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An inhibitory effect of calcium on citrate utilization by various tissues
BIOCHEMICAL
MEDICINE
An
1,
168477
Inhibitory
(1967)
Effect
Utilization
by Various DAVID
Department
of
Medicine,
of Calcium
Tissues’
P. SIMPSON
University Seattle,
on Citrate
of Washington
Washington
Received
July
School
of
Medicine,
98106 31, 1967
In metabolic alkalosis the renal utilization of citrate is markedly inhibited and citrate clearance is increased (l-3). In contrast in other organs metabolic alkalosis has little effect on citrate utilization (4, 5). Recently we have described an inhibitory effect of bicarbonate ion on citrate metabolism in vitro which has characteristics very similar to the in vivo effect of metabolic alkalosis on the metabolism of this substrate (6). In particular, the inhibitory effect of bicarbonate ion on the oxidation of CY4-labeled citrate is readily demonstrable in slices of renal cortex but there is little comparable effect in slices of liver or heart. In the present study we have investigated the organ specificity of this phenomenon in more detail and have found that the presence of calcium ion in the medium is responsible for the difference in radioactive citrate metabolism in liver and heart slices as compared with slices of renal cortex. MATERIALS
AND
METHODS
The techniques for tissue preparation, incubation, and organic acid analysis were the same as those described previously (6). Each medium contained 5 mM KCI, 1 mM KH,PO,, 2 mM Na pyruvate, 2 mM Na malate, 1 mM Na citrate, 10-50 mM NaHCO, and O-5 mM CaCL. NaCl was added in amounts calculated to maintain a constant osmolality of 290 m0smJliter. Magnesium was not added to the medium since it was found not to have any independent effect on the rate of citrate oxidation. Gas phase was 57% C02-95% 0, in all experiments. Incubation time was 18 minutes in experiments in which citrate-1,5-W was used and 30 minutes in experiments in which substrate utilization was measured. Citrate oxidation was determined by measuring the amount of CW, produced from citrate-1,5-Cl4 added to the medium; the term citrate ‘This Metabolic
investigation Diseases,
was supported U. S. Public Health
by National Service, grant 168
Institute No. 5ROl
of Arthritis AM 69822.
and
EFFECT
OF CALCIUM
Oh’
169
CITRATE
oxidation is used exclusively in this paper to describe results obtained from such experiments using labeled substrate. Rates of citrate, malate, and pyruvate utilization were determined by measuring substrate remaining in the medium at the end of the incubation period. Each result is expressed as mean + one standard deviation. RESULTS
Efiect of Calcium on the Oxidation of Citrate-1,5-P
In the experiments depicted in Figs. l-3 slices of various organs were incubated in media containing either no calcium or 2.5 mM Ca++. The concentration of bicarbonate ion in the media varied between 10 and 50 mM. In each tissue, in the absence of calcium, increasing the bicarbonate concent.ration and pH resulted in marked inhibition of citrate oxidation. 24-
.$
I
T
20-
e 2 16P 2. 12Qz 8-
KIDNEY h-0
I
[Ca*l=O [Ca*l=2.5mM
1
IO
I
20 30 CHCO-4 mM
40
50
FIG. 1. Renal cortex: effect of calcium on rate (R) of citrate oxidation at different concentrations of bicarbonate ion. The results of four experiments are shown.
When the concentration of calcium was 2.5 mM, the rate of citrate oxidation by slices of renal cortex was moderately reduced. At a bicarbonate concentration of 10 mM this reduction amounted to about one-third of the rate of oxidation found in the absence of calcium. Despite this decrease, inhibition of citrate oxidation by increasing bicarbonate ion and pH was still clearly discernible. In heart and liver the presence of calcium in the media caused a greater
reduction
in citrate
oxidation
than
it did in renal
cortex.
Calcium
170
DAVID
P.
SIMPSON
IO-
LIVER [Co-I:0 [Co*lz25mM
21 I
I
IO
20
I
30
[HCO,-I
40
50
mM
FIG. 2. Liver: effect of calcium on rate (R) of citrate oxidation at different. concentrations of bicarbonate ion. The results of three experiments are shown.
decreased the rate of oxidation of citrate at 10 mM HCO,- by 60% in liver and by 50% in heart. In addition, no inhibitory effect of bicarbonate ion occurred in slices of Ever when calcium was present in the medium and only a slight inhibitory effect occurred in heart slices. Thus, in heart and liver, a physiologic concentration of calcium in the medium lnarkedly inhibits citrate oxidation and masks the inhibitory effect of
HEART [Co*l~O CCo*I~2.5mM
_-IO
20 [HCO;I
I I-.----I
30
40
50
mM
FIG. 3. Heart: effect of calcium on rate (A!) of citrate oxidation at different concentrations of bicarbonate ion. The results of five experiments are shown.
