Symposium:

Current Concepts of Calcium Absorption

ULRICH KARBACH3 Medizinische Klinik, Klinikum Innenstadt, University of Munich, Munich, Germany effect" (8). In other words, intestinal calcium trans port, absorption or secretion, not only is determined by a cellular mechanism but is also the result of passive calcium flux along the paracellular shunt pathway. Therefore, calcium transport across the rat duodenum, jejunum and ileum was studied with special reference to the flux across the paracellular route (3, 6). Two criteria were used to differentiate between the transand the paracellular calcium (45CaCl2)transport: com parison with simultaneously measured paracellular flux as measured with 3H-mannitol and the voltage dependence of calcium flux across clamped prepara tions. Calcium transport was measured in controls and in rats pretreated with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) [250 ng-kg-'-d'1, 1,25-OH2D3 sub-

ABSTRACT Concentration and voltage dependence of unidirectional 45Ca transport measurements indicated that ~ 60-70% of the mucosa-to-serosa calcium flux measured across the short-circuited rat duodenum, je junum and ileum is paracellular, with only 30—40%of the mucosa-to-serosa calcium transport cellular. The calcium flux from serosa to mucosa was purely para cellular in all segments. Duodenal calcium serosal-tomucosal flux was of the same order of magnitude as the mucosal-to-serosal paracellular movement. How ever, the serosal-to-mucosal flux of jejunum and ileum was twice as high. Therefore, net calcium absorption occurs only in the duodenum, whereas calcium is se creted in the jejunum and ileum by a passive paracellular route, presumably involving an anomalous solvent drag effect. Administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) led to an increase in the transcellular mucosa-to-serosa flux in the duodenum only. It also led to a stimulation of paracellular calcium flux in both directions in all three intestinal segments, with no change in net paracellular calcium absorption. Thus, the only vitamin D-related increase in calcium absorp tion was due to the increase in duodenal transcellular absorption. The mechanism by which 1,25-(OH)2D3 in creased paracellular flux is not known but may have resulted from an osmotic effect on the intercellular spaces. J. Nutr. 122: 672-677, 1992.

cutaneously, given for 3 d).

RESULTS

Short circuit and voltage clamp calcium flux calcium flux across the duodenum, jejunum and ileum. Electrical parameters and the simultaneously measured calcium and mannitol fluxes across the short-circuited tissue are listed in Table 1. Calcium mucosa-to-serosa (ms) transport was approximately the same in all intestinal segments of the controls.

INDEXING KEY WORDS:

•calcium transport •rats •small intestine •voltage clamp •1,25-dihydroxycholecalciferol

1Presented as part of a symposium: Current Concepts of Calcium Absorption, given at the 75th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlanta, GA, April 22, 1991. The symposium was sponsored by the American Institute of Nutrition. Guest editor for this symposium was F. Bronner, De partment of BioStructure and Function, University of Connecticut Health Center, Farmington, CT. 2Supported by Deutsche Forschungsgemeinschaft (DFG Ka 639/ 1-3, SFB38 "Membrane Research") 3To whom correspondence should be addressed: Medizinische Klinik, UniversitätMünchen,Ziemssenstr. l, D-8000 München2, Germany. 4 Abbreviations: MS, mucosal to serosal; SM, serosal to mucosal; 1,25-|OH)2-D3, 1,25-dihydroxycholecalciferol; J, unidirectional flux; I, current; R, resistance; PD, potential difference.

In vitro flux measurements in the absence of elec trochemical gradients across intact rat small intestinal epithelium have shown that calcium is absorbed only in the duodenum but is secreted in jejunum and ileum (1-6). Net movement across the short-circuited tissue and its dependence on metabolic energy have been taken as evidence for the presence of an active mech anism for calcium secretion in these segments. How ever, Nellans and Kimberg (7) clearly demonstrated that calcium secretion in the ileum is not a cellular process but is the result of an "anomalous solvent drag 0022-3166/92 $3.00 ©1992 American Institute of Nutrition.

