Comp. Bioehem. Physioi., 1915, Vol. SIA, pp. 705 to 710. Pergamon Press. Printed in Great Britain
THE IN ~~~~~ TRANSPORT OF CALCIUM BY THE FROG INTESTINE AND THE EFFECT OF VITAMIN Da* D. R. ROBERTSON Department of Anatomy, State University of New York, Upstate Medical Center, Syracuse, N.Y. 13210, U.S.A. (Received 1 June 1974)
Abstract-l. Calcium transport of everted gut sacs of the frog &na pipierts was found to be higher in the duodenal than in the jejunal-&al segments. 2. Vitamin Ds with ingestion of caIcium ions increases calcium transport about 50 per cent higher in duodenum than in jejunurn-iieum resulting in hypercalcemia after 24 hr. 3. Sensitivity of gut to vitamin Ds is greater in September when plasma calcium IeveIs are high than in March when calcium levels are lower. 4. In normal animals a linear relationship exists between plasma calcium and capacity to transport calcium in small intestine.
MATERIAL!3 AND B4LETHODS
?-HE CAPACITYOf the small intestine t0 transport calcium across a con~n~ation gradient has been demonstrated in mammals (Schachter & Rosen, 1959; Harrison & Harrison, 1960; Schachter et al., 1960) and in birds (Sallis & Holdsworth, 1962). In both groups absorption is greatest in the proximal or duodenal segment of the small intestine although there are exceptions such as in the golden hamster where calcium is maximally absorbed in the ileum (Wilson, 3962). Vitamin D has been found to be the most important substance regulating intestinal calcium absorption in everted gut sacs of rabbits, rats and guinea pigs (Schachter & Rosen, 1959) and in sacs and in vim studies with chicks (Harwitz et ui., 1967). However, little is known about the absorption of calcium across the frog gut, although there have been studies on calcium movement in frog gastric mucosa (Forte & Nauss, 1966). In addition, studies on the effect of vitamin D in frogs have been limited to whole body responses of bones and calcium carbonate stores (Schlumberger & Burk, 1953). These authors demonstrated that with vitamin D8 and maintenance of frogs in water to which calcium salts had been added, calcium was deposited at storage sites and renal calcification occurred. They interpreted these responses as a demonstration that vitamin D was effective in enhancing the intestinal absorption of calcium while similar studies by Robertson (1968) substantiated these observations. However, there are no direct studies of calcium absorption in amphibian small intestine or the effects of vitamin D.
Adult male Rana pipiens (Northern variety) weighing 25-45 g body weight were obtained commercially from Gshkosh, Wisconsin, from August to March. Frogs were maintained unfed in a continual change of fresh water (05-1~0 m-equiv. calcium/l.) at 18-22°C and used within 2 weeks.
*Supported by Grant No. AM-14644 from the National Institutes of Health; Arthritis, Metabolism and Digestive Diseases.
Blood samples were collected from a leg artery in heparinized Natelson pipets, centrifuged and plasma calcium determined by microtitration (Robertson, 1969). calcium transport was studied using an everted g& sac technioue mod&d after Wilson & Wiseman (1954). The frog was first decapitated and the gut segment fro& midstomach to rectum removed and washed in buffer modified after Martin & DeLuca (1969) which was comaosed of: 100 mM sodium chloride. 10 mM fructose. 30 r&4 Tris buffer t&H 7.4) (Tris (hydromethyl) amino: methane) and 0125 mM calcium chloride except where noted. This simplified medium omits potassium and phosphate since these two ions have heen shown to inhibit calcium transport (Harrison & Harrison, 1963; Helbock et al., 1966; Martin & DeLuca, 1969). After washing, a length of polyethylene tubing (PE 50, ClayAdams) was passed through the hunen with the distal end tied to the tube and the gut everted. The intestine was flushed of bubbles and the sac filled with @2-0=4ml buffer and the proximal end tied at the pyloris. The sac was then ligated 0.5 cm distal to the junction of the biliary duct to provide a proximal or duodenal segment and a distal or jejunal-ileal segment. The sac was then placed in a 150-ml flask containing 100 ml buffer and incubated for 30min at 20-22°C under continual aeration with 95% oxygen. After this period the sac was removed, carefully blotted and the contents drained into small vials. Calcium inside the sac was determined by microtitration as for blood and the data expressed as the ratio (S/M) of caIcium ~n~nt~tion in the serosal medium
D. R. ROBERTSON
(inside the sac) to the concentration of calcium in the mucosal medium (outside the sac). This procedure estimates the “concentrative transport system” in which initially the serosal and mucosal surfaces are bathed in a medium containing the same concentration of calcium (0.125 mM calcium).
