- Email: [email protected]
Biphasic recovery of vitamin D-dependent Ca2+ uptake by rat intestinal Golgi membranes
GASTROENTEROLOGY
78325-332,
19RO
Biphasic Recovery of Vitamin DDependent Ca2+ Uptake by Rat Intestinal Golgi Membranes JULIA A. MacLAUGHLIN, MILTON and ROGER A. FREEDMAN
M. WEISER,
Department of Medicine, Harvard Medical School, Medical Massachusetts General Hospital, Boston, Massachusetts
A previous study had demonstrated that rat intestinal epithelial cell GoJgi, lateral-basal, and microvillus membrane vesicles translocated Ca’+, and that this process was markedly decreased in vitamin D-deficient rats. In the present study the kinetics of recovery of CaZ+ uptake by intestinal GoJgi vesicles was examined in vitamin D-deficient rats after a single i.v. injection of 1.25 ng 1,25-dihydroxycholecalciferol (1,25-(OH),D,). Calcium uptake was measured by determination of 4sCar+ associated with GoJgi membranes after collection by micropore filtration. GoJgi membranes recovered rapidly (66% in 15 min, 83% in 30 min) but then quickly returned to prerepletion levels by 8 hr. This was followed by a second phase of recovery starting at 48 hr, with approximately 80% recovery by 96 hr which was maintained at least to 120 hr. By comparison, partial recovery of calcium uptake by microvillus membrane vesicles was first detected at 15 hr and full recovery did not occur until 72 hr; a biphasic pattern to microvillus membrane recovery was not apparent. The vitamin D-dependent recovery of Ca”’ uptake by Goigi membrane vesicles was also compared to studies with gut sacs which showed 50% recovery by 3 hr and 100% recovery by 6 hr; gut sac recovery was sustained for at least 120 hr. The recovery of Co.‘+ uptake by GoJgi was shown to be specific for Received August 6, 1979. Accepted September 24, 1979. Address requests for reprints to: Milton M. Weiser, Division of Gastroenterology and Nutrition, State University of New York at Buffalo, Clinical Center Annex, CC 1186, 462 Grider Street, Buffalo, New York 14215. This research was supported by grants from the National Instltutes of Health (CA-16703) and the American Cancer Society (BC93). Research support was also provided by the Howard Hughes Medical Institute when M.M. Weiser was a Howard Hughes Investigator. The authors wish to thank Dr. Kurt J. Isselhacher for his generous support and advice, and Dr. M. Holick for his advice and discussions. 0 1980 by the American Gastroenterological Association OOlfi-5085/80/020325-07$02.25
Services
(Gastrointestinal
ITnit),
1,25-(OH),D,. The first peak of recovery of Caz+ uptake by intestinal GoJgi membrane vesicles is one of the earliest reported effects of 1,25-(OH),D,. The biphasic nature of recovery demonstrated by GoJgi membranes suggests that there may be more than one mechanism of action of 2,25-(OH),D, on the intestine. Absorption of Ca2+ through the intestinal epithelial cell appears to be a summation of passive diffusion and an active transport process’ with the latter process dependent on vitamin D.’ The prevailing concept is that the active metabolite of vitamin D, l,&dihydroxycholecalciferol (1,25-(OH),D,),* acts on the intestine in a way analogous to that described for steroid hormones.” Indeed, specific cytosolic receptors in the intestine have been described for 1,25(OH),D,, and the synthesis of a number of intestinal proteins thought to be related to CaZ+ transport has been shown to be stimulated by the administration of 1,25-(OH),D, to vitamin D-deficient animals.4-14 The problem has been to show a direct relationship between the synthesis of these proteins and the change in Ca’+ transport induced hy 1,25-(OH),D,,. The Ca”’ binding protein (CaBP) described by Wasserman and Taylor is the best characterized of these proteins, and its synthesis appeared to be correlated with vitamin D-dependent changes in intestinal transport”; however, more recent studies have seriously questioned this correlation.‘” CaBP is isolated from the soluble fraction while other proteins felt to be responsible for the vitamin D-dependent Ca’+ transport process have been identified with mitochondria”,‘“,” or lateral-basal membranes.‘” It has not been established where the vitamin Ddependent Ca2+ transport process is located within Abbreviations used in this paper: 1,25-(OH),D,, 1,25-dihydroxycholecalciferol: CaBP, calcium hinding protein; S/M ratio, ratio of [Ca2+J in serosal fluid to that in mucosal fluid. l
326
MacLAUGHLIN
ET AL.
the cell. It has been suggested that the vitamin D-dependent Ca’+ transport process requires sequestration of Ca’+ by a subcellular organelle such as mitochondria.” Electron probe microanalysis studies of Warner and Coleman’9 have detected intracellular calcium in the supranuclear region of the intestinal cell where Golgi are concentrated, and these discrete areas are reduced in vitamin D-deficient animals and increased after repletion. A previous study from our laboratory had demonstrated the feasibility of studying Ca’+ uptake in membrane vesicles prepared from subcellular fractions of rat intestine.” We were able to demonstrate that Golgi membranes have the most rapid initial rate and the highest equilibrium level of Ca2+ uptake when compared to lateral-basal and microvillus membranes. We have also shown that Ca’+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by extensive binding of Ca’+ to the vesicle; other data suggest the existence of a cation counter-transport system in these vesicles.Z1 Furthermore, Golgi membrane vesicles prepared from vitamin Ddeficient rats were shown to have a significantly decreased Ca2+ uptake capability.‘” We now present data on the kinetics of recovery of Ca’+ uptake by intestinal epithelial cell Golgi membrane vesicles prepared from vitamin D-deficient animals after i.v. injection with 1,25-(OH),D,. The recovery of Ca’+ uptake appeared to require 1,25-(OH),D, specifically, and the time-course of recovery by Golgi membranes was biphasic. The first peak of Ca2+ uptake recovery by Golgi membrane occurs within 15-30 min gfter repletion and represents one of the earliest reported effects of 1,25-(OH),D,.
Materials and Methods Animals Male weanling albino rats were obtained from the Holtzman Co. (Madison, Wisconsin) at 21 days of age and maintained from 3-4 wk on a complete pellet diet (Purina lab chow, Ralston Purina Co., St. Louis, MO.). In order to make the animals vitamin D depleted, weanling male rats of the same strain were maintained on a purified low calcium diet containing 0.02% (wt :wt) calcium and 0.30% (wt : wt) phosphorous with no vitamin D and housed under incandescent light for 3-4 wk. Animals were starved for 18 hr before death by cervical dislocation; the animals had water available at all times. Vitamin D-deficient animals were repleted by an intrajugular injection of 125 ng of 1,25-(OH),D, or 250 ng of 25-(OH)D, in 0.05 ml of 95% (vol : vol) ethanol, whereas control animals received 0.05 ml of 95% (vol: vol) ethanol. Both metabolites of vitamin D were generous gifts of Drs. Michael Holick and Mary Clark, Massachusetts General Hospital. Where indicated, nephrectomy was performed on the day preceding an experiment and the rats w&e returned to incandescent light-
GASTROENTEROLOGY
ing between tabolites.
nephrectomy
and injection
Vol. 78, No. 2
of vitamin
D me-
Tissue Preparations The small intestine was removed, washed with 0.154 M-NaCl, slit open longitudinally, and the mucosa obtained by scraping with glass microscope slides. A purified microvillus membrane was prepared by the method of Hopfer et al. ZZGolgi membrane vesicles were prepared as previously described.” Everted gut sacs were made from the first 6 cm of duodenum according to the method of Schachter et a1.23 Ca’+ transport studies across gut sacs were performed as described by Martin and DeLucaz4 and the results expressed as the ratio of serosal [Ca”] to mucosal [Ca”] (S/M ratio).
Assay
of Ca2+ Uptake
The ability of membrane vesicles to take up Ca2+ was measured within 24 hr of vesicle preparation. The assay for Ca2+ uptake was as previously described.“’ Briefly, the membranes (final protein concentration of 0.01-0.05 mg/ml) were incubated at 25°C in a medium containing 20 mM Na-Hepes buffer, pH 8, 100 mM NaCl, 2.5 mM MgCl,, and 0.5 mM “5CaC1, (usual specific activity = 1 &i/ymol) in a total volume of 4.2 ml. At designated time intervals, 1.0 ml aliquots were removed, filtered through nitrocellulose filters and the retained radioactivity determined as previously described.“’ All radioactive materials and Liquifluor were purchased from New England Nuclear (Boston, Mass.). Protein was determined by the method of Lowry et a1.25
Results We had previously demonstrated that Ca*+ uptake by Golgi and lateral-basal membranes is reduced when the membranes are prepared from vitamin D-deficient rats. To test whether this reduction of Ca’+ uptake was directly related to the action of vitamin D, 1,25-(OH),D, was injected intrajugularly (125 ng in 0.05 ml 95% (vol:vol) ethanol/rat) and the rate of recovery of Ca*+ uptake determined. Normal and vitamin D-deficient rats given 0.05 ml 95% (vol:vol) ethanol served as controls. Golgi membrane vesicles showed a recovery of 60-80% of equilibrium levels of Ca’+ uptake (uptake after 20 min) by 30 min after repletion but appeared to rapidly lose their ability to take up Ca2+ (Table 1 and Figure 1). Recovery of Ca2+ uptake by Golgi membrane vesicles was also manifested by increases in initial rate of uptake (Table 1 and Figure l), but the increase in uptake was not as great as the increase in equilibrium binding capacity. The time-course of recovery of Ca’+ uptake at equilibrium by Golgi membrane vesicles is shown in Figure 2 and is compared to the recovery of Ca’+ uptake by gut sac preparations.