672

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Paracellular Calcium Transport Across the Small Intestine12

673

SYMPOSIUM: CURRENT CONCEPTS OF CALCIUM ABSORPTION TABLE 1

Electrical parameters and unidirectional mucoaa-to-serosa ¡fmj, serosa-to-mucoaa {/.„! and net transport //„ell„. I.J of calcium ¡I1'*! and of the si multaneously measured paracellular marker mannitol (Jm*°! across the short-circuited tissues of controls (CO)and in rats pretreated with l^SfOH^D, (VDf*

DuodenumCOVDJejunumCOVDIleumCOVDP, 4*52+ (6)74 +22 (6)67+7 + 6* 2*91 ±-24 (9)103 (7)62 ±11*

7105± 1074+7103+ ±

+-2±-12

(6)83 ± 5 (6)100 ±13*

(9)127+ (6)137 lit

6-44 ± 1-37 ±1

±0.13.3 0.23.5 ±

6186 12*114+ ±

It28

(9)198 ±13 (7)74 ±26'

(10)243 ± 8 (6)106+ + 28*

8-43 ± 25-32±

0.23.1 ± ±0.33.8

15167+ 17§88

219+ 1*43 ±

±218±

3-38 ± ±-18±65710t33J"*°, 225+ (6)126 7 5122± (6)88 ± 6 ±0.33.0 (6)85+ ± 5 + 6* (6)•cm-2-h-'I«.84+7 ±0.3*R,/iA/cm2110± 5* (6)•crrr2-h-'JOB36+ 7*(9)(6)(10)(6)(6)(12)PDJnet14 ± 31KmV3.6 + 4* ± 7t(12)PDJ«,-33 ± 9tQcm233 1Number of experiments is given in parentheses; values are means + SEM. PD = transepithelial electrical potential difference; IK = shortcircuit current; R, = tissue resistance; negative sign indicates secretion. Significant difference when compared with the corresponding values of the controls: *P < 0.05, tP < 0.01, +P < 0.001, §P< 0.02. 2VD; 250 ng 1,25(OH)2D3given subcutaneously for 3 d.

The serosal-to-mucosal (sm) flux in the duodenum was smaller than the ms flux. Calcium absorption is there fore positive. In the jejunum and ileum, however, the sm calcium flux was more than twice as high as in the duodenum and ~ 20-30% higher when compared with the corresponding ms calcium transport in these seg ments. In contrast to what happens in the duodenum, calcium thus appears to be secreted across the jejunum and ileum. The concentration dependence of unidirectional calcium flux across the short-circuited tissue indicates that the ms calcium pathway in all segments consists of a saturable and a nonsaturable step. Sm flux, on the other hand, is a linear function of the calcium con centration throughout all segments (Fig. 1). The max imum transport capacity and the affinity of the carrier for calcium were about the same in duodenum, je junum and ileum. The nonsaturable component of ms calcium transport was about the same in the duo denum (19 ±5 nmol Ca-cm'^-h"1) and in the ileum (17 ±13 nmol Ca-cnT^rT1) but was less than half the value found in the jejunum (46 ±6 nmol Ca-cm~2'h~'). In the duodenum the nonsaturable component of ms calcium flux was approximately equal to the purely passive sm flux (19 ±5 vs 25 ±3 nmol Ca-cm"2-^1). The purely passive sm calcium flux in the jejunum, on the other hand, was only ~56% of the ms flux (72 ±4 vs 46 ±6 nmol Ca-cm"2-h"'). In the ileum the passive sm flux was more than three times higher than the corresponding ms flux (66 ±15 vs 17 ±13 nmol Ca-cnT^rT1). The calcium flux across the clamped tissue was evaluated by using the equation of Frizzell and Schultz (9): L = oJd-r1/2 + Jm, where £= exp (z-F-PD/R-T). J¡ is the unidirectional flux at any clamp PD and 0Jdis

