Experimental Physiology (1992), 77, 141 -152 Printed in Great Britain
EVIDENCE FOR Na'-Ca2" EXCHANGE AND Ca2+INDUCED Ca2+ RELEASE IN A CULTURED VASCULAR SMOOTH MUSCLE CELL LINE FROM THE RAT J. I. GILLESPIE, H. OTUN*t, J. R. GREENWELL AND W. DUNLOP* Departments of Physiological Sciences and * Obstetrics and Gynaecology, The Medical School, The University, Newcastle upon Tyne NE2 4HH (MANUSCRIPT RECEIVED 22 JUNE 1991, ACCEPTED
AUGUST 1991)
SUMMARY
Measurements of intracellular calcium (Ca2+) and sodium (Na+) have been made in single smooth muscle cells from the rat aortic cell line (AIO) using the Ca2+- and Na+-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Ca2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Ca2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na'-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca2+-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Ca2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca2+-induced Ca2+-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na+-Ca2+ exchanger operating in reverse. The possible modulation of the Na+-Ca2+ exchanger and Ca2+-induced Ca2+ release by internal Na+ is discussed. INTRODUCTION
The activation of contraction and the maintenance of tone in vascular smooth muscle is associated with changes in intracellular free calcium (Ca 2+) (Morgan & Morgan, 1984; Bond & Somlyo, 1989). The Ca2+ responsible for contraction is available from several different sources, for example from the extracellular environment via voltage-gated channels (Bean, Sturek, Puga & Hermsmeyer, 1986; Behman, Hess & Tsien, 1987; Yatani, Seidel, Allen & Brown, 1987) or via receptor-activated channels (Benham & Tsien, 1987). Alternatively, Ca2+ can be derived from stores in the cytoplasm (Somylo, Bond, Somylo & Scarpa, 1985). The intracellular concentration of Ca2+ is also influenced by mechanisms extruding Ca2+ from the cell or sequestering Ca21 to storage sites within the cytoplasm. In general terms, four main mechanisms have been identified in smooth muscle cells; uptake of Ca2+ into cytoplasmic stores, extrusion to the extracellular environment via Ca2+-ATPase pumping mechanisms, extrusion via Na+-Ca2+ exchange and intracellular buffering by proteins such as parvalbumin in skeletal muscle (Baylor, Chandler & Marshall, 1983). The interactions between these Ca2+-elevating and Ca2+-uptake mechanisms is complex. Subtle alterations to the activity of any one or combination of these mechanisms can have profound effects on Ca 2+ which in turn can affect muscle tone and vascular resistance. t To whom correspondence should be addressed.
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It has been proposed that changes in intracellular Na+ (Na+) can influence the levels of and consequently vessel tone (Friedman & Friedman, 1976; Webb & Bohr, 1979; Jones, 1981). The mechanism by which this change might be brought about is not known. One hypothesis is that an elevation of Na+ could exert its effect by reducing the effectiveness of the Na'-Ca2" exchange mechanism. In this paper we have used cultured smooth muscle cells derived from embryonic rat thoracic aorta (A 10; Kimes & Brandt, 1976) to determine the presence of any Na'-Ca2" exchange and its potential role in Ca 2' regulation. Using the inhibition of the Na+-K+ pump to elevate Na+ we have also explored the influence of Na+ on Ca 2 regulation. The results suggest that in resting control cells Na- Ca2' exchange plays little role in determining the resting Ca 2+ or the recovery from agonist-induced elevations of Ca2+. However, chronic elevation of Na+ can lead to a rise in Ca 2+ and the possible activation of Na+-Ca2+ exchange. A preliminary account of this work has been published (Dunlop, Gillespie, Greenwell & Otun, 1989).
Ca"2
METHODS
Cell preparation Cells from the rat aortic smooth muscle cell line AIO were obtained from American Culture Collection (ATCC, Bethesda, MD, USA) and grown on 22 mm diameter glass cover-slips (BDH, Dagenham, Essex) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (Flow Laboratories, High Wycombe, Bucks) until 80-90% confluent. The cover-slips were then washed and used to form the floor of a small Perspex chamber with a volume of approximately 100 /iml on the stage of the microspectrofluorimeter (Gillespie & Greenwell, 1988). Solution of the desired composition, at 22 °C or at 37 °C, was superfused through the chamber at a rate of 4-5 ml min'. All experiments were carried out in balanced salt solutions containing (mM): 140 NaCl, 5 KCI, 2 CaCl), 2 MgCl.,, 5 NaHPO,, 5 NaHCO3, 10 glucose and 10 HEPES-acid buffer (pH 7 4). Sodium-free balanced salt solution was made by substituting Na+ with N-methyl-D-glucamine (NMG), Tris, choline or potassium. Nominally calcium-free solutions were made without added calcium and with the addition of 10 mM-EGTA. The solutions were not gassed with CO., or 0,.
DYe loadling and calibration Cells were loaded with ion-sensitive fluorescent dye (Fura-2, or SBFI (sodium-binding benzofuran isophthalate); Haughland, 1989) by incubation at 37 °C in 1 ml of DMEM containing 5-10 /iM of the appropriate acetoxymethyl ester. Occasionally, the dispersing agent pluronic F-127 (Molecular probes, OR, USA) at a final concentration of 0-02 % (w/v) was added to the dye aliquots (1 mm dye in dry dimethyl sulphoxide) prior to dilution as this procedure often enhanced dye loading, particularly with SBFI (Haughland, 1988). The microspectrofluorimeter was based on a NIKON Diaphot fluorescence microscope fitted with a 75 W Xenon EPI-fluorescence attachment. The excitation wavelengths used were 350 + 7 and 380+ 7 nm for both Fura-2 and SBFI. The emitted light was filtered using a 520 nm long-pass filter.
The excitation filters were changed using a computer-driven filter changer (Newcastle Photometric Systems, Kingston House, Kingston Park, Newcastle,) and fluorescence measurements taken at each wavelengths over 0 5 s intervals. The intensity of emitted light was measured using a single photon counter (Newcastle Photometric Systems), displayed and stored by microcomputer. The ratio of emitted intensities were calculated approximately once every I -I s. The system was calibrated using 10 /4M solutions of the penta potassium salts of the dyes in buffers of known Ca2 or Naconcentration. These solutions were placed in the cell chamber (without cells) and the ratios noted. Concentration-ratio curves were constructed using this data. It is generally accepted that this type of calibration cannot be used to estimate absolute ion concentrations in cells loaded with the acetoxymethyl ester forms of the dyes (Williams & Fay, 1990). This is primarily because no account is taken of intracellular protein binding (Konishi, Olson, Holingworth & Baylor, 1988) or the uptake of the dyes into intracellular organelles (Goldman & Blaustein, 1989). However, this method can be used to detect transients within a single cell and to compare relative differences in ion concentration between cells.
