IONIC CONTROL OF 1,25-DIHYDROXY VITAMIN D-3 PRODUCTION BY ISOLATED CHICK RENAL MITOCHONDRIA
INFLUENCE OF ANIONS AND SUCROSE
DANIEL D. BIKLE a and HOWARD RASMUSSEN b
a The Department of Medicine, Letterman Army Institute o f Research, Presidio of San Francisco, Calif. 94129 and b Department of Medicine, Yale University, New Haven, Conn. 06510 (U.S.A.)
(Received May 10th, 1977)
Summary
The influence of three anions, chloride, acetate, and phosphate, on the activ- ity of the 25-hydroxy vitamin D-3 la -hydroxylase in chick renal mitochondria was studied in the presence and absence of calcium at different sucrose concen- trations.
1. An increase in the concentra t ion of either acetate or phosphate, but not chloride, stimulates l~-hydroxylase activity in the absence of calcium when the anion concentra t ion is raised above 10 mM.
2. Concentrat ions of calcium less than 10-6M stimulate la -hydroxylase activity more effectively in the presence of acetate and phosphate than in the presence of chloride.
3. Concentrat ions of calcium greater than 10-6M inhibit l a -hydroxylase activity. This effect is potent ia ted by concentrat ions of phosphate greater than 10 mM. This inhibition of l a -hydroxylase activity by calcium parallels an inhibition of malate oxidation to CO2.
4. Phosphate modest ly increases malate oxidation to CO2 in the absence of calcium at concentrat ions between 10 to 40 mM.
T h e o p i n i o n s o r a s s e r t i o n s c o n t a i n e d h e r e i n a re t h e p r i v a t e v i e w s o f t h e a u t h o r s a n d a re n o t t o b e c o n s t r u e d as o f f i c i a l o r as r e f l e c t i n g t h e v i e w s o f t h e D e p a r t m e n t o f t h e A r m y o r t h e D e p a r t m e n t o f D e f e n s e .
I n conduct ing the r e s e a r c h d e s c r i b e d in t h i s r e p o r t , t h e i n v e s t i g a t o r s a d h e r e d t o t h e G u i d e f o r L a b o r a t o r y A n i m a l F a c i l i t i e s a n d C a r e , as p r o m u l g a t e d b y t h e C o m m i t t e e o n t h e G u i d e f o r L a b o r a -
t o r y A n i m a l R e s o u r c e s , F a c i l i t i e s a n d C a r e , o f t h e I n s t i t u t e o f L a b o r a t o r y A n i m a l R e s o u r c e s , N a t i o n a l A c a d e m y o f S c i e n c e s - N a t i o n a l R e s e a r c h C o u n c i l . A b b r e v i a t i o n s : 2 5 - O H D - 3 , 2 5 - h y d r o x y v i t a m i n D - 3 ; 1 , 2 5 - ( O H ) 2 D - 3 , 1 , 2 5 - d i h y d r o x y v i t a m i n D - 3 .
128
5. Mersalyl blocks malate supported la-hydroxylase activity, an effect which is reversed by phosphate.
6. Pyruvate supports la-hydroxylase activity, and the stimulation of la-hy- droxylase activity by phosphate in the presence of pyruvate is not modified by mersalyl.
7. Increasing the sucrose concentration from 90 to 200 or 300 mM shifts the optimal calcium concentration for la-hydroxylase activity from 10 -6 to 10 -S M, reduces maximal stimulation by calcium, and blunts the inhibition of la-hy- droxylase activity by supraoptimal calcium concentrations. This pattern is seen regardless of the anion present.
8. High concentrations of sucrose reduce the stimulation of la-hydroxylase activity by acetate and phosphate in the absence of calcium.
9. High concentrations of sucrose reduce the accumulation of calcium by mitochondria unlike comparable increases in tonicity with sodium chloride which have no effect on calcium accumulation.
10. Malate oxidation to COs is modestly reduced by sucrose, but by no greater an amount than comparable increases in tonicity with sodium chloride.
We postulate that phosphate and, perhaps, acetate regulate la-hydroxylase activity by influencing energy substrate and cation entry into mitochondria, although an additional effect on some other parameter like intramitochondrial pH also seems likely. Sucrose alters the ionic control of la-hydroxylase activity by restricting ion flux across the mitochondrial membrane via a mechanism independent of its effects on tonicity of the incubation medium.
