Biochimica et Biophysica Acta. I 134 (1992) 7-16 © 1992 Elsevier Science Publishers B.V. All rights reserved OI67-4889/92/$05.l~t
7
BBAMCR 13100
Na+-Ca 2+ antiporter activity of rat hepatocytes. Effect of adrenalectomy on Ca z+ uptake and release from plasma membrane vesicles Rebecca K. Studer and Andr~ B. Borle Department of Physiology. Uni~'ersityof Piusburgh Sclu~l ~f Medicine. Pittsburgh. PA (U.S.A.)
(Received 27 June 1991) (Revised manuscript received 27 September 1991)
Key words: Sodium ion-calcium ion antiporter: Intracellular calcium: Plasma membrane vesicle: Calcium ion flux: (Hepatocyte)
The presence and mode of Na+-Ca2+ antiportcr activity were studi,.'d in hepatocytcs isolated from sham-operated or adrenalectomized rats and in inside-out plasma membrane vesicles isolated from rat liver. Decreasing extraeellular Na + (Na +) immediately increased eytosolic free calcium (Ca T÷ ). The rise in Ca T+ was proportional to the reduction in Nao+ and was caused by an increased calcium influx, presumably on the Na +-Ca2+ antiporter operating in the reverse mode. Perfusing the cells with Ca2+-free media stimulated Ca -'+ effiux and decreased CaT + , an effect dependent on Nao*. This suggests an activation of the forward mode of N a t - C a -'+ exchange. There was little difference in these parameters between sham and adx groups, in contrast, steady-state calcium uptake by inside-out plasma membrane vesicles was inhibited 40% after adrenalectomy. The decrcvscd calcium uptake was not caused by a deficiency in the ATP-dependent Ca 2+ pump+ who~e K m and V~, were unaffected by adrenaleetomy, but by an Nat-dependent leak from the vesicles. Ca -'+ cfflux was proportional to the extravesicular Na + concentration, suggesting that the calcium leak may take place on a Na +-Ca~"+ antiporter. This Na+-dependcnt calcium cffiux was significantly increased in vesicles prepared from adx rat livers. These results suggest that hepatocytes have functional N a t - C a 2+ antiporters that can operate in both forward and reverse modes. Under normal conditions, the Na+-Ca-'+ anfiporter apparently operates in the reverse mode as a Ca 2+ influx pathway. The increase in Nat-dependent Ca -'+ effiux evoked by adrenalectomy in plasma membrane vesicles could explain the recent results we obtained in hepatocytes isolated from adx rats, showing increased calcium influx, increased CAT++ increased intracellular calcium sequestration, and increased plasmalemmal calcium cycling.
Introduction W e have r e p o r t e d t h a t h e p a t o c y t e calcium h o m e o stasis a n d C a 2+ signalling a r e significantly a l t e r e d by
Abbreviations: adx, adrenalectomized; Ca~÷, cytosolic free calcium; Ca2o+, extracellular free calcium; Em, plasma membrane electrical potential difference; ER, reversal potential of the Na÷-Ca2+ antiporter ER-3ENa*-2Eca:*; KHB, Krebs-Henseleit bicarbonate buffer; Na +, intracellular Na ÷ concentration: Na~,, extracellular Na + concct,tration; SBFI, sodium binding benzofuran isophthalate; TMA, tetramethylammonium. Correspondence: A.B. Bode, Department of Physiolo~, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
a d r e n a l e c t o m y in the rat [1-3]: cytosolic free calcium (Ca~+), total cell calcium a n d the size o f the intracellular e x c h a n g e a b l e calcium pools a r e all significantly i n c r e a s e d from 30 to 2 5 0 % [1-3]. A f t e r a d r e n a l e c t o m y , the steady state e x c h a n g e o f calcium across the p l a s m a m e m b r a n e is increased 80%, the basal r a t e o f calcium influx is increased 3 0 % a n d the resting cytosolic free calcium (Ca~ +) is 3 0 % h i g h e r [3]. However, C a 2+ signalling evoked by e p i n e p h r i n e o r vasopressin is depressed a f t e r adrenalectomy: the rise in Ca~ + a n d the c o n c o m i t a n t rise in C a 2+ effiux a r e significantly decreased; moreover, the sensitivity o f the C a "+ effiux p a t h w a y to a given rise in C a 2+ a p p e a r s to b e diminished [2,3]. On the o t h e r h a n d , the stimulation o f C a -'+ influx by e p i n e p h r i n e is g r e a t e r in h e p a t o c y t e s from
8
adx rats than in sham controls [2]. The depressed sensitivity to Ca 2+ of the Ca 2~ efflux process, the increased basal and stimulated Ca 2+ influx, and the increased Ca,?+ suggest that adrenalectomy may have a major effect on Ca 2+ transport across the plasma membrane of rat hepatocytes. The specific Ca 2+ pathways affected by adrenalectomy are Unknown. In rat hepatocytes, the Ca 2+ transporters involved in Ca 2+ cycling across the plasmalemma are not as well defined as in other cells. A high affinity (Ca2+-Mg 2+) ATPase has been characterized in hepatoeyte plasma membranes [4], but is not currently accepted as the enzymic expression of the ATP-dependent Ca2+-transport [5]. However, ATP-dependent calcium accumulation by inside-out vesicles from hepatocyte plasma membranes is well documented [6-9]. The existence of a Na+-Ca 2+ antiporter in hepatocytes has been postulated by several investigators [10,11] and convincing experimental evidence has been published [7,12]. It appears, however, that the antiporter does not operate in the forward mode as a calcium efflux pathway but in the reverse mode as a calcium influx pathway [12]. On the other hand, cultured rat hepatocytcs have been reported to have lost their Na+-Ca 2+ antiporter activity [13]. Finally, the pres.:nee of voltage sensitive or receptor operated calcium chanilels in rat hepatocytes is still speculative. Although investigators found that some calcium channel blockers decrease the stimulation of Ca 2+ influx caused by Ca 2+ mobilizing hormones [11], the blocker concentrations used were 2 to 3 orders of magnitude greater than the concentration required tO block voltage-dependent Ca 2+ channels in cardiac or smooth muscle [14,15]. Concentrations of these blockers close to their EDs0 do not inhibit the Ca 2+ influx responses evoked by Ca 2+ mobilizing hormones [16]. The issue is further complicated by the fact that Ca 2+ channel blockers, and di- or trivalent cations inhibit Na+-Ca 2+ exchange as well as Ca 2+ channels [17-20], especially at high concentrations. The aim of this series of experiments was first to confirm the existence of the Na+-Ca 2+ antiporter, and to demonstrate its activation in the reverse and forward mode by changing the extracellular concentration of Na + (Nap+), of Ca z+ (Ca 2+) or both. The second aim of these studies was to compare the Ca 2+ transport properties of liver plasma membrane vesicles prepared from sham or adx rats. We found evidence of Na+-Ca 2+ exchange activity in isolated hepatocytes and of Na+-dependent Ca 2+ fluxes in liver plasma membrane vesicles. In intact cells, the antiporter seemed capable of operating in the forward and reverse mode. In vesicles, the kinetic properties of the ATP-dependent Ca 2+ transport were not changed after adrenalectomy, but the Na+-dependent efflux from the vesicles was significantly greater in vesicles from adx than from sham operated animals. Consequently,
Ca 2+ uptake by inside out plasma membrane vesicles isolated from adx animals was significantly decreased when compared to the sham-operated controls Materials and Methods
Bilaterally adrenalectomized or sham operated male rats (225-250 g) were obtained from Zivic-Miller Laboratories, Allison Park, PA. They were used for the preparation of hepatocytes or liver plasma membraee vesicles 7-14 days after surgery. The adrenalectomized (adx) animals were maintained on 0.9% NaCI while the shams received tap water. Hepatocytes were isolated from fed animals with collagenase (Sigma type IV, Sigma, St. Louis, M e ) and incubated in standard Krebs-Henseleit bicarbonate buffer (KHB) containing 1.3 mM Ca2+: 1% bovine serum albumin and 5 mM glucose, at 37"C. When appropriate, tetramethylammonium chloride (TMA) and choline bicarbonate were substituted for NaCI and NaHCO 3 to reduce the Na + concentration of the incubation media (Na+). Drugs, chemicals and materials were from the sources noted in our previous papers [1,2]. Measurement of cytosolic free calcium. Ca 2+ was determined with aeqm~rln. The photoprotein was incorporated by gravity loading, i.e., by centrifuging the cells at 5 0 x g f o r 60 s (500 rpm in a standard IEC centrifuge) in Ca2+-free medium containing 2 /zg of aequorin/ml cell suspension [21]. The aequorin loaded cells were imbedded into agarose gel threads, placed in a cuvette in an aequorin photometer and perfused at 0.6 m l / m i n with KHI] at 37"C. The light signal was calibrated as previously described [22] assuming an intr.acellular free Mg 2+ concentration of 0.5 raM. Calcium efflux. Hepatocytes were labeled with 45Ca, washed, and perfused in multi-channel peffusion chambers with nonradioactive media as previously described [2]. Calcium efflux is expressed as the experimental/control ratio, i.e., the ratio of calcium effiux from cells peffused with an experimental medium (24 mM Na +) to that of control cells perfused concurrently with standard KHB (144 mM Na+). Calcium influx. Calcium influx into hepatoeytes was measured with 45Ca as pre~,iously described [23]. Hepatocytes were incubated at 37"C in KHB as a stirred suspension. Aliquots of the suspension were centrifuged 15 s at 50 x g, and preiucobated for 10 rain in KHB or in media of different Ca 2+ a n d / o r Na + concentrations. After the preincubation, the cells were centrifuged 15 s, resuspended in 1 ml of the appropriate medium, and 45Ca (0.25 t t C i / m ) was added 30 s later. Aliquots of the cell suspension were taken at 6, 12, 18, 24 and 30 s after addition of aSCa to determine the initial rate of calcium influx following acute changes in medium Na + a n d / o r Ca 2+ concentrations.