Ca2+ REGULATION IN SMOOTH MUSCLE
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Data has been expressed as means+ standard error of the mean with statistical analyses being carried out using Student's t test. RESULTS
Brief applications of vasopressin (10- l-10-8 M) produced a rapid transient increase in Ca2+ (for example see Fig. I A). Previous studies have shown that in this cell line a fraction of this Ca2+ comes from cytoplasmic stores and a second component via an influx from the extracellular medium (Monck, Reynolds, Thomas & Williamson, 1988). Once Ca"2 has been elevated and on removal of the agonist, the rate of recovery of Ca2+ must primarily reflect the activity of Ca2+ sequestration and extrusion mechanisms. In order to examine the influence of one of these possible mechanisms, the Na+-Ca2+ exchanger, experiments were carried out on different cells under the conditions where the transmembrane Na+ gradient was reversed (for example see Fig. 1 B). Under these conditions the Na'-Ca2+ exchanger should be inhibited or even operate in the opposite direction to cause an influx of Ca2+ (Bingham Smith, Cragoe & Smith, 1987; Vigne, Breittmayer, Duval, Frelin & Lazdunski, 1988) and consequently impair the Ca2+-recovery mechanisms. Semilogarithmic plots of the recovery phases (Fig. 1 D) following a brief stimulation with arginine vasopressin (AVP) shows that the rates of recovery were similar in the Na+-containing and Na+-free media. There was no significant slope difference between these lines (P < 05) and the regression coefficients were -0923 and -0 758 respectively (n = 6). The mean time constants of recovery in Na+-containing and Na+-free solutions were 26-3 + 2-9 (n = 6) and 28 0 + 1-4 s-1 (n = 6) respectively, not significantly different (P < 0 5). This observation suggests that there is little functional Na+-Ca2+ exchange in these cells under these conditions. The absence of any active Na+-Ca2+ exchange is also supported by the observation that there is no change in Ca 2+ on removal of extracellular Na+ (Fig. 2). Under these conditions, where the Na+ ion concentration gradient is reversed, there should be an increase in Ca 2+ As shown, no significant change could be detected over the time scale studied in this and in a total of five other experiments.
Experiments on Na+-loaded cells If, in these particular cells, the resting intracellular Na+ concentration is low then on removal of extracellular Na+, the electrochemical driving force exchanging internal Na+ for external Ca2+ may not be sufficient to effect a measurable influx of Ca2+. This possibility was examined by increasing internal Na+ by pre-incubating the cells with ouabain (10-3 M for 180 min at 37 °C). The Na+-sensitive dye SBFI was used to measure Nat in order to check the effectiveness of the ouabain treatment. The resting Na+ was found to be increased from 112 +0-5 (n = 8) to 27-6 +21 mM (n = 8) after pre-incubation of the cells with ouabain (Fig. 3A). In ouabain-treated cells, the resting Ca 2+ was 186+ 11 nM (n = 30) significantly higher than control cells (106+7 nM; n = 30; P < 0-001). When the external Ca2+ was removed from ouabain-treated cells there was a rapid and significant fall in Ca 2+ from 188+ 14 to 92+ 14 nM (n = 10; P < 0-001). In control cells, removal of external Ca2+ from 108 + 9 to 83 + 8 nM (n = 10; P < 0 05). Comparing values of Ca2+ in the absence of external Ca2+ there was no significant difference (P > 0 5) between the ouabain and control cells. These data suggest that under both control conditions and after ouabain treatment there is an influx of Ca2+ from the external medium which contributes to the resting level of Ca'+. Furthermore, it would appear that there is a larger influx in ouabain-treated cells.
J. I. GILLESPIE AND OTHERS
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B
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Fig. 1. Stimulation of cultured AIO cells by AVP (10-9 M). A, in control cells in the presence of external Na+; B, in control cells in the absence of external Na+ and C, in ouabain-treated cells in the presence of external Na+. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca"t in nm. D, semilogarithmic plots of the recovery phase of Ca"t transients after stimulation by 5 s applications of 4 x 10-9 M-AVP in single AIO cells in the presence of (0) external Na+, in the absence of (Li1) external Na+ and in the presence of external Na+ after prior incubation with 10--3 M-ouabain (A) for 180 min. Ordinate, R (ratio at time T after peak response)- R (ratio at T > 500 s). Nat substitute NMG. Temperature 32-34 'C.
Ouabain-treated cells responded to AVP stimulation with a Ca"t transient similar to control cells (Fig. 1 C) but the mean value for the time constant for the recovery after AVP activation was 35 1 + 2-3 s-1 (n = 6; regression coefficient = - 0948). This was significantly slower than that found in untreated cells (26 3 + 2 9 s-'; n = 6; P < 0 05) (Fig. 1 D) with a significant difference between the slopes of the semilogarithmic regression plots (P < 0 001). Thus the elevation in Na+ must have had some affect on Ca`t homeostasis either directly or indirectly as a secondary consequence of the elevation in Ca". Such slowing of recovery may reflect interference with the cytoplasmic sequestration of Ca2` or the
Ca2+ REGULATION
145
IN SMOOTH MUSCLE
Na+
n
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.-1000
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c.)
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Fig. 2. The effect of external Na+ (Na+) substitution (NMG) on the intracellular Ca"+ transients induced in a single AI0 cell (5 s applications; 4 x 10 9 M-VP). Where indicated by the horizontal bars, solutions were changed or agonists added to the superfusion solution. Ordinate shows the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca"> in nm. Temperature 33 'C. B 200
A 40
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(mM)
100 -
±
I
50-
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0 Normal Ouabain untreated treated
II I ~~I I~ Normal untreated
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Fig. 3. Bar graphs illustrating the change in Na+ brought about by a 3 h incubation in 10 3 M-ouabain (A) and the change in Ca,' resulting from a similar incubation in 10 3 M-ouabain (B). Open bars show Cal in the presence of external Ca2- and filled bars show the concentration of Ca2+ in control and ouabain-treated cells washed in nominally Ca2--free medium. Temperature 32 35 'C.
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Nit+
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250 Ca.