Introduction
Parathyroid hormone and calcitonin modulate the production of 1,25-dihy- droxy vitamin D-3 (1,25-(OH)2D-3) by the kidney both in vivo and in vitro [1-- 6]. The enzyme, 25-OHD-3 l~-hydroxylase, responsible for 1,25-(OH)2D-3 pro- duction, is located in kidney mitochondria [7]. These hormones probably exert their effects in part, by altering the ion content of the kidney cell cytosol [8--10]. Several groups have reported that changes in the concentrations of cal- cium and phosphate influence 1,25-(OH)zD-3 production in isolated renal cells and mitochondria [2,8,11--19]. Our previously reported data indicate that physiologic concentrations of calcium and phosphate can stimulate 1,25- (OH)zD-3 production by isolated renal tubules [17] and mitochondria [18]. An unresolved question is whether such ions exert a direct effect on the l~- hydroxylase or an indirect effect through some other parameter of mitochon- drial function such as shape change, substrate entry, or proton efflux. An argument supporting the latter possibility is that potassium can stimulate 1,25- (OH)2D-3 production by isolated renal mitochondria as effectively as calcium [18,20]. Evidence favoring an action of phosphate on la-hydroxylase activity mediated by enhanced cation and energy substrate entry is presented in this report. The possibility that sucrose modifies the la-hydroxylase activity of iso- lated mitochondria [21] by osmotically induced shape change was explored, but our results indicate that the effect of sucrose cannot be duplicated by changes in the osmolarity of the medium per se.
129
Methods
Animals. White Leghorn cockerels were raised on a vitamin D deficient diet containing 0.43% calcium and 0.3% P as inorganic phosphate for 21 to 25 days [17].
Materials. 25-[26,27-SH]OHD-3, adjusted to a specific activity of 1 Ci/mmol, was obtained from Amersham Searle Corp., (Arlington Hts., Ill). Whatman SG81 chromatography paper was obtained from H. Reeve Angel Co. Inc., (Clif- ton, N.J.). Nonradioactive 25-OHD-3 was a gift from Dr. John Babcock, Up- john Pharmaceuticals.
Mitochondrial preparation and incubation were performed as previously described [18] except that sucrose and anion concentrations were varied as indicated in the figure legends. The typical incubation mixture of 1 ml con- tained 5 to 6 mg mitochondrial protein, 90 to 300 mM sucrose, 10 mM sodium malate, 2.0 mM magnesium sulfate, 10 mM EGTA with sufficient calcium chloride to give the desired free calcium concentration [22] and sodium chlo- ride to maintain a constant chloride concentration (22 mM) for each of the Ca-EGTA buffers, and 0.01 M Tris balanced by 0.01 M of the appropriate acid. Anion concentrations above 0.01 M were achieved with the addition of the respective sodium salts. The pH of the complete incubation medium was adjusted at 37°C to 6.8 with NaOH prior to the addition of the mitochondria. The mitochondria were preincubated for 10 min then incubated in the presence of 25 pmol of 25-OHD-3 for an additional 20 min at 37°C under an atmosphere of 02. The reaction was terminated by the addition of CHC1JMeOH (1 : 2, v/v). The amount of 1,25-(OH)2D-3 produced was determined as previously described [23]. For each experiment a single pooled mitochondrial preparation was used so that only during the incubation were mitochondria exposed to dif- ferent environments. Duplicate determinations revealed a variation in la-hy- droxylase activity consistently less than 10% and generally less than 5% for each point in each experiment reported.
Malate oxidation to CO2 was determined under conditions essentially iden- tical to those used for the 25-OHD-3 la-hydroxylase assay. No preincubation was employed, and the total incubation time was 30 min. Sodium [U-14C] - malate (0.5 Ci/10 mol sodium malate/ml incubation media) was included, and the ~4CO2 produced was detected by standard procedures [24]. For each expe- riment a single pooled mitochondrial preparation was used so that only during the incubation were mitochondria exposed to different environments.