9
Intracellulor free sodium. Na~" was measured with SBFI as previously described [24]. Hepatocytes were incubated as suspensions for 60 min with l0 p.M SBFI/AM at room temperature, in the presence of 0.1% pluronic acid. The cells were then washed, cast in agarose gel threads, placed in a quartz cuvette and perfused with KHB at a rate of 0.6 ml/min at 37°C. Fluorescence was measured in a SPEX dual excitation spectrofluorometer. The excitation monochromato, s were set at 340 and 385 nm, the emission at 505 nm and the cell fluorescence was recorded as the 340/385 nm ratio. At the end of each experiment, a calibration was performed by perfusing solutions of various Na + concentrations (50, 24, 12, 6, 3 and 0 mM) with 10 mM Hepes (pH 7.0), KCI, 2 p.M monensin, and 2 p.M gramicidin. The ionic strength was maintained constant at 0.15 by substituting KCI for NaCI. Isolation of liver plasma membranes. The plasma membrane preparations of Prpic et al. [6] were used. Livers were removed from adx or sham operated rats, rinsed, minced in cold 0.9% NaCI, an:l homogenized in a solution containing (raM): sucrose, 250; EGTA, 1; Hepes, 5 at pH 7. The homogenate was diluted to 6%, centrifuged at 1500×g for 10 min, and the pellet homogenized in 250 mM sucrose, 5 mM Hepes (pH 7.3), 0.1 mM EGTA. The pellet homogenate was diluted to 8% in the same solution, and mixed with Percoll at a ratio of 10.4 mi homogenate to 1.4 ml percoll. The plasma membranes were separated by centrifugation at 35000 × g for 30 rain, and washed once with 250 mM sucrose, 25 mM Hepes (pH 7.15) then centrifuged for 10 rain at 1500 × g. The membrane pellet was diluted 1 to 5 or 1 to 10 in 250 raM sucrose, 25 mM Hepes (pH 7.15), 1 mM dithiothreitol, 0.2 mM phenylmethanesulphonyl fluoride and kept on ice until used for calcium transport studies. The homogenate, the initial pellet and the final membrane preparation were assayed for 5' nucleotidase [25] and glucose.6-phosphatase [26] with phosphate determined by a modification of the Fiske-SubbaRow method [27]. Plasma membrane calcium transport. 0.025 ml of plasma membranes were added to 0.225 mi of medium containing (in raM): KCI, 100; NaCI, 20;, MgCI z, 5; KH2PO 4, 1; Hepes, 25 (pH 7.15); NaN 3, 5; phosphoereatine, 5; creatine kinase, 5 U/ml; EGTA, 0.5; 45CaCI2, 0.025/zCi/ml; ATP, 1.5. CaCI 2 was added at various concentrations, from 0.1 to 0.525 raM, to obtain a free calcium ranging from 0.06 to 35 /zM as determined with a calcium sensitive electrode (World Precision Instruments, New Haven, CT). lIte standard total Na + concentration of the incubating medium was 25 mM unless stated otherwise. The membranes were incubated at 37°C in a shaking water bath for different lengths of times, and calcium accumulation was terminated by the addition of 4 ml of an ice-cold wash solution containing 250 mM sucrose, 5 mM Hepes (pH
7.i). The membranes were collected by vacuum filtratiop on Gclman Metricel GN-6 filters with a pore size of 0.45 txm and washed three times with 4 ml of the same wash solution. The filt~:s were dissolved in scintillation fluid (3a70b, Research Products International, Mount Prospect, ILl and the 45Ca accumulated by the inside-out plasma membrane vesicles was determined by scintillation spectrophotometry. The ATP-dependent uptake was corrected for any passive accumulation of calcium by measuring the uptake without ATP and creatine kinase in identical aliquots of plasma membranes incubated concurrently. Finally, to measure Na+-dependent Ca z+ uptake, vesicles were first loaded with Na ÷ according to the expc.rimental procedure of Slaughter et al. [28] to establish a Na + gradient, then the vc~ieles were resuspended into buffered media containing 160 mM KCI and 60 ttM free calcium. Statistics. StatisticaEy significant differences between groups were determined using either Student's t-test or F statistics where necessary. Results
Effect of extracelhdar Na + on cytosolic free Ca" + and Ca: + efflttr in freshly isolated hepatocytes perfused with KHB, Ca~ + was found to be 148 _+ 12 nM (n = 8) and the intracellular Na + concentration cNa~) was 15.9 +_2.4 mM (n = 14). These values agree with those published previously for both Ca~ + [2,3] and Na~ [29]. The effect of lowering the extracellular Na + concentration (Nao+) on Ca~ + of peffused bepatocytes is shown in Fig. 1, which is a tracing obtained with cells from a sham operated animal. When the Na + concentration of the perfusate was decreased, Cai2+ increased. The peak rise in Ca~ + or dCa~ + was directly related to the decrease in Nao+. After the peak rise, Ca z+l slowly declined towards its resting control level. When Na + was returned to 144 mM by perfusing the cells with
is0
I~w
0
Fig. I. Effect of decreasingthe extracellular Na+ concentration on Ca?÷ of perfusedisolated rat hepatocytes.Ca~÷ was measuredwith aequorin. This representativetracingwas taken frc.n the lightsignal obtained fromcellsisolatedfroma sham-operatedanimal.TMA was used as substitute for Na+ and. below24 mM Nao, choline bicarbonate was substituted for NaHCO3. The 10 rain periodsof perfusion with low Nao+ are indicted by the hor~ntal lines below the tracingabovethe respectiveNao+ concentrations.
150[ ~ sham
140
~
o~
.so •lO0
70
40
24
•
100
0
l..................... is0 •
o0
30
12
40
eight sepav!lteexperiments.