Jejunum - ileum
.P 54 1. 2
Treatment procedures Crystalline vitamin D, (cholecalciferol, Sigma Chemical Co.) was dissolved in oil (Wesson) and administered i.m. at specified doses (25,000-100,000 units/ frog). In some groups, frogs were kept in fresh water to which 0.05 M calcium chloride was added and maintained for specified periods of time. Calcium ions enter
the frog through direct ingestion (Schlumberger & Burk, 1953) since calcium ions are not transported across skin (Huf & Wills, 1951). Analysis of data Significance of differences of data was analyzed by use of Student’s t-test and the linear regression curve was calculated by standard procedures (Snedecor & Cochran, 1967).
The entire small intestine of a 35-g frog is approximately 8 cm in length, and the duodenal segment about 1.5 cm. The duodenum is not sharply delineated from the remainder of the small intestine but near its distal point it has a mesenteric attachment to the liver and mesenteric arteries which serve as a landmark for ligature. Macroscopic observation of everted gut sacs studied showed no delamination of mucosa after 30 min of incubation, but changes were observed after 60 min which was accompanied by a decline in the rate of transport, indicating a departure from linear uptake. All
0 ,o z
=; .g E
s I 0.125
Fig. 1. Effect of various concentrations of calcium in the incubation buffer medium on the S/M calcium ratios of duodenal and jejunal-ileal segments of small intestine of adult male frogs in August. Maximal transport occurs in duodenum at lower calcium concentrations, while inhibition occurs at higher concentrations. (Four to six animals in each group.) Bars indicate standard deviation.
E .? 0
Fig. 2. Response of duodenal and jejunal-ileal gut segments from frogs in September with exposure to calcium ions alone, 25,000 units vitamin D, alone or vitamin D3 and calcium after 2 days’ treatment. Figures above column are pIasma calcium levels. Exposure to calcium alone caused a slight increase in S/M and plasma calcium while vitamin D3 alone increases S/M significantly (P = @Ol) with no increase in plasma calcium. Exposure to vitamin Da and calcium ions increased significantly (P = 0.01) the S/M ratio of both segments and blood calcium levels over initial levels. Duodenum was significantly elevated (P = 0.01) over jejunal-ileal segments except in the last group. (Four to six frogs in each group.) studies were performed with 30 min incubation to insure that the S/M ratio would not represent a final concentration. Figure 1 illustrates the difference in S/M ratios of duodenal and jejunal-ileal segments at various concentrations of calcium. Since an inhibitory effect was noted at higher concentrations the buffer medium of all studies contained 0.125 mM calcium chloride. Throughout this series the duodenal segment was significantly higher than the jejunalileal segment at the two lower concentrations. The difference between the two segments was greater with 0.25 mM calcium chloride but the greatest transport in both segments occurred with 0.125 mM CaCl,. In a separate series, frogs exposed to 0.05 M CaCl, alone showed an insignificant but consistent increase in the S/M ratio and plasma calcium; while 25,000 units vitamin D, alone was effective in elevating the S/M ratio but the plasma calcium was unchanged or occasionally lower after 2 days (Fig. 2). With administration of vitamin D, and calcium ions available the S/M ratio was significantly enhanced (P = 0.01) which was accompanied by a concomitant hypercalcemia. In each situation the S/M ratio of the duodenum was consistently higher than the jejunum-ileum although they were not significantly different with vitamin D, and calcium after 2 days’ treatment.