Calcium uptake was performed essentially under the same conditions as used for the 25-OHD-3 l~-hydroxylase assay except that 0.5--0.6 mg mitochondrial protein/ml was employed. No preincubation was included, and the total incu- bation time was 30 min. Tracer amounts of 4SCa (0.5 pCi/2.5 • 10 -9 mol/ml) and [3H]sucrose (1.0 g C i / 5 - 1 0 -~° mol/ml) were added to the incubation media. After 30 min of incubation, 100-pl aliquots were filtered through Milli- pore filters. The radioactivity of the sample trapped by the filters was deter- mined by liquid scintillation techniques, and the amount of calcium incorpo- rated into the space impermeant to sucrose was calculated.
130
R e s u l t s
The effects of changes in the concentrat ion of chloride, acetate, and phos- phate on 1,25-(OH)2D-3 product ion by isolated chick renal mitochondria are shown in Fig. 1. Increasing the chloride concentra t ion had little effect on la- hydroxylase activity. Increasing the concentrat ion of either acetate or phos- phate from 10 to 110 mM s t imu la t ed l a -hyd roxy la se activity 2--3-fold in the absence of calcium. Little stimulation was seen in other experiments at concen- trations of these anions less than 10 mM in the absence of calcium or potas- sium. Raising the [Ca > ] from less than 10 -s M to 10 -~ M caused a small stimu- lation in the presence of either acetate or phosphate but no stimulation in the chloride media. A rise in [Ca 2÷] from 10 -~ M to 10 -6 M caused a marked stimu- lation in all anionic media. When the concentra t ion of [Ca > ] was raised above 10 -6 M, an inhibition of enzyme activity was seen regardless of the anionic composi t ion of the medium. The absolute amount of la -hydroxylase activity obtained at the optimal calcium concentra t ion (10 -6 M) varied among mito- chondrial preparations and was not correlated to the anion present.
Malate was employed as the energy substrate in the preceding studies. To determine whether the ion effects observed could be explained by alterations in malate utilization, the rate of malate oxidation was measured (Table I). Phosphate concentrat ions from 10 to 40 mM stimulated oxidation of malate
F i g . 1. T h e e f f e c t s o f t h r e e d i f f e r e n t a n i o n s o n l c ~ - h y d r o x y l a s e a c t i v i t y in m i t o c h o n d r i a i n c u b a t e d in t h e p r e s e n c e o f d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s . T h e t h r e e d i f f e r e n t m i t o c h o n d r i a l s u s p e n s i o n s u s e d w e r e
p r e p a r e d in t h e a p p r o p r i a t e 0 . 0 1 M T r i s a n i o n b u f f e r a n d i n c u b a t e d in a 9 0 m M s u c r o s e / 0 . 0 1 M T r i s a n i o n
b u f f e r to w h i c h s o d i u m sa l t s o f t h e r e s p e c t i v e a n i o n s w e r e a d d e d . T h e e n e r g y s u b s t r a t e w a s s o d i u m
m a l a t e , 1 0 m M . T h e p H w a s 6 . 8 . T h e c a l c i u m c o n c e n t r a t i o n s w e r e c o n t r o l l e d w i t h C a - E G T A b u f f e r s .
1 3 1
by the mitochondria in the absence of calcium when compared to comparable chloride concentrations, although both anions are inhibitory at 110 mM. Rais- ing the calcium concentration from 0 to 10 -6 M had little consistent effect on malate oxidation in the absence of phosphate, but 10 -6 M calcium markedly inhibited malate oxidation at 110 mM phosphate. At 10 -3 M calcium an inhibi- tion of malate oxidation occurred regardless of anion species employed.
The next question to be examined was whether or not phosphate could alter l~-hydroxylase activity when energy substrates other than dicarboxylic acids were employed. Since high concentrations of phosphate (110 mM) inhibited malate oxidation (Table I), 10 mM potassium was added to the incubation medium so that in the absence of calcium the effect of much lower concentra- tions of phosphate (0.1 to 3.0 mM) could be evaluated (Fig. 2). Malate, 10 mM, did not support as much 1,25-(OH)2D-3 production at 0 to 0.1 mM phosphate as did 10 mM pyruvate, although this difference was not seen at 3 mM phos- phate. On the other hand, TMPD-ascorbate supported essentially no 1,25- (OH)2D-3 production at any phosphate concentration (data not shown). Essen- tially no difference in 1,25-(OH)2D-3 production was noted between the use of succinate and malate at any phosphate concentration (0 to 10 mM) (data not shown). Meralyl (10 -4 M), an inhibitor of the dicarboxylic acid shuttle, inhib- ited 1,25-(OH)~D-3 production when malate was the energy substrate in the absence of phosphate. This inhibition was reversed by 3 mM phosphate. Cal- cium did not reverse the mersalyl inhibition (data not shown). Mersalyl did not alter the ability of pyruvate to support 1,25-(OH)2D-3 production. Instead, in the presence of mersalyl, pyruvate supported l~-hydroxylase activity was modestly greater than in the absence of mersalyl at all phosphate concentra- tions (Fig. 2).