KHB, there was a fall in Ca~ + below baseline that was proportional to the previously imposed decrease in Na +. Fig. 2 shows ACa,2.+ , i.e., the peak Ca~ + rise evoked by various decreases in Na + and the maximal fall in Ca 2+ resulting from. the subsequent return to normal KHB in hepatocytes isolated from adx and sham operated animals. There was no significant difference between the two groups. In both groups, the rise in Ca,?+ in low Na,+, and its fall below baseline at recovery in KHB, were proportional to the decrease in Na +. W e cannot deduce from these experiments whether the initial rise in Ca 2+ was caused by a decreased Ca ~+ efflux or by an increased Ca 2+ influx. To discriminate between these two possibilities, the changes in calcium efflux and in Ca~ + were determined in the same hepatocyte preparations. The cells were perfused for 10 min with 24 mM Na + (from rain 40 to 50). Fig. 3 shows the relation between the change in Ca2+i or ACa,2.+ and Ca 2+ e f f l u x in h e p a t o c y t e s from sham operated rats. Ca~ + rose 100 nM ( P < 0 . 0 0 1 ) during the low Na + perfusion, while calcium efflux increased slightly. The 10% increase in Ca 2+ efflux during the first min of low Na + perfusion (min 41) was statistically significant ( P < 0.05), as was the transient depression observed at min 50. Between these two time points, Ca 2+ efflux was not significantly different from control. After min 50, when the cells were perfused with normal KHB (Na + = 144 mM), Ca~ + dropped below baseline as in Figs. 1 and 2 ( P < 0.001), and Ca 2+ efflux rose 35% ( P < 0.01). Similar results were obtained when Nao+ was reduced to 0 mM (results not shown). This indicates that the sudden rise in Ca 2+ i that reached its maximum in less than 5 min cannot be caused by a decreased transport of calcium out of the ceils, since, if anything, calcium efflux increased slightly. It must he caused instead by an increased calcium influx. And indeed, we measured an increased calcium
50
s0
70
so
Time (mint
Extracetlular Na + (raM) Fig. 2. Effect of progressive decreases in Na~ on ,dCa~+. the evoked peak rise in Ca~+, and the depression in Ca~ 4 that follows the return 1o 144 mM Na~. The cells isolated from sham (hatched bars) and adx (shaded bars) animals were prepared and perfused as described in Fig. I. The data represent the mean_+S.E, of three to
~oo
Fig. 3. Effect of lowering intracellular Na + to 24 mM on Ca~ + and
on calcium efflux from isolated hepatocytes. After a control period of 40 rain, the cells were perfused from rain 40 to 50 with 24 mM Na~ media followedwith KHB (Na~ = 144) from rain 50 to 70. The changes in Ca~+ (ACa~ +, closed circles) relate to the right ordinate; the changes in calcium eft'lax (open circles) relate to the left ordinate. The changes in calcium effiux are expressed as% of a control group peffused concurrently and continuously with KHB. The data
represent the mean±S.E, of 6-8 experiments. * difference statistically significant( P < 0.01).
influx, 12 s after changing the m e d i u m from control KHB to 0 mM Na + . When the results are expressed as the ratio of calcium influx in 0 Na + over that in control KHB, we found a ratio of 1.75 + 0.21 in shams and 1.24 + 0.07 in cells from adx rats. Since in control conditions, calcium influx is 30% faster in adx than in shams, or 1.98 + 0.09 vs. 1.52 + 0.07 n m o l / m i n per mg protein [3], there was no significant difference between the two groups in the increased calcium influx measured when Na + was decreased from 144 to 0 mM: 2.66 vs. 2.46 n m o l / m i n per mg in sham and adx group,
TABLE I Effect of low extracellular Na + and of Ca z +-free medium on Ca" + influx i n isolated rat hepatocytes
Values are mean ± S.E. of the number of experiments shown in parenthesis. Ca"* influx (nmol/min per m s protein) sham
adx
1.52±0.07
1.98 ± 0.09 b
(8)
(8)
N a ~ = flm M
2.66±0.34 c (5)
2.46±0.18 d (5)
Control
1.21±0.06 (4) 4,1 :i:0,66 ~ (4)
N a ~ = 144 m M "
Ca~)+ Recovery in 1,3 mM Ca~)+ after 10 min in 0 Ca~)+
before 0
h c d c
From Borle and Studer [3]. p < 0.01 compared to sham. p < 0.05 compared to sham, 144 m M Na~. p < 0.01 compared to adx, 144 m M Na~. p < 0.01 compared to control before 0 Ca(2)+ .
TABLE II
15o
B
Effect~ ~,( 0 Ca~' aml u[ O Ca o ~ and 0 Na,; on hepatvo'tes O'toso/ic fre calcium
Values art: mean_+S.E, of the number of experiments shown in parenthesis; n.s.. not significant. Ca," Change nM 148.+ 12 (8) - Ifi6+_15 - 72~(4) +216_+28 +514%
Control Ca~" (nM) .ACa~' in 0 C'a~( Fig. 4. Effect of Ca2 ~-frec and Na ~-frec media on Ca~ ~ in hcpatocytcs isolated frova sham operated rats. Ca~* was vacasured with aequorin in ceils perfused with KHB as described in Fig. I and in Methods. This tracing, representative of fimr to six separate cxpcrivaents, shows the effects of perfusing the cells with I) Ca~," for 5 vain and with 0 Ca~,+ +0 Na,,~ for 5 vain. immediately followed with I) Na~ + 1.3 yaM Ca~,÷. The experimental periods of perfusiop with Ca2+-free and Na ~-frc¢ media are indicated by the horizontal lines below the tracing above the value for medium compositi~va.
respectively (Table i). O n the o t h e r h a n d , the close t e m p o r a l relation b e t w e e n the r a p i d rise in calcium efflux a n d the fast d r o p in Ca~ + , w h e n Na~ is r e s t o r e d to 144 m M in the s h a m g r o u p (Fig. 3), suggests that d u r i n g recovery the fall in C a 2 + is c a u s e d by the rise .in calcium efflux. E f f e c t s o f C a " +-free m e d i a o n Ca~ +
W e recently d e m o n s t r a t e d t h a t the f o r w a r d m o d e o f N a + - C a 2+ e x c h a n g e c o u l d be activated by perfusing k i d n e y cells with Ca2+-free m e d i a [24]. T o f u r t h e r investigate the p r e s e n c e of N a + - C a z+ e x c h a n g e in liver, we studied the effects o f Ca2+-frec a n d o f C a +- a n d N a + - f r e e m e d i a o n Ce 2+ in isolated hepatocytes. Simply omitting CaCI 2 f r o m K H B usually lowers the m e d i u m free c a l c i u m (Ca2,+) to 1 0 - * - 1 0 -5 M [24]. T o achieve a virtually CaZ+-free milieu a n d to t r a p the significant a m o u n t o f C a 2+ b o u n d to the surface o f isolated cells o r a n y C a 2+ t r a n s p o r t e d o u t o f the cells, 0.1 m M E G T A w a s a d d e d to all Ca2+-free p e r f u s a t e s d u r i n g the e x p e r i m e n t a l period. Fig. 4 is representative o f f o u r e x p e r i m e n t s in 0 C a 2+ a n d six e x p e r i m e n t s in b o t h 0 C a 2+ a n d 0 N a +, w h o s e results a r e s h o w n in T a b l e I1. Fig. 4 shows t h a t p e r f u s i n g rat h e p a t o c y t e s with a CaZ+-free m e d i u m , immediately lowered Ca.2.+ to less t h a n 3 0 % o f its resting value (Table 11). A t the s a m e time, Fig. 5 shows t h a t C a 2+ efflux increased significantly ( P < 0.01) d u r i n g the few m i n u t e s following the removal o f extracellular calcium. T h i s strongly s u p p o r t s the idea t h a t the i n c r e a s e d C a 2+ efflux w a s responsible for *~he fail in C a 2+. Indeed, it is h a r d to imagine how C a 2+ efflux o n the C a 2+ p u m p could increase w h e n C a 2÷ d e c r e a s e s m o r e t h a n 70%. W h e n Ca2o+ w a s r e s t o r e d to 1.3 raM, C a 2+ increased m o r e t h a n 5 0 0 % , significantly a b o v e the basal levels, t h e n r e t u r n e d t o control values (Fig. 4 a n d T a b l e lit. T h e
ACa~" at recovery
P value
< 0.IM)I < I).iM)I
(4)
ACa~ ~ in o Ca~," and 0 Na,; ACa~ ' at recovery in 0 Na,; anti 1.3 mM Ca~~
n.s. +91 .+ 17 (6)
n.s. + 78~-
< 0.(iOI
rise in Ca~ ÷ w h e n C a -`+ was r e i n t r o d u c e d into the perfusate was c a u s e d by a n increased C a 2+ i n f u x . Indeed, 30 s a f t e r a d d i n g control K H B to cells incub a t e d in Ca2÷-free m e d i u m for 10 rain, C a 2+ influx increased 3.4-fold, from a control value o f 1.21 + 0.06 to 4.1 + 0 . 6 6 n m o l / m i n p e r m g protein ( P < 0 . 0 1 ) as ~hown in T a b l e 1. W h e n N a + was omitted from the Ca2+-fre¢ p e r f u s a t e (Na+-free a n d Ca2~-free K H B ) , the fall in Ca~ + was abolished (Table !1 a n d s e c o n d stimulation o f Fig. 4). T h i s strongly suggests that extracellular Na + is necessary for the fall in Ca~ +. T h e large rise in C a , + w h e n C a 2+ is r e i n t r o d u c e d in the Na+-free p e r f u s a t e (second stimulation of Fig. 4) is c o m p a r a b l e to t h a t shown in Fig. 1 a f t e r the shift to N a * - f r e e media. In t h a t instance, C a 2+ influx was increased 33%. T h e s e results support the idea that hepatocytes possess a functional N a + - C a 2+ a n t i p o r t e r in t h e i r p l a s m a m e m b r a n e capable o f o p e r a t i n g in both forward a n d 1.2
gl
•
-
•
t t
°~ v Time (min)
Fig. 5. Effect of a Ca2+-free medium on calcium efflux in rat hepatocytes. From min 40 to 50, the ceils were perfused with a Ca2~-free KHB containing 0.1 yaM EGTA. Calcium emux is expressed as the ratio of the Ca-'+ efflux of the group pcrfused with 0 Ca~,+ to the Ca2+ effiux of controls peffused concurrently with KHB. The data represent the mean+S.E, of ten separate experiments.
