AMERICAN
JOURNAL
OF PHYSIOLOGY
Vol. 229, No. 6, December
1975.
Ptinted in U.S.A.
Effect of calcium JOSE BEHAR Department of Mediche,
on magnesium
Veterans Hospital
and Yale University School of Medicine,
BEHAR, JOSE. Effect of calcium on magnesium absorption. Am. J. Physiol. 229(6) : 1590-l 595. 1975.-The effect of calcium on magnesium absorption was studied in the rat ileum in vivo. The increase in the luminal concentration of calcium led to a progressive decrease in magnesium absorption, which was accomparried by a parallel decrease in net sodium absorption. This calcium effect was also observed when sodium chloride was replaced by urea. However, a consistent correlation was observed between the magnitude of net magnesium absorption and the rates of net water absorption at all calcium concentrations. These findings suggest that calcium decreases magnesium absorption by a nonspecific reduction in membrane permeability to solutes that induce net water flow and are consistent with the concept that magnesium is transported by solvent “drag.’ The increase in the luminal concentration of calcium resulted in an increase in tissue accumulation of magnesium. This increase in tissue accumulation of magnesium was associated with a decrease in net sodium absorption and in the negativity of the transmural PD. These findings suggest an additional mechanism of magnesium transport operating independently of net water flow.
bulk water accumulation;
flow; solvent transmural
drag; PD
membrane
permeability;
absorption
tissue
CLINICAL AND EXPERIMENTAL STUDIES have shown that calcium inhibits magnesium absorption (4, 8). These findings have suggested that magnesium shares or competes with calcium for a common step in their transport pathway (13). However, recent studies have indicated that magnesium is transported by passive forces (2). The magnitude and direction of bulk water flow appears to determine the magnitude of net magnesium absorption, suggesting that solvent “drag” is the main transport mechanism (3). These observations are in conflict with the concept of a common divalent pathway, the reciprocal inhibition observed with divalent cations such as calcium and magnesium (l), and thus require additional explanations. Calcium and magnesium are known to influence membrane permeability and reduce the absorption of solutes other than divalent cations (9, 15). Since calcium decreases net sodium and water absorption, it is conceivable that calcium could decrease magnesium absorption by reducing the magnitude of solvent drag. The present studies were designed to investigate the effect of calcium on magnesium absorption in relation to changes in sodium and water absorption in the rat ileum.
West Haven, Connecticut 06516
Mg 0.19 %, Ca 0.94 %, and vitamin D 3.3 IU/g) were used. A 20-cm intestinal loop of terminal ileum was perfused in vivo by a method previously reported (3). All test solutions prior to perfusion were isotonic (290-3 10 mosM) and the pH ranged from 5.5 to 7.4. The composition of the test solutions, unless indicated, consisted of 150 mM of NaCl and 5 mM of KCl. The concentration of MgClz ranged from 0.5 to 32 mM. Calcium was added in the form of CaCl2 and its concentration ranged from 0 to 4 mM. At high magnesium concentrations, the concentration of NaCl was slightly reduced to maintain isotonicity. 2sMg was added to the test solutions immediately before the experiments. The tissue content of 28Mg and 24Mg were determined according to methods previously reported (2, 3). Transmural potential difference (PD) determination was performed with Ringer-agar bridges (PE-10 tubing). One bridge was placed into the distal end of the ileal loop and the other on its serosal, exactly opposite to the mucosal bridge. The bridges were connected to a calomel electrode, each one immersed in a beaker in the Ringer solution. The resulting voltage from the calomel half-cells was determined in a differential high-input impedance amplifier (Orion Research). Before each measurement, the system was shortcircuited by connecting a Ringer-agar bridge between the two Ringer beakers to determine cumulative drift. Methods of calculation. Polyethylene glycol-14C ([‘“CIPEG) from New England Nuclear Corporation was used as a marker whose validity has been previously established (3, 10). Lumen-to-plasma (L-P) movement and net absorption of magnesium were calculated using the equations of Wasserman, Kallfelz, and Comar (16). The assumptions in these calculations are that the backflow of 28Mg from plasma to lumen is small. Serum specific activity (SA) at the end of 1 h of perfusion was less than 100 counts/min per pmol of magnesium, in contrast to the perfusate which averaged 6,000 counts/min per pmol of magnesium, SO that the backdiffusion of radioactive magnesium was small. The results of magnesium transport and net water and sodium absorption are given in millimicromoles per liter per centimeter per hour for Mg++, micromoles per liter per centimeter per hour for Na+, and microliters per centimeter per hour for water. net water absorption lumen-to-plasma = PR
[28Mg] 28Mgr -
net absorption [24Mg] = PR [24MgI
(I)
(28Mg,
(‘)
WI
METHODS
Albino male rats of Charles River strain, weighing 150200 g and fed with Purina laboratory chow (content of
= PR (1 - PEGI/PEGF) x
PEGI/PEGF)
-I- S&/2) (4
-
(24Mg,x
PEG/PEG)]
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MAGNESIUM
1591
ABSORPTION
When SA is specific activity (defined as counts/min per mpmol of 24Mg), PR is pump rate, I is initial solution, and F is final solution. Transport of chemical sodium was also calculated by equation 3. Tissue accumulation of chemical determined at the end of each perfusion, was magnesium, calculated as the difference between tissue magnesium of the perfused loops and the mean of six nonperfused loops of full-thickness ileum. The results were expressed as micromoles per liter of 24Mg in 1 g of dry tissue. 28Mg was obtained as magnesium chloride from Brookhaven National Laboratory (Upton, N.Y.). Three milliliters of this solution contained approximately 50 PCi at the time of arrival. Chemical measurements of total magnesium (24Mg) were determined by atomic absorption spectroscopy (model 290, the Perkin-Elmer Corporation, Norwalk, Conn.). Radioactivity of initial and final samples and of full-thickness ileum was determined with the Packard gamma well counter. Because of the short half-life of 28Mg (2 1.3 h), all counts to be compared were corrected to a standard reference time. [14C]PEG was counted when the counts per minute of the 28Mg standard had returned to that of background. Sodium was measured with a flame photometer, and osmolarities of the solutions were determined with an osmometer from Advance Instruments, Boston. All samples were determined in duplicates, and the coefficient of variation was less than 3 %.