In an experiment performed without EGTA or calcium in the incubation medium, a marked inhibition of la-hydroxylase activity by sucrose was noted when the concentration of sucrose was increased from 100 to 200 or 300 mM in the presence of either chloride or acetate (Fig. 3). The amount of l~-hy- droxylase activity observed in this experiment at 100 mM sucrose (with no added calcium or potassium) equaled the amount of la-hydroxylase activity observed in the presence of optimal calcium concentrations (10 -6 M) controlled by Ca • EGTA buffers in other experiments. Therefore, we investigated the pos-
T A B L E I
OXIDATION OF MALATE TO CO 2 BY MITOCHONDRIA INCUBATED IN THE PRESENCE OF DIF-
FERENT CONCENTRATIONS OF CALCIUM, CHLORIDE, OR PHOSPHATE
Each value is the mean of duplicate determinations. Units are nmol malate oxidized to CO 2/rag mito-
chondrial protein. The range of variability of the duplicates did not exceed 3 nmol malate oxidized/rag
Fig , 2. T h e e f f e c t o f p h o s p h a t e c o n c e n t r a t i o n o n l ~ - h y d r o x y l a s e a c t i v i t y i n m i t o c h o n d r i a i n c u b a t e d w i t h
10 m M m a i a t e o r 10 m M s o d i u m p y r u v a t e as e n e r g y s u b s t r a t e . Al l i n c u b a t i o n m e d i a c o n t a i n e d 10 m M
p o t a s s i u m , a d d e d as KC1 + K H 2 P O 4. T h e m i t o c h o n d r i a were p r e p a r e d in 0 . 0 2 M Tr i s • C1 b u f f e r s and
i n c u b a t e d in t he p r e s e n c e o f 1 5 0 m M s uc ros e in a 0 . 0 1 6 M Tr i s • Cl b u f f e r . Mersa ly l , 10 -4 M, was a d d e d
t o t h e i n d i c a t e d s a m p l e s p r i o r t o t h e i n c u b a t i o n .
1 4
12
~ 10
0
z
~ 6 0
x g 4
(~k A = O.O1 M OAc
~ i A B = 0.01 M C1 12
t~ 10
z ~ 8
x £
~ 4 o ~ o
o 0 v_ a.
2q x
Oot, o12 o125 o'3 [~u¢~o~]~
A ~ 90 mM SUCROSE i / ~ [3-'0 300 mM SUCROSE
:I..\~m-i- /
7 6 5 4 3
pCa Fig . 3. T h e e f f e c t o f s u c r o s e o n l c ~ - h y d r o x y l a s e a c t i v i t y i n m i t o c h o n d r i a p r e p a r e d in e i t h e r Tr is c h l o r i d e ( C l - ) o r Tr is a c e t a t e ( O A c ) a n d i n c u b a t e d in t h e a b s e n c e o f E G T A or a d d e d c a l c i u m .
F ig . 4. T h e e f f e c t o f s u c r o s e c o n c e n t r a t i o n o n t he r e s p o n s e o f l ~ - h y d r o x y l a s e a c t i v i t y t o v a r i o u s c a l c i u m
c o n c e n t r a t i o n s . T h e s a m e m i t o c h o n d r i a l p r e p a r a t i o n ( in Tr i s • C1) was u s e d fo r al l p o i n t s w i t h e x p o s u r e t o d i f f e r e n c e s in [ s u c r o s e ] o r [Ca 2+] o n l y d u r i n g t h e i n c u b a t i o n a n d p r e i n c u b a t i o n p e r i o d s .
1 3 3
sibility that sucrose modifies the effect of different calcium concentrat ions on la -hydroxylase activity. It was of special interest to determine whether or no t the data shown in Fig. 3 could be explained by postulating that at reduced sucrose concentrat ions, sufficient endogenous mitochondrial calcium is present in the absence of EGTA to stimulate maximally l a -hydroxylase activity.