12
reverse modes. Although no significant differences were observed between sham and adx animals when the hepatocytes were pcrfused with 0 Na + or with 0 Ca2o+, the possibility remains that adrcnalectomy may alter the properties of the antiporter in more physiological conditions [1-3]. Thus, we evaluated calcium transport in inside-out plasma membrane vesicles isolated from the liver of sham and adx rats. We verified the enrichment of these vesicle preparations by measuring the plasma membrane enzyme marker 5'-nucleotidase, and the degree of contamination from endoplasmic reticulure by measuring the activity of glucose-6-phosphatase. From homogenate to plasma membrane, *.he vesicles displayed a 20-fold enrichment in 5'-nucleotidase from 32 + 3 to 658 + 30 mg P~/mg protein per 20 rain and a 50% reduction in glucose-6-phosphatas¢ from 61 + 8 to 32 + 4 mg P~/mg protein per 20 rain. There was no significant difference between sham and adx rats.
Effect of adrenalectomy on ATP-dependent Ca 2+ uptake and Ca z+ efflux in inside-out plasma membrane vesicles ATP-dependent Ca 2+ uptake was measured in inside-out plasma membrane vesicles isolated from the livers of sham and adx rats in the presence of 25 mM Na+. First, to assess the passive Ca 2+ binding to the vesicles, we measured the ATP-independent Ca 2+ uptake. There was no difference between sham and adx: 1.87 + 0.14 vs. 1.76 + 0.14 n m o l / m g protein per 30 rain, respectively. On the other hand, there was a statistically significant difference between the two groups in the ATP-dependent Ca 2. uptake. Fig. 6 shows that, in the presence o f A T P , the vesicles iso-
30
:
. . sham
~
.
+
.
.
•
E"6
0
o!•
o.~"
0
1'0
'
2'0
'
30
Time (min)
Fig. 6, Effect of adrcn deetomyon ATP-depend=.nt Ca2+ accumulation by plasma membrane inside-out vesicles isolated from rat liver. Plasma membranes were prepared from sham (e) and adx (o) rat livers as described in Materials and Methods and incubated for various times at 37¢C in media containing 8.5 p.M ionized Ca=* and 25 mM Na +. Ca2+ uptake is corrected for ATP-independent binding and uptake. The data represent the mean~S.E, of 5-15 separate experiments. * P < 0.05 when compared to sham group.
~E
~
-=: :~.°o ~tlc 4
8 10
CaZ+ (~tM) Fig. 7. Initial rate of ATP-depeodent Ca2. uptake as a function of the medium free Caz+ concentration by inside-out plasma membrane vesicles isolated from the liver of sham (e) and adx (o) rats. The incubation medium contained 0.5 mM EGTA and Caz+ concentrations ranging from 0.1 to 0.525 raM. 4SCauptake was measured at
37°C for exactly 60 s. The inset showsan Hofstee plot of the data. The equations for the line fitting the data are: sham, y = 4.2-0.29x (%R2 = 0.g7): adx, y = 4.7-0.32x (R2 = 0.83). The data represent the mean+ S.E. of g-12 separate experiments.
lated from adx animals ac:umulated or retained 64% less calcium than the shams. When the ionophore A23187 (10 -H M) was added after 25 rain of incubation, the 4SCa accumulated immediately decreased to 1.8 nmol/mg protein, the levels measured in vesicles incubated without ATP, indicating that the 4SCa taken up was transported inside the vesicles (data not shown). The difference between the two groups in the calcium accumulated in the presence of ATP was statistically significant (P < 0.05). The decreased uptake of calcium in vesicles from adx rats could be caused by a decreased affinity a n d / o r a lesser capacity of the ATPdependent calcium pump or to an increased efflux of calcium from the vesicles. These possibilities were investigated. First, the initial rate of calcium uptake was determined in vesicles which had been preincubated at 37°C then exposed for 1 rain to media of various Ca 2÷ concentrations containing 45Ca. 45Ca uptake was linear between 12 and 60 s in both sham and adx groups (data not shown). Fig. 7 shows tile initial rate of Ca 2+ uptake by inside-out plasma membrane vesicles incubated in media with Ca 2+ concentrations ranging from 0.14 to 8.5/~M. The inset of Fig. 7 shows an Hofstee plot of the data. The Ca ~'+ affinity (K m) was 3.0 + 0.4.10 -7 and 3.2 + 0.4.10 -7 M in sham and adx, respectively, and the maximum rate of ATP-dependent Ca 2+ accumulation (Vm~) by vesicles from sham and adx rats was 4.17 =l=0.15 and 4.36 ± 0.31 nmoi/min per mg protein, respectively. The lack of significant difference in K m and Vmx between the two groups suggests that the decreased Ca 2+ uptake by vesicles from adx rats was not caused by a decreased affinity or a lesser capacity of the ATP-dependent Ca z+ pump.
In another series of experiments, the passive efflux of Ca ~ ~ from plasma membrane vesicles was initiated by adding 50 u n i t s / m l hexokinase and l0 mM glucose to vesicles loaded for 25 rain in the presence of ATP and 8.5 /LM Ca 2~. This caused a rapid disappearance of ATP and blocked any further active accumulation of calcium. As a result, calcium leaked out of the vesicles and their calcium content decreased. Fig. 8 shows that the calcium retained in the vesicles decreased with time. when expressed in absolute terms or as the percent of the calcium accumulated after 30 rain of uptake (inset of Fig. 8). The loss of calcium from vesicles isolated from adx animals was significantly greater ( P < 0.05) during the first 5 rain that followed the inhibition of the active uptake (inset of Fig. 8). This suggests that the difference in steady state calcium accumulation between the two groups shown in Fig. 6, was due to a greater leak of calcium from the vesicles isolated from adx rats. To determine whether calcium efflux from inside-out plasma membrane vesicles was d e p e n d e n t on the Na + concentration of the incubating medium, ATP-dependent calcium uptake by the vesicles was measured in media of increasing Na* concentrations (5-55 raM). All media contained 5 mM NaN3 and 8.5 p.M Ca 2+, with the sum of the Na + and K + concentrations kept constant to maintain the same osmolarity. Fig. 9 shows the influence of the m e d i u m Na + concentration on the A T P - d e p e n d e n t calcium uptake expressed both in absolute terms and as a percent of the amount accumulated in 5 m M Na + medium. At the lowest Na + concentration (5 mM), there was no leak of Ca :+ from 20 • sham
~100 .
E~~ .~_
~
o
1o
20
3o
Time (rmn~
'o" ) o
o o
o
10 Time (rain) ]Fig. 8. Effect of adrenalnctomy on ~Ca efflux from plasma membrane vesicles isolated from the liver of sham (e) and adx (o) rats. Membrane vesiclns were first incubated with ~SCa for 25 min as described in Fig. 6. Alter 25 min, hesokinase (50 U / m l ) and glucose (tO raM) were added to deplete th~ preparation of AI"P and to block
ATP-dependent calcium uptake (time 0). The 45Ca retained in the vesicles was then determined from time 0 (addition of hexokinaso+ glucose) to min 30. In the inset, the same data are expressed as the% of the calcium accumulated after 25 min of uptake. The data represent the mean+S.E, of 5-13 separate experiments. * P < 0.05 for the difference between sham and adx groups.
30
t
. ,'0
;o
3'o
2o
~'o
~0
Sodium (mM) Fig. 9. Effect of adrenalectomy on the 4SCa accumulated and re*ained by liver plasma membrane inside-out vesich~sas a function of the ¢xtravesicular Na ~ concentration. The vesich~s isolated from sham and adx rat liver were incubated for 25 min in media containing 5 mM NaN:~and various additions of Na ÷ (0-50 raM). Na * was substituted for K" to maintain the .sameosmolarity.~Ca uptake was performed as described in Fig. 6. In the inset, the same data are expressed as the percent of the accumulation measured in the control medium that contained 5 mM NAN,. The data represent the mean_+S.E, of 0-12 separate experiments. * P
7
BBAMCR 13100
Na+-Ca 2+ antiporter activity of rat hepatocytes. Effect of adrenalectomy on Ca z+ uptake and release from plasma membrane vesicles Rebecca K. Studer and Andr~ B. Borle Department of Physiology. Uni~'ersityof Piusburgh Sclu~l ~f Medicine. Pittsburgh. PA (U.S.A.)