EFFECT
increasing caortex. Effect
OF
CALCIUM
1)I-E and bicarbonate of Varying
ON
concentration
of
Concentrations
171
CITRATE
seen in slices of renal
Calcium
on Citrate
Oxidation
When the calcium concentration in the medium was varied from 0 to 5 rnM and a bicarbonate concentration of 20 mM was used in all flasks, we obtained the results shown in Fig. 4. The rate of citrate oxidation at each concentration of calcium is expressed as the percentage
L
I
0
IO
20
30
40
I
5.0
CCa*I mM FIG. 4. Effect of increasing concentrations of calcium on the rate (I<) of citraPc oxidation in different tissues. [HCO,-1 was 20 rn~ in each medium.
of the rate measured for that tissue when no calcium was present in the medium. Increasing concentrations of calcium caused decreasing rates of citrate oxidation in each tissue, but the magnitude of the inhibitory effect was considerably greater in heart and liver than in kidney. Ejfect of Calcium on Net Substrate Utilization
When we measured the amount of each organic acid substrate remaining in the medium after 30 minutes of incubation, we obtained the results shown in Table 1 for slices of heart and liver. The amount of citrate measured represents a balance between citrate synthesized and released into the medium and citrate metabolized by the slices. When citrate synthesis exceeded citrate utilization (net synthesis), the citrate after incubation exceeded that present in the medium at the start of the experiment. Conversely when citrate utilization was more rapid than citrate synthesis, less citrate was found after incubation than before (net utilization). In each tissue increasing bicarbonate concentration was accompanied by a steady decrease in citrate utilization; in heart net citrate production was present at 40 and 50 mM HCO,- in the
-14 -9.0 -4.3 +0.7 +5.0
-0.3
50
10 20 30 40 50
-14 -11 -4.7 -1.0
10 20 30 40
* zk + f f
f
+ + rt f
Ob
5.0 4.0 1.3 5.3 2.7
9.3
6.7 6.3 6.0 7.3
Citrate
* Net substrate utilization = --; net substrate experiments with liver and of four experiments b Calcium concentration (mM) in medium.
Heart
Liver
Tissue
[HCOa-I mu
TABLE
1
9.7 6.0 4.7 8.0
I!z + & 31 *
0.7 4.0 4.0 6.0 3.3
+ 5.0
+ f 31 5
production with heart
+7.7 +12 $13 +13 +15
+6.3
-1.7 +0.7 +2.3 +4.3
2.5”
-38 -44 -44 -49 -56
-33
-28 -28 -29 -29
8.7 3.7 3.3 3.7
+ + rt f f represent,
8.0 6.0 4.7 4.3 1.0
Malate
7.3 5.3 4.7 3.0
5 zk zk + f
2.1 2.7 1.3 2.7 6.7
+ 3.0
f f + f
m~moles/minute/200-mg
-40 -43 -43 -47 -47
-29
-22 -22 -27 -27
2.5”
or production”
-79 -95 -93 -95 -101
-63
-34 -42 -41 -60
rt f + * f
slices.
8.3 3.3 4.7 5.7 7.0
+ 11
The
Pyruvate
Lk 9.0 * 8.7 f 9.0 I!C 14
Ob
BY SLICES OF LIVER AND HEART
utilization
+ 2.7
f * + f
Ob
substrate
= +; valrles are given.
Net
EFFECT OF CALCIUM ON SUBSTRATE METABOLISM
resrllts
-64 -74 -80 -83 -80
-39
-27 -29 -34 -37
2.7 0.3 0.i 1.0
4.3 12 8.i 3.i 7.7 of three
31 + f IL IL
rt 1.0
+ i * *
2.5*
g
r2 5: z
.?
8
$
EFFECT
OF
CALCTUM
ON
173
CITRATE
absence of calcium. In the presence of 2.5 m-v calcium a similar steady decrease in citrate utilization occurred but net citrate production was found at all bicarbonate concentrations. In liver calcium decreased malate utilization slightly; in heart it had no effect on malate utilization. In both organs pyruvate utilization decreased 2533% in the presence of calcium. Effect
of Calcium
on Total
Chate
Content
We measured the total amount of citrate present after 15 minutes of incubation when slices of heart or liver were incubated with pyruvate and malate but no citrate in the media at the start of the experiment (Table 2). The citrate measured in this way represents both citrate contained in the slices and citrate which escapes from the cells into the medium. In kidney, as previously reported by Simpson (6), the amount TABLE 2 EFFECT OF CALCIUM ON TOTAL CITRATE CONTENT OF SLICES AND MEDIUM INCUBATED WITH HIGH AND Low CONCENTRATIONS OF BICARBONATEO [Ca++] = 0 Ti88Ue
Liver Heart
[HCOI-I mM 10 40 10 40
Experiment 0.20 0.40 0.29 0.52
IL + f +
0.012 0.037 0.025 0.010
1
[Ca++] = 2.5 rnM
Experiment 0.18 0.41 0.29 0.49
+ f IL +
0.003 0.003 0.013 0.012
2
Experiment 0.22 0.39 0.56 0.83
+ lk rk +
n Results represent rmoles citrate in 2-ml medium containing value is the mean citrate content of three flasks.