/ / /
0
/
_ 2.5 t
0.5 1
/
I
I
I
4
8
16
MAGNESIUM
CONCENTRATION
/
/
T
1
32 (mM)
2. Effect of 2 mM calcium on L-P transport of magnesium. Relation between L-P transport and initial magnesium concentration from 0.5 to 16 mM is shown as a broken line (Y = 0.96). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means & SE of 6 experiments. FIG.
RESULTS
Effect of calcium on magnesium absorption. creasing the concentration of magnesium
The effect of infrom 0.5 to 32
0.5 1
4.0
4
8 MAGNESIUM
16 CONCENTRATION
32 (mM)
FIG. 3. Effect of 4 mM calcium on L-P transport of magnesium. Relationship between L-P transport and initial magnesium concentration from 0.5 to 16 mM is shown as a broken line (r = 0.95). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means & SE of 6 experiments.
Yc = -149.89
+ 248.00X
r = 0.98
0.5 1
4
8 MAGNESIUM
16 CONCENTRATION
32 (mM)
FIG. 1. Effect of increasing concentration of magnesium on L-P transport of magnesium. Correlation between initial magnesium concentration of 0.5-16 mM and L-P transport is expressed as a broken line (r = 0.98). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means =t SE of 6 experiments.
mM on lumen-to-plasma transport of magnesium is shown in Fig. 1. The relationship between L-P transport of magnesium and magnesium concentration, in the absence of calcium, was linear between 0.5 and 16 mM (r = 0.98, P < 0.001). The increase in magnesium concentration from 16 to 32 mM, however, did not lead to a further increase in L-P transport of magnesium. The increase in calcium concentration of the test solutions to 2 and 4 mM led to a progressive decrease in the L-P transport of magnesium at all magnesium concentrations (Figs. 2 and 3). Th e association between L-P transport of magnesium and magnesium concentration from 0.5 to 16 mM remained linear for calcium concentration of 2 (T = 0.96, P < 0.001) and 4 mM (r = 0.95, P < 0.001) and continued linear through magnesium concentration of 32 mM at a calcium concentration of 4 mM (r = 0.98, P < 0.001). In addition, trend lines drawn by joining the
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1592
J. BEHAR
means of L-P transport of magnesium at each magnesium concentration tended to approach linearity as calcium concentration was increased from 0 to 4 mM (Figs. 1, 2, and 3). Eject of magnesium and calcium on net sodium and water absorption. Figure 4 indicates that net sodium absorption decreases when the concentration of calcium is increased from 0 to 4 mM in the test solutions. In the absence of calcium, net sodium absorption was influenced by the increase in concentration when an analysis-of-variance magnesium technique was applied to these data (Table 1, F = 4.01, P < 0.01). The magnitude of net sodium absorption, however, was only different between 16 and 32 mM magnesium (P < 0.05, multiple comparison test). At 2 mM calcium, a significant variation tias also observed between net sodium absorption and magnesium concentration for the en tire experiment (Av = 3.46, P < O.Ol), but multiple comparison tests revealed no significant differences among the varying levels of magnesium concentrations (P > 0.05). At 4 mM calcium, the relationship between net sodium absorption and magnesium concentration was also significant (F = 4.15, P < o-01), and the net sodium absorption observed at 8 mM magnesium was statistically different from magnesium concentrations of 16 and 32 mM (P < 0.05). The magnitude of net water absorption also decreased 30
--2 \ -E
250
-
5 200 E 0 \ -x V
-
150
-
100
-
z F E 2
m a
2
II
II
,I
II
1
0 mM [Cal++
a W 2
50-
3 I:
I I
0.5
1
I
I
I
J
4
0
16
32
MAGNESIUM
FIG.
nesium ments.