(Received 27 June 1991) (Revised manuscript received 27 September 1991)
Key words: Sodium ion-calcium ion antiporter: Intracellular calcium: Plasma membrane vesicle: Calcium ion flux: (Hepatocyte)
The presence and mode of Na+-Ca2+ antiportcr activity were studi,.'d in hepatocytcs isolated from sham-operated or adrenalectomized rats and in inside-out plasma membrane vesicles isolated from rat liver. Decreasing extraeellular Na + (Na +) immediately increased eytosolic free calcium (Ca T÷ ). The rise in Ca T+ was proportional to the reduction in Nao+ and was caused by an increased calcium influx, presumably on the Na +-Ca2+ antiporter operating in the reverse mode. Perfusing the cells with Ca2+-free media stimulated Ca -'+ effiux and decreased CaT + , an effect dependent on Nao*. This suggests an activation of the forward mode of N a t - C a -'+ exchange. There was little difference in these parameters between sham and adx groups, in contrast, steady-state calcium uptake by inside-out plasma membrane vesicles was inhibited 40% after adrenalectomy. The decrcvscd calcium uptake was not caused by a deficiency in the ATP-dependent Ca 2+ pump+ who~e K m and V~, were unaffected by adrenaleetomy, but by an Nat-dependent leak from the vesicles. Ca -'+ cfflux was proportional to the extravesicular Na + concentration, suggesting that the calcium leak may take place on a Na +-Ca~"+ antiporter. This Na+-dependcnt calcium cffiux was significantly increased in vesicles prepared from adx rat livers. These results suggest that hepatocytes have functional N a t - C a 2+ antiporters that can operate in both forward and reverse modes. Under normal conditions, the Na+-Ca-'+ anfiporter apparently operates in the reverse mode as a Ca 2+ influx pathway. The increase in Nat-dependent Ca -'+ effiux evoked by adrenalectomy in plasma membrane vesicles could explain the recent results we obtained in hepatocytes isolated from adx rats, showing increased calcium influx, increased CAT++ increased intracellular calcium sequestration, and increased plasmalemmal calcium cycling.
Introduction W e have r e p o r t e d t h a t h e p a t o c y t e calcium h o m e o stasis a n d C a 2+ signalling a r e significantly a l t e r e d by
Abbreviations: adx, adrenalectomized; Ca~÷, cytosolic free calcium; Ca2o+, extracellular free calcium; Em, plasma membrane electrical potential difference; ER, reversal potential of the Na÷-Ca2+ antiporter ER-3ENa*-2Eca:*; KHB, Krebs-Henseleit bicarbonate buffer; Na +, intracellular Na ÷ concentration: Na~,, extracellular Na + concct,tration; SBFI, sodium binding benzofuran isophthalate; TMA, tetramethylammonium. Correspondence: A.B. Bode, Department of Physiolo~, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
a d r e n a l e c t o m y in the rat [1-3]: cytosolic free calcium (Ca~+), total cell calcium a n d the size o f the intracellular e x c h a n g e a b l e calcium pools a r e all significantly i n c r e a s e d from 30 to 2 5 0 % [1-3]. A f t e r a d r e n a l e c t o m y , the steady state e x c h a n g e o f calcium across the p l a s m a m e m b r a n e is increased 80%, the basal r a t e o f calcium influx is increased 3 0 % a n d the resting cytosolic free calcium (Ca~ +) is 3 0 % h i g h e r [3]. However, C a 2+ signalling evoked by e p i n e p h r i n e o r vasopressin is depressed a f t e r adrenalectomy: the rise in Ca~ + a n d the c o n c o m i t a n t rise in C a 2+ effiux a r e significantly decreased; moreover, the sensitivity o f the C a "+ effiux p a t h w a y to a given rise in C a 2+ a p p e a r s to b e diminished [2,3]. On the o t h e r h a n d , the stimulation o f C a -'+ influx by e p i n e p h r i n e is g r e a t e r in h e p a t o c y t e s from
8
adx rats than in sham controls [2]. The depressed sensitivity to Ca 2+ of the Ca 2~ efflux process, the increased basal and stimulated Ca 2+ influx, and the increased Ca,?+ suggest that adrenalectomy may have a major effect on Ca 2+ transport across the plasma membrane of rat hepatocytes. The specific Ca 2+ pathways affected by adrenalectomy are Unknown. In rat hepatocytes, the Ca 2+ transporters involved in Ca 2+ cycling across the plasmalemma are not as well defined as in other cells. A high affinity (Ca2+-Mg 2+) ATPase has been characterized in hepatoeyte plasma membranes [4], but is not currently accepted as the enzymic expression of the ATP-dependent Ca2+-transport [5]. However, ATP-dependent calcium accumulation by inside-out vesicles from hepatocyte plasma membranes is well documented [6-9]. The existence of a Na+-Ca 2+ antiporter in hepatocytes has been postulated by several investigators [10,11] and convincing experimental evidence has been published [7,12]. It appears, however, that the antiporter does not operate in the forward mode as a calcium efflux pathway but in the reverse mode as a calcium influx pathway [12]. On the other hand, cultured rat hepatocytcs have been reported to have lost their Na+-Ca 2+ antiporter activity [13]. Finally, the pres.:nee of voltage sensitive or receptor operated calcium chanilels in rat hepatocytes is still speculative. Although investigators found that some calcium channel blockers decrease the stimulation of Ca 2+ influx caused by Ca 2+ mobilizing hormones [11], the blocker concentrations used were 2 to 3 orders of magnitude greater than the concentration required tO block voltage-dependent Ca 2+ channels in cardiac or smooth muscle [14,15]. Concentrations of these blockers close to their EDs0 do not inhibit the Ca 2+ influx responses evoked by Ca 2+ mobilizing hormones [16]. The issue is further complicated by the fact that Ca 2+ channel blockers, and di- or trivalent cations inhibit Na+-Ca 2+ exchange as well as Ca 2+ channels [17-20], especially at high concentrations. The aim of this series of experiments was first to confirm the existence of the Na+-Ca 2+ antiporter, and to demonstrate its activation in the reverse and forward mode by changing the extracellular concentration of Na + (Nap+), of Ca z+ (Ca 2+) or both. The second aim of these studies was to compare the Ca 2+ transport properties of liver plasma membrane vesicles prepared from sham or adx rats. We found evidence of Na+-Ca 2+ exchange activity in isolated hepatocytes and of Na+-dependent Ca 2+ fluxes in liver plasma membrane vesicles. In intact cells, the antiporter seemed capable of operating in the forward and reverse mode. In vesicles, the kinetic properties of the ATP-dependent Ca 2+ transport were not changed after adrenalectomy, but the Na+-dependent efflux from the vesicles was significantly greater in vesicles from adx than from sham operated animals. Consequently,
Ca 2+ uptake by inside out plasma membrane vesicles isolated from adx animals was significantly decreased when compared to the sham-operated controls Materials and Methods
Bilaterally adrenalectomized or sham operated male rats (225-250 g) were obtained from Zivic-Miller Laboratories, Allison Park, PA. They were used for the preparation of hepatocytes or liver plasma membraee vesicles 7-14 days after surgery. The adrenalectomized (adx) animals were maintained on 0.9% NaCI while the shams received tap water. Hepatocytes were isolated from fed animals with collagenase (Sigma type IV, Sigma, St. Louis, M e ) and incubated in standard Krebs-Henseleit bicarbonate buffer (KHB) containing 1.3 mM Ca2+: 1% bovine serum albumin and 5 mM glucose, at 37"C. When appropriate, tetramethylammonium chloride (TMA) and choline bicarbonate were substituted for NaCI and NaHCO 3 to reduce the Na + concentration of the incubation media (Na+). Drugs, chemicals and materials were from the sources noted in our previous papers [1,2]. Measurement of cytosolic free calcium. Ca 2+ was determined with aeqm~rln. The photoprotein was incorporated by gravity loading, i.e., by centrifuging the cells at 5 0 x g f o r 60 s (500 rpm in a standard IEC centrifuge) in Ca2+-free medium containing 2 /zg of aequorin/ml cell suspension [21]. The aequorin loaded cells were imbedded into agarose gel threads, placed in a cuvette in an aequorin photometer and perfused at 0.6 m l / m i n with KHI] at 37"C. The light signal was calibrated as previously described [22] assuming an intr.acellular free Mg 2+ concentration of 0.5 raM. Calcium efflux. Hepatocytes were labeled with 45Ca, washed, and perfused in multi-channel peffusion chambers with nonradioactive media as previously described [2]. Calcium efflux is expressed as the experimental/control ratio, i.e., the ratio of calcium effiux from cells peffused with an experimental medium (24 mM Na +) to that of control cells perfused concurrently with standard KHB (144 mM Na+). Calcium influx. Calcium influx into hepatoeytes was measured with 45Ca as pre~,iously described [23]. Hepatocytes were incubated at 37"C in KHB as a stirred suspension. Aliquots of the suspension were centrifuged 15 s at 50 x g, and preiucobated for 10 rain in KHB or in media of different Ca 2+ a n d / o r Na + concentrations. After the preincubation, the cells were centrifuged 15 s, resuspended in 1 ml of the appropriate medium, and 45Ca (0.25 t t C i / m ) was added 30 s later. Aliquots of the cell suspension were taken at 6, 12, 18, 24 and 30 s after addition of aSCa to determine the initial rate of calcium influx following acute changes in medium Na + a n d / o r Ca 2+ concentrations.