February
Table
VITAMIN
1980
1.
Effect of 1,25-(OH),D,
on ca*+ Uptake by Intestinal
D REPLETION
Go@ Membrane Ca’+ Uptake
Time after administration 1,25-(OH)* cholecalciferol 0
of (min)
Normal [14]” Vit-D Deficient
15
I 31
30
I 31
60
1 31
120
I 51
240
I 21
480
I 31
15 set 161 + 8.8 43 f 5.0 71 f 24 (24%)” 87 -t 11 (37%) 83 f 9.6 (34%) 69 + 8.4 (22%) 64 f 7 (18%) 38 + 8.7 (-0%)
[18]
OF CA”
Vesicles
(nmoles/mg
from
protein
LJPTAKE
Vitamin
BY GOLGI
D-Deficient
327
Rats
-f SEM) at
1 min
5 min
20 min
303 + 14 79 f 7.4 161 f 50 (37%) 193 -t 43 (51Y0) 201 f 42 (54Y”) 111 * 5.4 (14%) 103 -+ 10 (11%) 88 f 13
493 + 21 137 f 21 354 -t 67 (61%) 386 + 50 (70%) 294 + 35 (44%) 216 + 5.2 (22%) 172 -c 7.5 (10%) 140 + 13 (-0%)
587 f 19 175 f 14.5 447 -+ 45 (66%) 517 * 39 (83%) 331+ 36 (38%) 254 -t 9.7 (19%) 211 + 12 (9%) 167 f 18.5 (-0%)
(-0%) ’ Percent
recovery
to “normal,”
i.e., 100 x (uptake
” No. of animals: Golgi were prepared from individual rats and were repleted animals - uptake by D-deficient animals)/uptake by normal
not pooled. animals.
by
Uptake by Golgi membrane vesicles showed a fractional recovery of 0.66 by 15 min after repletion, appeared to peak at 30 min and returned to prerepletion levels by 8 hr (Figure 2). Gut sac preparations tested for their ability to transport Ca2+ against a concentration gradient after repletion with I,25 (OH),D, showed full recovery by 6 hr (Figure 2). These results were interpreted as being compatible with a process that had begun in the Golgi and had then resulted in recovery of transport across the gut sac; this interpretation suggested that recovery of Ca2+ uptake by another membrane fraction might more precisely coincide with gut sac recovery. Therefore, microvillus membrane vesicles were tested for Ca’+ uptake despite earlier evidence that these membranes had a relatively low capacity for
Ca’+ uptake when compared to Golgi and lateralbasal membranes.2” memFigure 3 shows Ca2+ uptake by microvillus brane vesicles from normal and D-deficient rats and illustrates again the relatively lower Ca*+ uptake by microvillus membranes. This lower Ca”+ uptake did not appear to be due to a failure to form vesicles since the microvillus membrane vesicles prepared for these experiments retained their ability to translocate glucose (data not presented) to the same degree as previously reported.” Microvillus membranes prepared from vitamin D-deficient animals
Normal 30 ml” 15 ml”
I hr 2 hrs D-Deflclent
Figure Figure
1. Ca2+ uptake by intestinal Golgi membrane vesicles from normal, vitamin D-deficient, and 1,25-(OH),D,-repleted rats. Vitamin D-deficient animals were sacrificed at different intervals after a single injection of 125 ng of 1,25(OH),D, in 0.05 ml of 95% (vol:vol) ethanol; normal and vitamin D-deficient control rats received 0.05 ml of 95% (vol:vol) ethanol. Normal (0). vitamin D-deficient (A); repleted: 15 min (U), 30 min (O), 1 hr (m, 2 hr (A).
2. Comparison of times of recovery of Ca2+ uptake by Golgi membrane vesicles, and of Ca” transport across everted gut sacs after repletion with 1,25-(OH),D,. Fractional recovery was calculated from the 20 min uptake value (see Table 1) using the ratio (repleted-vitamin Ddeficient)/(normal-vitamin D-deficient). Fractional recovery of Ca2+ transport across gut sacs was calculated after determination of serosal/mucosal Ca2+ concentrations using the same ratio above. Golgi (0); everted gut sacs (0).
328
MacLAIJGHLIN
I
ET AL.
2 MINUTES
Figure
3. Ca2+ uptake by intestinal microvillus membrane vesicles from normal and vitamin D-deficient rats. Normal (O), vitamin D-deficient (0). Values represent the mean k SEM (18 different preparations of 18 different rats).
showed a significantly lower Ca’+ uptake, but the decrease was not as great as that found for Golgi membrane (Figure 3). It was difficult to obtain any statistically significant stimulation of Ca’+ uptake by microvillus membrane vesicles for the earlier time points following repletion with 1,25-(OH),D,. In particular, no significant recovery of Ca’+ uptake by microvillus membranes was detected at the time of gut sac recovery shown in Figure 2. ability by Golgi The total loss of Ca*+ uptake membranes 8 hr after repletion with 1,25-(OH),D, prompted an evaluation of later time points since it had been previously shown’” that recovery of intestinal Ca”+ transport is maintained for up to 7 days after one injection of 125 ng of 1,25-(OH),D,. As illustrated in Figure 4, Golgi membranes exhibited a second recovery of Ca’+ uptake starting 48-72 hr after repletion. The rate of this recovery was slower than that observed at 15-30 min postrepletion, but the increased uptake was sustained for up to 120 hr. Microvillus membranes also showed a slow recovrepletion with vitamin ery of Ca’+ uptake following D. They reached full recovery by 72 hr at which time the Golgi membranes showed a fractional recovery of only 0.3-0.45. Throughout this time, transport by
GASTROENTEROLOGY
Vol. 78, No. 2
gut sacs was maintained at full recovery levels (Figure 4). To test whether the recovery of Ca2+ uptake by Golgi membrane vesicles was specific for 1,25(OH),D,, results were compared to those from animals repleted with 25-(OH)D,. Ca’+ uptake by Golgi membranes and Ca’+ transport by gut sacs were evaluated after an intrajugular injection of 250 ng of 25-(OH)D,. The results for Golgi membranes are illustrated in Figure 5 which shows that 25-(OH)D, repletion, in contrast to repletion with 1,25-(OH),D,, caused a later recovery of Ca2+ uptake by Golgi membranes, with the first peak of full recovery occurring at 3 hr instead of 30 min. The recovery of uptake induced by either derivative of vitamin D rapidly disappeared after the peak response. Gut sacs also showed a delayed response to 25-(OH)D, repletion. Furthermore, while nephrectomy had no effect on the recovery of Ca*+ uptake after administration of 1,25-(OH),D,, it abolished the recovery that could be seen after giving 25-(OH)D, (Figure 6). These results suggest that the recovery of Ca2+ uptake by Golgi membrane vesicles which is observed after repletion with 25-(OH)D:, occurs only after renal Lxhydroxylation of 25-(OH)D, to 1,25-(OH),D,.
Discussion The properties of intestinal Ca2+ transport have been defined primarily by in vivo studies and in experiments using gut sac preparations.2’~Z4.“7.28 Recently some progress has been made toward characterizing the subcellular basis of intestinal Ca2+ transport, particularly the vitamin D-dependent processes. However, it is not known whether the vitamin D-dependent step is located in the microvillus membrane, the lateral-basal membrane, an intracellular organelle, the cytoplasm, or, indeed, in more than one cellular location. Several vitamin D-dependent Ca’+ binding proteins and Ca’+ dependent en-
Figure
4. Comparison of times of recovery of Ca’+ uptake by Golgi, microvillus membrane vesicles, and of Ca2+ transport across everted gut sacs after intrajugular administration of 1,25-(OH),D,. Microvillus membrane (A), everted gut sac (a), Golgi (0).
February
1980
zymes have been localized in the microvillus membrane, including CaBP,” a particulate Ca2+ binding complex,“’ and a Ca”+-stimulated ATPase.‘” Their proposed role in Ca2+ transport is based on their dependence on vitamin D for activity and their interaction with Ca’+ in vitro. More recently, three laboratories,“.“,14 have detected synthesis of microvillus membrane proteins in response to 1,25-(OH),D,. The times after vitamin D administration at which the synthesis of these proteins was detected varied from 4 to 15 hr. Rasmussen et a1.17 have described a vitamin D-dependent Ca*+ uptake by chick intestinal microvillus membrane vesicles which responds to repletion by 7 hr, and this response was unaffected by cycloheximide, a protein synthesis inhibitor. In addition to the microvillus membrane, other parts of the intestinal cell’R~2R~30-32 have been implicated as the site of the vitamin D-dependent transport process. The possibility that calcium absorption may be related to secretion, along with the supranuclear sequestration of calcium detected by x-ray microanalysis,‘” prompted our laboratory to examine Ca2+ uptake by membrane vesicles prepared from the Golgi apparatus and lateral-basal membranes as well as from microvillus membranes. The earlier studies had indicated that Golgi membrane vesicles had the highest initial rate and equilibrium level of Ca’+ uptake when compared to lateral-basal and microvillus membrane vesicles and that this uptake was significantly reduced in vitamin D-deficient aniwork showed that vitamin D mals.“’ The present deficiency caused a significant decrease in Ca2+ uptake by microvillus membranes prepared from vitamin D-deficient rats (Figure 3). In order to determine if these decreases in Ca’+ uptake by membrane vesicles prepared from vitamin D-deficient animals could be reversed by the administration of 1,25(OH),D,, the repletion studies described in this paper were performed. Our data indicate that Golgi membranes show two distinct phases of recovery of Ca2+ uptake after repletion with 1,25-(OH),D:, (Table 1, Figures 2. and 4). The early phase consists of a rapid rise to almost by an almost 85% recovery of Ca2+ uptake followed equally rapid decline to the prerepletion levels (Figure 2). The late phase is characterized by a slower approach to recovery (Figure 4). The early phase of to be specific for recovery of Ca2+ uptake appears 1,25-(OH),D,, in that there was a delay in recovery in Ca’+ uptake when 25-(OH)D, was used (Figure 5) and nephrectomy prevented the repletion effects of 25-(OH)D, but not that of 1,25-(OH),D, (Figure 6). These data are compatible with the known requirement for la-hydroxylation of 25-(OH)D, by the kidney to produce the metabolite most active in the intestine, 1,25-(OH),D,.“” Our data showed that after a
VITAMIN
D REPLETION
OF CA’+
UPTAKE
BY GOLGI
329
1 I
J HOURS
Figure
5.