The effect of different calcium concentrat ions on 25-OHD-3 la -hydroxylase activity in mitochondria incubated at 90 mM sucrose compared to 300 mM sucrose in the absence of st imulatory anions is shown in Fig. 4. Sucrose reduced the sensitivity of the la -hydroxylase to calcium in three ways, by (1) shifting the optimal calcium concentrat ion for la -hydroxylase activity from 10 -6 M to 10 -s M, (2) reducing the amount of stimulation by optimal calcium concentra t ion from an increment of 95 • 10 -15 mol 1,25-(OH):D-3 produced/ min per mg protein to 3 2 . 1 0 -is mol /min per mg protein and (3) decreasing the amount of inhibition by 10 -3 M calcium from a decrement of 100 • 10 -is mol 1,25-(OH)2D-3 produced/min per mg protein to 15 • 10 -Is mol/min per mg pro- tein. The amount of l~-hydroxylase activity in the absence of calcium (10 mM EGTA) was also modest ly reduced by sucrose. Similar results were obtained with 200 mM sucrose.
If sodium chloride instead of sucrose was used to increase the tonici ty of the incubation medium, little change in the sensitivity of the la -hydroxylase to calcium could be demonstrated (Fig. 1). Acetate and phosphate, which might be expected to reduce the osmotic effect of sucrose by their ability to pene- trate the mitochondrial membrane [25], were not capable of reversing the effects of sucrose with respect to altered calcium sensitivity of the l a -hydroxy- lase (data not shown). In fact, stimulation of la -hydroxylase activity by these anions in the absence of calcium was also reduced by sucrose (Table II).
A reduct ion in ion uptake by mitochondria could explain these effects of sucrose. Fig. 5A indicates that mi tochondr ia incubated in 90 mM sucrose were capable of accumulating twice as much calcium as mi tochondr ia incubated in 200 mM sucrose. Increasing the sodium chloride concentra t ion in the incuba- t ion medium by 100 mM did not reduce the ability of the mitochondr ia to accumulate calcium (Fig. 5B). In fact, a small increase in calcium uptake at the higher chloride concentra t ion was observed in this experiment.
Reduct ion in energy substrate utilization by mitochondria in the presence of sucrose secondary to an inhibition of either t ransport or subsequent oxidat ion
T A B L E II
T H E E F F E C T O F S U C R O S E ON T H E S T I M U L A T I O N OF l a - H Y D R O X Y L A S E A C T I V I T Y BY I N C R E A S I N G C O N C E N T R A T I O N S O F A C E T A T E OR P H O S P H A T E IN T H E A B S E N C E O F C A L C I U M
Each value is the m e a n o f dup l i ca t e d e t e r m i n a t i o n s . Un i t s are f m o l 1 , 2 5 - ( O H ) 2 D 3 p r o d u c e d / r a i n pe r m g m i t o c h o n d r i a l p ro t e in . A single p o o l e d m i t o c h o n d r i a l p r e p a r a t i o n was used. Dup l i ca t e d e t e r m i n a t i o n s
were w i t h i n 5% of t he m e a n s in all cases.
l C]--r12oo mM SUCROSE 0--'0 0.062 M Cl" 600 600 ~ , m l A , 0.132 M Cl"
I . p , JA
soo soo l l ~ ' s " ~
O 400 400 / 0 1 ~ _ _
o u 3 0 0 3 0 0
0o~ 6 5 4 3 6 5 4 3
pCa pCa F i g . 5. ( A ) T h e e f f e c t o f s u c r o s e c o n c e n t r a t i o n o n c a l c i u m a c c u m u l a t i o n b y m i t o c h o n d r i a i n c u b a t e d in m e d i a c o n t a i n i n g v a r i o u s e x t r a m i t o c h o n d r i a l c a l c i u m c o n c e n t r a t i o n s . (B) T h e e f f e c t o f c h l o r i d e c o n c e n -
t r a t i o n o n c a l c i u m a c c u m u l a t i o n b y m i t o c h o n d r i a i n c u b a t e d in m e d i a c o n t a i n i n g v a r i o u s e x t r a m i t o c h o n -
d r i a l c a l c i u m c o n c e n t r a t i o n s . E a c h d a t a p o i n t is t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s w h i c h in all c a s e s
w e r e w i t h i n 1 0 % o f t h e m e a n .