CONCENTRATION
5. Effect of increasing on net water absorption.
hM)
concentration of calcium and magValues are means & SE of 6 experi-
TABLE 2. Analysis of variance of effect of magnesium concentration on net water absorption __ --______ -
Scurce of Variation
SL
Total Variation
df
Averape Variation ----p-e.-
’
P
was calculated as a percentage of the luminal concentration of magnesium [(J&J&C& X 100 as suggested by Hakim and Lifson (7). Where JMg is net magnesium absorption, Jw is net water absorption
r
350
I
BULK DO 5
:
75
: A
0
3
LINE
0 0 0 A
2
0
X 3 -3 \ I”
50
0 0 mM [Co]++ 0 II II
2
f,
a4
11
II
11 4.0
FIG. 8. tration in tration of calculated
l
FLOW
100
8.0 MAGNESIUM
16.0 CONCENTRATION
32.0
Effect of concentration of calcium on magnesium concenwater transported (J&JW) as function of luminal concenmagnesium [(J&JW)/Chg] X 100. Points are means of individual values.
300 8 1 \ E -0 250
1 E”
0 ‘l
I
‘.
‘\
8
z iti
200
k
150
A
4mM
[~g]++
YC = 3.05+ r = 0.79
0
1 mM
[Mg]”
YC r Yc r
t
\
o 0.5mM
\\
+0.37X
0.68 0.92
+ 0.20
x
M
t
Q 1
0’ 0
NaCl 50
0
-
Yc = 183.6
-19.7x
r = -0.84 Urea
l
Yc = 275.7
-103.3x
+ 15.2
x2
5: =12 0.3
r = -0.95 I
0
I
0
CALCIUM
6. Correlation plasma magnesium and urea (- - - -).
2 CONCENTRATION
between calcium concentration transport from isotonic solutions
FIG.
I
4 tmM)
and lumen-toof NaCl (--)
a w
2 m F:
1
Or0
2 CALCIUM
300
CONCENTRATION
4 (mM)
FIG. 9. Correlation between luminal calcium concentration and tissue accumulation of magnesium. Luminal concentration of magnesium was 0.5, 1.O, and 4.0 mM.
2 \ E z 250 z
E”
0 0
50 NET
FIG.
1.22 0.89
t
2 cn
from
[Mg]”
6
= = = =
0.43~
too WATER
150 200 250 ABSORPTION (pl/cm/hr
300 1
7. Relation between net magnesium and water isotonic solutions of NaCl (--) and urea (- - - -)*
absorption
and CM, is luminal concentration of magnesium. Figure 8 shows that with increasing calcium concentration, magnesium concentration in the water transported decreases. However, this reduction is relatively smaller than the overall reduction in magnesium absorption. Efect of calcium on tissue accumulation of magnesium. The effect of calcium on tissue accumulation was studied with relatively low luminal concentrations of magnesium of 0.5, 1.0, and 4.0 mM. Increasing the concentration of calcium led to an increase in tissue accumulation of magnesium. Figure 9 shows that tissue accumulation of magnesium increased with increasing luminal calcium concentration. In contrast, Fig. 10 shows that tissue magnesium correlated inversely with net sodium absorption, which was progressively reduced by increasing the concentration of calcium (at Mgff concentration of 0.5 mM,
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J. BEHAR
1594
53 G
w 2 ,m
A 4mM 8
0 1 mM l
0.5mM
[Mg]“’
Yc = 6.71
[Mg]++
r = -0.81 Yc = 3.98-0.12x r = -0.75
[Mg]*+
-0.18x
Yc = 2.32 - 0.08x r = - 0.81
W 3 m cn F
0
0
10 NET
SODIUM
20 ABSORPTION
30
(pM/cm/hr)
FIG. 10. Correlation between tissue accumulation of magnesium Concentration of magnesium in test and net sodium absorption. solutions was increased from 0.5 to 4 mM. I = -0.81, P < 0.001; at 1 mM, r = -0.75, P < 0.001; P < 0.001). Further, the adand, at 4 mM, r = -0.81, dition of calcium decreased the negativity of the transmural PD. In the absence of calcium, the transmural PD was - 3.2 + 0.8 compared to - 1.2 & 0.4 with 2 mM and to +0.8 rt 0.5 mV with 4 mM of calcium, respectively. DISCUSSION
The results of this study confirm previous observations that calcium inhibits magnesium absorption in the small intestine (4, 8). The magnitude of inhibition of magnesium absorption was dependent on the concentration of calcium and was associated with a decrease in the rates of net sodium This calcium effect, however, was and water absorption. also observed when sodium chloride was replaced by urea, suggesting that the inhibitory action of calcium on magnesium absorption is not specifically associated to changes in net sodium absorption. Although sodium cannot be entirely eliminated from in vivo systems, sodium does not promote magnesium absorption at low concentrations (3). Changes in the magnitude of magnesium absorption induced by calcium, however, correlated consistently with the rates of net water absorption. These findings are compatible with the hypothesis that magnesium is transported by solvent drag (3). The effect of calcium does not seem to be specific on the transport of any solute examined. It is possible that calcium decreases the relative permeability of the transport membrane to solute diffusion, regardless of their type of transport. This interpretation is in complete agreement with previous studies on the effect of calcium on transport in a variety of tissues. Dumont, Curran, and Solomon (6) found that calcium decreases net sodium and water absorption in the rat ileum by a nonspecific “stiffening” of the cellular membrane. Curran and Gill (5) made similar observations