9
Intracellulor free sodium. Na~" was measured with SBFI as previously described [24]. Hepatocytes were incubated as suspensions for 60 min with l0 p.M SBFI/AM at room temperature, in the presence of 0.1% pluronic acid. The cells were then washed, cast in agarose gel threads, placed in a quartz cuvette and perfused with KHB at a rate of 0.6 ml/min at 37°C. Fluorescence was measured in a SPEX dual excitation spectrofluorometer. The excitation monochromato, s were set at 340 and 385 nm, the emission at 505 nm and the cell fluorescence was recorded as the 340/385 nm ratio. At the end of each experiment, a calibration was performed by perfusing solutions of various Na + concentrations (50, 24, 12, 6, 3 and 0 mM) with 10 mM Hepes (pH 7.0), KCI, 2 p.M monensin, and 2 p.M gramicidin. The ionic strength was maintained constant at 0.15 by substituting KCI for NaCI. Isolation of liver plasma membranes. The plasma membrane preparations of Prpic et al. [6] were used. Livers were removed from adx or sham operated rats, rinsed, minced in cold 0.9% NaCI, an:l homogenized in a solution containing (raM): sucrose, 250; EGTA, 1; Hepes, 5 at pH 7. The homogenate was diluted to 6%, centrifuged at 1500×g for 10 min, and the pellet homogenized in 250 mM sucrose, 5 mM Hepes (pH 7.3), 0.1 mM EGTA. The pellet homogenate was diluted to 8% in the same solution, and mixed with Percoll at a ratio of 10.4 mi homogenate to 1.4 ml percoll. The plasma membranes were separated by centrifugation at 35000 × g for 30 rain, and washed once with 250 mM sucrose, 25 mM Hepes (pH 7.15) then centrifuged for 10 rain at 1500 × g. The membrane pellet was diluted 1 to 5 or 1 to 10 in 250 raM sucrose, 25 mM Hepes (pH 7.15), 1 mM dithiothreitol, 0.2 mM phenylmethanesulphonyl fluoride and kept on ice until used for calcium transport studies. The homogenate, the initial pellet and the final membrane preparation were assayed for 5' nucleotidase [25] and glucose.6-phosphatase [26] with phosphate determined by a modification of the Fiske-SubbaRow method [27]. Plasma membrane calcium transport. 0.025 ml of plasma membranes were added to 0.225 mi of medium containing (in raM): KCI, 100; NaCI, 20;, MgCI z, 5; KH2PO 4, 1; Hepes, 25 (pH 7.15); NaN 3, 5; phosphoereatine, 5; creatine kinase, 5 U/ml; EGTA, 0.5; 45CaCI2, 0.025/zCi/ml; ATP, 1.5. CaCI 2 was added at various concentrations, from 0.1 to 0.525 raM, to obtain a free calcium ranging from 0.06 to 35 /zM as determined with a calcium sensitive electrode (World Precision Instruments, New Haven, CT). lIte standard total Na + concentration of the incubating medium was 25 mM unless stated otherwise. The membranes were incubated at 37°C in a shaking water bath for different lengths of times, and calcium accumulation was terminated by the addition of 4 ml of an ice-cold wash solution containing 250 mM sucrose, 5 mM Hepes (pH
7.i). The membranes were collected by vacuum filtratiop on Gclman Metricel GN-6 filters with a pore size of 0.45 txm and washed three times with 4 ml of the same wash solution. The filt~:s were dissolved in scintillation fluid (3a70b, Research Products International, Mount Prospect, ILl and the 45Ca accumulated by the inside-out plasma membrane vesicles was determined by scintillation spectrophotometry. The ATP-dependent uptake was corrected for any passive accumulation of calcium by measuring the uptake without ATP and creatine kinase in identical aliquots of plasma membranes incubated concurrently. Finally, to measure Na+-dependent Ca z+ uptake, vesicles were first loaded with Na ÷ according to the expc.rimental procedure of Slaughter et al. [28] to establish a Na + gradient, then the vc~ieles were resuspended into buffered media containing 160 mM KCI and 60 ttM free calcium. Statistics. StatisticaEy significant differences between groups were determined using either Student's t-test or F statistics where necessary. Results
Effect of extracelhdar Na + on cytosolic free Ca" + and Ca: + efflttr in freshly isolated hepatocytes perfused with KHB, Ca~ + was found to be 148 _+ 12 nM (n = 8) and the intracellular Na + concentration cNa~) was 15.9 +_2.4 mM (n = 14). These values agree with those published previously for both Ca~ + [2,3] and Na~ [29]. The effect of lowering the extracellular Na + concentration (Nao+) on Ca~ + of peffused bepatocytes is shown in Fig. 1, which is a tracing obtained with cells from a sham operated animal. When the Na + concentration of the perfusate was decreased, Cai2+ increased. The peak rise in Ca~ + or dCa~ + was directly related to the decrease in Nao+. After the peak rise, Ca z+l slowly declined towards its resting control level. When Na + was returned to 144 mM by perfusing the cells with
is0
I~w
0
Fig. I. Effect of decreasingthe extracellular Na+ concentration on Ca?÷ of perfusedisolated rat hepatocytes.Ca~÷ was measuredwith aequorin. This representativetracingwas taken frc.n the lightsignal obtained fromcellsisolatedfroma sham-operatedanimal.TMA was used as substitute for Na+ and. below24 mM Nao, choline bicarbonate was substituted for NaHCO3. The 10 rain periodsof perfusion with low Nao+ are indicted by the hor~ntal lines below the tracingabovethe respectiveNao+ concentrations.
150[ ~ sham
140
~
o~
.so •lO0
70
40
24
•
100
0
l..................... is0 •
o0
30
12
40
eight sepav!lteexperiments.