the unidirectional flux under short-circuit conditions. Thus, Ji should be a linear function of £~l/2, with an intercept on the y-axis that corresponds to Jra and a slope that corresponds to 0Jd.Accordingly, the voltageindependent fraction of transport, which represents the nondiffusive, i.e., cellular transport, is given by the intercept with the y-axis (Jm).The slope of the line is a measure of the voltage-dependent, i.e., the diffu sive and probably paracellular, component of the flux. The voltage-clamp experiments also demonstrate that only ms calcium transport involves a voltage-in dependent cellular component that is similar in duo denum, jejunum, and ileum (15±9vs27±5vsl9 ±6 nmol Ca-cm"2-!!"1) (Fig. 1). From the voltage clamp data one can calculate that in the jejunum ~40% (67 ±7 vs 27 ±5 nmol Ca • cm 2• h '), and in the duo denum (50 ±9vs 15±9nmolCa'cm~2'h~1) and ileum (62 ±5 vs 19 ±6 nmol Ca-cm-2-^1) ~30%, of the total ms calcium flux is cellular, with the remainder passive and probably paracellular. The calculated frac tion of paracellular ms calcium flux in the jejunum (40 ±4-r1/2) and in the ileum (38 ±4-T1/2) is slightly higher than in the duodenum (29 ±9-£~1/2)In the duodenum paracellular calcium flux is equal in both directions (29 ±9 vs 32 ±5 • £~1/2|, but in the jejunum (40 ±4 vs 90 ±12 • r1/2) and in the ileum (38 ±4 vs 69 ±8 • £~1/2) the purely paracellular sm flux is about twice as high as the paracellular component and two to three times higher than the corresponding paracel lular calcium flux across the duodenum. Effect of 1,25-iOH^Dj on calcium flux across the short-circuited and clamped duodenum, jejunum and ileum. As shown in Table 1, transepithelial po tential difference (PD) across the ileum was slightly decreased as a result of 1,25-(OH)2D3 treatment, but

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FIGURE 1 Concentration (upperpanel) and voltage dependence (lowerpanel) of the unidirectional ms (Jms/solid symbols) and sm (Jsm,open symbols) calcium flux across the rat duodenum, jejunum and ileum. The concentration-dependent fluxes are measured across the short-circuited tissue. The saturable and nonsaturable fraction of the concentration-dependent ms calcium flux is given as solid lines; the dotted line represents the sm calcium flux. The sign of PD in the voltage clamp experiments is given with reference to that side of the tissue from which the flux is measured. The calcium concentration in the clamp experiments was 1.25 mmol/L; £= exp(z-F-PD/R-T). Values given are means ±SEM,n = 5-13.

no effect on PD was noted in the duodenum or je junum. Short-circuit current (Isc),however, was sig nificantly increased and tissue resistance (Rt)decreased some 40% in all segments after 1,25-(OH)2D3 treat ments. Treatment with 1,25-(OH)2D3 increased bidi rectional calcium flux throughout the intestine, as did flux of the paracellular marker, mannitol (Table 1). In the duodenum, calcium absorption was increased slightly, but 1,25-(OH)2D3 has virtually no effect on net calcium transport across the ileum. In the jejunum, 1,25-(OH)2D3 activated ms calcium flux by ~55% and bidirectional mannitol flux by ~100%, but 1,25(OH)2D3 was without effect on sm calcium flux. As a result jejunal calcium secretion was abolished. The voltage-clamp experiments demonstrate that 1,25-(OH)2D3 increases cellular ms calcium flux across the duodenum to only a minor degree, whereas the cellular fraction of ms calcium flux across the jejunum or the ileum was unaffected by the vitamin treatment (Fig. 2). However, the voltage-dependent, i.e., the para-

cellular fraction of ms calcium transport in the duo denum, the jejunum and the ileum, went up by ~ 100%. Paracellular sm calcium flux in the duodenum went up by 74% and by ~50% in the ileum. However, 1,25-(OH)2D3 had no significant effect on paracellular sm calcium flux across the jejunum (90 ±12 vs 81

±i8-r1/2).