Comparison of the effects of 1.25~(OH),D, with 25(OH)D, on the times for recovery of Ca2+ uptake by Golgi membrane vesicles. Vitamin D-deficient animals were killed at different intervals after a single intrajugular injection of either 125 ng of 1,25-(OH),D, or 250 ng of 25-(OH),D, in 0.05 ml of 95% (vol:vol) ethanol. Animals injected with 1,25-(OH)2D9 (0) or 25-(OH)D, (0).
single injection of 1,25-(OH),D,, maximal recovery of Ca2+ transport by gut sacs occurred 6 hr later. Transport ability declines slightly between 12 and 16 hr but rose again to the maximum and was maintained for up to 120 hr (Figures 2 and 4). This biphasic recovery across gut sacs is also evident in the data of Holick et a1.“4 and was first noted by Walling and Kimberg.“” The early phase of recovery of Ca2+ uptake by Golgi membranes precedes the recovery of Ca’+ transport across gut sacs, suggesting that these processes are related. However, against this concept is the observation that recovery of Ca2+ uptake by Golgi membranes was not maintained but returned to prerepletion levels at a time when Ca2+ transport
600 -
Figure
6. Effect of nephrectomy on the induction by 1,25-(OH),D, and 25-(OH)D, of recovery of CaZ+ uptake by Golgi membrane vesicles. Rats given 1.25-(OH),D, were killed 30 min after, and those given 25-(OH)D,, 3 hr after repletion. Normal (0); vitamin D-deficient (A); vitamin Ddeficient + nephrectomy + 25(OH)D,, (A); vitamin Ddeficient + nephrectomy + l,ZS-(OH)pD, (0).
330
MacLAUGHLIN
ET AL.
GASTROENTEROLOGY
across gut sacs was approaching full recovery. One possible explanation is that a process which begins in the Golgi is subsequently localized in another part of the plasma membrane, e.g., the microvillus membrane. This possibility is consistant with evidence for redistribution of proteins after their insertion into the plasma membrane.36 However, recovery of Ca’+ uptake by microvillus membrane vesicles did not parallel but occurred after gut sac recovery (Figure 4). It is possible that, because of difficulties in evaluating the relatively low Ca’+ uptake by microvillus membranes, we failed to detect a small but significant amount of recovery that paralleled, and was sufficient to explain, gut sac recovery. Rasmussen et a1.17has been able to demonstrate full recovery of Ca’+ uptake by chick microvillus membrane vesicles by 7 hr after repletion. The observation that there are two phases of recovery of Ca*+ uptake by Golgi membranes in response to 1,25-(OH),D, suggests that there may be two separate effects on the intestine by this metabolite of vitamin D. The early peak of Golgi recovery occurred 15-30 min after repletion (Figure 2) and represents the earliest reported effect of 1,25(OH),D, when given to an intact animal. Corradino,37
Vol. 78. No. 2
using embryonic chick intestine maintained in organ culture, has reported an increase in CAMP within 30 min after placing the tissue in a culture medium containing relatively high concentrations (65 nM) of 1,25-(OH),D,. It has also been shown that vitamin D causes a biphasic increase in intestinal CAMP content in this organ culture system3’ and in in vivo experiments on chick intestine.“” These experiments, as well as those of other investigators,“-‘* suggest an interrelationship between the action of 1,25-(OH),D, on the intestine and a CAMP-mediated alteration in Ca2+ transport. It has been shown that Golgi membranes as well as lateral-basal membranes are rich in adenylate cyclase.*” It is possible that the early phase of recovery is due to the direct participation of adenylate cyclase in Ca’+ uptake or is the result of CAMP-dependent activation (i.e., via a protein kinase) of a specific Ca2+ carrier as has been implicated in the regulation of Ca2+ transport in sarcoplasmic reticulum.44.45 action on The prevailing theory of 1,25-(OH),D, the intestine in that it acts in a manner analogous to steroid hormones.3 Support for a steroid hormonelike action includes the detection of 1,25-(OH),D,specific receptor proteins in the cytoplasm and the
a
b
I,25(OH),D, Figure 7. Theoretical considerations: 1,25-(OH),D,.
CI.transcellular
route for calcium absorption,
I,25(OH),D, b. intracellular
sites, and c. cellular sites of action for
February
1980
temperature-dependent translocation of the receptor-hormone complex to the nucleus.46 In the nucleus, the complex associates with chromatin and stimulates mRNA synthesis.47-49 It is assumed that this mRNA codes for a specific protein necessary for Ca*+ transport. Against a strictly steroid-hormone effect is the report by Bikle et al.5o that actinomycin D and cycloheximide inhibit the synthesis of CaBP and alkaline phosphatase but do not affect recovery after administration of l,25of Ca’+ transport these re(OH),D,. Rasmussen et al. I7 have confirmed sults. If the early recovery of Ca’+ uptake by Golgi membranes is not caused by de novo protein synthesis, then 1,25-(OH),D, may act by effecting a posttranslational modification of a protein, perhaps through a CAMP-mediated protein kinase reaction as suggested above. Alternatively, 1,25-(OH),D, may be inserted into the membrane and form a Ca*+ channel as has been demonstrated for filipin.“’ Rasmussen et al.” have suggested a change in membrane lipid structure but have provided no evidence in support of this hypothesis. How may these be interpreted and how does the Golgi possibly participate in vitamin D-dependent Ca’+ transport across the intestine? Figure 7a schematically outlines the three major barriers to Ca*+ transport across the intestine. The Golgi may simply function between step 2 and 3 as a Ca’+ sink or Golgi may be the site for the assembly or final glycosylation of a Ca2+ transport protein destined for the lateral-basal or microvillus membranes (Figure 7b, solid line). A third possibility is that 1,25-(OH),D, localizes briefly to the Golgi and acts to form a Ca’+ channel (Figure 7b, dotted line). In working with intestine, it must be borne in mind that there are functional differences not only along the length of the intestine, but also from crypt to villus.“” The upper third of the villus is represented by what has been termed a differentiated cell. There are no data to indicate whether the upper villus cells are capable of transcription, but there is some evidence that new protein synthesis is mainly a property of the cells of the lower villus and the upper half of the crypts.“” Thus there may be a necessity for two modes of action of 1,25-(OH),D, in the control of Ca2+ transport, one that can act rapidly to alter the differentiated upper villus cell (Figure i’c, n), and another that acts on the upper crypt or lower villus cells to alter transcription (Figure 7c, 0). The late phase of recovery of Ca2+ uptake observed in this report may, indeed, be a reflection of a steroid hormone-like action of 1,25-(OH),D, on the upper crypt-lower villus cells. In summary, we have demonstrated a biphasic pattern of the recovery of Ca’+ uptake by Golgi membrane vesicles from rat intestinal epithelial cells
VITAMIN
D REPLETION
OF CA”
IJPTAKE
BY GOLGI
331
following repletion with a single injection of 1,25(OH),D,. The early phase of recovery by Golgi occurred within 15-30 min after repletion and preceded recovery of Ca’+ transport across gut sacs. The early recovery of Golgi membrane vesicle uptake, as well as the stimulation of gut sac transport, was shown to be specific for 1,25-(OH),D,. Assuming that recovery of Ca’+ uptake by Golgi membranes reflects subcellular changes directly leading to the reacross the intestine, the covery of Ca’+ transport data suggest that there may be two different ways in which 1,25-(OH),D, can effect Ca’+ transport.