1
0.016 0.013 0.021 0.006
Experiment 0.17 0.31 0.54 0.74
2
f 0.024 f 0.079 f 0.015 4_ 0.071
200-mg slices. Each
of citrate present is much greater at high bicarbonate concentrations than at low. Citrate content in slices of heart and liver was similarly affected by bicarbonate whether or not calcium was present in the medium. In liver, calcium had no effect on citrate content at either bicarbonate concentration. In heart, however, calcium increased citrate content at both bicarbonate concentrations. DISCUSSION
In the intact animal different organs use different substrates to obtain their energy. For example, brain uses only glucose (7). Kidney on the other hand derives about 55% of its energy from free fatty acids, 35% from lactate, and 10% from citrate (8). Heart uses about equal amounts of free fatty acids, glucose, and lactate but little citrate (9). Substrate utilization by liver depends on hormonal and dietary factors; citrate is
174
DAVID
P.
SIMPSON
used by liver but the amount is considerably less per gram of tissue than in renal cortex (10, 11). Data such as these, derived from measurements of arterio-venous differences across organs, describe net substrate utilization but do not reveal how much substrate is produced and hence do not indicate the absolute utilization rate of a substance. Since practically all organs contain the enzyme systems necessary to metabolize glucose, fatty acids, and citric acid cycle int’ermediates, there must be factors unrelated to the presence or absence of an enzyme which determine the preference of an organ for a particular substrate. We know little about the mechanisms responsible for these differences between organs. The present study suggests that the extracellular concentration of calcium ion is one factor which plays a role in determining the amount of citrate used by heart, liver and kidney. In the absence of calcium, slices of all three organs can clearly use more citrate than they can when this cation is present in physiologic amounts. While calcium causes a moderate decrease in the oxidation of citrate by renal cortex, its effect on liver and heart is marked. Metabolic alkalosis inhibits renal cortical citrate utilization but has little effect on utilization of this substrate elsewhere in the body. When C4-labeled citrate was administered to normal and alkalotic rats, the recovery of respiratory C’“0, was only slightly lower in the alkalotic animals (5). This decrease in citrate oxidation could be quantitabively accounted for by increased urinary excretion of unmetabolized labeled citrate which occurred because of inhibition by the metabolic alkalosis of citrate reabsorption in the renal tubules. Thus metabolic alkalosis decreased the reabsorption and oxidation of citrat.e in the kidney but had little effect on citrate oxidation by other organs. Also, metabolic alkalosis increases citrate content in kidney but not in other organs (4, 5). These results indicate that, the effect of metabolic alkalosis on citrate utilization and oxidation is limited to the kidney in the intact animal. In vitro this effect is reflected by inhibition of citrate oxidation in slices of renal cortex by increasing pH and bicarbonate ion (6). Liver and heart slices also show a similar phenomenon in the absence of calcium in the medium but, in its presence, little or no inhibitory effect of bicarbonate occurs. The inhibitory effect of calcium therefore overshadows that of bicarbonate ion and pH in these latter two tissues and is responsible, at least in part, for the organ specificity of the bicarbonate-pH effect. The tlata do not indicate whether calcium also prevents the increase in intracellular citrate in alkalosis in heart and liver. High bicarbonate concentrations in the media did increase total citrate content in medium and slices in these tissues in the presence of calcium. Since citrate concentrations within cells were not directly measured, this increase may
EFFECT
OF CAIXITJM
OX
(‘ITHATE
I 7.;
rcl)rcsent an increase in intracellular citrate or it may represent an increase: in citrate synthesis in t8he cell with subsequent rcl~ase 0I eitratc! into the medium. If an increase in intracellular citrate o(*curF rmtlcr these conditions then some factor other than calcium is reaponsiblc in ~,Gvo for the failure of citrate concentrat,ion t.0 change significantly in heart and liver during metabolic alkalosis. Alternatively. it may 1)~~ that the tissue concentration of &rate in these tissues does not rise iI\ lnct~abolic alkalosis because of rapid diffusion of cit,rate out of cells. Several possibilities exist which might be responsible for this effect. 01 c~alcium on citrate metabolism. Calcium chelates citrate and if the (‘oncentration of free citrate ion in the medium were greatly reduced, t)hi> might. affect the rate of citrate oxidation. T’nder the conditions of th(* experiments reported here, with 1 mu citrate an11 2.5 IBM calcium in the llredia, about 2.5%) of the citrate is present as free citrate ion (121. ;t cluant,ity which, in the absence of calcium, is sufficient to clenlonstratcb the inhibitory effect. of pH and bicarbonate ion. Thcreforc chelation d ritratcl in the medium cannot account by itself for the c4fect of caalcium on citrate oxidation. However, chelation of citrate by calcium within tl~c‘ ccl1 may be responsible for this effect. Since iicGthcr the cytoplarniic* concentration of calcium nor that of citrate is known, it is not po+ sible to judge the likelihood of this mechanism. The known effects of calcium on physiologic processes are nulnt’rou> and many undiscovered influences undoubtedly exist. Of the known effects only two suggest themselves as possible esplanations for the markecl effect of calcium on citrate oxidation by heart, and liver. Calcium has l~~cn shown to inhibit Na-K-activated adcnosinc triphosphatasr ( 13). the enzyme believed to provide energy for the ubiquitous sodium pun~l), If citrate transport i&o cells were linked to sodium t’ransport, the rat<% of uptake of extracellular citrate might be limitrd by the rate of sodium influx. In this case calcium, by inhibitin g sodium transport, woulc-I indirectJy inhibit citrate entry into the cell and hence its rate of oxidation, Another effect of calcium ion which might affect citrate tiictabolisn~ is its effect on mitochondrial metabolism. In recent years a number 01’ investigators have shown that mitochondria possess a remarkable ability t’o concentrate calcium from the surrounding medium (14-16,. At th(~ same time oxidative phosphorylation is inhibit’ed by calcium concentratjions approximating those of extracellular fluid. If physiologic COII~~IItratons of calcium inside the cell decreased the rate of oxidative pho+ phorylation, citrate oxidation would be reduced. The concentration of calcium ion in the cytoplasm is uncertain but certainly it must, be COIIsidcrably lower than in the extracellular fluid; otherwise effective mitechondrial metabolism would cease. The present. investigation does nor
176
DAVID
P.