(nM)
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o
J
F
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Time (s) Fig. 4. The effect of external Na' substitution on Ca 2+ in a single ouabain-treated (3 h; 10 M) A1O cell. Where indicated the external solution containing Na+ was replaced with one containing NMG. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca2 in nm. Temperature 33 'C.
extrusion of Ca21 from the cytoplasm. A more dramatic effect of the elevated Na' can be seen in experiments where the Na+ gradient was reversed (Fig. 4). On removing external Na+, Ca2+ increased at a rate of 673 + 173 nm min-1 (n = 9) which reached a peak value of 402 + 60 nM (n = 9) which then fell to a plateau value above baseline (225 + 58 nM; n = 9). This complex transient pattern of Ca2+ mobilization was seen in a total of nine ouabaintreated cells. This is in contrast to control cells (Fig. 2) where the removal of extracellular Na+ had no effect on Cai . Similar responses were seen in ouabain-treated cells with other Na+ substitutes (Tris and choline) suggesting that the mobilization of Ca2+ is not a consequence of a pharmacological action of NMG and represents a true effect of Na+ removal. These complex increases in Cai , seen on Na+ removal from ouabain-treated cells, cannot simply be explained on the basis of inhibition or reversal of Na+-Ca2+ exchange. Simplistically, reversal of the exchanger should lead to a steady influx and gradual rise in Ca 2' and not a transient rise, fall and secondary increase. It is therefore more than likely that several mechanisms become activated on removal of external Na+. The source of the Ca2+ involved in these changes was determined by reducing the external Ca2+ to nominally zero. Under these conditions the rapid increase in Ca 2+ seen on Na+ removal is completely absent (Fig. 5A). When Ca2+ was returned to the bathing medium with the cells still in sodium-free solution the large transient influx of Ca2,+ was activated. This increase had the same time course and shape as responses seen on removal of Na+ in the presence of extracellular Ca 2. The rate of rise in Ca 2+ in these transients was 713+104 nm min-1 (n = 6), not significantly different to that found on removal of Na+ in the presence of Cao In a separate series of experiments the extracellular Ca2+ was removed during the exposure to low external Na+. Cai2 fell rapidly. In the experiment illustrated Cai2 fell to the baseline value. Returning the extracellular Ca2+ restored the Ca2+ influx but on such occasions the rate of rise of Ca 2+ was 158 +41 nM min-1 (n = 7), considerably slower than that measured on initial Na+ removal (Fig. 4) or on the addition of Ca 2+ in Na+-free solution (Fig. 5A). Furthermore on no occasion was an overshoot of Ca2+ seen on
Ca'+
147
REGULATION IN SMOOTH MUSCLE
A
Na' > Ca'+ 0
1-5
500
1 3 _
C) S-
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Na+
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Ca`
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,
,
200
,
I
,
400
I
600
,
800
1000
Time (s)
Fig. 5. The effect of external Ca" removal on the mobilization of Ca"+ by external Na+ substitution (NMG) in a ouabain-pre-treated cell (3 h, 10-3 M). In A, the cell was first washed in Ca2"-free solution and then Na+-free solution. Where indicated the extracellular Ca" was returned to the superfusion solution, still in the absence of external Na+. B, the effect of extracellular Ca" removal in a cell in which Ca"+ has been elevated by prior exposure to Na+-free solution. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca> in nm. Temperature 34 'C.
returning extracellular Ca2" to such cells. These observations could suggest that component of Ca2" mobilization is modified after its initial activation.
an
early
This inactivation was investigated further using repetitive activation of the Ca2" influx (Figs 6 and 7). An ouabain-treated cell was initially washed in Ca2"-free and then Na+-free solution. Where indicated Ca2" was returned to the bathing solution for short periods of time. In the experiment shown in Fig. 6, during the first exposure the rise in Ca"+ was large but on subsequent identical exposures the response gradually diminished. The collected data from nine such experiments is illustrated in Fig. 6B. In Fig. 7A two consecutive 30 s exposures to Ca2" produced increases in Ca"2 of similar amplitude. Increasing the duration of the pulse to 50 s produced a larger increase than might be expected from such a small
148
J. I. GILLESPIE AND OTHERS A
Na 0 2+ Ca 0
1-5
500
1.3 250 Ca (nM)
c) 1A1 0.9
IVy
0~~~~~~~~~~~~~~5 -~~~~~~~~~~~5 0.7
075 0
400
200
600
Time (s)
B 100
2+
A Ca. 50 50
A(%.)'2
(0o)
1
2 3 Pulse number
4
Fig. 6. A, shows a record from a series of experiments to determine the effect of repeated brief applications of external Ca2" to a single ouabain-treated cell superfused in Na+-free solution (NMG). Horizontal bars indicate where solution changes were made. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca2" in nm. Temperature 34 'C. B, the collected data from nine experiments illustrating the progressive reduction in amplitude of the response observed on successive brief applications of media containing Ca2+.
relative time difference. A second 50 s exposure brought about a similar large response but a third and fourth 50 s pulse produced much smaller rises in Ca'+. This was seen in a total of nine similar experiments. Both these series of experiments point to a system within these cells where a small elevation of Ca2` leads to a secondary rise in Ca2+. This system therefore demonstrates the properties of threshold and inactivation. The gradual reduction in the response on repeated brief elevations of extracellular Ca2` may point to a depletion of the source of Ca2` responsible for the initial rise in Ca"+. If part of the initial rise in Ca2` came from an internal store of Ca2` the data is consistent with the hypothesis that the initial influx of Ca2" activates a further release of intracellular Ca2+. This mechanism would be similar to the Ca2`-induced Ca2+-release mechanism described in other tissues (Endo, Tanaka & Ogawa, 1970; Ford & Podolski, 1970; Fabiato & Fabiato, 1978; Fabiato, 1983). There is an internal store of Ca2" in these cells which can be released by application of the agonist AVP (Somlyo et al. 1985). The question then arises, is this the same store as that postulated to be discharged by brief elevations of Ca` ? Figure 7 B shows an example of
149
Ca2+ REGULATION IN SMOOTH MUSCLE A
Na+ Ca2+ 0
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F
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100
Q 09
r-
C
50 07
05 0
200 400 600 800 1000 1200 1400 Time (s)
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I n
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.j
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200
l
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Time (s) Fig. 7. A, Ca2" transients in a single ouabain-treated AIO cell in the absence of external Na+ resulting from short and long exposure to Ca2"-containing media. The first two pulses were of 30 s duration, followed by four pulses of 50 s duration. B, an experiment to explore the possible interaction between the AVP 'sensitive' intracellular store of Ca2" and the Ca"+ mobilized by Ca2" influx in Na+-free solution. In both records, the horizontal bars indicate where solution changes were made. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca2" in nm. Temperature 34 °C in both experiments.