References Norman AW: Calcium and phosphorous absorption. In: Vitamin Il. Edited by DEM Lawson. London, Academic Press, Inc.. 1978, p 93-132 2 Schachter D. Rosen SM: Active transport of 4”Ca by the small intestine and its dependence on vitamin D. Am J Physiol 196:357, 1959 3. Haussler MR, McCain TA: Basic and clinical concepts related to vitamin D metabolism and action. N Engl J Med 297:974, 297:1041,1977 4. Brumbaugh PF, Haussler MR: la,25dihydroxycholecalciferol receptors in intestine. I. Association of lu,Z!%dihydroxycholecalciferol with intestinal mucosa. J Biol Chem 249:1251, 1974 5. Kream B, Suchiko Y, Schnoes HK, et al: Specific cytosol-binding protein for 1,25_dihydroxyvitamin D, in rat intestine. J Biol Chem 252:4501, 1977 6. Tsai HC, Wong RG, Norman AW: Studies on calciferol metabolism. IV. Subcellular localization of 1,25_dihydroxyvitamin D, in intestinal mucosa and correlation with increased calcium transport. J Biol Chem 247:5511, 1972 7. Krawitt EL, Korson R: Effect of vitamin D on brush border alkaline phosphatase in the rat small intestine. Enzyme 13278, 1972 a. Wasserman RH, Taylor AN: Vitamin D-dependent calciumbinding protein. Response to some physiological and nutritional variables. J Biol Chem 243:3987.1968 9. Wilson PW. Lawson DEM: 1,25-dihydroxyvitamin D stimulation of specific membrane proteins in chick intestine. Biochim Biophys Acta 497:805, 1977 10. Melancon MJ, DeLuca HF: Vitamin D stimulation of calcium dependent adenosive triphosphatase in chick intestinal brush borders. Biochemistry 9:1658, 1970 11. Haussler MR, Nagoda LA, Rasmussen H: Induction of intestinal brush border alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature (Lond) 228:1199, 1970 H, Max EE, Goodman DBP: The effect of la-OH12. Rasmussen D, treatment on the structure and function of chick intestine brush border membrane. In: Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Edited by AW Norman, K Schaefer, JW Coburn, HF DeLuca, D Fraser, HG Grigoleit. Dv Herrath. Berlin, New York, Walter de Gruyter, 1977,p 913-926 13. Kowarski S, Schachter D: Vitamin D and adcnosine triphosphatase dependent on divalent cations in rat intestinal mucosa. J Clin Invest 522765, 1973 14. Moriuchi S, DeLuca HF: The effect of vitamin D metabolites on membrane proteins of chick intestinal brush border. Arch Biochem Biophys 174:367,1976 1
332
MacLAUGHLIN
ET AL.
GASTROENTEROLOGY
15. Spencer R, Charman M, Wilson P, et al: Vitamin D-stimulated intestinal calcium absorption may not involve calcium-binding protein directly. Nature (Lond) 263:161, 1976 16. Borle AB: Calcium metabolism at the cellular level. Fed Proc 32:1944. 1973 17. Rasmussen H. Fontaine 0, Max EE, et al: The effect of la-hydroxyvitamin D, administration on calcium transport in chick intestine brush border membrane vesicles. J Biol Chem 254:2993, 1979 18. Parkinson DK, Radde IC: Ca++ sensitive ATPase in duodenal mucosa: localization in basal membranes. In: Cellular Mechanisms for Calcium Transfer and Homeostasis. Edited by G Nichols Jr, RH Wasserman. New York and London, Academic Press, Inc. 1971, p 506 19. Warner RR, Coleman JR: Electron probe analysis of calcium transport by small intestine. J Cell Biol 64:54, 1975 20. Freedman RA, Weiser MM, Isselbacher KJ: Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine:decrease in vitamin D deficient rats. Proc Nat1 Acad Sci USA 74:3612, 1977 21. Freedman RA, MacLaughlin J, Weiser MM: Evidence for H+Ca2+ countertransport in vitamin D dependent calcium uptake by Golgi membrane vesicles. Gastroenterology 74:1123, 1978 22. Hopfer V, Nelson K, Perrotto J, et al: Glucose transport in isolated brush border membranes from rat small intestine. J Biol Chem 248:25, 1973 23. Schachter D, Dowdle EB, Schenker H: Active transport of calcium by the small intestine of the rat. Am J Physiol 198:263, 1960 24. Martin DL, DeLuca HF: Influence of sodium on calcium transport by the rat small intestine. Am J Physiol 216:1351, 1969 25. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265,1951 26. Holick MF, Kleiner-Bosaller A, Schnoes HK, et al: 1,24,25-trihydroxyvitamin D,, A metabolite of vitamin D, effective on intestine. J Biol Chem 248:6691, 1973 27. Schachter D, Kimberg DV, Schenker H: Active transport of calcium by intestine: action and bioassay of vitamin D. Am J Physiol 200:1263, 1961 28. Schachter D, Kowarski S, Finkelstein JD, et al: Tissue concentration differences during active transport of calcium by intestine. Am J Physiol 211:1131, 1966 29. Kowarski S, Schachter D: Vitamin D-dependent, particulate calcium-binding activity and intestinal calcium transport. Am J Physiol 229:1198, 1975 30. Sampson HW, Mathews JL, Martin JH, et al: An electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin-D-treated rats. Calcif Tissue Res 5:305, 1970 31. Morrissey RL, Empson RN Jr, Zolock DT, et al: Intestinal response to la-25 dihydroxycholecalciferol. II. A timed study of the intracellular localization of calcium binding protein. Biochim Biophys Acta 538:34, 1978 32. Taylor AN, Wasserman RH: Immunofluorescent localization of vitamin D-dependent calcium-binding protein. J Histothem Cytochem 18:107, 1970 33. DeLuca HF. Schnoes HK: Metabolism tion of vitamin D. Ann Rev Biochem
and Mechanism 45:631, 1976
35. Walling MW, Kimberg DV: Effects of la,25_dihydroxyvitamin D, and Solanum glaucophyllum on intestinal calcium and phosphate transport and on plasma Ca, Mg, and P levels in the rat. Endocrinology 97:1567, 1975 G: Migration
of glycoprotein
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
48.
49.
50.
of ac-
34. Holick MF, Tavela TE, Holick SA, et al: Synthesis of 1 alphahydroxy [6-“H] vitamin D, and its metabolism to l-alpha, 25dihydroxy [6-3H] vitamin D, in the rat. J Biol Chem 251:1020, 1976
36. Bennett
37.
from Golgi apparatus
to
51.
52.
53.
Vol. 78, No. 2
cell coat in the columnar cells of the duodenal epithelium. J Cell Biol 45:668, 1970 Corradino RA: Embryonic chick intestine in organ culture: earliest biological action of la,25-(OH),D, is the stimulation of cyclic AMP production. Fed Proc 35:339, 1976 Corradino RA: Embryonic chick intestine in organ culture: interaction of adenylate cyclase system and vitamin D,-mediated calcium absorptive mechanism. Endocrinology 94:X07, 1974 Wasserman RH, Corradino Ra, Feher J, et al: Temporal patterns of response of the intestinal calcium absorptive system and related parameters to 1,25-dihydroxycholecalciferol. In: Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Edited by AW Norman, K Schaefer, JW Coburn. HF DeLuca, D Fraser, GH Grigoleit, Dv Herrath. New York, Walter de Gruyter, 1977, p 331-340 Harrison HE, Harrison HC: Dibutyryl cyclic AMP, vitamin D and intestinal permeability to calcium. Endocrinology 86:756, 1970 Neville E, Holdsworth ES: A second messenger for vitamin D. FEBS (Fed Eur Biochem Sot) Lett 2:313, 1969 Brostrom CO, Huang YC, Breckenridge B McL, et al: Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc Nat1 Acad Sci USA 7264, 1975 Weiser MM, Neumeier MM, Quaroni A, et al: Synthesis of plasmalemmal glycoproteins in intestinal cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane. J Cell Biol 77:722, 1978 Kirchberger MA, Tada M, Katz AM: Adenosine 3’:5’-monophosphate dependent protein kinase-catalyzed phosphorylation reaction and its relationship to CaZ+ transport in cardiac sarcoplasmic reticulum. J Biol Chem 249:6166, 1974 Her1 WH, Jennissen HP, Heilmeyer LMG Jr: Evidence for the participation of a Ca *+-dependent protein kinase and a protein phosphatase in the regulation of the Ca’+ transport ATPase of the sarcoplasmic reticulum. 1. Effect of inhibitors of the Ca*+-dependent protein kinase and protein phosphorylase. Biochemistry 17:759, 1978 Brumbaugh PF, Haussler MR: la,25-dihydroxycholecalciferol receptors in intestine. I. Association of la,25-dihydroxycholecalciferol with intestinal mucosa chromatin. J Biol Chem 249:1251, 1974 Tsai HC, Norman AW: Studies on the mode of action of calciferol. VI. Effect of 1,25-dihydroxy-vitamin D, on RNA synthesis in the intestinal mucosa. Biochem Biophys Res Commun 54:622, 1973 Zerwekh JE, Haussler MR, Lindell TJ: Rapid enhancement of chick intestinal DNA-dependent RNA polymerase II activity by la,25_dihydroxyvitamin D, in vivo. Proc Nat1 Acad Sci USA 71:2337, 1974 Zerwekh JE, Lindell TJ, Haussler MR: Increased intestinal chromatin template activity: influence of la,25-dihydroxyvitamin D, and hormonereceptor complexes. J Biol Chem 251:2388, 1976 Bikle DD, Zolock DT, Morrissey RL, et al: Independence of 1,25-dihydroxyvitamin Da-mediated calcium transport from de novo RNA and protein synthesis. J Biol Chem 253:4&l, 1978 Wong RG, Norman AW: Studies on the mechanism of action of calciferol. VIII. The effects of dietary vitamin D and the polyene antibiotic, filipin. in vitro, on the intestinal cellular uptake of calcium. J Biol Chem 250:2411, 1975 Weiser MM: Intestinal epithelial cell surface membrane glycoprotein synthesis. 1. An indicator of cellular differentiation. J Biol Chem 248:2536, 1973 Dubs P, Gitzelman R, Steinman B, et al: Catalytically inactive sucrase antigen of rabbit small intestine-the enzyme precursor. Helv Paediatr Acta 30:89, 1975
78325-332,
19RO
Biphasic Recovery of Vitamin DDependent Ca2+ Uptake by Rat Intestinal Golgi Membranes JULIA A. MacLAUGHLIN, MILTON and ROGER A. FREEDMAN
M. WEISER,
Department of Medicine, Harvard Medical School, Medical Massachusetts General Hospital, Boston, Massachusetts