A B O . ~ O 90 mM SUCROSE C 3 0 . 0 3 2 M Cl"
123- '42] 2 0 0 ,,,M SUCROSE 0--'0 0 . 0 6 2 M C," J A ~ . , A 0.132 M Cl"
100
8O
.~ 20
0 6 5 4 3 ~ 6 S 4 3
pCa pCa Fig. 6. (A) The effect of sucrose concentration on m~late oxidat ion by mitochond~a incubated in media c o n t a i n i n g d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s . ( B ) T h e e f f e c t o f c h l o r i d e c o n c e n t r a t i o n o n m a l a t e o x i d a t i o n b y m i t o c h o n d r i a i n c u b a t e d in m e d i a c o n t a i n i n g d i f f e r e n t c a l c i u m c o n c e n t r a t i o n s . E a c h d a t a p o i n t is t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s w h i c h in all e a s e s w e r e w i t h i n 2 . 5 n m o l m a l a t e o x i d i z e d p e r m g p r o t e i n o f t h e m e a n .
135
might also explain the sucrose effects. The data shown in Fig. 6A indicate that malate oxidation was reduced in the mitochondria incubated in 200 mM sucrose compared to 90 mM sucrose. This inhibition was more apparent at cal- cium concentrations less than 10 -s M. However, increasing the sodium chloride concentration in the incubation medium inhibited malate oxidation just as effectively (Fig. 6B) as sucrose. A striking inhibition of malate oxidation by calcium concentrations greater than 10 -6 M was noted in all circumstances.
Discussion
The ionic control of 25-OHD-3 la-hydroxylase has been studied in a number of laboratories [2,8,11--21]. Most at tention has been given to the role for cal- cium and phosphate in controlling the production of 1,25-(OH)2D-3 since this potent metabolite of vitamin D-3 appears to be central to the regulation of cal- cium and phosphate homeostasis [26].
However, calcium and phosphate are not the only ions capable of regulating 1,25-(OH)2D-3 production. Potassium appears to be as effective as calcium in stimulating la-hydroxylase activity in isolated mitochondria [20] especially in the presence of valinomycin, a specific potassium ionophore [27]. In this report we have shown that acetate, like phosphate, stimulates la-hydroxylase activity in isolated mitochondria.
Other laboratories have obtained results indicating that calcium [3,15,16] and phosphate [16] do not stimulate la-hydroxylase. However, these investi- gators failed to prevent the exposure of the mitochondria to extracellular cal- cium during preparation or to regulate the concentration of calcium in the incubation medium. The amount of endogenous calcium found in even care- fully prepared mitochondria [18] is sufficient to stimulate maximally or inhibit the la-hydroxylase activity unless appropriately buffered. That this occurs is indicated by our own results (Figs. 3 and 4) demonstrating that the la-hy- droxylase activity of mitochondria incubated in the absence of added calcium or EGTA is inhibited by sucrose (Fig. 3) similarly to the activity of the la - hydroxylase of mitochondria incubated in 10-6M calcium controlled by Ca- EGTA buffers (Fig. 4). Likewise, in the presence of calcium concentrations sufficient to inhibit the la-hydroxylase activity, phosphate potentiates this inhibition ([17,18] and Fig. 1). Since calcium and phosphate concentrations which inhibit la-hydroxylase activity also inhibit malate oxidation (Table I), calcium and phosphate at these supraphysiologic concentrations may act as nonspecific inhibitors of mitochondrial metabolism and not as specific regu- lators of la-hydroxylase activity. Therefore, our attention should be focused on lower and physiologic concentrations of calcium achieved with the use of Ca-EGTA buffers.
The studies included in our present report represent a continued a t tempt to determine the general mechanism(s) by which ions control 1,25-(OH)2D-3 pro- duction by the mitochondrion. Our evidence shows that changes in the concen- trations of certain anions can alter la-hydroxylase activity and that increasing sucrose concentration in the medium modifies the ionic control of this enzyme.