SIMPSON
indicate the mechanism by which calcium affects citrate utilization. One of the above possibilities may be responsible but’ a tlircct effect. of cnlcium on citrate transport or metabolism seems equally likely.
We studied the effect’ of calcium on t,hc oxidation of C”-labeled citrate in slices of heart, liver and kidney. In each of these tissues, in the absence of calcium, citrate oxidation was rapid at low bicarbonate concentrations; when pH and bicarbonate concentration increased, citrate oxidation was inhibited, as has been previously described for kidney. In the presence of 2.5 rnM calcium citrate oxidation was markedly inhibited in slices of heart and liver so that little or no additional inhibition by bicarbonate ion and pH occurred. In slices of renal cortex calcium inhibited citrate oxidation to a lesser degree than in t’he other two tissues and the inhibitory effect of bicarbonate ion and pH was still quite noticeable. We also measured substrate utilization in the presence and absence of calcium in heart and liver slices. In general, with concentrations of bicarbonate from 10 to 50 mM net citrate utilization in the absence of calcium changed to net citrate production when calcium was added to the medium. As the concentratBion of birarbonate ion increased the amount of net citrate utilization decreased or net citrate production increased. Calcium had minor effects on net, pyruvate and malate utilization under these circumstances. High bicarbonate concentrations caused citrate content of medium and slices to increase both in the presence and absence of calcium. These results suggest that calcium plays a role in t’he intact animal in limiting the amount of citrate utilized by heart and liver and in determining the organ specificity of the effect of metabolic alkalosis on citrate met,abolism. ACRNOWLEDGMEST The nut,hor is indchhl nssistancc. 1. 2. 3.
CHA~FORD, HERRIN, SIMPSON,
4. CRAWFORD, SIMPSON, SIMPSON, GOTTSTEIN,
5. 6. 7.
( 1963).
to Patricia
Dsida md Carol Ralph for vomlw~ rnt twhnicxl
REFERENCES M. A., MILNE, M. D., AND SCRIBNER, B. H.. .I. Physiol. 149, 413 (1959) R. C., AND LARDINOIS, C. C., Proc. Sot. Ezptl. Biol. 97, 294 (1958). D. P.. Amer. J. Physiol. 206, 875 (1964). M. A., Biochem. J. 88, 115 (1963). D. P., Proc. Sot. Erptl. Biol. 114, 263 (1963). D. P., .I. Ch. hvest. 46, 225 (1967). U., BERNSMEIER, A., AND SEDLMEYER, J., K&n. Tl’ochschr. 41, 943
XFFECT
S. KuT~, 9. &TILL,
H., AND J.. DOLL,
f’jfiigers
SCWLLMEYER, E., ST~IK,
OF
H.,
CALCIUM P., Suture HOMBCRGER,
ON
CITRATE;
177
209, 1244 (1966).
282, 43 (1965). SROIIMAKEH, IIT. C.,
H.,
&RN,
H.,
.\NI)
RIIISI)IILI~.
II .
Arch. Ges. Physiol.