A previous study had demonstrated that rat intestinal epithelial cell GoJgi, lateral-basal, and microvillus membrane vesicles translocated Ca’+, and that this process was markedly decreased in vitamin D-deficient rats. In the present study the kinetics of recovery of CaZ+ uptake by intestinal GoJgi vesicles was examined in vitamin D-deficient rats after a single i.v. injection of 1.25 ng 1,25-dihydroxycholecalciferol (1,25-(OH),D,). Calcium uptake was measured by determination of 4sCar+ associated with GoJgi membranes after collection by micropore filtration. GoJgi membranes recovered rapidly (66% in 15 min, 83% in 30 min) but then quickly returned to prerepletion levels by 8 hr. This was followed by a second phase of recovery starting at 48 hr, with approximately 80% recovery by 96 hr which was maintained at least to 120 hr. By comparison, partial recovery of calcium uptake by microvillus membrane vesicles was first detected at 15 hr and full recovery did not occur until 72 hr; a biphasic pattern to microvillus membrane recovery was not apparent. The vitamin D-dependent recovery of Ca”’ uptake by Goigi membrane vesicles was also compared to studies with gut sacs which showed 50% recovery by 3 hr and 100% recovery by 6 hr; gut sac recovery was sustained for at least 120 hr. The recovery of Co.‘+ uptake by GoJgi was shown to be specific for Received August 6, 1979. Accepted September 24, 1979. Address requests for reprints to: Milton M. Weiser, Division of Gastroenterology and Nutrition, State University of New York at Buffalo, Clinical Center Annex, CC 1186, 462 Grider Street, Buffalo, New York 14215. This research was supported by grants from the National Instltutes of Health (CA-16703) and the American Cancer Society (BC93). Research support was also provided by the Howard Hughes Medical Institute when M.M. Weiser was a Howard Hughes Investigator. The authors wish to thank Dr. Kurt J. Isselhacher for his generous support and advice, and Dr. M. Holick for his advice and discussions. 0 1980 by the American Gastroenterological Association OOlfi-5085/80/020325-07$02.25
Services
(Gastrointestinal
ITnit),
1,25-(OH),D,. The first peak of recovery of Caz+ uptake by intestinal GoJgi membrane vesicles is one of the earliest reported effects of 1,25-(OH),D,. The biphasic nature of recovery demonstrated by GoJgi membranes suggests that there may be more than one mechanism of action of 2,25-(OH),D, on the intestine. Absorption of Ca2+ through the intestinal epithelial cell appears to be a summation of passive diffusion and an active transport process’ with the latter process dependent on vitamin D.’ The prevailing concept is that the active metabolite of vitamin D, l,&dihydroxycholecalciferol (1,25-(OH),D,),* acts on the intestine in a way analogous to that described for steroid hormones.” Indeed, specific cytosolic receptors in the intestine have been described for 1,25(OH),D,, and the synthesis of a number of intestinal proteins thought to be related to CaZ+ transport has been shown to be stimulated by the administration of 1,25-(OH),D, to vitamin D-deficient animals.4-14 The problem has been to show a direct relationship between the synthesis of these proteins and the change in Ca’+ transport induced hy 1,25-(OH),D,,. The Ca”’ binding protein (CaBP) described by Wasserman and Taylor is the best characterized of these proteins, and its synthesis appeared to be correlated with vitamin D-dependent changes in intestinal transport”; however, more recent studies have seriously questioned this correlation.‘” CaBP is isolated from the soluble fraction while other proteins felt to be responsible for the vitamin D-dependent Ca’+ transport process have been identified with mitochondria”,‘“,” or lateral-basal membranes.‘” It has not been established where the vitamin Ddependent Ca2+ transport process is located within Abbreviations used in this paper: 1,25-(OH),D,, 1,25-dihydroxycholecalciferol: CaBP, calcium hinding protein; S/M ratio, ratio of [Ca2+J in serosal fluid to that in mucosal fluid. l
326
MacLAUGHLIN
ET AL.
the cell. It has been suggested that the vitamin D-dependent Ca’+ transport process requires sequestration of Ca’+ by a subcellular organelle such as mitochondria.” Electron probe microanalysis studies of Warner and Coleman’9 have detected intracellular calcium in the supranuclear region of the intestinal cell where Golgi are concentrated, and these discrete areas are reduced in vitamin D-deficient animals and increased after repletion. A previous study from our laboratory had demonstrated the feasibility of studying Ca’+ uptake in membrane vesicles prepared from subcellular fractions of rat intestine.” We were able to demonstrate that Golgi membranes have the most rapid initial rate and the highest equilibrium level of Ca2+ uptake when compared to lateral-basal and microvillus membranes. We have also shown that Ca’+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by extensive binding of Ca’+ to the vesicle; other data suggest the existence of a cation counter-transport system in these vesicles.Z1 Furthermore, Golgi membrane vesicles prepared from vitamin Ddeficient rats were shown to have a significantly decreased Ca2+ uptake capability.‘” We now present data on the kinetics of recovery of Ca’+ uptake by intestinal epithelial cell Golgi membrane vesicles prepared from vitamin D-deficient animals after i.v. injection with 1,25-(OH),D,. The recovery of Ca’+ uptake appeared to require 1,25-(OH),D, specifically, and the time-course of recovery by Golgi membranes was biphasic. The first peak of Ca2+ uptake recovery by Golgi membrane occurs within 15-30 min gfter repletion and represents one of the earliest reported effects of 1,25-(OH),D,.
Materials and Methods Animals Male weanling albino rats were obtained from the Holtzman Co. (Madison, Wisconsin) at 21 days of age and maintained from 3-4 wk on a complete pellet diet (Purina lab chow, Ralston Purina Co., St. Louis, MO.). In order to make the animals vitamin D depleted, weanling male rats of the same strain were maintained on a purified low calcium diet containing 0.02% (wt :wt) calcium and 0.30% (wt : wt) phosphorous with no vitamin D and housed under incandescent light for 3-4 wk. Animals were starved for 18 hr before death by cervical dislocation; the animals had water available at all times. Vitamin D-deficient animals were repleted by an intrajugular injection of 125 ng of 1,25-(OH),D, or 250 ng of 25-(OH)D, in 0.05 ml of 95% (vol : vol) ethanol, whereas control animals received 0.05 ml of 95% (vol: vol) ethanol. Both metabolites of vitamin D were generous gifts of Drs. Michael Holick and Mary Clark, Massachusetts General Hospital. Where indicated, nephrectomy was performed on the day preceding an experiment and the rats w&e returned to incandescent light-
GASTROENTEROLOGY
ing between tabolites.
nephrectomy
and injection
Vol. 78, No. 2
of vitamin
D me-
Tissue Preparations The small intestine was removed, washed with 0.154 M-NaCl, slit open longitudinally, and the mucosa obtained by scraping with glass microscope slides. A purified microvillus membrane was prepared by the method of Hopfer et al. ZZGolgi membrane vesicles were prepared as previously described.” Everted gut sacs were made from the first 6 cm of duodenum according to the method of Schachter et a1.23 Ca’+ transport studies across gut sacs were performed as described by Martin and DeLucaz4 and the results expressed as the ratio of serosal [Ca”] to mucosal [Ca”] (S/M ratio).
Assay
of Ca2+ Uptake
The ability of membrane vesicles to take up Ca2+ was measured within 24 hr of vesicle preparation. The assay for Ca2+ uptake was as previously described.“’ Briefly, the membranes (final protein concentration of 0.01-0.05 mg/ml) were incubated at 25°C in a medium containing 20 mM Na-Hepes buffer, pH 8, 100 mM NaCl, 2.5 mM MgCl,, and 0.5 mM “5CaC1, (usual specific activity = 1 &i/ymol) in a total volume of 4.2 ml. At designated time intervals, 1.0 ml aliquots were removed, filtered through nitrocellulose filters and the retained radioactivity determined as previously described.“’ All radioactive materials and Liquifluor were purchased from New England Nuclear (Boston, Mass.). Protein was determined by the method of Lowry et a1.25
Results We had previously demonstrated that Ca*+ uptake by Golgi and lateral-basal membranes is reduced when the membranes are prepared from vitamin D-deficient rats. To test whether this reduction of Ca’+ uptake was directly related to the action of vitamin D, 1,25-(OH),D, was injected intrajugularly (125 ng in 0.05 ml 95% (vol:vol) ethanol/rat) and the rate of recovery of Ca*+ uptake determined. Normal and vitamin D-deficient rats given 0.05 ml 95% (vol:vol) ethanol served as controls. Golgi membrane vesicles showed a recovery of 60-80% of equilibrium levels of Ca’+ uptake (uptake after 20 min) by 30 min after repletion but appeared to rapidly lose their ability to take up Ca2+ (Table 1 and Figure 1). Recovery of Ca2+ uptake by Golgi membrane vesicles was also manifested by increases in initial rate of uptake (Table 1 and Figure l), but the increase in uptake was not as great as the increase in equilibrium binding capacity. The time-course of recovery of Ca’+ uptake at equilibrium by Golgi membrane vesicles is shown in Figure 2 and is compared to the recovery of Ca’+ uptake by gut sac preparations.