data from a series of experiments designed to examine this question. A cell was initially exposed to nominally Ca2"-free solution and maximally stimulated with AVP to deplete any internal store. Subsequent brief or prolonged exposure to Ca2"-containing solution did not activate a large influx of Ca" similar to that illustrated in Fig. 6A. Also, there was no overshoot seen on returning Ca" to the external medium. These observations suggest that the source of Ca" mobilized on Na+ removal and by AVP are the same. 6
EPH 77
150
J. I. GILLESPIE AND OTHERS DISCUSSION
This paper presents results obtained from a cultured cell line initially derived from embryonic rat vascular smooth muscle. These cells possess several features in common with vascular smooth muscle (Kimes & Brandt, 1976). Although they lack a functional contractile mechanism, some basic observations can be made which may have a bearing on the function of both normal and pathological smooth muscle cells. AIO cells demonstrate an agonist-induced increase in Ca". The initial rapid rise in Ca"+ occurs in the absence of extracellular Ca2" unlike the second slower component of the response which is dependent upon extracellular Ca2". This has been interpreted to mean that the initial fast transient represent the mobilization of an intracellular store whilst the second slow component represents an influx across the plasma membrane (Monck et al. 1988). In many cell types, reversal of the Na+ gradient reverses the Na'-Ca2" exchange mechanism (Bingham Smith et al. 1987; Carafoli, 1987; Vigne et al. 1988). If a Na'-Ca2" exchange mechanism was functioning to regulate Ca"2 the reversal of the electrochemical driving force for Na+ should affect the resting Ca"+ and the time course of recovery of any brief elevation ofCa". In the present experiments on control cells removal of extracellular Na+ had no effect on the resting Ca"2 or on the AVP-induced response. Therefore, it is unlikely the Na'-Ca2" exchanger is involved in the rapid regulation of Ca"2 after stimulation with AVP. In this respect, A10 cells are very similar to another vascular smooth muscle cell line, A7r5 (Vigne et al. 1988) in which Na'-Ca2" exchange appears to play very little if any part in the recovery from AVP stimulation. A1O cells with elevated Na1+ (after incubation with ouabain) behave differently. The first change is that the resting level of Ca"2 is increased. Since, the fall in Ca"2 on removal of external Ca2" is much larger in the ouabain-treated cells than control this suggests that there is an increase in the resting influx. The mechanism underlying this influx is not known. One simple explanation would be that ouabain-treated cells are depolarized and consequently have a high resting influx of Ca2" through non-inactivating voltage-operated channels. On removal of external Na+ the treated cells show changes in Ca"+ which are dependent on the presence of external Ca2". This altered responsiveness when the cells are loaded with Na+ points to a potentially important role for Nat, either directly or indirectly, in the functioning of this particular cell type. One possible explanation for the apparent lack of functional Na'-Ca2" exchange in the control cells is that the intracellular Na+ concentration is very low such that when the external Na+ concentration is reduced to zero there is insufficient electrochemical driving force available to reverse the exchanger. This may not be the case since estimates of Nat using the fluorescent dye SBFI suggest a resting level of II mm. If this is the case then Na'-Ca2" exchange may be present but inactive in the control cells. The elevation of Nat could lead to the unmasking, activation or insertion of additional Na'-Ca2" exchange mechanisms. Alternatively Nat may act at some other as yet unidentified mechanism in the cytoplasm. Increasing Nat using ouabain would lead to a large transmembrane concentration difference on subsequent removal of external Na+. This increased electrochemical driving force may be sufficient to reverse any Na'-Ca2+ exchange with a consequent influx of Ca2 . 2+ seen on removal of Na' from ouabain-treated cells are The complex changes in Ca unlikely to be due to the simple reversal of any Na'-Ca2' exchange. The response appears to be divided into three components. An initial rapid entry of Ca21 followed, some 200-300 s later, by a much slower increase. Superimposed on the early and rapid rise there
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Ca2+ REGULATION IN SMOOTH MUSCLE
appears to be a transient elevation in Ca". This transient appears to be activated by the initial Ca2" influx. This component appears to require a threshold level of Ca"+ (Fig. 7A) and is readily depleted by repetitive activation and prior AVP stimulation (Fig. 7 B). These observations are consistent with the idea that there is an internal store of Ca"+ which is released on the rapid elevation of cytoplasmic Ca2". Such Ca2"-induced Ca2" release is well documented in many other cell types (Endo et al. 1970; Ford & Podolski, 1970; Fabiato & Fabiato, 1978; Fabiato, 1983) and may therefore be present in these A1O cells. The initial Ca2" entry to activate such Ca2"-induced Ca2" release could come from different mechanisms acting independently or together. For example, Na+ removal should hyperpolarize the cells but, if for some unforseen effect of the Na+ substitute, it led to a depolarization this would open Ca2" channels and result in a rapid influx of Ca2". Alternatively, Na+ replacement may reverse the operation of the Na'-Ca2" exchanger resulting in the inward transport of Ca2". The observation that application of AVP to mobilize the intracellular stores of Ca2" interferes with the possible Ca2"-induced Ca2" release could indicate that the same intracellular store can be accessed by cytoplasmic Ca2" and inositol 1,4,5-trisphosphate. Once the internal store is depleted of Ca"+ then the effect of removing extracellular Na+ should be simplified. However, a rapid and slow phase still remain (see Fig. 5). The absence of any mobilization of Ca"2 in control cells on removal of external Na+ may be accounted for by the conditions outlined above. However, the possibility cannot be excluded that the elevation of Na+ by ouabain treatment sensitizes the Ca2"-induced Ca2"-release mechanism, such that under these abnormal conditions the sarcoplasmic reticulum is activated by smaller changes in internal Ca2". REFERENCES
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ENDO, M., TANAKA, M. & OGAWA, Y. (1970). Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature 228, 34-36. FABIATO, A. (1983). Calcium-induced release of calcium ions from the cardiac sarcoplasmic reticulum. American Journal of Ph 'siologi' 245, Cl 14. FABIATO, A. & FABIATO, F. (1978). Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat, and frog hearts and from fetal and newborn rat ventricles. Annals of the New York Academy of Sciences 307, 491-522. 6-2
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FORD, L. E. & PODOLSKI, R. J. (1970). Regenerative calcium release within muscle cells. Science 167. 58-59. FRIEDMAN, S. M. & FRIEDMAN, C. L. (1976). Cell permeability, sodium transport and the hypertensive process in the rat. Circulation Research 39, 433-441. GILLESPIE, J. I. & GREENWELL, J. R. (1988). Changes in intracellular pH and pH regulating mechanisms in somatic cells of the early chick embryo: a study using fluorescent pH-sensitive dye. Journal of Phy siologj 405, 385-395. GOLDMAN, W. F. & BLAUSTEIN, M. P. (1989). Distribution of Ca2+ in cultured vascular smooth muscle cells at rest and during activation: comparison of two fura-2 loading methods. PhYsiologist 32, 148. HAUGLAND, R. P. (1989). Molecular Probes. Handbook of Fluorescent Probes and Research Chemicals. R. P. Haugland, Molecular Probes Inc., Eugene, OR, USA. JONES, A. W. (1981). Kinetics of active sodium transport in aortas from control and deoxycorticosterone hypertensive rats. Hlypertension 3, 631-640. KIMES, B. W. & BRANDT, B. L. (1976). Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Experimental Cell Research 98, 349-366. KONISHI, M., OLSON, A., HOLINGWORTH, S. & BAYLOR, S. M. (1988). Myoplasmic binding of Fura-2 investigated by steady state fluorescence and absorbance measurements. Biophpsical Journal 54, 1089-1104. MONCK, J. R., REYNOLDS, E. E., THOMAS, A. P. & WILLIAMSON, J. R. (1988). Novel kinetics of single cell Ca2- transients in stimulated hepatocytes and AI0 cells measured using Fura-2 and fluorescent videomicroscopy. Journal of Biological Chemistry 263, 4569-4575. MORGAN, J. P. & MORGAN, K. G. (1984). Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. Journal of Ph'siologj 351, 155-167. SOMLYO, A. V., BOND, M., SOMLYO, A. P. & SCARPA, A. (1985). Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle. Proceedings of the National Academy of Sciences of the USA 82, 5231-5235. VIGNE, P., BREITTMAYER, J. P., DUVAL, D., FRELIN, C. & LAZDUNSKI, M. (1988). The Na-/Ca2+ antiporter in aortic smooth muscle cells: Characterization and demonstration of an activation by phorbol esters. Journal of Biological ChemistrY 263, 8078-8083. WEBB, R. C. & BOHR, D. F. (1979). Potassium relaxation of vascular smooth muscle from spontaneously hypertensive rats. Blood Vessels 16, 71-79. WILLIAMS, D. A. & FAY, F. S. (1990). Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium 11, 75-83. YATANI, A., SEIDEL, C. L., ALLEN, J. & BROWN, A. M. (1987). Whole cell and single channel calcium currents of isolated smooth muscles cells from saphenous vein. Circulation Research 60, 523-533.