Several explanations for the observed effects of anions on la-hydroxylase
136
activity were considered. First, phosphate and acetate may exert their effects by modifying cation, calcium and potassium in particular, availability inside the mitochondrion. As noted in this report and elsewhere [18,19], calcium is a potent modulator of la-hydroxylase activity. The ability of phosphate and ace- tate to enhance the stimulation by low concentrations of calcium (10 -~ M) of l(~-hydroxylase activity and of phosphate to potentiate the inhibition by higher concentrations of calcium (10 -S to 10 -3 M) (Fig. 1) is consistent with an ability of phosphate and acetate to increase calcium concentration inside the mito- chondrion. However, both phosphate and acetate stimulated la-hydroxylase activity when the mitochondria were incubated in the presence of 10 mM EGTA, conditions in which calcium flux across the mitochondrial membrane should be essentially zero (Fig. 1). Therefore, this cannot be the entire explana- tion for the effects of these anions.
A second possibility is that phosphate exerts its effects by stimulating the dicarboxylic acid shuttle [25], and thereby, increasing the supply of energy substrate for the production of 1,25-(OH)2D-3. That energy is required for 1,25-(OH)2D-3 production has been demonstrated previously by noting the antagonistic effects of antimycin A and dinitrophenol, inhibitors of oxidative phosphorylation, on la-hydroxylase activity [16,18,21]. Two types of evi- dence suggest that this second possibility may be operative. First, phosphate at concentrations from 10 to 40 mM increased malate oxidation, but comparable concentrations of chloride did not (Table I). Second, mersalyl, an inhibitor of the dicarboxylic acid shuttle [28], blocked the ability of malate (a dicar- boxylic acid) but not pyruvate (a monocarboxylic acid) to support 1,25- (OH)2D-3 production (Fig. 2). This inhibition could be reversed by phosphate (Fig. 2). However, an effect of phosphate on the dicarboxylic acid shuttle does not explain why phosphate also stimulates 1,25-(OH)2D-3 production when pyruvate is the energy substrate (Fig. 2), nor does it explain the observation that chloride and phosphate above 40 mM inhibit malate oxidation (Table I) even though these anions at these concentrations do not inhibit 1,25(OH)2D-3 production in the absence of calcium (Fig. 1). However, this effect of phos- phate may account for part of its effect on la-hydroxylase activity.
The results obtained with a number of anions suggest a third possibility, namely, that intramitochondrial hydrogen ion concentration regulates la-hy- droxylase activity. Lehninger [ 29], on the basis of calcium uptake and swelling experiments, concluded that acetate and other weak monocarboxylic acids as well as phosphate enter the mitochondrion in a form capable of donating a hydrogen ion to the matrix. The hydrogen ion inside the mitochondrion could be used to exchange for calcium, as in Lehninger's studies, or to stimulate 1,25- (OH)2D-3 production in some fashion. Such a mechanism might explain the stimulation of 1,25-(OH)2D-3 production by high concentrations of phosphate and acetate and the lack of effect of chloride. The increased production of 1,25-(OH)2D-3 when pyruvate (a monocarboxylic acid) rather than malate is used as energy substrate is also consistent with this notion (Fig. 2). However, the uncertainty in determining which molecular form enters the mitochondrion makes this hypothesis difficult to prove.
Since our results suggest that the entry of various anions and cations into the mitochondrion is necessary for these ions to exert their effects, it was not
1 3 7
surprising that an increase in the extramitochondrial osmolarity with sucrose blocked the ability of calcium, phosphate, and acetate to stimulate the l~- hydroxylase activity. The inhibition occurred in parallel with a reduction in cal- cium accumulation and malate oxidation. However, comparable increases in osmolarity with sodium chloride did not alter calcium accumulation or block the calcium modulat ion of la-hydroxylase activity even though such increases in osmolarity did reduce malate oxidation. Furthermore, the influence of sucrose on calcium regulated la-hydroxylase activity was little influenced by the presence of permeant anions. Therefore, we conclude that sucrose reduced ion flux by a mechanism other than simply an osmotically-induced change in mitochondrial conformation.
In conclusion, we have found that phosphate and acetate can modify l~- hydroxylase activity. The mechanism by which this regulation occurs may be related to the effects of such anions on cation (specifically calcium and potas- sium) entry, energy substrate (malate) entry, or proton availability inside the mitochondrion. Sucrose blunts such ion effects by a mechanism other than its effects on mitochondrial swelling.
Acknowledgement
We wish to acknowledge the technical support of Lillian Johnson and the secretarial support of Lorraine Carlson and Sue Davis. This work was supported in part by a grant from the National Institute of Arthritic, Metabolic and Diges- tive Diseases (AM 19813).
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