February
Table
VITAMIN
1980
1.
Effect of 1,25-(OH),D,
on ca*+ Uptake by Intestinal
D REPLETION
Go@ Membrane Ca’+ Uptake
Time after administration 1,25-(OH)* cholecalciferol 0
of (min)
Normal [14]” Vit-D Deficient
15
I 31
30
I 31
60
1 31
120
I 51
240
I 21
480
I 31
15 set 161 + 8.8 43 f 5.0 71 f 24 (24%)” 87 -t 11 (37%) 83 f 9.6 (34%) 69 + 8.4 (22%) 64 f 7 (18%) 38 + 8.7 (-0%)
[18]
OF CA”
Vesicles
(nmoles/mg
from
protein
LJPTAKE
Vitamin
BY GOLGI
D-Deficient
327
Rats
-f SEM) at
1 min
5 min
20 min
303 + 14 79 f 7.4 161 f 50 (37%) 193 -t 43 (51Y0) 201 f 42 (54Y”) 111 * 5.4 (14%) 103 -+ 10 (11%) 88 f 13
493 + 21 137 f 21 354 -t 67 (61%) 386 + 50 (70%) 294 + 35 (44%) 216 + 5.2 (22%) 172 -c 7.5 (10%) 140 + 13 (-0%)
587 f 19 175 f 14.5 447 -+ 45 (66%) 517 * 39 (83%) 331+ 36 (38%) 254 -t 9.7 (19%) 211 + 12 (9%) 167 f 18.5 (-0%)
(-0%) ’ Percent
recovery
to “normal,”
i.e., 100 x (uptake
” No. of animals: Golgi were prepared from individual rats and were repleted animals - uptake by D-deficient animals)/uptake by normal
not pooled. animals.
by
Uptake by Golgi membrane vesicles showed a fractional recovery of 0.66 by 15 min after repletion, appeared to peak at 30 min and returned to prerepletion levels by 8 hr (Figure 2). Gut sac preparations tested for their ability to transport Ca2+ against a concentration gradient after repletion with I,25 (OH),D, showed full recovery by 6 hr (Figure 2). These results were interpreted as being compatible with a process that had begun in the Golgi and had then resulted in recovery of transport across the gut sac; this interpretation suggested that recovery of Ca2+ uptake by another membrane fraction might more precisely coincide with gut sac recovery. Therefore, microvillus membrane vesicles were tested for Ca’+ uptake despite earlier evidence that these membranes had a relatively low capacity for
Ca’+ uptake when compared to Golgi and lateralbasal membranes.2” memFigure 3 shows Ca2+ uptake by microvillus brane vesicles from normal and D-deficient rats and illustrates again the relatively lower Ca*+ uptake by microvillus membranes. This lower Ca”+ uptake did not appear to be due to a failure to form vesicles since the microvillus membrane vesicles prepared for these experiments retained their ability to translocate glucose (data not presented) to the same degree as previously reported.” Microvillus membranes prepared from vitamin D-deficient animals
Normal 30 ml” 15 ml”
I hr 2 hrs D-Deflclent
Figure Figure
1. Ca2+ uptake by intestinal Golgi membrane vesicles from normal, vitamin D-deficient, and 1,25-(OH),D,-repleted rats. Vitamin D-deficient animals were sacrificed at different intervals after a single injection of 125 ng of 1,25(OH),D, in 0.05 ml of 95% (vol:vol) ethanol; normal and vitamin D-deficient control rats received 0.05 ml of 95% (vol:vol) ethanol. Normal (0). vitamin D-deficient (A); repleted: 15 min (U), 30 min (O), 1 hr (m, 2 hr (A).
2. Comparison of times of recovery of Ca2+ uptake by Golgi membrane vesicles, and of Ca” transport across everted gut sacs after repletion with 1,25-(OH),D,. Fractional recovery was calculated from the 20 min uptake value (see Table 1) using the ratio (repleted-vitamin Ddeficient)/(normal-vitamin D-deficient). Fractional recovery of Ca2+ transport across gut sacs was calculated after determination of serosal/mucosal Ca2+ concentrations using the same ratio above. Golgi (0); everted gut sacs (0).
328
MacLAIJGHLIN
I
ET AL.
2 MINUTES
Figure
3. Ca2+ uptake by intestinal microvillus membrane vesicles from normal and vitamin D-deficient rats. Normal (O), vitamin D-deficient (0). Values represent the mean k SEM (18 different preparations of 18 different rats).
showed a significantly lower Ca’+ uptake, but the decrease was not as great as that found for Golgi membrane (Figure 3). It was difficult to obtain any statistically significant stimulation of Ca’+ uptake by microvillus membrane vesicles for the earlier time points following repletion with 1,25-(OH),D,. In particular, no significant recovery of Ca’+ uptake by microvillus membranes was detected at the time of gut sac recovery shown in Figure 2. ability by Golgi The total loss of Ca*+ uptake membranes 8 hr after repletion with 1,25-(OH),D, prompted an evaluation of later time points since it had been previously shown’” that recovery of intestinal Ca”+ transport is maintained for up to 7 days after one injection of 125 ng of 1,25-(OH),D,. As illustrated in Figure 4, Golgi membranes exhibited a second recovery of Ca’+ uptake starting 48-72 hr after repletion. The rate of this recovery was slower than that observed at 15-30 min postrepletion, but the increased uptake was sustained for up to 120 hr. Microvillus membranes also showed a slow recovrepletion with vitamin ery of Ca’+ uptake following D. They reached full recovery by 72 hr at which time the Golgi membranes showed a fractional recovery of only 0.3-0.45. Throughout this time, transport by
GASTROENTEROLOGY
Vol. 78, No. 2
gut sacs was maintained at full recovery levels (Figure 4). To test whether the recovery of Ca2+ uptake by Golgi membrane vesicles was specific for 1,25(OH),D,, results were compared to those from animals repleted with 25-(OH)D,. Ca’+ uptake by Golgi membranes and Ca’+ transport by gut sacs were evaluated after an intrajugular injection of 250 ng of 25-(OH)D,. The results for Golgi membranes are illustrated in Figure 5 which shows that 25-(OH)D, repletion, in contrast to repletion with 1,25-(OH),D,, caused a later recovery of Ca2+ uptake by Golgi membranes, with the first peak of full recovery occurring at 3 hr instead of 30 min. The recovery of uptake induced by either derivative of vitamin D rapidly disappeared after the peak response. Gut sacs also showed a delayed response to 25-(OH)D, repletion. Furthermore, while nephrectomy had no effect on the recovery of Ca*+ uptake after administration of 1,25-(OH),D,, it abolished the recovery that could be seen after giving 25-(OH)D, (Figure 6). These results suggest that the recovery of Ca2+ uptake by Golgi membrane vesicles which is observed after repletion with 25-(OH)D:, occurs only after renal Lxhydroxylation of 25-(OH)D, to 1,25-(OH),D,.
Discussion The properties of intestinal Ca2+ transport have been defined primarily by in vivo studies and in experiments using gut sac preparations.2’~Z4.“7.28 Recently some progress has been made toward characterizing the subcellular basis of intestinal Ca2+ transport, particularly the vitamin D-dependent processes. However, it is not known whether the vitamin D-dependent step is located in the microvillus membrane, the lateral-basal membrane, an intracellular organelle, the cytoplasm, or, indeed, in more than one cellular location. Several vitamin D-dependent Ca’+ binding proteins and Ca’+ dependent en-
Figure
4. Comparison of times of recovery of Ca’+ uptake by Golgi, microvillus membrane vesicles, and of Ca2+ transport across everted gut sacs after intrajugular administration of 1,25-(OH),D,. Microvillus membrane (A), everted gut sac (a), Golgi (0).
February
1980
zymes have been localized in the microvillus membrane, including CaBP,” a particulate Ca2+ binding complex,“’ and a Ca”+-stimulated ATPase.‘” Their proposed role in Ca2+ transport is based on their dependence on vitamin D for activity and their interaction with Ca’+ in vitro. More recently, three laboratories,“.“,14 have detected synthesis of microvillus membrane proteins in response to 1,25-(OH),D,. The times after vitamin D administration at which the synthesis of these proteins was detected varied from 4 to 15 hr. Rasmussen et a1.17 have described a vitamin D-dependent Ca*+ uptake by chick intestinal microvillus membrane vesicles which responds to repletion by 7 hr, and this response was unaffected by cycloheximide, a protein synthesis inhibitor. In addition to the microvillus membrane, other parts of the intestinal cell’R~2R~30-32 have been implicated as the site of the vitamin D-dependent transport process. The possibility that calcium absorption may be related to secretion, along with the supranuclear sequestration of calcium detected by x-ray microanalysis,‘” prompted our laboratory to examine Ca2+ uptake by membrane vesicles prepared from the Golgi apparatus and lateral-basal membranes as well as from microvillus membranes. The earlier studies had indicated that Golgi membrane vesicles had the highest initial rate and equilibrium level of Ca’+ uptake when compared to lateral-basal and microvillus membrane vesicles and that this uptake was significantly reduced in vitamin D-deficient aniwork showed that vitamin D mals.“’ The present deficiency caused a significant decrease in Ca2+ uptake by microvillus membranes prepared from vitamin D-deficient rats (Figure 3). In order to determine if these decreases in Ca’+ uptake by membrane vesicles prepared from vitamin D-deficient animals could be reversed by the administration of 1,25(OH),D,, the repletion studies described in this paper were performed. Our data indicate that Golgi membranes show two distinct phases of recovery of Ca2+ uptake after repletion with 1,25-(OH),D:, (Table 1, Figures 2. and 4). The early phase consists of a rapid rise to almost by an almost 85% recovery of Ca2+ uptake followed equally rapid decline to the prerepletion levels (Figure 2). The late phase is characterized by a slower approach to recovery (Figure 4). The early phase of to be specific for recovery of Ca2+ uptake appears 1,25-(OH),D,, in that there was a delay in recovery in Ca’+ uptake when 25-(OH)D, was used (Figure 5) and nephrectomy prevented the repletion effects of 25-(OH)D, but not that of 1,25-(OH),D, (Figure 6). These data are compatible with the known requirement for la-hydroxylation of 25-(OH)D, by the kidney to produce the metabolite most active in the intestine, 1,25-(OH),D,.“” Our data showed that after a
VITAMIN
D REPLETION
OF CA’+
UPTAKE
BY GOLGI
329
1 I
J HOURS
Figure
5.