in the frog skin and suggested that calcium is possibly bound to negative charged sites at the pore wall that block the pore membranes reducing diffusion through them. Furthermore, Tidball ( 15), using phenol red as an indicator, has shown that calcium and magnesium regulate the aqueous permeability of the small intestine in vitro. In agreement with previous in vitro studies (12), saturation of magnesium transport was observed at a high magnesium concentration and in the absence of calcium. Although this saturation could suggest that magnesium is transported by facilitated or active transport, it is conceivable that high magnesium concentrations could decrease membrane permeability ( 15). This assumption is supported by the finding that high magnesium concentrations also decrease net water absorption. At these high concentrations, magnesium could limit its own transport by reducing the solvent drag effect. Calcium lowered the concentration of magnesium in the water transported, suggesting an additional and more direct effect on magnesium absorption independent of changes in net water flow. The calcium effect on membrane permeability could explain these observations. Calcium could increase the sieving of magnesium by decreasing the effective size of the aqueous “pores” of the mem brane. However, an alternate hypothesis, one that assumes that a fraction of magnesium is actively transported (13, 14), could also explain this finding. Calcium could compete with magnesium for a divalent cation pump and specifically inhibit magnesium transport at the basal lateral membrane. This specific inhibition of magnesium could reduce its movement at the basal lateral membrane, resulting in greater tissue accumulation, which is independent of the rates of net water absorption. As previously suggested (3), however, greater tissue accumulation of magnesium could result from changes in the electric gradients of the transport cells. Rose and Schultz (11) have shown in the rabbit ileum in vitro that a reduction in the lumen to cell movement of sodium leads to an increase in the intracellular negativity, which in turn promotes the diffusion of the divalent cations into the cell. It is conceivable that calcium stimulates tissue accumulation of magnesium by decreasing the rates of net sodium transport. This hypothesis is supported by the findings that the increase in luminal calcium and the decrease in net sodium absorption are coupled with a decrease in the negativity of the transmural PD. Further definition of the mechanisms involved in the decrease of this fraction of magnesium absorption will require more detailed in vitro studies. The author is indebted to Mr. Emanuel statistical analysis and to Ann Bartiss and valuable technical assistance. This work was supported by a Veterans grant. Received
for publication
3 June
Lerner for handling Allyn Moore for their Administration
the in-
research
1974.
REFERENCES 1. ALCOCK, N., AND I. MACINTYRE. Inter-relation of calcium magnesium absorption. C&z. Sci. 22 : 185-l 93, 1962. 2. ALDOR, T. M., AND E. W. MOORE. Magnesium absorption everted sacs of rat intestine and colon. Gastroenterology 59: 753, 1970.
and by 745-
3. BEHAR, J. Magnesium absorption by the rat ileum Am. J. Physiol. 227: 334-340, 1974. 4. CARE, A. D., AND A. T. VAN’T KLOOSTER. In vivo
magnesium and other cations testinal tract of sheep. J. Physiol.,
across London
and
colon.
transport of the wall of the gastroin177 : 174-l 9 1, 1965.
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MAGNESIUM
1595
ABSORPTION
P. F., AND J. R. GILL. The effect of calcium on sodium 5. CURRAN, transport by frog skin. J. Gen. Physiol. 45: 625-641, 1962. P. A., P. F. CURRAN, AND A. F. SOLOMON. Calcium and 6. DUHONT, strontium in the rat small intestine. Their fluxes and their effect on Na flux. J. Gen. Physiof. 43 : 1119- 1135, 1960. A. A., AND N. LIFSON. Urea transport across dog in7. HAKIM, testinal mucosa in vitro. Am. J. Physiol. 206: 13 15-l 320, 1964. 8. HANNA, S., M. HARRISON, I. MACINTYRE, AND R. FRASER. Syndrome of magnesium deficiency in man. Lancet 2 : 172-l 75, 1960. J. F. Effects of Ca+” ions on membranes. Federation 9. MANERY, Proc. 25 : 1804-l 8 10, 1966. 10. MILLER, D. L., AND H. P. SCHEDL. Total recovery studies of nonabsorbable indicators in the rat small intestine. Gastroenterology 58: 40-46, 1970. 11. ROSE, R. C., AND S. G. SCHULTZ. Studies on the electrical potential profile across rabbit ileum. Effect of sugars and amino
12.
13.
14.
15. 16.
acids on transmural and mucosal electrical potential differences. J. Gen. Physiol. 57 : 639-663, 1971. ROSS, D. B. In vitro studies on the transport of magnesium across the intestinal wall of the rat. J. Physiol., London 160: 417-428, 1962. SCHACHTER, D., AND S. M. ROSEN. Active transport of 46Ca by the small intestine and its dependence on vitamin D. Am. J. Physioi. 196 : 357-362, 1959. SCHACHTER, D., E. B. DOWDLE, AND H, SCHENKER. Active transport of calcium by small intestine of the rat. Am. J. Physiol. 198: 263-268, 1960. TIDBALL, C. S. Magnesium and calcium as regulators of intestinal permeability. Am. J. Physiol. 206 : 243-246, 1964. WASSERMAN, R. H., F. A. KALLFELZ, AND C. L. COMAR. Active transport of calcium by rat duodenum in uivo. Scienct 133: 833884, 1961.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on July 30, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
JOURNAL
OF PHYSIOLOGY
Vol. 229, No. 6, December
1975.