Thus, as shown in Table 1, net calcium flux was increased in the duodenum after treatment with 1,25(OH)2D3. There was no effect in the ileum, whereas in the jejunum calcium secretion was virtually stopped.

DISCUSSION It is apparent from the above results that net calcium absorption in these animals was restricted to the duo denum, with calcium secreted in jejunum and ileum

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FIGURE 2 Voltage dependence of unidirectional ms (Jms/solid signs) and sm (Jsra,open signs) calcium flux across the duodenum, jejunum and ileum of controls (circles) and of rats pretreated with 1,25-(OH)2D3 150 ng-kg^-d"1 sc given for 3 d (VD3, triangles). The sign of PD is given with reference to that side of the tissue from which the flux is measured. calcium concentration was 1.25 mmol/L; £= exp(z-F-PD/R-T). Values given are means ±SEM,n = 6-10.

(Table 1). Of total transepithelial flux, only about onethird is routed transcellularly, whereas the remainder is moved paracellularly. In all likelihood all sm move ment is paracellular, inasmuch as calcium and man-

The

nitol movements from sm are parallel in quantity. This movement cannot be down an electrochemical gra dient under the present experimental set-up. An al ternate mechanism may involve an osmotic gradient.

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Õ-r.

120 -i

676

KARBACH ACTIVE Na*TRANSPORT

HYDROSTATIC GRADIENT

WATER ABSORPTION

WATER RECYCLING (ANOMALOUS SOLVENT DRAG)

FIGURE3 Working model to explainthe asymmetry of the convectively driven calciumfluxacrossthe paracellularshunt pathway in various segments of the rat intestine (for details see Discussion). Such a gradient would cause water to move from the lumen into the intercellular space. The resulting hy drostatic pressure in the intercellular space not only drives water out of the basal end of the space but may also induce water to flow back into the lumen. Net paracellular transport of a convectively driven solute depends on the hydraulic conductivity of the junctions for the solute and is determined by the amount and direction of paracellular water flow. As shown in Figure 3, a solvent drag may result if water is absorbed across the paracellular way. Such behavior, i.e., prevalence of paracellular ms calcium flux, has been reported for the rat proximal colon (10). On the other hand, an anomalous solvent drag may become effective if water is absorbed across the epithelial cell. In that case, the recycling of water flow may induce secretion of calcium across the tight junctions in a di rection opposite to transepithelial fluid absorption (8). This anomalous solvent drag effect, which has been described as the mechanism for the secretion of extra cellular markers in the rat jejunum (11) and for the secretory flux of calcium in the descending colon (12), may also be responsible for the secretion of calcium across the jejunum and ileum, as observed in the pres ent experiments. Studies of intestinal calcium transport have in gen eral focused on cellular mechanisms and their hor monal regulation. This is surprising because Wasserman and coworkers (13, 14), by studying the effect of cholecalciferol on unidirectional in vivo calcium flux across the chick and rat duodenum 25 years ago, con cluded that their "data are not consistent with the the ory that vitamin D3 operates through an unidirectional orientated active transport system but can be more readily explained on the basis of a permeability change at the mucosal cell." The present results seem to con firm this conclusion. The simultaneous increase of bi directional calcium flux and of the paracellular marker mannitol, as well as the decrease in electrical resis tance, strongly suggest that 1,25-(OH)2D3 enhances