Comparison of the effects of 1.25~(OH),D, with 25(OH)D, on the times for recovery of Ca2+ uptake by Golgi membrane vesicles. Vitamin D-deficient animals were killed at different intervals after a single intrajugular injection of either 125 ng of 1,25-(OH),D, or 250 ng of 25-(OH),D, in 0.05 ml of 95% (vol:vol) ethanol. Animals injected with 1,25-(OH)2D9 (0) or 25-(OH)D, (0).
single injection of 1,25-(OH),D,, maximal recovery of Ca2+ transport by gut sacs occurred 6 hr later. Transport ability declines slightly between 12 and 16 hr but rose again to the maximum and was maintained for up to 120 hr (Figures 2 and 4). This biphasic recovery across gut sacs is also evident in the data of Holick et a1.“4 and was first noted by Walling and Kimberg.“” The early phase of recovery of Ca2+ uptake by Golgi membranes precedes the recovery of Ca’+ transport across gut sacs, suggesting that these processes are related. However, against this concept is the observation that recovery of Ca2+ uptake by Golgi membranes was not maintained but returned to prerepletion levels at a time when Ca2+ transport
600 -
Figure
6. Effect of nephrectomy on the induction by 1,25-(OH),D, and 25-(OH)D, of recovery of CaZ+ uptake by Golgi membrane vesicles. Rats given 1.25-(OH),D, were killed 30 min after, and those given 25-(OH)D,, 3 hr after repletion. Normal (0); vitamin D-deficient (A); vitamin Ddeficient + nephrectomy + 25(OH)D,, (A); vitamin Ddeficient + nephrectomy + l,ZS-(OH)pD, (0).
330
MacLAUGHLIN
ET AL.
GASTROENTEROLOGY
across gut sacs was approaching full recovery. One possible explanation is that a process which begins in the Golgi is subsequently localized in another part of the plasma membrane, e.g., the microvillus membrane. This possibility is consistant with evidence for redistribution of proteins after their insertion into the plasma membrane.36 However, recovery of Ca’+ uptake by microvillus membrane vesicles did not parallel but occurred after gut sac recovery (Figure 4). It is possible that, because of difficulties in evaluating the relatively low Ca’+ uptake by microvillus membranes, we failed to detect a small but significant amount of recovery that paralleled, and was sufficient to explain, gut sac recovery. Rasmussen et a1.17has been able to demonstrate full recovery of Ca’+ uptake by chick microvillus membrane vesicles by 7 hr after repletion. The observation that there are two phases of recovery of Ca*+ uptake by Golgi membranes in response to 1,25-(OH),D, suggests that there may be two separate effects on the intestine by this metabolite of vitamin D. The early peak of Golgi recovery occurred 15-30 min after repletion (Figure 2) and represents the earliest reported effect of 1,25(OH),D, when given to an intact animal. Corradino,37
Vol. 78. No. 2
using embryonic chick intestine maintained in organ culture, has reported an increase in CAMP within 30 min after placing the tissue in a culture medium containing relatively high concentrations (65 nM) of 1,25-(OH),D,. It has also been shown that vitamin D causes a biphasic increase in intestinal CAMP content in this organ culture system3’ and in in vivo experiments on chick intestine.“” These experiments, as well as those of other investigators,“-‘* suggest an interrelationship between the action of 1,25-(OH),D, on the intestine and a CAMP-mediated alteration in Ca2+ transport. It has been shown that Golgi membranes as well as lateral-basal membranes are rich in adenylate cyclase.*” It is possible that the early phase of recovery is due to the direct participation of adenylate cyclase in Ca’+ uptake or is the result of CAMP-dependent activation (i.e., via a protein kinase) of a specific Ca2+ carrier as has been implicated in the regulation of Ca2+ transport in sarcoplasmic reticulum.44.45 action on The prevailing theory of 1,25-(OH),D, the intestine in that it acts in a manner analogous to steroid hormones.3 Support for a steroid hormonelike action includes the detection of 1,25-(OH),D,specific receptor proteins in the cytoplasm and the
a
b
I,25(OH),D, Figure 7. Theoretical considerations: 1,25-(OH),D,.
CI.transcellular
route for calcium absorption,
I,25(OH),D, b. intracellular
sites, and c. cellular sites of action for
February
1980
temperature-dependent translocation of the receptor-hormone complex to the nucleus.46 In the nucleus, the complex associates with chromatin and stimulates mRNA synthesis.47-49 It is assumed that this mRNA codes for a specific protein necessary for Ca*+ transport. Against a strictly steroid-hormone effect is the report by Bikle et al.5o that actinomycin D and cycloheximide inhibit the synthesis of CaBP and alkaline phosphatase but do not affect recovery after administration of l,25of Ca’+ transport these re(OH),D,. Rasmussen et al. I7 have confirmed sults. If the early recovery of Ca’+ uptake by Golgi membranes is not caused by de novo protein synthesis, then 1,25-(OH),D, may act by effecting a posttranslational modification of a protein, perhaps through a CAMP-mediated protein kinase reaction as suggested above. Alternatively, 1,25-(OH),D, may be inserted into the membrane and form a Ca*+ channel as has been demonstrated for filipin.“’ Rasmussen et al.” have suggested a change in membrane lipid structure but have provided no evidence in support of this hypothesis. How may these be interpreted and how does the Golgi possibly participate in vitamin D-dependent Ca’+ transport across the intestine? Figure 7a schematically outlines the three major barriers to Ca*+ transport across the intestine. The Golgi may simply function between step 2 and 3 as a Ca’+ sink or Golgi may be the site for the assembly or final glycosylation of a Ca2+ transport protein destined for the lateral-basal or microvillus membranes (Figure 7b, solid line). A third possibility is that 1,25-(OH),D, localizes briefly to the Golgi and acts to form a Ca’+ channel (Figure 7b, dotted line). In working with intestine, it must be borne in mind that there are functional differences not only along the length of the intestine, but also from crypt to villus.“” The upper third of the villus is represented by what has been termed a differentiated cell. There are no data to indicate whether the upper villus cells are capable of transcription, but there is some evidence that new protein synthesis is mainly a property of the cells of the lower villus and the upper half of the crypts.“” Thus there may be a necessity for two modes of action of 1,25-(OH),D, in the control of Ca2+ transport, one that can act rapidly to alter the differentiated upper villus cell (Figure i’c, n), and another that acts on the upper crypt or lower villus cells to alter transcription (Figure 7c, 0). The late phase of recovery of Ca2+ uptake observed in this report may, indeed, be a reflection of a steroid hormone-like action of 1,25-(OH),D, on the upper crypt-lower villus cells. In summary, we have demonstrated a biphasic pattern of the recovery of Ca’+ uptake by Golgi membrane vesicles from rat intestinal epithelial cells
VITAMIN
D REPLETION
OF CA”
IJPTAKE
BY GOLGI
331
following repletion with a single injection of 1,25(OH),D,. The early phase of recovery by Golgi occurred within 15-30 min after repletion and preceded recovery of Ca’+ transport across gut sacs. The early recovery of Golgi membrane vesicle uptake, as well as the stimulation of gut sac transport, was shown to be specific for 1,25-(OH),D,. Assuming that recovery of Ca’+ uptake by Golgi membranes reflects subcellular changes directly leading to the reacross the intestine, the covery of Ca’+ transport data suggest that there may be two different ways in which 1,25-(OH),D, can effect Ca’+ transport.