Calcium transport in frog intestine
Table 1. Dose sensitivity of R. pipiens to vitamin DI1 in the presence or absence of calcium (O-05 M CaCla) after 2 days of treatment during the months of September to March 'UmePeriod
Dose wt. D3 Units/frog
During the course of these studies a pattern developed which suggested a relationship of sensitivity of gut to vitamin DI with the time of year (Table 1). A dose of 25,000 units D8 was sticient during September-January to significantly (P = 0.01) elevate the S/M ratio with calcium available after 2 days; while the same dose in March with calcium was without effect. A signifkant response (P = 0.01) was seen at this time with 100,000 units DI with calcium in the surrounding water. Also apparent during this 7-month period was a gradual decline in plasma calcium from the high of 7.4 mg/lOO ml in September to 6.6 mg/lOO ml in March. During the September period frogs were injected with 25,000 units DI, and maintained in calcium water with the response observed over a period of 6 days. A signikant increase (P = 0.05) in the S/M ratio of both duodenal and jejunal-ikal segments was observed after 1 day concomitant with hypercalcemia 1.2 mg/lOO ml above initial levels (Fig. 3). On the second day the S/M ratio continued to rise, the duodenal segment attaining a maximum ratio of 6.1 and the jejunal-ileal segment 4.5. However, the plasma calcium levels continued to decline to a level about @4 mg/lOOml over initial levels after the sixth day coincident with a decline in the S/M ratio of both gut segments. A similar study was made in March over a S-day period when frogs were relatively less sensitive to vitamin Dg and only responded to high doses (Table 1). In this series (Fig. 4) with a dose of 100,000 units D8 slight differences were seen in the S/M ratios of duodenal and jejunal-ileal segments during the tirst 2 days although both segments were elevated from initial levels. At the fifth day the duodenal segment had a significantly higher S/M
ratio (P = 0.01) when compared to the jejunal-ikal segment. Further, maximal hypercalcemia occurred on the second day from an initial plasma calcium of 6.7 mg/lOO ml to 8.2 mg/lOO ml while after 5 days it fell to 7.4 mg/lOO ml.
0 Jejunum- ileum
Fig. 3. Response of frogs to 25,000 units vitamin D8 and calcium ions during September. The S/M ratio of duodenal segment was signikantly higher (P = O-05) than jejunal-ileal segment on the first and second day which was concomitant with a maximal hypercakemia after the first day. (Four animals in each group.) Bars represent standard deviation.
D. R. ROBERTSON . Duodenum urn- iIeu*i 3.0
I I 0
y 90.4666 2.0
Fig. 4. Response of frogs to 100,000 units vitamin D, and calcium ions during March. The S/M ratio of the duodenal segment is significantly higher than the jejunalileal segment after 2 days (P = O-01). Plasma calcium levels show no change after 1 day but rise to maximal levels (1.2 mg/lOO ml above initial values; P = 0.01) after 2 days. After 5 days plasma calcium levels remain slightly elevated (P= 0.05). (Four to six animals in each
group.) Bars represent standard deviation. In view of the spectra of responses to vitamin D, and changing plasma calcium values, the whole gut S/M response (Y) of normal untreated frogs from September to March was plotted as a function of plasma calcium (X) (Fig. 5). The data represent the means of various groups of frogs (12-28 animals/point) acquired during this period, which can be expressed by the linear regression equation Y = 0.4666X- O-66. Low plasma calcium is associated with a reduced capacity to transport calcium. DISCUSSION The small intestine of the frog, while not grossly differentiated along its length, does show a greater capacity to transport calcium across the duodenal segment. The decreasing capacity of the everted frog gut to transport calcium above 0.125 mM CaCl, suggests that the calcium transport mechanism may become saturated resulting in maximal transport. A similar situation observed in dogs (Cramer, 1963) has been cited as an argument for a carrier-mediated calcium transport mechanism. With the injection of vitamin D3 both segments show a variable degree of sensitivity with the duodenum exhibiting about a 50 per cent increased capacity when compared to jejunal-ileal segments during periods of maximal absorption. Forte &
mg / 100 ml
Fig. 5. Linear regression curve of S/M ratio of whole small intestine (Y) and plasma calcium levels (X) from September to March. The relationship indicates that low plasma calcium is associated with a reduced capacity to transport calcium. Number of animals indicated for each point with S.E.M. indicated by vertical bar.