EVIDENCE FOR Na'-Ca2" EXCHANGE AND Ca2+INDUCED Ca2+ RELEASE IN A CULTURED VASCULAR SMOOTH MUSCLE CELL LINE FROM THE RAT J. I. GILLESPIE, H. OTUN*t, J. R. GREENWELL AND W. DUNLOP* Departments of Physiological Sciences and * Obstetrics and Gynaecology, The Medical School, The University, Newcastle upon Tyne NE2 4HH (MANUSCRIPT RECEIVED 22 JUNE 1991, ACCEPTED
AUGUST 1991)
SUMMARY
Measurements of intracellular calcium (Ca2+) and sodium (Na+) have been made in single smooth muscle cells from the rat aortic cell line (AIO) using the Ca2+- and Na+-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Ca2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Ca2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na'-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca2+-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Ca2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca2+-induced Ca2+-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na+-Ca2+ exchanger operating in reverse. The possible modulation of the Na+-Ca2+ exchanger and Ca2+-induced Ca2+ release by internal Na+ is discussed. INTRODUCTION
The activation of contraction and the maintenance of tone in vascular smooth muscle is associated with changes in intracellular free calcium (Ca 2+) (Morgan & Morgan, 1984; Bond & Somlyo, 1989). The Ca2+ responsible for contraction is available from several different sources, for example from the extracellular environment via voltage-gated channels (Bean, Sturek, Puga & Hermsmeyer, 1986; Behman, Hess & Tsien, 1987; Yatani, Seidel, Allen & Brown, 1987) or via receptor-activated channels (Benham & Tsien, 1987). Alternatively, Ca2+ can be derived from stores in the cytoplasm (Somylo, Bond, Somylo & Scarpa, 1985). The intracellular concentration of Ca2+ is also influenced by mechanisms extruding Ca2+ from the cell or sequestering Ca21 to storage sites within the cytoplasm. In general terms, four main mechanisms have been identified in smooth muscle cells; uptake of Ca2+ into cytoplasmic stores, extrusion to the extracellular environment via Ca2+-ATPase pumping mechanisms, extrusion via Na+-Ca2+ exchange and intracellular buffering by proteins such as parvalbumin in skeletal muscle (Baylor, Chandler & Marshall, 1983). The interactions between these Ca2+-elevating and Ca2+-uptake mechanisms is complex. Subtle alterations to the activity of any one or combination of these mechanisms can have profound effects on Ca 2+ which in turn can affect muscle tone and vascular resistance. t To whom correspondence should be addressed.
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It has been proposed that changes in intracellular Na+ (Na+) can influence the levels of and consequently vessel tone (Friedman & Friedman, 1976; Webb & Bohr, 1979; Jones, 1981). The mechanism by which this change might be brought about is not known. One hypothesis is that an elevation of Na+ could exert its effect by reducing the effectiveness of the Na'-Ca2" exchange mechanism. In this paper we have used cultured smooth muscle cells derived from embryonic rat thoracic aorta (A 10; Kimes & Brandt, 1976) to determine the presence of any Na'-Ca2" exchange and its potential role in Ca 2' regulation. Using the inhibition of the Na+-K+ pump to elevate Na+ we have also explored the influence of Na+ on Ca 2 regulation. The results suggest that in resting control cells Na- Ca2' exchange plays little role in determining the resting Ca 2+ or the recovery from agonist-induced elevations of Ca2+. However, chronic elevation of Na+ can lead to a rise in Ca 2+ and the possible activation of Na+-Ca2+ exchange. A preliminary account of this work has been published (Dunlop, Gillespie, Greenwell & Otun, 1989).
Ca"2
METHODS
Cell preparation Cells from the rat aortic smooth muscle cell line AIO were obtained from American Culture Collection (ATCC, Bethesda, MD, USA) and grown on 22 mm diameter glass cover-slips (BDH, Dagenham, Essex) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (Flow Laboratories, High Wycombe, Bucks) until 80-90% confluent. The cover-slips were then washed and used to form the floor of a small Perspex chamber with a volume of approximately 100 /iml on the stage of the microspectrofluorimeter (Gillespie & Greenwell, 1988). Solution of the desired composition, at 22 °C or at 37 °C, was superfused through the chamber at a rate of 4-5 ml min'. All experiments were carried out in balanced salt solutions containing (mM): 140 NaCl, 5 KCI, 2 CaCl), 2 MgCl.,, 5 NaHPO,, 5 NaHCO3, 10 glucose and 10 HEPES-acid buffer (pH 7 4). Sodium-free balanced salt solution was made by substituting Na+ with N-methyl-D-glucamine (NMG), Tris, choline or potassium. Nominally calcium-free solutions were made without added calcium and with the addition of 10 mM-EGTA. The solutions were not gassed with CO., or 0,.
DYe loadling and calibration Cells were loaded with ion-sensitive fluorescent dye (Fura-2, or SBFI (sodium-binding benzofuran isophthalate); Haughland, 1989) by incubation at 37 °C in 1 ml of DMEM containing 5-10 /iM of the appropriate acetoxymethyl ester. Occasionally, the dispersing agent pluronic F-127 (Molecular probes, OR, USA) at a final concentration of 0-02 % (w/v) was added to the dye aliquots (1 mm dye in dry dimethyl sulphoxide) prior to dilution as this procedure often enhanced dye loading, particularly with SBFI (Haughland, 1988). The microspectrofluorimeter was based on a NIKON Diaphot fluorescence microscope fitted with a 75 W Xenon EPI-fluorescence attachment. The excitation wavelengths used were 350 + 7 and 380+ 7 nm for both Fura-2 and SBFI. The emitted light was filtered using a 520 nm long-pass filter.