Ptinted in U.S.A.
Effect of calcium JOSE BEHAR Department of Mediche,
on magnesium
Veterans Hospital
and Yale University School of Medicine,
BEHAR, JOSE. Effect of calcium on magnesium absorption. Am. J. Physiol. 229(6) : 1590-l 595. 1975.-The effect of calcium on magnesium absorption was studied in the rat ileum in vivo. The increase in the luminal concentration of calcium led to a progressive decrease in magnesium absorption, which was accomparried by a parallel decrease in net sodium absorption. This calcium effect was also observed when sodium chloride was replaced by urea. However, a consistent correlation was observed between the magnitude of net magnesium absorption and the rates of net water absorption at all calcium concentrations. These findings suggest that calcium decreases magnesium absorption by a nonspecific reduction in membrane permeability to solutes that induce net water flow and are consistent with the concept that magnesium is transported by solvent “drag.’ The increase in the luminal concentration of calcium resulted in an increase in tissue accumulation of magnesium. This increase in tissue accumulation of magnesium was associated with a decrease in net sodium absorption and in the negativity of the transmural PD. These findings suggest an additional mechanism of magnesium transport operating independently of net water flow.
bulk water accumulation;
flow; solvent transmural
drag; PD
membrane
permeability;
absorption
tissue
CLINICAL AND EXPERIMENTAL STUDIES have shown that calcium inhibits magnesium absorption (4, 8). These findings have suggested that magnesium shares or competes with calcium for a common step in their transport pathway (13). However, recent studies have indicated that magnesium is transported by passive forces (2). The magnitude and direction of bulk water flow appears to determine the magnitude of net magnesium absorption, suggesting that solvent “drag” is the main transport mechanism (3). These observations are in conflict with the concept of a common divalent pathway, the reciprocal inhibition observed with divalent cations such as calcium and magnesium (l), and thus require additional explanations. Calcium and magnesium are known to influence membrane permeability and reduce the absorption of solutes other than divalent cations (9, 15). Since calcium decreases net sodium and water absorption, it is conceivable that calcium could decrease magnesium absorption by reducing the magnitude of solvent drag. The present studies were designed to investigate the effect of calcium on magnesium absorption in relation to changes in sodium and water absorption in the rat ileum.
West Haven, Connecticut 06516
Mg 0.19 %, Ca 0.94 %, and vitamin D 3.3 IU/g) were used. A 20-cm intestinal loop of terminal ileum was perfused in vivo by a method previously reported (3). All test solutions prior to perfusion were isotonic (290-3 10 mosM) and the pH ranged from 5.5 to 7.4. The composition of the test solutions, unless indicated, consisted of 150 mM of NaCl and 5 mM of KCl. The concentration of MgClz ranged from 0.5 to 32 mM. Calcium was added in the form of CaCl2 and its concentration ranged from 0 to 4 mM. At high magnesium concentrations, the concentration of NaCl was slightly reduced to maintain isotonicity. 2sMg was added to the test solutions immediately before the experiments. The tissue content of 28Mg and 24Mg were determined according to methods previously reported (2, 3). Transmural potential difference (PD) determination was performed with Ringer-agar bridges (PE-10 tubing). One bridge was placed into the distal end of the ileal loop and the other on its serosal, exactly opposite to the mucosal bridge. The bridges were connected to a calomel electrode, each one immersed in a beaker in the Ringer solution. The resulting voltage from the calomel half-cells was determined in a differential high-input impedance amplifier (Orion Research). Before each measurement, the system was shortcircuited by connecting a Ringer-agar bridge between the two Ringer beakers to determine cumulative drift. Methods of calculation. Polyethylene glycol-14C ([‘“CIPEG) from New England Nuclear Corporation was used as a marker whose validity has been previously established (3, 10). Lumen-to-plasma (L-P) movement and net absorption of magnesium were calculated using the equations of Wasserman, Kallfelz, and Comar (16). The assumptions in these calculations are that the backflow of 28Mg from plasma to lumen is small. Serum specific activity (SA) at the end of 1 h of perfusion was less than 100 counts/min per pmol of magnesium, in contrast to the perfusate which averaged 6,000 counts/min per pmol of magnesium, SO that the backdiffusion of radioactive magnesium was small. The results of magnesium transport and net water and sodium absorption are given in millimicromoles per liter per centimeter per hour for Mg++, micromoles per liter per centimeter per hour for Na+, and microliters per centimeter per hour for water. net water absorption lumen-to-plasma = PR
[28Mg] 28Mgr -
net absorption [24Mg] = PR [24MgI
(I)
(28Mg,
(‘)
WI
METHODS
Albino male rats of Charles River strain, weighing 150200 g and fed with Purina laboratory chow (content of
= PR (1 - PEGI/PEGF) x
PEGI/PEGF)
-I- S&/2) (4
-
(24Mg,x
PEG/PEG)]
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MAGNESIUM
1591
ABSORPTION
When SA is specific activity (defined as counts/min per mpmol of 24Mg), PR is pump rate, I is initial solution, and F is final solution. Transport of chemical sodium was also calculated by equation 3. Tissue accumulation of chemical determined at the end of each perfusion, was magnesium, calculated as the difference between tissue magnesium of the perfused loops and the mean of six nonperfused loops of full-thickness ileum. The results were expressed as micromoles per liter of 24Mg in 1 g of dry tissue. 28Mg was obtained as magnesium chloride from Brookhaven National Laboratory (Upton, N.Y.). Three milliliters of this solution contained approximately 50 PCi at the time of arrival. Chemical measurements of total magnesium (24Mg) were determined by atomic absorption spectroscopy (model 290, the Perkin-Elmer Corporation, Norwalk, Conn.). Radioactivity of initial and final samples and of full-thickness ileum was determined with the Packard gamma well counter. Because of the short half-life of 28Mg (2 1.3 h), all counts to be compared were corrected to a standard reference time. [14C]PEG was counted when the counts per minute of the 28Mg standard had returned to that of background. Sodium was measured with a flame photometer, and osmolarities of the solutions were determined with an osmometer from Advance Instruments, Boston. All samples were determined in duplicates, and the coefficient of variation was less than 3 %.