Nauss (1966) demonstrated that calcium flux across bullfrog gastric mucosa was greater in the serosa to mucosa direction and that it was not possible to determine unequivocally that calcium transport occurred from lumen to blood. Thus the duodenal segment would appear to represent the most active site of calcium absorption in frogs, following the general pattern of most mammals studied (Avioli, 1972). However, since no data are available regarding the transit time of food in any segment, the jejunal-ileal segments may be more important in the long-term absorption of calcium. Previous studies by Schlumberger & Burk (1953) and Robertson (1968) indicated that vitamin D alone was not sufiicient to induce hypercalcemia but with calcium ions available in the surrounding media, hypercalcemia occurred. In the present study vitamin D3 was effective in increasing the whole gut S/M ratio and calcium ions enhanced this response within 24 hr in September frogs. With a fourfold increase in the dose of vitamin D, in March frogs, hypercalcemia was not as pronounced and maximum expression was delayed until the second day. Thus the dose-response of frogs after 2 days’ treatment (Table 1) may be misleading since the maximal response will vary with time of year. Examination of Figs. 3 and 4 reveals that the response of the jejunal-ileal segment was similar in both cases although the dose was different. However, the duodenal segment showed a signilicant delay in the rise in S/M ratio during the March series which was coincident with the delay in acute hypercalcemia. It would appear that the duodenal segment was the primary or limiting factor in the expression of hypercalcemia under these conditions. Further, it represents the segment with the greater capacity to absorb calcium in addition to its greater sensitivity
Calcium transport in frog intestine to vitamin Ds. Non-injected frogs exposed only to calcium also exhibited a slight but consistent elevation in the S/M ratio which may only be an apparent increase since it could represent intracellular calcium released during incubation. However, the slight elevation in plasma calcium in these frogs does indicate that absorption of calcium was probably increased. The failure of vitamin Ds treated frogs kept in fresh water to exhibit au elevation in plasma calcium, and in some cases a slight depression, has been noted by others (Schlumberger & Burk, 1953). This non-response or depressant effect may be due to calcitonin release during vitamin Ds administration which has been demonstrated in mammals (Capen SL Young, 1969). Recent evidence suggests that parathyroid hormone facilitates the conversion of the vitamin Ds metabolite 25hydroxycholecalciferol to the active 1,25 dihydroxycholecalciferol form (Rasmussen et al., 1972; Fraser & Kocieck, 1373). This activated metabolite is necessary for the formation of an intestinal calcium binding protein (Norman et al., 1971) required for intestinal calcium transport (Wasserman & Taylor, 1966). It is thought that with increasing hypercalcemia the calcium ion inhibits this conversion by the direct effect of calcium on the rates of parathyroid and calcitonin secretion (Boyle et al., 1971). The response of frogs over several days both in September and in March when injected with vitamin Ds is compatible with this suggestion since the resultant hypercalcemia is followed by a decline in the S/M ratio. This might suggest that secretion of parathyroid hormone may have been suppressed when blood calcium was silently elevated or that calcitonin was released in response to the initial hypercalcemia. However, a significant feature of anurans is the atrophy and decreased cytological activity displayed by the frog parathyroid glands during the winter 1929). Waggener, 1926; months (Romeis, McWhinnie & Lehrer (1972) have presented evidence to suggest that the seasonal depression in plasma phosphorus and urinary calcium concentrations during the winter months are due to the depressed activity of the parathyroids. The linear relationship in the capacity of frogs to absorb calcium over a wide range of blood calcium levels strongly suggests that the depressed blood calcium levels in late winter may in part be a reflection of a decreased capacity to transport calcium from the intestine. Further, the need for higher doses of vitamin D3 in March and the delay in the maximal expression of hypercal~emia and S/M ratios of gut when compared to September animals may reflect a depression of general metabolic activity. On the other hand, in view of the evidence regarding the role of parathyroid hormone and vitamin D metabolism these associated phenomena may represent the collective expression of a decreased capacity to convert
vitamin Ds into the active metabolities required for intestinal transport of calcium ~ckn~w~~~~~nt-~he author appreciates the technical assistance of Mrs. N. Cheever.