The excitation filters were changed using a computer-driven filter changer (Newcastle Photometric Systems, Kingston House, Kingston Park, Newcastle,) and fluorescence measurements taken at each wavelengths over 0 5 s intervals. The intensity of emitted light was measured using a single photon counter (Newcastle Photometric Systems), displayed and stored by microcomputer. The ratio of emitted intensities were calculated approximately once every I -I s. The system was calibrated using 10 /4M solutions of the penta potassium salts of the dyes in buffers of known Ca2 or Naconcentration. These solutions were placed in the cell chamber (without cells) and the ratios noted. Concentration-ratio curves were constructed using this data. It is generally accepted that this type of calibration cannot be used to estimate absolute ion concentrations in cells loaded with the acetoxymethyl ester forms of the dyes (Williams & Fay, 1990). This is primarily because no account is taken of intracellular protein binding (Konishi, Olson, Holingworth & Baylor, 1988) or the uptake of the dyes into intracellular organelles (Goldman & Blaustein, 1989). However, this method can be used to detect transients within a single cell and to compare relative differences in ion concentration between cells.
Ca2+ REGULATION IN SMOOTH MUSCLE
143
Data has been expressed as means+ standard error of the mean with statistical analyses being carried out using Student's t test. RESULTS
Brief applications of vasopressin (10- l-10-8 M) produced a rapid transient increase in Ca2+ (for example see Fig. I A). Previous studies have shown that in this cell line a fraction of this Ca2+ comes from cytoplasmic stores and a second component via an influx from the extracellular medium (Monck, Reynolds, Thomas & Williamson, 1988). Once Ca"2 has been elevated and on removal of the agonist, the rate of recovery of Ca2+ must primarily reflect the activity of Ca2+ sequestration and extrusion mechanisms. In order to examine the influence of one of these possible mechanisms, the Na+-Ca2+ exchanger, experiments were carried out on different cells under the conditions where the transmembrane Na+ gradient was reversed (for example see Fig. 1 B). Under these conditions the Na'-Ca2+ exchanger should be inhibited or even operate in the opposite direction to cause an influx of Ca2+ (Bingham Smith, Cragoe & Smith, 1987; Vigne, Breittmayer, Duval, Frelin & Lazdunski, 1988) and consequently impair the Ca2+-recovery mechanisms. Semilogarithmic plots of the recovery phases (Fig. 1 D) following a brief stimulation with arginine vasopressin (AVP) shows that the rates of recovery were similar in the Na+-containing and Na+-free media. There was no significant slope difference between these lines (P < 05) and the regression coefficients were -0923 and -0 758 respectively (n = 6). The mean time constants of recovery in Na+-containing and Na+-free solutions were 26-3 + 2-9 (n = 6) and 28 0 + 1-4 s-1 (n = 6) respectively, not significantly different (P < 0 5). This observation suggests that there is little functional Na+-Ca2+ exchange in these cells under these conditions. The absence of any active Na+-Ca2+ exchange is also supported by the observation that there is no change in Ca 2+ on removal of extracellular Na+ (Fig. 2). Under these conditions, where the Na+ ion concentration gradient is reversed, there should be an increase in Ca 2+ As shown, no significant change could be detected over the time scale studied in this and in a total of five other experiments.
Experiments on Na+-loaded cells If, in these particular cells, the resting intracellular Na+ concentration is low then on removal of extracellular Na+, the electrochemical driving force exchanging internal Na+ for external Ca2+ may not be sufficient to effect a measurable influx of Ca2+. This possibility was examined by increasing internal Na+ by pre-incubating the cells with ouabain (10-3 M for 180 min at 37 °C). The Na+-sensitive dye SBFI was used to measure Nat in order to check the effectiveness of the ouabain treatment. The resting Na+ was found to be increased from 112 +0-5 (n = 8) to 27-6 +21 mM (n = 8) after pre-incubation of the cells with ouabain (Fig. 3A). In ouabain-treated cells, the resting Ca 2+ was 186+ 11 nM (n = 30) significantly higher than control cells (106+7 nM; n = 30; P < 0-001). When the external Ca2+ was removed from ouabain-treated cells there was a rapid and significant fall in Ca 2+ from 188+ 14 to 92+ 14 nM (n = 10; P < 0-001). In control cells, removal of external Ca2+ from 108 + 9 to 83 + 8 nM (n = 10; P < 0 05). Comparing values of Ca2+ in the absence of external Ca2+ there was no significant difference (P > 0 5) between the ouabain and control cells. These data suggest that under both control conditions and after ouabain treatment there is an influx of Ca2+ from the external medium which contributes to the resting level of Ca'+. Furthermore, it would appear that there is a larger influx in ouabain-treated cells.
J. I. GILLESPIE AND OTHERS
144
B
A
C 1-3
17
17
015
15
013
13
I0 0
1.2 06 10
40
0.9
0.9
0-7
0.7 -0-7
0-6
0.51
0-5 0
200 Time (s)
400
200 Time (s)
0
400
0.5 0
200 Time (s)
400
D
1*0
0.5
R- R-.
0.1
0
10
20
30
40 Time (s)
50
60
7'0
80
Fig. 1. Stimulation of cultured AIO cells by AVP (10-9 M). A, in control cells in the presence of external Na+; B, in control cells in the absence of external Na+ and C, in ouabain-treated cells in the presence of external Na+. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca"t in nm. D, semilogarithmic plots of the recovery phase of Ca"t transients after stimulation by 5 s applications of 4 x 10-9 M-AVP in single AIO cells in the presence of (0) external Na+, in the absence of (Li1) external Na+ and in the presence of external Na+ after prior incubation with 10--3 M-ouabain (A) for 180 min. Ordinate, R (ratio at time T after peak response)- R (ratio at T > 500 s). Nat substitute NMG. Temperature 32-34 'C.
Ouabain-treated cells responded to AVP stimulation with a Ca"t transient similar to control cells (Fig. 1 C) but the mean value for the time constant for the recovery after AVP activation was 35 1 + 2-3 s-1 (n = 6; regression coefficient = - 0948). This was significantly slower than that found in untreated cells (26 3 + 2 9 s-'; n = 6; P < 0 05) (Fig. 1 D) with a significant difference between the slopes of the semilogarithmic regression plots (P < 0 001). Thus the elevation in Na+ must have had some affect on Ca`t homeostasis either directly or indirectly as a secondary consequence of the elevation in Ca". Such slowing of recovery may reflect interference with the cytoplasmic sequestration of Ca2` or the
Ca2+ REGULATION
145
IN SMOOTH MUSCLE
Na+
n
AVP
I
1600
30 25
.-1000
0 L-
c)
20
Ca- (nM)
c.)