/ / /
0
/
_ 2.5 t
0.5 1
/
I
I
I
4
8
16
MAGNESIUM
CONCENTRATION
/
/
T
1
32 (mM)
2. Effect of 2 mM calcium on L-P transport of magnesium. Relation between L-P transport and initial magnesium concentration from 0.5 to 16 mM is shown as a broken line (Y = 0.96). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means & SE of 6 experiments. FIG.
RESULTS
Effect of calcium on magnesium absorption. creasing the concentration of magnesium
The effect of infrom 0.5 to 32
0.5 1
4.0
4
8 MAGNESIUM
16 CONCENTRATION
32 (mM)
FIG. 3. Effect of 4 mM calcium on L-P transport of magnesium. Relationship between L-P transport and initial magnesium concentration from 0.5 to 16 mM is shown as a broken line (r = 0.95). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means & SE of 6 experiments.
Yc = -149.89
+ 248.00X
r = 0.98
0.5 1
4
8 MAGNESIUM
16 CONCENTRATION
32 (mM)
FIG. 1. Effect of increasing concentration of magnesium on L-P transport of magnesium. Correlation between initial magnesium concentration of 0.5-16 mM and L-P transport is expressed as a broken line (r = 0.98). A trend line drawn by joining means of L-P transport at all magnesium concentrations (0.5-32 mM) is shown as a continuous line. Values are means =t SE of 6 experiments.
mM on lumen-to-plasma transport of magnesium is shown in Fig. 1. The relationship between L-P transport of magnesium and magnesium concentration, in the absence of calcium, was linear between 0.5 and 16 mM (r = 0.98, P < 0.001). The increase in magnesium concentration from 16 to 32 mM, however, did not lead to a further increase in L-P transport of magnesium. The increase in calcium concentration of the test solutions to 2 and 4 mM led to a progressive decrease in the L-P transport of magnesium at all magnesium concentrations (Figs. 2 and 3). Th e association between L-P transport of magnesium and magnesium concentration from 0.5 to 16 mM remained linear for calcium concentration of 2 (T = 0.96, P < 0.001) and 4 mM (r = 0.95, P < 0.001) and continued linear through magnesium concentration of 32 mM at a calcium concentration of 4 mM (r = 0.98, P < 0.001). In addition, trend lines drawn by joining the
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1592
J. BEHAR
means of L-P transport of magnesium at each magnesium concentration tended to approach linearity as calcium concentration was increased from 0 to 4 mM (Figs. 1, 2, and 3). Eject of magnesium and calcium on net sodium and water absorption. Figure 4 indicates that net sodium absorption decreases when the concentration of calcium is increased from 0 to 4 mM in the test solutions. In the absence of calcium, net sodium absorption was influenced by the increase in concentration when an analysis-of-variance magnesium technique was applied to these data (Table 1, F = 4.01, P < 0.01). The magnitude of net sodium absorption, however, was only different between 16 and 32 mM magnesium (P < 0.05, multiple comparison test). At 2 mM calcium, a significant variation tias also observed between net sodium absorption and magnesium concentration for the en tire experiment (Av = 3.46, P < O.Ol), but multiple comparison tests revealed no significant differences among the varying levels of magnesium concentrations (P > 0.05). At 4 mM calcium, the relationship between net sodium absorption and magnesium concentration was also significant (F = 4.15, P < o-01), and the net sodium absorption observed at 8 mM magnesium was statistically different from magnesium concentrations of 16 and 32 mM (P < 0.05). The magnitude of net water absorption also decreased 30
--2 \ -E
250
-
5 200 E 0 \ -x V
-
150
-
100
-
z F E 2
m a
2
II
II
,I
II
1
0 mM [Cal++
a W 2
50-
3 I:
I I
0.5
1
I
I
I
J
4
0
16
32
MAGNESIUM
FIG.
nesium ments.