10. HESNEMAN, D. H., .~ND E/rt/oc,~idogy 68, 8SO (1960). 11. MARTP:XSSOX, J. Acfa Ph ysiol. Scutd. Suppl. 2, I (1940). 12. H.~STIXGS, A. B., McI,E.~N, F. C., EI~~I-~~:LHISRC:ER, R.. w.4~1,. ,I. J,. .ISL) D.+COST 1, ,I., J. Biol. rhenz. 107, 351 (1934). 13. EPSTKIN. F. H., .4x1 WIIITTAM, R., Biuchem. J. 99, 232 (1966). 14. SLATKK, E. C.. .4x1) CLELAND, K. W., Biochem. J. 55, 566 (1953). 15. VASINGTON, F. D., AND MURPHY, J. V., J. Bid. Chern. 237, 2670 (1962). 16. I
MEDICINE
An
1,
168477
Inhibitory
(1967)
Effect
Utilization
by Various DAVID
Department
of
Medicine,
of Calcium
Tissues’
P. SIMPSON
University Seattle,
on Citrate
of Washington
Washington
Received
July
School
of
Medicine,
98106 31, 1967
In metabolic alkalosis the renal utilization of citrate is markedly inhibited and citrate clearance is increased (l-3). In contrast in other organs metabolic alkalosis has little effect on citrate utilization (4, 5). Recently we have described an inhibitory effect of bicarbonate ion on citrate metabolism in vitro which has characteristics very similar to the in vivo effect of metabolic alkalosis on the metabolism of this substrate (6). In particular, the inhibitory effect of bicarbonate ion on the oxidation of CY4-labeled citrate is readily demonstrable in slices of renal cortex but there is little comparable effect in slices of liver or heart. In the present study we have investigated the organ specificity of this phenomenon in more detail and have found that the presence of calcium ion in the medium is responsible for the difference in radioactive citrate metabolism in liver and heart slices as compared with slices of renal cortex. MATERIALS
AND
METHODS
The techniques for tissue preparation, incubation, and organic acid analysis were the same as those described previously (6). Each medium contained 5 mM KCI, 1 mM KH,PO,, 2 mM Na pyruvate, 2 mM Na malate, 1 mM Na citrate, 10-50 mM NaHCO, and O-5 mM CaCL. NaCl was added in amounts calculated to maintain a constant osmolality of 290 m0smJliter. Magnesium was not added to the medium since it was found not to have any independent effect on the rate of citrate oxidation. Gas phase was 57% C02-95% 0, in all experiments. Incubation time was 18 minutes in experiments in which citrate-1,5-W was used and 30 minutes in experiments in which substrate utilization was measured. Citrate oxidation was determined by measuring the amount of CW, produced from citrate-1,5-Cl4 added to the medium; the term citrate ‘This Metabolic
investigation Diseases,
was supported U. S. Public Health
by National Service, grant 168
Institute No. 5ROl
of Arthritis AM 69822.
and
EFFECT
OF CALCIUM
Oh’
169
CITRATE
oxidation is used exclusively in this paper to describe results obtained from such experiments using labeled substrate. Rates of citrate, malate, and pyruvate utilization were determined by measuring substrate remaining in the medium at the end of the incubation period. Each result is expressed as mean + one standard deviation. RESULTS
Efiect of Calcium on the Oxidation of Citrate-1,5-P
In the experiments depicted in Figs. l-3 slices of various organs were incubated in media containing either no calcium or 2.5 mM Ca++. The concentration of bicarbonate ion in the media varied between 10 and 50 mM. In each tissue, in the absence of calcium, increasing the bicarbonate concent.ration and pH resulted in marked inhibition of citrate oxidation. 24-
.$
I
T
20-
e 2 16P 2. 12Qz 8-
KIDNEY h-0
I
[Ca*l=O [Ca*l=2.5mM
1
IO
I
20 30 CHCO-4 mM
40
50
FIG. 1. Renal cortex: effect of calcium on rate (R) of citrate oxidation at different concentrations of bicarbonate ion. The results of four experiments are shown.
When the concentration of calcium was 2.5 mM, the rate of citrate oxidation by slices of renal cortex was moderately reduced. At a bicarbonate concentration of 10 mM this reduction amounted to about one-third of the rate of oxidation found in the absence of calcium. Despite this decrease, inhibition of citrate oxidation by increasing bicarbonate ion and pH was still clearly discernible. In heart and liver the presence of calcium in the media caused a greater
reduction
in citrate
oxidation
than
it did in renal
cortex.
Calcium
170
DAVID
P.
SIMPSON
IO-
LIVER [Co-I:0 [Co*lz25mM
21 I
I
IO
20
I
30
[HCO,-I
40
50
mM
FIG. 2. Liver: effect of calcium on rate (R) of citrate oxidation at different. concentrations of bicarbonate ion. The results of three experiments are shown.
decreased the rate of oxidation of citrate at 10 mM HCO,- by 60% in liver and by 50% in heart. In addition, no inhibitory effect of bicarbonate ion occurred in slices of Ever when calcium was present in the medium and only a slight inhibitory effect occurred in heart slices. Thus, in heart and liver, a physiologic concentration of calcium in the medium lnarkedly inhibits citrate oxidation and masks the inhibitory effect of
HEART [Co*l~O CCo*I~2.5mM
_-IO
20 [HCO;I
I I-.----I
30
40
50
mM
FIG. 3. Heart: effect of calcium on rate (A!) of citrate oxidation at different concentrations of bicarbonate ion. The results of five experiments are shown.