calcium flux in both directions across the paracellular route in all small intestinal segments (Table 1, Fig. 2). Inasmuch as 1,25-(OH)2D3 increases paracellular calcium flux in both directions to the same extent, this effect cannot be seen when measuring net Ca transport in vivo (15). Yet the data reported here suggest that the major effect of 1,25-(OH)2D3 is not on the cellular transport process of calcium but on paracellular cal cium permeability (16). The mechanism underlying this effect is not clear. 1,25-(OH)2D3has been reported to increase calcium permeability by changing the phospholipid composition of the brush border mem brane (17). Conceivably the vitamin may also change the chemical structure of the junctional complex and thereby increase paracellular calcium flux. LITERATURE CITED 1. Walling, W.V.W.,Favus,M. J. &Kimberg,D.V. (1974) Effects of 25-hydroxyvitamin D3 on rat duodenum, jejunum, and ileum. Correlation of calcium active transport with tissue levels of vi tamin D3 metabolites. J. Biol. Chem. 249: 213-217. 2. Favus, M. J., Angeid-Backman, E., Breyer, M. D. & Coe, F. L. (1983) Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium transport. Am. J. Physiol. 244: Gl 11-G115. 3. Karbach, U. (1991) Segmental heterogeneity of cellular and paracellular calcium transport across the rat duodenum and je junum. Gastroenterology 100: 47-58. 4. Nellans, H. N. & Kimberg, D. V. (1978) Cellular and para cellular calcium transport in rat ileum: Effect of dietary calcium. Am. J. Physiol. 235: E726-E737. 5. Peters, J. & Binswanger, U. (1988) Calcium and inorganic phosphate secretion of rat ileum in vitro. Res. Exp. Med. 188: 139-149. 6. Karbach, U. & Rummel, W. (1987) Cellular and paracellular calcium transport in the rat ileum and the influence of 1,25dihydroxyvitamin D3 and dexamethasone. Naunyn-Schmiedeberg's Arch. Pharmakol. 336: 117-124. 7. Nellans, H. N. &. Kimberg, D. V. (1979) Anomalous calcium secretion in rat ileum: Role of paracellular pathway. Am. J. Physiol. 264: E473-E481. 8. Ussing, H. H. &. Johansen, B. (1969) Anomalous transport of sucrose and urea in toad skin. Nephron 6: 317-328.

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OSMOTIC GRADIENT

OSMOTIC WATER FLOW [SOLVENT DRAG)

SYMPOSIUM:

CURRENT

CONCEPTS

ABSORPTION

677

13. Wasserman, R. H. & Kallfelz, F. A. (1962) Vitamin D3 and unidirectional calcium fluxes across the rachitic chick duo denum. Am. J. Physiol. 203: 221-224. 14. Wasserman, R. H., Taylor, A. N. & Kallfelz, F. A. (1966) Vi tamin D and transfer of plasma calcium to intestinal lumen in chicks and rats. Am. J. Physiol. 211: 419-423. 15. Pansu, D., Bellaton, C., Roche, C. & Bronner, F. (1983) Duo denal and ileal calcium absorption in the rat and effects of vi tamin D. Am. J. Physiol. 244: G695-G700. lé.Karbach, U. & Rummel, W. (1986) Trans- and paracellular calcium transport across the colonie mucosa after short and long term treatment with 1,25-dihydroxyvitamin D3. Eur. J. Clin. Invest. 16:347-351. 17. Rasmussen, H., Matsumoto, T., Fontaine, O. & Goodman, D. B. P. (1982) Role of changes in membrane lipid structure in action of 1,25-dihydroxyvitamin D3. Fed. Proc. 41: 72-77.

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9. Frizzell, R. A. & Schultz, S. G. (1971) Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differ ence. J. Gen. Physiol. 59: 318-340. 10. Karbach, U. &.Rummell, W. (1987) Calcium transport across the mucosa of the colon ascendens and the influence of 1,25dihydroxyvitamin D3 and dexamethasone. Eur. J. Clin. Invest. 17:368-374. 11. Munck, B. G. & Rasmussen, S. N. (1977) Paracellular perme ability of extracellular space markers across rat jejunum in vitro. Indications of a transepithelial fluid circuit. J. Physiol. (Lond) 271: 473-488. 12. Karbach, U., Bridges, R. J. &.Rummel, W. (1986) The role of the paracellular pathway in the net transport of calcium across the colonie mucosa. Naunyn-Schmiedeberg's Arch. Pharmakol. 334: 525-530.

OF CALCIUM

Paracellular calcium transport across the small intestine.

Concentration and voltage dependence of unidirectional 45Ca transport measurements indicated that approximately 60-70% of the mucosa-to-serosa calcium...
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