0
500
0 1-
300 200
10
100
50 05 0
200
400
600 Time (s)
800
1000
1200
Fig. 2. The effect of external Na+ (Na+) substitution (NMG) on the intracellular Ca"+ transients induced in a single AI0 cell (5 s applications; 4 x 10 9 M-VP). Where indicated by the horizontal bars, solutions were changed or agonists added to the superfusion solution. Ordinate shows the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca"> in nm. Temperature 33 'C. B 200
A 40
150
Na+
Ca. (nM)
(mM)
100 -
±
I
50-
0
0 Normal Ouabain untreated treated
II I ~~I I~ Normal untreated
Ouabain treated
Fig. 3. Bar graphs illustrating the change in Na+ brought about by a 3 h incubation in 10 3 M-ouabain (A) and the change in Ca,' resulting from a similar incubation in 10 3 M-ouabain (B). Open bars show Cal in the presence of external Ca2- and filled bars show the concentration of Ca2+ in control and ouabain-treated cells washed in nominally Ca2--free medium. Temperature 32 35 'C.
146
J. I. GILLESPIE AND OTHERS
Nit+
1.5
500
250 Ca.
(nM)
10
o
J
F
~~~~~~~~~~~~100 50
0
100
200
300
Time (s) Fig. 4. The effect of external Na' substitution on Ca 2+ in a single ouabain-treated (3 h; 10 M) A1O cell. Where indicated the external solution containing Na+ was replaced with one containing NMG. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of Ca2 in nm. Temperature 33 'C.
extrusion of Ca21 from the cytoplasm. A more dramatic effect of the elevated Na' can be seen in experiments where the Na+ gradient was reversed (Fig. 4). On removing external Na+, Ca2+ increased at a rate of 673 + 173 nm min-1 (n = 9) which reached a peak value of 402 + 60 nM (n = 9) which then fell to a plateau value above baseline (225 + 58 nM; n = 9). This complex transient pattern of Ca2+ mobilization was seen in a total of nine ouabaintreated cells. This is in contrast to control cells (Fig. 2) where the removal of extracellular Na+ had no effect on Cai . Similar responses were seen in ouabain-treated cells with other Na+ substitutes (Tris and choline) suggesting that the mobilization of Ca2+ is not a consequence of a pharmacological action of NMG and represents a true effect of Na+ removal. These complex increases in Cai , seen on Na+ removal from ouabain-treated cells, cannot simply be explained on the basis of inhibition or reversal of Na+-Ca2+ exchange. Simplistically, reversal of the exchanger should lead to a steady influx and gradual rise in Ca 2' and not a transient rise, fall and secondary increase. It is therefore more than likely that several mechanisms become activated on removal of external Na+. The source of the Ca2+ involved in these changes was determined by reducing the external Ca2+ to nominally zero. Under these conditions the rapid increase in Ca 2+ seen on Na+ removal is completely absent (Fig. 5A). When Ca2+ was returned to the bathing medium with the cells still in sodium-free solution the large transient influx of Ca2,+ was activated. This increase had the same time course and shape as responses seen on removal of Na+ in the presence of extracellular Ca 2. The rate of rise in Ca 2+ in these transients was 713+104 nm min-1 (n = 6), not significantly different to that found on removal of Na+ in the presence of Cao In a separate series of experiments the extracellular Ca2+ was removed during the exposure to low external Na+. Cai2 fell rapidly. In the experiment illustrated Cai2 fell to the baseline value. Returning the extracellular Ca2+ restored the Ca2+ influx but on such occasions the rate of rise of Ca 2+ was 158 +41 nM min-1 (n = 7), considerably slower than that measured on initial Na+ removal (Fig. 4) or on the addition of Ca 2+ in Na+-free solution (Fig. 5A). Furthermore on no occasion was an overshoot of Ca2+ seen on
Ca'+
147
REGULATION IN SMOOTH MUSCLE
A
Na' > Ca'+ 0
1-5
500
1 3 _
C) S-
Ct
Q
0
250
Ca'+ (nM)
09
100 07
50
05 0
200
400 Time (s)
600
800
B
Na+
Ca2+0
1
1-5 500 1-3
250
Ca`
(nM)
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~~~~~~~~~~~~~~~~~100 0-7
0-5 #.
0
50
,
,
200
,
I
,
400
I
600
,
800
1000
Time (s)
Fig. 5. The effect of external Ca" removal on the mobilization of Ca"+ by external Na+ substitution (NMG) in a ouabain-pre-treated cell (3 h, 10-3 M). In A, the cell was first washed in Ca2"-free solution and then Na+-free solution. Where indicated the extracellular Ca" was returned to the superfusion solution, still in the absence of external Na+. B, the effect of extracellular Ca" removal in a cell in which Ca"+ has been elevated by prior exposure to Na+-free solution. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca> in nm. Temperature 34 'C.
returning extracellular Ca2" to such cells. These observations could suggest that component of Ca2" mobilization is modified after its initial activation.
an
early
This inactivation was investigated further using repetitive activation of the Ca2" influx (Figs 6 and 7). An ouabain-treated cell was initially washed in Ca2"-free and then Na+-free solution. Where indicated Ca2" was returned to the bathing solution for short periods of time. In the experiment shown in Fig. 6, during the first exposure the rise in Ca"+ was large but on subsequent identical exposures the response gradually diminished. The collected data from nine such experiments is illustrated in Fig. 6B. In Fig. 7A two consecutive 30 s exposures to Ca2" produced increases in Ca"2 of similar amplitude. Increasing the duration of the pulse to 50 s produced a larger increase than might be expected from such a small
148
J. I. GILLESPIE AND OTHERS A
Na 0 2+ Ca 0
1-5
500
1.3 250 Ca (nM)
c) 1A1 0.9
IVy
0~~~~~~~~~~~~~~5 -~~~~~~~~~~~5 0.7
075 0
400
200
600
Time (s)
B 100
2+
A Ca. 50 50
A(%.)'2
(0o)
1
2 3 Pulse number
4
Fig. 6. A, shows a record from a series of experiments to determine the effect of repeated brief applications of external Ca2" to a single ouabain-treated cell superfused in Na+-free solution (NMG). Horizontal bars indicate where solution changes were made. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca2" in nm. Temperature 34 'C. B, the collected data from nine experiments illustrating the progressive reduction in amplitude of the response observed on successive brief applications of media containing Ca2+.
relative time difference. A second 50 s exposure brought about a similar large response but a third and fourth 50 s pulse produced much smaller rises in Ca'+. This was seen in a total of nine similar experiments. Both these series of experiments point to a system within these cells where a small elevation of Ca2` leads to a secondary rise in Ca2+. This system therefore demonstrates the properties of threshold and inactivation. The gradual reduction in the response on repeated brief elevations of extracellular Ca2` may point to a depletion of the source of Ca2` responsible for the initial rise in Ca"+. If part of the initial rise in Ca2` came from an internal store of Ca2` the data is consistent with the hypothesis that the initial influx of Ca2" activates a further release of intracellular Ca2+. This mechanism would be similar to the Ca2`-induced Ca2+-release mechanism described in other tissues (Endo, Tanaka & Ogawa, 1970; Ford & Podolski, 1970; Fabiato & Fabiato, 1978; Fabiato, 1983). There is an internal store of Ca2" in these cells which can be released by application of the agonist AVP (Somlyo et al. 1985). The question then arises, is this the same store as that postulated to be discharged by brief elevations of Ca` ? Figure 7 B shows an example of
149
Ca2+ REGULATION IN SMOOTH MUSCLE A
Na+ Ca2+ 0
j=
F
1I5 C
500
13
0
250 Ca. (nM)
100
Q 09
r-
C
50 07
05 0
200 400 600 800 1000 1200 1400 Time (s)
B
Na+0
I
Ca2+JT
AVP
I n
---
500
0 19
250 Ca+ (nM)
0
.c)Co_ 09 2
100
.j
50
0.