EFFECT
increasing caortex. Effect
OF
CALCIUM
1)I-E and bicarbonate of Varying
ON
concentration
of
Concentrations
171
CITRATE
seen in slices of renal
Calcium
on Citrate
Oxidation
When the calcium concentration in the medium was varied from 0 to 5 rnM and a bicarbonate concentration of 20 mM was used in all flasks, we obtained the results shown in Fig. 4. The rate of citrate oxidation at each concentration of calcium is expressed as the percentage
L
I
0
IO
20
30
40
I
5.0
CCa*I mM FIG. 4. Effect of increasing concentrations of calcium on the rate (I<) of citraPc oxidation in different tissues. [HCO,-1 was 20 rn~ in each medium.
of the rate measured for that tissue when no calcium was present in the medium. Increasing concentrations of calcium caused decreasing rates of citrate oxidation in each tissue, but the magnitude of the inhibitory effect was considerably greater in heart and liver than in kidney. Ejfect of Calcium on Net Substrate Utilization
When we measured the amount of each organic acid substrate remaining in the medium after 30 minutes of incubation, we obtained the results shown in Table 1 for slices of heart and liver. The amount of citrate measured represents a balance between citrate synthesized and released into the medium and citrate metabolized by the slices. When citrate synthesis exceeded citrate utilization (net synthesis), the citrate after incubation exceeded that present in the medium at the start of the experiment. Conversely when citrate utilization was more rapid than citrate synthesis, less citrate was found after incubation than before (net utilization). In each tissue increasing bicarbonate concentration was accompanied by a steady decrease in citrate utilization; in heart net citrate production was present at 40 and 50 mM HCO,- in the
-14 -9.0 -4.3 +0.7 +5.0
-0.3
50
10 20 30 40 50
-14 -11 -4.7 -1.0
10 20 30 40
* zk + f f
f
+ + rt f
Ob
5.0 4.0 1.3 5.3 2.7
9.3
6.7 6.3 6.0 7.3
Citrate
* Net substrate utilization = --; net substrate experiments with liver and of four experiments b Calcium concentration (mM) in medium.
Heart
Liver
Tissue
[HCOa-I mu
TABLE
1
9.7 6.0 4.7 8.0
I!z + & 31 *
0.7 4.0 4.0 6.0 3.3
+ 5.0
+ f 31 5
production with heart
+7.7 +12 $13 +13 +15
+6.3
-1.7 +0.7 +2.3 +4.3
2.5”
-38 -44 -44 -49 -56
-33
-28 -28 -29 -29
8.7 3.7 3.3 3.7
+ + rt f f represent,
8.0 6.0 4.7 4.3 1.0
Malate
7.3 5.3 4.7 3.0
5 zk zk + f
2.1 2.7 1.3 2.7 6.7
+ 3.0
f f + f
m~moles/minute/200-mg
-40 -43 -43 -47 -47
-29
-22 -22 -27 -27
2.5”
or production”
-79 -95 -93 -95 -101
-63
-34 -42 -41 -60
rt f + * f
slices.
8.3 3.3 4.7 5.7 7.0
+ 11
The
Pyruvate
Lk 9.0 * 8.7 f 9.0 I!C 14
Ob
BY SLICES OF LIVER AND HEART
utilization
+ 2.7
f * + f
Ob
substrate
= +; valrles are given.
Net
EFFECT OF CALCIUM ON SUBSTRATE METABOLISM
resrllts
-64 -74 -80 -83 -80
-39
-27 -29 -34 -37
2.7 0.3 0.i 1.0
4.3 12 8.i 3.i 7.7 of three
31 + f IL IL
rt 1.0
+ i * *
2.5*
g
r2 5: z
.?
8
$
EFFECT
OF
CALCTUM
ON
173
CITRATE
absence of calcium. In the presence of 2.5 m-v calcium a similar steady decrease in citrate utilization occurred but net citrate production was found at all bicarbonate concentrations. In liver calcium decreased malate utilization slightly; in heart it had no effect on malate utilization. In both organs pyruvate utilization decreased 2533% in the presence of calcium. Effect
of Calcium
on Total
Chate
Content
We measured the total amount of citrate present after 15 minutes of incubation when slices of heart or liver were incubated with pyruvate and malate but no citrate in the media at the start of the experiment (Table 2). The citrate measured in this way represents both citrate contained in the slices and citrate which escapes from the cells into the medium. In kidney, as previously reported by Simpson (6), the amount TABLE 2 EFFECT OF CALCIUM ON TOTAL CITRATE CONTENT OF SLICES AND MEDIUM INCUBATED WITH HIGH AND Low CONCENTRATIONS OF BICARBONATEO [Ca++] = 0 Ti88Ue
Liver Heart
[HCOI-I mM 10 40 10 40
Experiment 0.20 0.40 0.29 0.52
IL + f +
0.012 0.037 0.025 0.010
1
[Ca++] = 2.5 rnM
Experiment 0.18 0.41 0.29 0.49
+ f IL +
0.003 0.003 0.013 0.012
2
Experiment 0.22 0.39 0.56 0.83
+ lk rk +
n Results represent rmoles citrate in 2-ml medium containing value is the mean citrate content of three flasks.