KHB, there was a fall in Ca~ + below baseline that was proportional to the previously imposed decrease in Na +. Fig. 2 shows ACa,2.+ , i.e., the peak Ca~ + rise evoked by various decreases in Na + and the maximal fall in Ca 2+ resulting from. the subsequent return to normal KHB in hepatocytes isolated from adx and sham operated animals. There was no significant difference between the two groups. In both groups, the rise in Ca,?+ in low Na,+, and its fall below baseline at recovery in KHB, were proportional to the decrease in Na +. W e cannot deduce from these experiments whether the initial rise in Ca 2+ was caused by a decreased Ca ~+ efflux or by an increased Ca 2+ influx. To discriminate between these two possibilities, the changes in calcium efflux and in Ca~ + were determined in the same hepatocyte preparations. The cells were perfused for 10 min with 24 mM Na + (from rain 40 to 50). Fig. 3 shows the relation between the change in Ca2+i or ACa,2.+ and Ca 2+ e f f l u x in h e p a t o c y t e s from sham operated rats. Ca~ + rose 100 nM ( P < 0 . 0 0 1 ) during the low Na + perfusion, while calcium efflux increased slightly. The 10% increase in Ca 2+ efflux during the first min of low Na + perfusion (min 41) was statistically significant ( P < 0.05), as was the transient depression observed at min 50. Between these two time points, Ca 2+ efflux was not significantly different from control. After min 50, when the cells were perfused with normal KHB (Na + = 144 mM), Ca~ + dropped below baseline as in Figs. 1 and 2 ( P < 0.001), and Ca 2+ efflux rose 35% ( P < 0.01). Similar results were obtained when Nao+ was reduced to 0 mM (results not shown). This indicates that the sudden rise in Ca 2+ i that reached its maximum in less than 5 min cannot be caused by a decreased transport of calcium out of the ceils, since, if anything, calcium efflux increased slightly. It must he caused instead by an increased calcium influx. And indeed, we measured an increased calcium
50
s0
70
so
Time (mint
Extracetlular Na + (raM) Fig. 2. Effect of progressive decreases in Na~ on ,dCa~+. the evoked peak rise in Ca~+, and the depression in Ca~ 4 that follows the return 1o 144 mM Na~. The cells isolated from sham (hatched bars) and adx (shaded bars) animals were prepared and perfused as described in Fig. I. The data represent the mean_+S.E, of three to
~oo
Fig. 3. Effect of lowering intracellular Na + to 24 mM on Ca~ + and
on calcium efflux from isolated hepatocytes. After a control period of 40 rain, the cells were perfused from rain 40 to 50 with 24 mM Na~ media followedwith KHB (Na~ = 144) from rain 50 to 70. The changes in Ca~+ (ACa~ +, closed circles) relate to the right ordinate; the changes in calcium eft'lax (open circles) relate to the left ordinate. The changes in calcium effiux are expressed as% of a control group peffused concurrently and continuously with KHB. The data
represent the mean±S.E, of 6-8 experiments. * difference statistically significant( P < 0.01).
influx, 12 s after changing the m e d i u m from control KHB to 0 mM Na + . When the results are expressed as the ratio of calcium influx in 0 Na + over that in control KHB, we found a ratio of 1.75 + 0.21 in shams and 1.24 + 0.07 in cells from adx rats. Since in control conditions, calcium influx is 30% faster in adx than in shams, or 1.98 + 0.09 vs. 1.52 + 0.07 n m o l / m i n per mg protein [3], there was no significant difference between the two groups in the increased calcium influx measured when Na + was decreased from 144 to 0 mM: 2.66 vs. 2.46 n m o l / m i n per mg in sham and adx group,
TABLE I Effect of low extracellular Na + and of Ca z +-free medium on Ca" + influx i n isolated rat hepatocytes
Values are mean ± S.E. of the number of experiments shown in parenthesis. Ca"* influx (nmol/min per m s protein) sham
adx
1.52±0.07
1.98 ± 0.09 b
(8)
(8)
N a ~ = flm M
2.66±0.34 c (5)
2.46±0.18 d (5)
Control
1.21±0.06 (4) 4,1 :i:0,66 ~ (4)
N a ~ = 144 m M "
Ca~)+ Recovery in 1,3 mM Ca~)+ after 10 min in 0 Ca~)+
before 0
h c d c
From Borle and Studer [3]. p < 0.01 compared to sham. p < 0.05 compared to sham, 144 m M Na~. p < 0.01 compared to adx, 144 m M Na~. p < 0.01 compared to control before 0 Ca(2)+ .
TABLE II
15o
B
Effect~ ~,( 0 Ca~' aml u[ O Ca o ~ and 0 Na,; on hepatvo'tes O'toso/ic fre calcium
Values art: mean_+S.E, of the number of experiments shown in parenthesis; n.s.. not significant. Ca," Change nM 148.+ 12 (8) - Ifi6+_15 - 72~(4) +216_+28 +514%
Control Ca~" (nM) .ACa~' in 0 C'a~( Fig. 4. Effect of Ca2 ~-frec and Na ~-frec media on Ca~ ~ in hcpatocytcs isolated frova sham operated rats. Ca~* was vacasured with aequorin in ceils perfused with KHB as described in Fig. I and in Methods. This tracing, representative of fimr to six separate cxpcrivaents, shows the effects of perfusing the cells with I) Ca~," for 5 vain and with 0 Ca~,+ +0 Na,,~ for 5 vain. immediately followed with I) Na~ + 1.3 yaM Ca~,÷. The experimental periods of perfusiop with Ca2+-free and Na ~-frc¢ media are indicated by the horizontal lines below the tracing above the value for medium compositi~va.
respectively (Table i). O n the o t h e r h a n d , the close t e m p o r a l relation b e t w e e n the r a p i d rise in calcium efflux a n d the fast d r o p in Ca~ + , w h e n Na~ is r e s t o r e d to 144 m M in the s h a m g r o u p (Fig. 3), suggests that d u r i n g recovery the fall in C a 2 + is c a u s e d by the rise .in calcium efflux. E f f e c t s o f C a " +-free m e d i a o n Ca~ +
W e recently d e m o n s t r a t e d t h a t the f o r w a r d m o d e o f N a + - C a 2+ e x c h a n g e c o u l d be activated by perfusing k i d n e y cells with Ca2+-free m e d i a [24]. T o f u r t h e r investigate the p r e s e n c e of N a + - C a z+ e x c h a n g e in liver, we studied the effects o f Ca2+-frec a n d o f C a +- a n d N a + - f r e e m e d i a o n Ce 2+ in isolated hepatocytes. Simply omitting CaCI 2 f r o m K H B usually lowers the m e d i u m free c a l c i u m (Ca2,+) to 1 0 - * - 1 0 -5 M [24]. T o achieve a virtually CaZ+-free milieu a n d to t r a p the significant a m o u n t o f C a 2+ b o u n d to the surface o f isolated cells o r a n y C a 2+ t r a n s p o r t e d o u t o f the cells, 0.1 m M E G T A w a s a d d e d to all Ca2+-free p e r f u s a t e s d u r i n g the e x p e r i m e n t a l period. Fig. 4 is representative o f f o u r e x p e r i m e n t s in 0 C a 2+ a n d six e x p e r i m e n t s in b o t h 0 C a 2+ a n d 0 N a +, w h o s e results a r e s h o w n in T a b l e I1. Fig. 4 shows t h a t p e r f u s i n g rat h e p a t o c y t e s with a CaZ+-free m e d i u m , immediately lowered Ca.2.+ to less t h a n 3 0 % o f its resting value (Table 11). A t the s a m e time, Fig. 5 shows t h a t C a 2+ efflux increased significantly ( P < 0.01) d u r i n g the few m i n u t e s following the removal o f extracellular calcium. T h i s strongly s u p p o r t s the idea t h a t the i n c r e a s e d C a 2+ efflux w a s responsible for *~he fail in C a 2+. Indeed, it is h a r d to imagine how C a 2+ efflux o n the C a 2+ p u m p could increase w h e n C a 2÷ d e c r e a s e s m o r e t h a n 70%. W h e n Ca2o+ w a s r e s t o r e d to 1.3 raM, C a 2+ increased m o r e t h a n 5 0 0 % , significantly a b o v e the basal levels, t h e n r e t u r n e d t o control values (Fig. 4 a n d T a b l e lit. T h e
ACa~" at recovery
P value
< 0.IM)I < I).iM)I
(4)
ACa~ ~ in o Ca~," and 0 Na,; ACa~ ' at recovery in 0 Na,; anti 1.3 mM Ca~~
n.s. +91 .+ 17 (6)
n.s. + 78~-
< 0.(iOI
rise in Ca~ ÷ w h e n C a -`+ was r e i n t r o d u c e d into the perfusate was c a u s e d by a n increased C a 2+ i n f u x . Indeed, 30 s a f t e r a d d i n g control K H B to cells incub a t e d in Ca2÷-free m e d i u m for 10 rain, C a 2+ influx increased 3.4-fold, from a control value o f 1.21 + 0.06 to 4.1 + 0 . 6 6 n m o l / m i n p e r m g protein ( P < 0 . 0 1 ) as ~hown in T a b l e 1. W h e n N a + was omitted from the Ca2+-fre¢ p e r f u s a t e (Na+-free a n d Ca2~-free K H B ) , the fall in Ca~ + was abolished (Table !1 a n d s e c o n d stimulation o f Fig. 4). T h i s strongly suggests that extracellular Na + is necessary for the fall in Ca~ +. T h e large rise in C a , + w h e n C a 2+ is r e i n t r o d u c e d in the Na+-free p e r f u s a t e (second stimulation of Fig. 4) is c o m p a r a b l e to t h a t shown in Fig. 1 a f t e r the shift to N a * - f r e e media. In t h a t instance, C a 2+ influx was increased 33%. T h e s e results support the idea that hepatocytes possess a functional N a + - C a 2+ a n t i p o r t e r in t h e i r p l a s m a m e m b r a n e capable o f o p e r a t i n g in both forward a n d 1.2
gl
•
-
•
t t
°~ v Time (min)
Fig. 5. Effect of a Ca2+-free medium on calcium efflux in rat hepatocytes. From min 40 to 50, the ceils were perfused with a Ca2~-free KHB containing 0.1 yaM EGTA. Calcium emux is expressed as the ratio of the Ca-'+ efflux of the group pcrfused with 0 Ca~,+ to the Ca2+ effiux of controls peffused concurrently with KHB. The data represent the mean+S.E, of ten separate experiments.