0. 7 Il
0
200
l
400
600
Time (s) Fig. 7. A, Ca2" transients in a single ouabain-treated AIO cell in the absence of external Na+ resulting from short and long exposure to Ca2"-containing media. The first two pulses were of 30 s duration, followed by four pulses of 50 s duration. B, an experiment to explore the possible interaction between the AVP 'sensitive' intracellular store of Ca2" and the Ca"+ mobilized by Ca2" influx in Na+-free solution. In both records, the horizontal bars indicate where solution changes were made. Ordinates show the ratio of emitted fluorescence (350/380 nm excitation) and the estimates of intracellular Ca2" in nm. Temperature 34 °C in both experiments.
data from a series of experiments designed to examine this question. A cell was initially exposed to nominally Ca2"-free solution and maximally stimulated with AVP to deplete any internal store. Subsequent brief or prolonged exposure to Ca2"-containing solution did not activate a large influx of Ca" similar to that illustrated in Fig. 6A. Also, there was no overshoot seen on returning Ca" to the external medium. These observations suggest that the source of Ca" mobilized on Na+ removal and by AVP are the same. 6
EPH 77
150
J. I. GILLESPIE AND OTHERS DISCUSSION
This paper presents results obtained from a cultured cell line initially derived from embryonic rat vascular smooth muscle. These cells possess several features in common with vascular smooth muscle (Kimes & Brandt, 1976). Although they lack a functional contractile mechanism, some basic observations can be made which may have a bearing on the function of both normal and pathological smooth muscle cells. AIO cells demonstrate an agonist-induced increase in Ca". The initial rapid rise in Ca"+ occurs in the absence of extracellular Ca2" unlike the second slower component of the response which is dependent upon extracellular Ca2". This has been interpreted to mean that the initial fast transient represent the mobilization of an intracellular store whilst the second slow component represents an influx across the plasma membrane (Monck et al. 1988). In many cell types, reversal of the Na+ gradient reverses the Na'-Ca2" exchange mechanism (Bingham Smith et al. 1987; Carafoli, 1987; Vigne et al. 1988). If a Na'-Ca2" exchange mechanism was functioning to regulate Ca"2 the reversal of the electrochemical driving force for Na+ should affect the resting Ca"+ and the time course of recovery of any brief elevation ofCa". In the present experiments on control cells removal of extracellular Na+ had no effect on the resting Ca"2 or on the AVP-induced response. Therefore, it is unlikely the Na'-Ca2" exchanger is involved in the rapid regulation of Ca"2 after stimulation with AVP. In this respect, A10 cells are very similar to another vascular smooth muscle cell line, A7r5 (Vigne et al. 1988) in which Na'-Ca2" exchange appears to play very little if any part in the recovery from AVP stimulation. A1O cells with elevated Na1+ (after incubation with ouabain) behave differently. The first change is that the resting level of Ca"2 is increased. Since, the fall in Ca"2 on removal of external Ca2" is much larger in the ouabain-treated cells than control this suggests that there is an increase in the resting influx. The mechanism underlying this influx is not known. One simple explanation would be that ouabain-treated cells are depolarized and consequently have a high resting influx of Ca2" through non-inactivating voltage-operated channels. On removal of external Na+ the treated cells show changes in Ca"+ which are dependent on the presence of external Ca2". This altered responsiveness when the cells are loaded with Na+ points to a potentially important role for Nat, either directly or indirectly, in the functioning of this particular cell type. One possible explanation for the apparent lack of functional Na'-Ca2" exchange in the control cells is that the intracellular Na+ concentration is very low such that when the external Na+ concentration is reduced to zero there is insufficient electrochemical driving force available to reverse the exchanger. This may not be the case since estimates of Nat using the fluorescent dye SBFI suggest a resting level of II mm. If this is the case then Na'-Ca2" exchange may be present but inactive in the control cells. The elevation of Nat could lead to the unmasking, activation or insertion of additional Na'-Ca2" exchange mechanisms. Alternatively Nat may act at some other as yet unidentified mechanism in the cytoplasm. Increasing Nat using ouabain would lead to a large transmembrane concentration difference on subsequent removal of external Na+. This increased electrochemical driving force may be sufficient to reverse any Na'-Ca2+ exchange with a consequent influx of Ca2 . 2+ seen on removal of Na' from ouabain-treated cells are The complex changes in Ca unlikely to be due to the simple reversal of any Na'-Ca2' exchange. The response appears to be divided into three components. An initial rapid entry of Ca21 followed, some 200-300 s later, by a much slower increase. Superimposed on the early and rapid rise there
151
Ca2+ REGULATION IN SMOOTH MUSCLE
appears to be a transient elevation in Ca". This transient appears to be activated by the initial Ca2" influx. This component appears to require a threshold level of Ca"+ (Fig. 7A) and is readily depleted by repetitive activation and prior AVP stimulation (Fig. 7 B). These observations are consistent with the idea that there is an internal store of Ca"+ which is released on the rapid elevation of cytoplasmic Ca2". Such Ca2"-induced Ca2" release is well documented in many other cell types (Endo et al. 1970; Ford & Podolski, 1970; Fabiato & Fabiato, 1978; Fabiato, 1983) and may therefore be present in these A1O cells. The initial Ca2" entry to activate such Ca2"-induced Ca2" release could come from different mechanisms acting independently or together. For example, Na+ removal should hyperpolarize the cells but, if for some unforseen effect of the Na+ substitute, it led to a depolarization this would open Ca2" channels and result in a rapid influx of Ca2". Alternatively, Na+ replacement may reverse the operation of the Na'-Ca2" exchanger resulting in the inward transport of Ca2". The observation that application of AVP to mobilize the intracellular stores of Ca2" interferes with the possible Ca2"-induced Ca2" release could indicate that the same intracellular store can be accessed by cytoplasmic Ca2" and inositol 1,4,5-trisphosphate. Once the internal store is depleted of Ca"+ then the effect of removing extracellular Na+ should be simplified. However, a rapid and slow phase still remain (see Fig. 5). The absence of any mobilization of Ca"2 in control cells on removal of external Na+ may be accounted for by the conditions outlined above. However, the possibility cannot be excluded that the elevation of Na+ by ouabain treatment sensitizes the Ca2"-induced Ca2"-release mechanism, such that under these abnormal conditions the sarcoplasmic reticulum is activated by smaller changes in internal Ca2". REFERENCES
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