CONCENTRATION
5. Effect of increasing on net water absorption.
hM)
concentration of calcium and magValues are means & SE of 6 experi-
TABLE 2. Analysis of variance of effect of magnesium concentration on net water absorption __ --______ -
Scurce of Variation
SL
Total Variation
df
Averape Variation ----p-e.-
’
P
was calculated as a percentage of the luminal concentration of magnesium [(J&J&C& X 100 as suggested by Hakim and Lifson (7). Where JMg is net magnesium absorption, Jw is net water absorption
r
350
I
BULK DO 5
:
75
: A
0
3
LINE
0 0 0 A
2
0
X 3 -3 \ I”
50
0 0 mM [Co]++ 0 II II
2
f,
a4
11
II
11 4.0
FIG. 8. tration in tration of calculated
l
FLOW
100
8.0 MAGNESIUM
16.0 CONCENTRATION
32.0
Effect of concentration of calcium on magnesium concenwater transported (J&JW) as function of luminal concenmagnesium [(J&JW)/Chg] X 100. Points are means of individual values.
300 8 1 \ E -0 250
1 E”
0 ‘l
I
‘.
‘\
8
z iti
200
k
150
A
4mM
[~g]++
YC = 3.05+ r = 0.79
0
1 mM
[Mg]”
YC r Yc r
t
\
o 0.5mM
\\
+0.37X
0.68 0.92
+ 0.20
x
M
t
Q 1
0’ 0
NaCl 50
0
-
Yc = 183.6
-19.7x
r = -0.84 Urea
l
Yc = 275.7
-103.3x
+ 15.2
x2
5: =12 0.3
r = -0.95 I
0
I
0
CALCIUM
6. Correlation plasma magnesium and urea (- - - -).
2 CONCENTRATION
between calcium concentration transport from isotonic solutions
FIG.
I
4 tmM)
and lumen-toof NaCl (--)
a w
2 m F:
1
Or0
2 CALCIUM
300
CONCENTRATION
4 (mM)
FIG. 9. Correlation between luminal calcium concentration and tissue accumulation of magnesium. Luminal concentration of magnesium was 0.5, 1.O, and 4.0 mM.
2 \ E z 250 z
E”
0 0
50 NET
FIG.
1.22 0.89
t
2 cn
from
[Mg]”
6
= = = =
0.43~
too WATER
150 200 250 ABSORPTION (pl/cm/hr
300 1
7. Relation between net magnesium and water isotonic solutions of NaCl (--) and urea (- - - -)*
absorption
and CM, is luminal concentration of magnesium. Figure 8 shows that with increasing calcium concentration, magnesium concentration in the water transported decreases. However, this reduction is relatively smaller than the overall reduction in magnesium absorption. Efect of calcium on tissue accumulation of magnesium. The effect of calcium on tissue accumulation was studied with relatively low luminal concentrations of magnesium of 0.5, 1.0, and 4.0 mM. Increasing the concentration of calcium led to an increase in tissue accumulation of magnesium. Figure 9 shows that tissue accumulation of magnesium increased with increasing luminal calcium concentration. In contrast, Fig. 10 shows that tissue magnesium correlated inversely with net sodium absorption, which was progressively reduced by increasing the concentration of calcium (at Mgff concentration of 0.5 mM,
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J. BEHAR
1594
53 G
w 2 ,m
A 4mM 8
0 1 mM l
0.5mM
[Mg]“’
Yc = 6.71
[Mg]++
r = -0.81 Yc = 3.98-0.12x r = -0.75
[Mg]*+
-0.18x
Yc = 2.32 - 0.08x r = - 0.81
W 3 m cn F
0
0
10 NET
SODIUM
20 ABSORPTION
30
(pM/cm/hr)
FIG. 10. Correlation between tissue accumulation of magnesium Concentration of magnesium in test and net sodium absorption. solutions was increased from 0.5 to 4 mM. I = -0.81, P < 0.001; at 1 mM, r = -0.75, P < 0.001; P < 0.001). Further, the adand, at 4 mM, r = -0.81, dition of calcium decreased the negativity of the transmural PD. In the absence of calcium, the transmural PD was - 3.2 + 0.8 compared to - 1.2 & 0.4 with 2 mM and to +0.8 rt 0.5 mV with 4 mM of calcium, respectively. DISCUSSION
The results of this study confirm previous observations that calcium inhibits magnesium absorption in the small intestine (4, 8). The magnitude of inhibition of magnesium absorption was dependent on the concentration of calcium and was associated with a decrease in the rates of net sodium This calcium effect, however, was and water absorption. also observed when sodium chloride was replaced by urea, suggesting that the inhibitory action of calcium on magnesium absorption is not specifically associated to changes in net sodium absorption. Although sodium cannot be entirely eliminated from in vivo systems, sodium does not promote magnesium absorption at low concentrations (3). Changes in the magnitude of magnesium absorption induced by calcium, however, correlated consistently with the rates of net water absorption. These findings are compatible with the hypothesis that magnesium is transported by solvent drag (3). The effect of calcium does not seem to be specific on the transport of any solute examined. It is possible that calcium decreases the relative permeability of the transport membrane to solute diffusion, regardless of their type of transport. This interpretation is in complete agreement with previous studies on the effect of calcium on transport in a variety of tissues. Dumont, Curran, and Solomon (6) found that calcium decreases net sodium and water absorption in the rat ileum by a nonspecific “stiffening” of the cellular membrane. Curran and Gill (5) made similar observations