1
0.016 0.013 0.021 0.006
Experiment 0.17 0.31 0.54 0.74
2
f 0.024 f 0.079 f 0.015 4_ 0.071
200-mg slices. Each
of citrate present is much greater at high bicarbonate concentrations than at low. Citrate content in slices of heart and liver was similarly affected by bicarbonate whether or not calcium was present in the medium. In liver, calcium had no effect on citrate content at either bicarbonate concentration. In heart, however, calcium increased citrate content at both bicarbonate concentrations. DISCUSSION
In the intact animal different organs use different substrates to obtain their energy. For example, brain uses only glucose (7). Kidney on the other hand derives about 55% of its energy from free fatty acids, 35% from lactate, and 10% from citrate (8). Heart uses about equal amounts of free fatty acids, glucose, and lactate but little citrate (9). Substrate utilization by liver depends on hormonal and dietary factors; citrate is
174
DAVID
P.
SIMPSON
used by liver but the amount is considerably less per gram of tissue than in renal cortex (10, 11). Data such as these, derived from measurements of arterio-venous differences across organs, describe net substrate utilization but do not reveal how much substrate is produced and hence do not indicate the absolute utilization rate of a substance. Since practically all organs contain the enzyme systems necessary to metabolize glucose, fatty acids, and citric acid cycle int’ermediates, there must be factors unrelated to the presence or absence of an enzyme which determine the preference of an organ for a particular substrate. We know little about the mechanisms responsible for these differences between organs. The present study suggests that the extracellular concentration of calcium ion is one factor which plays a role in determining the amount of citrate used by heart, liver and kidney. In the absence of calcium, slices of all three organs can clearly use more citrate than they can when this cation is present in physiologic amounts. While calcium causes a moderate decrease in the oxidation of citrate by renal cortex, its effect on liver and heart is marked. Metabolic alkalosis inhibits renal cortical citrate utilization but has little effect on utilization of this substrate elsewhere in the body. When C4-labeled citrate was administered to normal and alkalotic rats, the recovery of respiratory C’“0, was only slightly lower in the alkalotic animals (5). This decrease in citrate oxidation could be quantitabively accounted for by increased urinary excretion of unmetabolized labeled citrate which occurred because of inhibition by the metabolic alkalosis of citrate reabsorption in the renal tubules. Thus metabolic alkalosis decreased the reabsorption and oxidation of citrat.e in the kidney but had little effect on citrate oxidation by other organs. Also, metabolic alkalosis increases citrate content in kidney but not in other organs (4, 5). These results indicate that, the effect of metabolic alkalosis on citrate utilization and oxidation is limited to the kidney in the intact animal. In vitro this effect is reflected by inhibition of citrate oxidation in slices of renal cortex by increasing pH and bicarbonate ion (6). Liver and heart slices also show a similar phenomenon in the absence of calcium in the medium but, in its presence, little or no inhibitory effect of bicarbonate occurs. The inhibitory effect of calcium therefore overshadows that of bicarbonate ion and pH in these latter two tissues and is responsible, at least in part, for the organ specificity of the bicarbonate-pH effect. The tlata do not indicate whether calcium also prevents the increase in intracellular citrate in alkalosis in heart and liver. High bicarbonate concentrations in the media did increase total citrate content in medium and slices in these tissues in the presence of calcium. Since citrate concentrations within cells were not directly measured, this increase may
EFFECT
OF CAIXITJM
OX
(‘ITHATE
I 7.;
rcl)rcsent an increase in intracellular citrate or it may represent an increase: in citrate synthesis in t8he cell with subsequent rcl~ase 0I eitratc! into the medium. If an increase in intracellular citrate o(*curF rmtlcr these conditions then some factor other than calcium is reaponsiblc in ~,Gvo for the failure of citrate concentrat,ion t.0 change significantly in heart and liver during metabolic alkalosis. Alternatively. it may 1)~~ that the tissue concentration of &rate in these tissues does not rise iI\ lnct~abolic alkalosis because of rapid diffusion of cit,rate out of cells. Several possibilities exist which might be responsible for this effect. 01 c~alcium on citrate metabolism. Calcium chelates citrate and if the (‘oncentration of free citrate ion in the medium were greatly reduced, t)hi> might. affect the rate of citrate oxidation. T’nder the conditions of th(* experiments reported here, with 1 mu citrate an11 2.5 IBM calcium in the llredia, about 2.5%) of the citrate is present as free citrate ion (121. ;t cluant,ity which, in the absence of calcium, is sufficient to clenlonstratcb the inhibitory effect. of pH and bicarbonate ion. Thcreforc chelation d ritratcl in the medium cannot account by itself for the c4fect of caalcium on citrate oxidation. However, chelation of citrate by calcium within tl~c‘ ccl1 may be responsible for this effect. Since iicGthcr the cytoplarniic* concentration of calcium nor that of citrate is known, it is not po+ sible to judge the likelihood of this mechanism. The known effects of calcium on physiologic processes are nulnt’rou> and many undiscovered influences undoubtedly exist. Of the known effects only two suggest themselves as possible esplanations for the markecl effect of calcium on citrate oxidation by heart, and liver. Calcium has l~~cn shown to inhibit Na-K-activated adcnosinc triphosphatasr ( 13). the enzyme believed to provide energy for the ubiquitous sodium pun~l), If citrate transport i&o cells were linked to sodium t’ransport, the rat<% of uptake of extracellular citrate might be limitrd by the rate of sodium influx. In this case calcium, by inhibitin g sodium transport, woulc-I indirectJy inhibit citrate entry into the cell and hence its rate of oxidation, Another effect of calcium ion which might affect citrate tiictabolisn~ is its effect on mitochondrial metabolism. In recent years a number 01’ investigators have shown that mitochondria possess a remarkable ability t’o concentrate calcium from the surrounding medium (14-16,. At th(~ same time oxidative phosphorylation is inhibit’ed by calcium concentratjions approximating those of extracellular fluid. If physiologic COII~~IItratons of calcium inside the cell decreased the rate of oxidative pho+ phorylation, citrate oxidation would be reduced. The concentration of calcium ion in the cytoplasm is uncertain but certainly it must, be COIIsidcrably lower than in the extracellular fluid; otherwise effective mitechondrial metabolism would cease. The present. investigation does nor
176
DAVID
P.