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BoYJ_EI. T., GRAY R. W. & bhJCA H. F. (1971) Regulation by calcium of in uivo synthesis of’ 1,25~y~oxvchol~ciferol and 21.2S~~vdroxvlcholecaliiferoi Proc. natn. Acad. Sci. U.S.A. &,2131-2134. CAPENC. C!. & YOUNGD. M. (1969) Fine structural alterations in thyroid parafollicular cells in cows in response to experimental hypercalcemia induced by vitamin D. Am. J. Path. 55, 365-382. CRAMER C. F, (1963) Qualitative studies on the absorption and excretion of calcinm from Thiry-vella intestinal loops in the dog. In The Transfer of Calcium and Strontium Across Biological Membranes (Edited by WHEN R. H.), pp. 75-84. Academic Press, New York. FORTEJ. G. & NAUSSA. H. (1966) Calcium movements in isolated bullfrog gastric mucosa. Am. J. Physiol. 211, 239-242.
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MAR~N D. L. & DELUCA H. F. (1969) Intluence of sodium on calcium transport by the rat small intestine. Am. J. Physiol. 216, 1351-1359. NORMAN A. W., MYRTLEJ. R., M~GET R. J., NO~ICKI H. G., WILL&S F. & POPJAKG. (1971) 1,25 Dihv~oxvchole~lciferol: ident~~tion of the oroposed active form of vitamin D, in the intestine.- s&ence, Wash. 173, 51-54.
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D. R. ROBER’ISON
ROBERTSOND. R. (1969) The ultimobranchial body of Rana pipiens-VIII. Effects of extirpation upon calcium distribution and bone cell types. Gen. & compar. Endocr. 12,479-490. ROBERTSOND. R. (1968) The ultimobranchial body in Rana pipiens-IV. Hypercalcemia and glandular hypertrophy. Z. Zelt’forsch. mikrosk. Anat. 85, 441452. ROMEIS B. (1926) Morphologische und experimentelle Studien tiber Epithelkorperchen der Amphibien-I. Die Morphologie der Epithelkorper der Anuren. Z. Anat. Entwickl. Geschichte. 80, 547-578. SALLISJ. D. & HOLDSWORTHE. S. (1962) Influence of vitamin D on calcium absorption in the chick. Am. J. Physiol. 203, 497-505. SCHACHTERD., DOWDLE E. B. & SCHENKERH. (1960) Active absorption of calcium by the small intestine of the rat. Am. J. Physiol. 198, 263-268. SCHACH~ERD. & ROSENS. M. (1959) Active transport of Ca*6 by the small intestine and its dependence on vitamin D. Am. J. Physiol. 196, 357-362.
SCHLUMBERGER H. G. & BURK D. H. (1953) Comparative study of the reaction to injury-II. Hypervitaminosis D in the frog with special reference to the lime sacs. Arch. Parh. 56, 103-124. SNEDECORG. W. & C~CHRAN W. G. (1967) Statistical Methods, 6th Edn. Iowa State University Press, Ames. WAGGENERR. A. (1929) A histological study of the parathyroids of anura. J. Morphol. 48, 143. WASSERMANR. H. & TAYLORA. N. (1966) Vitamin D induced calcium binding protein in chick intestinal muwsa. Science, Wash. 152, 791-793. WILSON T. H. (1962) Intestinal Absorption. Saunders, Philadelphia. WILSONT. H. & WI~EMANG. (1954) The use of sacs of everted small intestine for the study of the transference of substances from mucosal to serosal surface. J. Physiol., Land. 123, 116-119. Key Word Index-Amphibian; anuran; frog; Rana pipiens; calcium transport; duodenum; small intestine; vitamin D,; plasma calcium.