in the frog skin and suggested that calcium is possibly bound to negative charged sites at the pore wall that block the pore membranes reducing diffusion through them. Furthermore, Tidball ( 15), using phenol red as an indicator, has shown that calcium and magnesium regulate the aqueous permeability of the small intestine in vitro. In agreement with previous in vitro studies (12), saturation of magnesium transport was observed at a high magnesium concentration and in the absence of calcium. Although this saturation could suggest that magnesium is transported by facilitated or active transport, it is conceivable that high magnesium concentrations could decrease membrane permeability ( 15). This assumption is supported by the finding that high magnesium concentrations also decrease net water absorption. At these high concentrations, magnesium could limit its own transport by reducing the solvent drag effect. Calcium lowered the concentration of magnesium in the water transported, suggesting an additional and more direct effect on magnesium absorption independent of changes in net water flow. The calcium effect on membrane permeability could explain these observations. Calcium could increase the sieving of magnesium by decreasing the effective size of the aqueous “pores” of the mem brane. However, an alternate hypothesis, one that assumes that a fraction of magnesium is actively transported (13, 14), could also explain this finding. Calcium could compete with magnesium for a divalent cation pump and specifically inhibit magnesium transport at the basal lateral membrane. This specific inhibition of magnesium could reduce its movement at the basal lateral membrane, resulting in greater tissue accumulation, which is independent of the rates of net water absorption. As previously suggested (3), however, greater tissue accumulation of magnesium could result from changes in the electric gradients of the transport cells. Rose and Schultz (11) have shown in the rabbit ileum in vitro that a reduction in the lumen to cell movement of sodium leads to an increase in the intracellular negativity, which in turn promotes the diffusion of the divalent cations into the cell. It is conceivable that calcium stimulates tissue accumulation of magnesium by decreasing the rates of net sodium transport. This hypothesis is supported by the findings that the increase in luminal calcium and the decrease in net sodium absorption are coupled with a decrease in the negativity of the transmural PD. Further definition of the mechanisms involved in the decrease of this fraction of magnesium absorption will require more detailed in vitro studies. The author is indebted to Mr. Emanuel statistical analysis and to Ann Bartiss and valuable technical assistance. This work was supported by a Veterans grant. Received
for publication
3 June
Lerner for handling Allyn Moore for their Administration
the in-
research
1974.
REFERENCES 1. ALCOCK, N., AND I. MACINTYRE. Inter-relation of calcium magnesium absorption. C&z. Sci. 22 : 185-l 93, 1962. 2. ALDOR, T. M., AND E. W. MOORE. Magnesium absorption everted sacs of rat intestine and colon. Gastroenterology 59: 753, 1970.
and by 745-
3. BEHAR, J. Magnesium absorption by the rat ileum Am. J. Physiol. 227: 334-340, 1974. 4. CARE, A. D., AND A. T. VAN’T KLOOSTER. In vivo
magnesium and other cations testinal tract of sheep. J. Physiol.,
across London
and
colon.
transport of the wall of the gastroin177 : 174-l 9 1, 1965.
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MAGNESIUM
1595
ABSORPTION
P. F., AND J. R. GILL. The effect of calcium on sodium 5. CURRAN, transport by frog skin. J. Gen. Physiol. 45: 625-641, 1962. P. A., P. F. CURRAN, AND A. F. SOLOMON. Calcium and 6. DUHONT, strontium in the rat small intestine. Their fluxes and their effect on Na flux. J. Gen. Physiof. 43 : 1119- 1135, 1960. A. A., AND N. LIFSON. Urea transport across dog in7. HAKIM, testinal mucosa in vitro. Am. J. Physiol. 206: 13 15-l 320, 1964. 8. HANNA, S., M. HARRISON, I. MACINTYRE, AND R. FRASER. Syndrome of magnesium deficiency in man. Lancet 2 : 172-l 75, 1960. J. F. Effects of Ca+” ions on membranes. Federation 9. MANERY, Proc. 25 : 1804-l 8 10, 1966. 10. MILLER, D. L., AND H. P. SCHEDL. Total recovery studies of nonabsorbable indicators in the rat small intestine. Gastroenterology 58: 40-46, 1970. 11. ROSE, R. C., AND S. G. SCHULTZ. Studies on the electrical potential profile across rabbit ileum. Effect of sugars and amino
12.
13.
14.
15. 16.
acids on transmural and mucosal electrical potential differences. J. Gen. Physiol. 57 : 639-663, 1971. ROSS, D. B. In vitro studies on the transport of magnesium across the intestinal wall of the rat. J. Physiol., London 160: 417-428, 1962. SCHACHTER, D., AND S. M. ROSEN. Active transport of 46Ca by the small intestine and its dependence on vitamin D. Am. J. Physioi. 196 : 357-362, 1959. SCHACHTER, D., E. B. DOWDLE, AND H, SCHENKER. Active transport of calcium by small intestine of the rat. Am. J. Physiol. 198: 263-268, 1960. TIDBALL, C. S. Magnesium and calcium as regulators of intestinal permeability. Am. J. Physiol. 206 : 243-246, 1964. WASSERMAN, R. H., F. A. KALLFELZ, AND C. L. COMAR. Active transport of calcium by rat duodenum in uivo. Scienct 133: 833884, 1961.
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