SIMPSON
indicate the mechanism by which calcium affects citrate utilization. One of the above possibilities may be responsible but’ a tlircct effect. of cnlcium on citrate transport or metabolism seems equally likely.
We studied the effect’ of calcium on t,hc oxidation of C”-labeled citrate in slices of heart, liver and kidney. In each of these tissues, in the absence of calcium, citrate oxidation was rapid at low bicarbonate concentrations; when pH and bicarbonate concentration increased, citrate oxidation was inhibited, as has been previously described for kidney. In the presence of 2.5 rnM calcium citrate oxidation was markedly inhibited in slices of heart and liver so that little or no additional inhibition by bicarbonate ion and pH occurred. In slices of renal cortex calcium inhibited citrate oxidation to a lesser degree than in t’he other two tissues and the inhibitory effect of bicarbonate ion and pH was still quite noticeable. We also measured substrate utilization in the presence and absence of calcium in heart and liver slices. In general, with concentrations of bicarbonate from 10 to 50 mM net citrate utilization in the absence of calcium changed to net citrate production when calcium was added to the medium. As the concentratBion of birarbonate ion increased the amount of net citrate utilization decreased or net citrate production increased. Calcium had minor effects on net, pyruvate and malate utilization under these circumstances. High bicarbonate concentrations caused citrate content of medium and slices to increase both in the presence and absence of calcium. These results suggest that calcium plays a role in t’he intact animal in limiting the amount of citrate utilized by heart and liver and in determining the organ specificity of the effect of metabolic alkalosis on citrate met,abolism. ACRNOWLEDGMEST The nut,hor is indchhl nssistancc. 1. 2. 3.
CHA~FORD, HERRIN, SIMPSON,
4. CRAWFORD, SIMPSON, SIMPSON, GOTTSTEIN,
5. 6. 7.
( 1963).
to Patricia
Dsida md Carol Ralph for vomlw~ rnt twhnicxl
REFERENCES M. A., MILNE, M. D., AND SCRIBNER, B. H.. .I. Physiol. 149, 413 (1959) R. C., AND LARDINOIS, C. C., Proc. Sot. Ezptl. Biol. 97, 294 (1958). D. P.. Amer. J. Physiol. 206, 875 (1964). M. A., Biochem. J. 88, 115 (1963). D. P., Proc. Sot. Erptl. Biol. 114, 263 (1963). D. P., .I. Ch. hvest. 46, 225 (1967). U., BERNSMEIER, A., AND SEDLMEYER, J., K&n. Tl’ochschr. 41, 943
XFFECT
S. KuT~, 9. &TILL,
H., AND J.. DOLL,
f’jfiigers
SCWLLMEYER, E., ST~IK,
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10. HESNEMAN, D. H., .~ND E/rt/oc,~idogy 68, 8SO (1960). 11. MARTP:XSSOX, J. Acfa Ph ysiol. Scutd. Suppl. 2, I (1940). 12. H.~STIXGS, A. B., McI,E.~N, F. C., EI~~I-~~:LHISRC:ER, R.. w.4~1,. ,I. J,. .ISL) D.+COST 1, ,I., J. Biol. rhenz. 107, 351 (1934). 13. EPSTKIN. F. H., .4x1 WIIITTAM, R., Biuchem. J. 99, 232 (1966). 14. SLATKK, E. C.. .4x1) CLELAND, K. W., Biochem. J. 55, 566 (1953). 15. VASINGTON, F. D., AND MURPHY, J. V., J. Bid. Chern. 237, 2670 (1962). 16. I