytosol after rapid, repetitive 5-HT stimuli.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AS-oligos inhibit NCX-mediated Ca2+ extrusion from VSM cells. Previously, we reported that AS-oligos targeted to the NCX inhibit, selectively, the extracellular Na+-dependent component of Ca2+ influx, which is mediated by the NCX, in primary cultured rat VSM cells (24). The AS-oligos did not interfere with normal growth or other physiological functions of these cells. Moreover, other studies have demonstrated that low concentrations of La3+ (0.03-0.25 mM) block, selectively, the extracellular Na+-independent component of Ca2+ efflux from arterial myocytes that is mediated by the PMCA pump (9, 22). Thus, by employing La3+ in conjunction with AS-oligos, we were able to test the role of the NCX in Ca2+ extrusion from VSM cells.

As shown in Figs. 1 and 2, there are two main components of Ca2+ efflux. When Ca2+ was mobilized from the SR and resequestration was prevented, with CPA plus caffeine, [Ca2+]cyt remained elevated if both efflux mechanisms were inhibited (with Na+-free media and La3+). In initial experiments (Fig. 1), we observed that treatment with either AS- or NS-oligos did not significantly affect the La3+-inhibitable (PMCA pump) component of Ca2+ efflux.

Selective La3+ inhibition of the PMCA pump enabled us to evaluate the extracellular Na+-dependent Ca2+ efflux directly (Fig. 2). After an evoked rise in [Ca2+]cyt, replacement of extracellular Na+ did not accelerate the removal of Ca2+ from the cytosol of AS-oligo-treated cells. In marked contrast, untreated control, NS-oligo-treated, and S-oligo-treated cells exhibited a 10- to 16-fold increase in the rate of decline of [Ca2+]cyt after the replacement of extracellular Na+. These results indicate that the AS-oligos knocked down the extracellular Na+-dependent Ca2+ efflux. The fact that the NCX functioned as well in S-oligo- as in NS-oligo-treated cells implies that AS-oligos are sequence specific and that NCX knockdown by AS-oligos was not a result of differences in oligo secondary structure (29).

The knockdown of NCX activity by AS-oligos was reversible (Fig. 3). This is a further indication that the effect of the AS-oligos was specific. Whether this recovery, which was observed at 5 days, but not at 1 or 2 days, was the result of a relatively long AS-oligo half-life, a long NCX protein half-life, and/or other factors is unknown. This result is, however, consistent with the evidence, from neonatal rat cardiac myocytes, that the NCX protein half-life is ~33-36 h (23, and W. J. Lederer and T. B. Rogers, personal communication).

The preceding observations confirm that the MA myocytes have two major mechanisms for extruding Ca2+. This apparent redundancy may, at first, seem surprising. Other studies, however, demonstrate that the PMCA pump and the NCX are distributed differently in the plasma membrane of MA myocytes: the NCX is confined to "junctional" domains of the plasma membrane that overlie junctional SR (11), whereas the PMCA pump appears to be more ubiquitously distributed over the cell surface (12). This differential distribution of these two Ca2+ transport systems is circumstantial evidence that they may play different roles in the physiology (and, perhaps, pathophysiology) of the arterial myocytes.

Physiological role of the NCX in arterial myocytes. To elucidate the physiological role of the NCX, the influence of AS-oligos on the responses to a physiological agonist, 5-HT, was studied. The AS-oligos had no detectable effects on the Ca2+ transients evoked by single brief exposures to 10 µM 5-HT or on transients evoked by repetitive applications of 5-HT when the stimuli were presented at a low frequency (every 15 min).

The experiments with La3+ (Fig. 5 and Table 2) also demonstrate that recovery after 5-HT-evoked [Ca2+]cyt elevation was only minimally slowed when either the PMCA pump was inhibited (Fig. 5C, control + La3+) or the NCX was knocked down as a result of AS-oligo treatment (Fig. 5C, AS-oligos, no La3+). When, however, the PMCA pump was blocked in AS-oligo-treated myocytes, the recovery of cytosolic Ca2+ was greatly slowed. These results all appear to indicate that the NCX may play only a limited role in Ca2+ homeostasis when the cells are not burdened with a large Ca2+ load. On the contrary, however, the augmentation of 5-HT-evoked Ca2+ tranients by removal of external Na+ in controls, but not AS-oligo-treated cells (Fig. 6), implies that NCX normally mediates the Na+ electrochemical gradient-dependent modulation of SR Ca2+ stores and the resultant cell responsiveness to agonists.

In another series of experiments, 5-HT was applied repetitively at short (3 min) intervals. The amplitudes of the Ca2+ transients declined progressively (i.e., peak 1 > peak 2 > peak 3) under these circumstances, but the decline in peak amplitude was attenuated in the AS-oligo-treated cells. Moreover, [Ca2+]cyt returned to baseline more slowly in the AS-oligo-treated cells than in controls. These observations are consistent with the view that Ca2+ was extruded from the cells more slowly when the NCX was knocked down. The smaller decline in peak height in the AS-oligo-treated cells (in standard PSS) can be attributed to more complete refilling of the SR Ca2+ stores. The larger (relative to control and NS-oligo) second and third responses in the AS-oligo-treated cells (Fig. 4B) indicate that downregulation of the 5-HT receptor/inositol trisphosphate signaling pathway is probably not the explanation for the marked decline in amplitude of the Ca2+ transients in the control and NS-oligo-treated cells.

Taken together, our results with AS-oligo knockdown of the NCX (this report and 24) and La3+ inhibition of the PMCA pump (22, and this report) indicate that quiescent arterial myocytes, and cells that are stimulated infrequently, can maintain reasonable Ca2+ balance with only one of the two Ca2+ transport systems functioning. When the myocytes are activated at short intervals, however, the effects of NCX knockdown become much more obvious. Under normal physiological conditions, the smooth muscle in most arterial beds is tonically activated by local, neurogenic, and hormonal factors (16). Thus the present results suggest that the NCX plays an important role in Ca2+ homeostasis in arterial smooth muscle in vivo.

    ACKNOWLEDGEMENTS

We thank Dr. Magdalena Juhaszova for critical comments on an early version of this manuscript.

    FOOTNOTES

This work was supported by National Institutes of Health (NIH) Grant HL-45215, by NIH Training Grant GM-025-22580, and by intramural research funds from the University of Maryland School of Medicine and the University of Maryland-Baltimore Graduate School.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. P. Blaustein, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 West Baltimore St., Baltimore, MD 21201.

Received 6 January 1998; accepted in final form 6 April 1998.

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