12
reverse modes. Although no significant differences were observed between sham and adx animals when the hepatocytes were pcrfused with 0 Na + or with 0 Ca2o+, the possibility remains that adrcnalectomy may alter the properties of the antiporter in more physiological conditions [1-3]. Thus, we evaluated calcium transport in inside-out plasma membrane vesicles isolated from the liver of sham and adx rats. We verified the enrichment of these vesicle preparations by measuring the plasma membrane enzyme marker 5'-nucleotidase, and the degree of contamination from endoplasmic reticulure by measuring the activity of glucose-6-phosphatase. From homogenate to plasma membrane, *.he vesicles displayed a 20-fold enrichment in 5'-nucleotidase from 32 + 3 to 658 + 30 mg P~/mg protein per 20 rain and a 50% reduction in glucose-6-phosphatas¢ from 61 + 8 to 32 + 4 mg P~/mg protein per 20 rain. There was no significant difference between sham and adx rats.
Effect of adrenalectomy on ATP-dependent Ca 2+ uptake and Ca z+ efflux in inside-out plasma membrane vesicles ATP-dependent Ca 2+ uptake was measured in inside-out plasma membrane vesicles isolated from the livers of sham and adx rats in the presence of 25 mM Na+. First, to assess the passive Ca 2+ binding to the vesicles, we measured the ATP-independent Ca 2+ uptake. There was no difference between sham and adx: 1.87 + 0.14 vs. 1.76 + 0.14 n m o l / m g protein per 30 rain, respectively. On the other hand, there was a statistically significant difference between the two groups in the ATP-dependent Ca 2. uptake. Fig. 6 shows that, in the presence o f A T P , the vesicles iso-
30
:
. . sham
~
.
+
.
.
•
E"6
0
o!•
o.~"
0
1'0
'
2'0
'
30
Time (min)
Fig. 6, Effect of adrcn deetomyon ATP-depend=.nt Ca2+ accumulation by plasma membrane inside-out vesicles isolated from rat liver. Plasma membranes were prepared from sham (e) and adx (o) rat livers as described in Materials and Methods and incubated for various times at 37¢C in media containing 8.5 p.M ionized Ca=* and 25 mM Na +. Ca2+ uptake is corrected for ATP-independent binding and uptake. The data represent the mean~S.E, of 5-15 separate experiments. * P < 0.05 when compared to sham group.
~E
~
-=: :~.°o ~tlc 4
8 10
CaZ+ (~tM) Fig. 7. Initial rate of ATP-depeodent Ca2. uptake as a function of the medium free Caz+ concentration by inside-out plasma membrane vesicles isolated from the liver of sham (e) and adx (o) rats. The incubation medium contained 0.5 mM EGTA and Caz+ concentrations ranging from 0.1 to 0.525 raM. 4SCauptake was measured at
37°C for exactly 60 s. The inset showsan Hofstee plot of the data. The equations for the line fitting the data are: sham, y = 4.2-0.29x (%R2 = 0.g7): adx, y = 4.7-0.32x (R2 = 0.83). The data represent the mean+ S.E. of g-12 separate experiments.
lated from adx animals ac:umulated or retained 64% less calcium than the shams. When the ionophore A23187 (10 -H M) was added after 25 rain of incubation, the 4SCa accumulated immediately decreased to 1.8 nmol/mg protein, the levels measured in vesicles incubated without ATP, indicating that the 4SCa taken up was transported inside the vesicles (data not shown). The difference between the two groups in the calcium accumulated in the presence of ATP was statistically significant (P < 0.05). The decreased uptake of calcium in vesicles from adx rats could be caused by a decreased affinity a n d / o r a lesser capacity of the ATPdependent calcium pump or to an increased efflux of calcium from the vesicles. These possibilities were investigated. First, the initial rate of calcium uptake was determined in vesicles which had been preincubated at 37°C then exposed for 1 rain to media of various Ca 2÷ concentrations containing 45Ca. 45Ca uptake was linear between 12 and 60 s in both sham and adx groups (data not shown). Fig. 7 shows tile initial rate of Ca 2+ uptake by inside-out plasma membrane vesicles incubated in media with Ca 2+ concentrations ranging from 0.14 to 8.5/~M. The inset of Fig. 7 shows an Hofstee plot of the data. The Ca ~'+ affinity (K m) was 3.0 + 0.4.10 -7 and 3.2 + 0.4.10 -7 M in sham and adx, respectively, and the maximum rate of ATP-dependent Ca 2+ accumulation (Vm~) by vesicles from sham and adx rats was 4.17 =l=0.15 and 4.36 ± 0.31 nmoi/min per mg protein, respectively. The lack of significant difference in K m and Vmx between the two groups suggests that the decreased Ca 2+ uptake by vesicles from adx rats was not caused by a decreased affinity or a lesser capacity of the ATP-dependent Ca z+ pump.
In another series of experiments, the passive efflux of Ca ~ ~ from plasma membrane vesicles was initiated by adding 50 u n i t s / m l hexokinase and l0 mM glucose to vesicles loaded for 25 rain in the presence of ATP and 8.5 /LM Ca 2~. This caused a rapid disappearance of ATP and blocked any further active accumulation of calcium. As a result, calcium leaked out of the vesicles and their calcium content decreased. Fig. 8 shows that the calcium retained in the vesicles decreased with time. when expressed in absolute terms or as the percent of the calcium accumulated after 30 rain of uptake (inset of Fig. 8). The loss of calcium from vesicles isolated from adx animals was significantly greater ( P < 0.05) during the first 5 rain that followed the inhibition of the active uptake (inset of Fig. 8). This suggests that the difference in steady state calcium accumulation between the two groups shown in Fig. 6, was due to a greater leak of calcium from the vesicles isolated from adx rats. To determine whether calcium efflux from inside-out plasma membrane vesicles was d e p e n d e n t on the Na + concentration of the incubating medium, ATP-dependent calcium uptake by the vesicles was measured in media of increasing Na* concentrations (5-55 raM). All media contained 5 mM NaN3 and 8.5 p.M Ca 2+, with the sum of the Na + and K + concentrations kept constant to maintain the same osmolarity. Fig. 9 shows the influence of the m e d i u m Na + concentration on the A T P - d e p e n d e n t calcium uptake expressed both in absolute terms and as a percent of the amount accumulated in 5 m M Na + medium. At the lowest Na + concentration (5 mM), there was no leak of Ca :+ from 20 • sham
~100 .
E~~ .~_
~
o
1o
20
3o
Time (rmn~
'o" ) o
o o
o
10 Time (rain) ]Fig. 8. Effect of adrenalnctomy on ~Ca efflux from plasma membrane vesicles isolated from the liver of sham (e) and adx (o) rats. Membrane vesiclns were first incubated with ~SCa for 25 min as described in Fig. 6. Alter 25 min, hesokinase (50 U / m l ) and glucose (tO raM) were added to deplete th~ preparation of AI"P and to block
ATP-dependent calcium uptake (time 0). The 45Ca retained in the vesicles was then determined from time 0 (addition of hexokinaso+ glucose) to min 30. In the inset, the same data are expressed as the% of the calcium accumulated after 25 min of uptake. The data represent the mean+S.E, of 5-13 separate experiments. * P < 0.05 for the difference between sham and adx groups.
30
t
. ,'0
;o
3'o
2o
~'o
~0
Sodium (mM) Fig. 9. Effect of adrenalectomy on the 4SCa accumulated and re*ained by liver plasma membrane inside-out vesich~sas a function of the ¢xtravesicular Na ~ concentration. The vesich~s isolated from sham and adx rat liver were incubated for 25 min in media containing 5 mM NaN:~and various additions of Na ÷ (0-50 raM). Na * was substituted for K" to maintain the .sameosmolarity.~Ca uptake was performed as described in Fig. 6. In the inset, the same data are expressed as the percent of the accumulation measured in the control medium that contained 5 mM NAN,. The data represent the mean_+S.E, of 0-12 separate experiments. * P