References Norman AW: Calcium and phosphorous absorption. In: Vitamin Il. Edited by DEM Lawson. London, Academic Press, Inc.. 1978, p 93-132 2 Schachter D. Rosen SM: Active transport of 4”Ca by the small intestine and its dependence on vitamin D. Am J Physiol 196:357, 1959 3. Haussler MR, McCain TA: Basic and clinical concepts related to vitamin D metabolism and action. N Engl J Med 297:974, 297:1041,1977 4. Brumbaugh PF, Haussler MR: la,25dihydroxycholecalciferol receptors in intestine. I. Association of lu,Z!%dihydroxycholecalciferol with intestinal mucosa. J Biol Chem 249:1251, 1974 5. Kream B, Suchiko Y, Schnoes HK, et al: Specific cytosol-binding protein for 1,25_dihydroxyvitamin D, in rat intestine. J Biol Chem 252:4501, 1977 6. Tsai HC, Wong RG, Norman AW: Studies on calciferol metabolism. IV. Subcellular localization of 1,25_dihydroxyvitamin D, in intestinal mucosa and correlation with increased calcium transport. J Biol Chem 247:5511, 1972 7. Krawitt EL, Korson R: Effect of vitamin D on brush border alkaline phosphatase in the rat small intestine. Enzyme 13278, 1972 a. Wasserman RH, Taylor AN: Vitamin D-dependent calciumbinding protein. Response to some physiological and nutritional variables. J Biol Chem 243:3987.1968 9. Wilson PW. Lawson DEM: 1,25-dihydroxyvitamin D stimulation of specific membrane proteins in chick intestine. Biochim Biophys Acta 497:805, 1977 10. Melancon MJ, DeLuca HF: Vitamin D stimulation of calcium dependent adenosive triphosphatase in chick intestinal brush borders. Biochemistry 9:1658, 1970 11. Haussler MR, Nagoda LA, Rasmussen H: Induction of intestinal brush border alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature (Lond) 228:1199, 1970 H, Max EE, Goodman DBP: The effect of la-OH12. Rasmussen D, treatment on the structure and function of chick intestine brush border membrane. In: Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Edited by AW Norman, K Schaefer, JW Coburn, HF DeLuca, D Fraser, HG Grigoleit. Dv Herrath. Berlin, New York, Walter de Gruyter, 1977,p 913-926 13. Kowarski S, Schachter D: Vitamin D and adcnosine triphosphatase dependent on divalent cations in rat intestinal mucosa. J Clin Invest 522765, 1973 14. Moriuchi S, DeLuca HF: The effect of vitamin D metabolites on membrane proteins of chick intestinal brush border. Arch Biochem Biophys 174:367,1976 1
332
MacLAUGHLIN
ET AL.
GASTROENTEROLOGY
15. Spencer R, Charman M, Wilson P, et al: Vitamin D-stimulated intestinal calcium absorption may not involve calcium-binding protein directly. Nature (Lond) 263:161, 1976 16. Borle AB: Calcium metabolism at the cellular level. Fed Proc 32:1944. 1973 17. Rasmussen H. Fontaine 0, Max EE, et al: The effect of la-hydroxyvitamin D, administration on calcium transport in chick intestine brush border membrane vesicles. J Biol Chem 254:2993, 1979 18. Parkinson DK, Radde IC: Ca++ sensitive ATPase in duodenal mucosa: localization in basal membranes. In: Cellular Mechanisms for Calcium Transfer and Homeostasis. Edited by G Nichols Jr, RH Wasserman. New York and London, Academic Press, Inc. 1971, p 506 19. Warner RR, Coleman JR: Electron probe analysis of calcium transport by small intestine. J Cell Biol 64:54, 1975 20. Freedman RA, Weiser MM, Isselbacher KJ: Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine:decrease in vitamin D deficient rats. Proc Nat1 Acad Sci USA 74:3612, 1977 21. Freedman RA, MacLaughlin J, Weiser MM: Evidence for H+Ca2+ countertransport in vitamin D dependent calcium uptake by Golgi membrane vesicles. Gastroenterology 74:1123, 1978 22. Hopfer V, Nelson K, Perrotto J, et al: Glucose transport in isolated brush border membranes from rat small intestine. J Biol Chem 248:25, 1973 23. Schachter D, Dowdle EB, Schenker H: Active transport of calcium by the small intestine of the rat. Am J Physiol 198:263, 1960 24. Martin DL, DeLuca HF: Influence of sodium on calcium transport by the rat small intestine. Am J Physiol 216:1351, 1969 25. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265,1951 26. Holick MF, Kleiner-Bosaller A, Schnoes HK, et al: 1,24,25-trihydroxyvitamin D,, A metabolite of vitamin D, effective on intestine. J Biol Chem 248:6691, 1973 27. Schachter D, Kimberg DV, Schenker H: Active transport of calcium by intestine: action and bioassay of vitamin D. Am J Physiol 200:1263, 1961 28. Schachter D, Kowarski S, Finkelstein JD, et al: Tissue concentration differences during active transport of calcium by intestine. Am J Physiol 211:1131, 1966 29. Kowarski S, Schachter D: Vitamin D-dependent, particulate calcium-binding activity and intestinal calcium transport. Am J Physiol 229:1198, 1975 30. Sampson HW, Mathews JL, Martin JH, et al: An electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin-D-treated rats. Calcif Tissue Res 5:305, 1970 31. Morrissey RL, Empson RN Jr, Zolock DT, et al: Intestinal response to la-25 dihydroxycholecalciferol. II. A timed study of the intracellular localization of calcium binding protein. Biochim Biophys Acta 538:34, 1978 32. Taylor AN, Wasserman RH: Immunofluorescent localization of vitamin D-dependent calcium-binding protein. J Histothem Cytochem 18:107, 1970 33. DeLuca HF. Schnoes HK: Metabolism tion of vitamin D. Ann Rev Biochem
and Mechanism 45:631, 1976
35. Walling MW, Kimberg DV: Effects of la,25_dihydroxyvitamin D, and Solanum glaucophyllum on intestinal calcium and phosphate transport and on plasma Ca, Mg, and P levels in the rat. Endocrinology 97:1567, 1975 G: Migration
of glycoprotein
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
48.
49.
50.
of ac-
34. Holick MF, Tavela TE, Holick SA, et al: Synthesis of 1 alphahydroxy [6-“H] vitamin D, and its metabolism to l-alpha, 25dihydroxy [6-3H] vitamin D, in the rat. J Biol Chem 251:1020, 1976
36. Bennett
37.
from Golgi apparatus
to
51.
52.
53.
Vol. 78, No. 2
cell coat in the columnar cells of the duodenal epithelium. J Cell Biol 45:668, 1970 Corradino RA: Embryonic chick intestine in organ culture: earliest biological action of la,25-(OH),D, is the stimulation of cyclic AMP production. Fed Proc 35:339, 1976 Corradino RA: Embryonic chick intestine in organ culture: interaction of adenylate cyclase system and vitamin D,-mediated calcium absorptive mechanism. Endocrinology 94:X07, 1974 Wasserman RH, Corradino Ra, Feher J, et al: Temporal patterns of response of the intestinal calcium absorptive system and related parameters to 1,25-dihydroxycholecalciferol. In: Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism. Edited by AW Norman, K Schaefer, JW Coburn. HF DeLuca, D Fraser, GH Grigoleit, Dv Herrath. New York, Walter de Gruyter, 1977, p 331-340 Harrison HE, Harrison HC: Dibutyryl cyclic AMP, vitamin D and intestinal permeability to calcium. Endocrinology 86:756, 1970 Neville E, Holdsworth ES: A second messenger for vitamin D. FEBS (Fed Eur Biochem Sot) Lett 2:313, 1969 Brostrom CO, Huang YC, Breckenridge B McL, et al: Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc Nat1 Acad Sci USA 7264, 1975 Weiser MM, Neumeier MM, Quaroni A, et al: Synthesis of plasmalemmal glycoproteins in intestinal cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane. J Cell Biol 77:722, 1978 Kirchberger MA, Tada M, Katz AM: Adenosine 3’:5’-monophosphate dependent protein kinase-catalyzed phosphorylation reaction and its relationship to CaZ+ transport in cardiac sarcoplasmic reticulum. J Biol Chem 249:6166, 1974 Her1 WH, Jennissen HP, Heilmeyer LMG Jr: Evidence for the participation of a Ca *+-dependent protein kinase and a protein phosphatase in the regulation of the Ca’+ transport ATPase of the sarcoplasmic reticulum. 1. Effect of inhibitors of the Ca*+-dependent protein kinase and protein phosphorylase. Biochemistry 17:759, 1978 Brumbaugh PF, Haussler MR: la,25-dihydroxycholecalciferol receptors in intestine. I. Association of la,25-dihydroxycholecalciferol with intestinal mucosa chromatin. J Biol Chem 249:1251, 1974 Tsai HC, Norman AW: Studies on the mode of action of calciferol. VI. Effect of 1,25-dihydroxy-vitamin D, on RNA synthesis in the intestinal mucosa. Biochem Biophys Res Commun 54:622, 1973 Zerwekh JE, Haussler MR, Lindell TJ: Rapid enhancement of chick intestinal DNA-dependent RNA polymerase II activity by la,25_dihydroxyvitamin D, in vivo. Proc Nat1 Acad Sci USA 71:2337, 1974 Zerwekh JE, Lindell TJ, Haussler MR: Increased intestinal chromatin template activity: influence of la,25-dihydroxyvitamin D, and hormonereceptor complexes. J Biol Chem 251:2388, 1976 Bikle DD, Zolock DT, Morrissey RL, et al: Independence of 1,25-dihydroxyvitamin Da-mediated calcium transport from de novo RNA and protein synthesis. J Biol Chem 253:4&l, 1978 Wong RG, Norman AW: Studies on the mechanism of action of calciferol. VIII. The effects of dietary vitamin D and the polyene antibiotic, filipin. in vitro, on the intestinal cellular uptake of calcium. J Biol Chem 250:2411, 1975 Weiser MM: Intestinal epithelial cell surface membrane glycoprotein synthesis. 1. An indicator of cellular differentiation. J Biol Chem 248:2536, 1973 Dubs P, Gitzelman R, Steinman B, et al: Catalytically inactive sucrase antigen of rabbit small intestine-the enzyme precursor. Helv Paediatr Acta 30:89, 1975