BIOCHIMICA ET BIOPHYSICA ACTA
`
`8i
`
`BBA 45 366
`
`THE MECHANISM OF CALCIUM TRANSPORT BY RAT INTESTINE
`
`HAROLD J. HELBOCK, JOHN G. FORTE * AND PAUL SALTMAN
`
`The Department of Biological Sciences, and The Graduate Program in Biochemistry, University of
`Southern California, Los Angeles, Calif. (U.S.A.)
`
`(Received February 14th, 1966)
`
`SUMMARY
`
`Short-circuit techniques for the study of ion transport by isolated membrane
`systems have been applied to defining the mechanism for calcium ion transport in
`the small intestine of the rat. In the absence of phosphate, the movement of calcium
`is passive. There is no evidence for a membrane-bound carrier to facilitate its trans-
`port. The calcium flux is a linear function of its concentration. However, phosphate
`ion is actively transported from the mucosal to the serosal surface. In the presence
`of phosphate, calcium appears to be actively transported, possibly as a counter-ion
`to the phosphate. The role of chelation in the transport of calcium has also been
`clarified.
`
`INTRODUCTION
`
`There has been widespread interest in calcium* * transport and metabolism by
`investigators with diverse concerns including nutrition, bone metabolism, effects of
`various hormones and vitamins, the metal's role in enzyme action, its movement
`during muscle contraction, and its accumulation by mitochondria, to cite but a few.
`A comprehensive review of the dynamics and function of calcium has been given
`by BRONNER1. The research presented here is directed to a single aspect of calcium
`transport —the mechanism by which the metal ion is moved across the small intestine
`of the rat. There have been two excellent reviews on this subject which should be
`consulted for perspective2,3.
`Our own interest in calcium metabolism began as a consequence of the studies
`carried out in this laboratory on the role of chelation in the passive transport of
`Fe3+ (ref. 4) and other trace metals into a wide variety of tissues and across many
`different cellular membranes. One particular class of metal chelates was both novel
`and of nutritional significance, the metal—sugar complexes5. We were intrigued par-
`ticularly by the concept that the nutritional stimulation of calcium metabolism by
`lactose and other sugars could be attributed to the formation of a calcium—lactose
`
`Abbreviations: Pi refers to the multiple species of inorganic phosphate present. Flux from
`mucosal to serosal surface, (M
`S) ; from serosal to mucosal, (S --> M).
`* Present address : Department of Physiology, University of California, Berkeley, Calif., U.S.A.
`** Calcium refers to all of the charged and uncharged species of calcium that are present
`under the conditions described.
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`MYLAN EXHIBIT - 1042
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`H. J. HELBOCK, J. O. FORTE, P. SALTMAN
`
`chelate6. We therefore began a series of experiments specifically to determine the
`mechanisms by which this metal is transported across the intestinal membrane.
`A systematic attempt to demonstrate active transport in vitro using rat small
`intestine was made by SCHACTER and his colleagues7,8. Fundamentally their technique
`consisted of everting a section of the small intestine, suspending the everted sac in
`a physiological buffer solution and studying the distribution of radioactive 45Ca intro-
`duced in both the serosal and mucosal compartments. Since the ratio of 45Ca within
`the sac compared to that of the bathing medium exceeded r.o, they proposed that
`active transport of calcium, per se, was involved. This asymmetric distribution was
`inhibited by metabolic poisons. Confirmation of the active transport hypothesis was
`presented by WASSERMAN et al. who studied calcium movement against a concen-
`tration and electropotential gradient in vivo using rat",16 and chicken intestines. In
`these experiments, all calcium removed from the duodenal lumen was considered to
`be transported actively.
`A careful analysis of data from WASSERMAN'S group12 indicated that there was
`a linear relationship between transport rate and the concentration of calcium in the
`lumen. This would suggest a mechanism controlled fundamentally by diffusion, in-
`volving no membrane-bound carrier to facilitate ion transport, or a carrier system
`far from saturation. Evidence for a passive mechanism has been presented by
`Mc K Ex NEv".
`One of the most direct, sensitive, and unambiguous methods currently available
`for the study of membrane transport has been provided by ll.Tssim; and his co-
`workers14,i' This method, known as the short-circuit technique, permits continuous
`measurement of trans-epithelial ion flux in the absence of a membrane potential.
`Using this technique, we have demonstrated that movement of calcium ion, per se,
`is a passive process and is not mediated by an active carrier system in the rat intestine.
`Further, chelating agents which stabilize the calcium as a soluble low-molecular-
`weight complex in the presence of competing ligands enhance the rate of movement
`of the ion. In the absence of Pi, we find no evidence for a facilitating carrier in the
`membrane and must conclude that the calcium moves either as the divalent cation,
`or, more likely, complexed to one of many endogenous or exogenous ligands. We have
`been able to demonstrate that Pi is actively transported by the intestinal mucosa.
`This active movement of P1 can result in an apparent active transport of calcium.
`
`-METHODS
`
`In all experiments in vitro where buffer solutions were required, a modified
`Krebs-Henseleit—Ringer solution was employed16. The modifications consisted of the
`removal of Mg2+ from the buffer as well as the addition of 2 mg/ml glucose to provide
`a metabolic substrate. As indicated in the specific experiments, the concentration of
`calcium and/or Pi was altered as needed to investigate specific aspects of the transport
`process. The pH of all solutions was 7.4.
`The "Ca used as a tracer in the studies in vitro was obtained from New England
`Nuclear Laboratories and diluted to a specific activity of r mC/m1 and further diluted
`as needed with carrier CaC12. Approx. 2.106 counts/min per ml were initially present
`in the compartment to which isotope was added. For whole animal experiments, the
`gamma-emitting, short-lived isotope 47Ca was obtained from Abbott Laboratories as
`
`Biochim. Biophys. Acta, 126 (2966) 81-93
`
`

`

`CALCIUM TRANSPORT BY RAT INTESTINE
`
`83
`
`a CaCl2 solution and diluted with appropriate amounts of carrier CaC12 before ad-
`ministration to an animal. Radioactivity of the 45Ca was determined using a Packard
`liquid scintillation counter. 47Ca was measured using a whole animal counter em-
`ploying two 3-inch NaI crystals and a pulse-height analyzer peaked at 1.29 meV.
`In experiments designed to measure active transport of sodium, "Na (obtained from
`New England Nuclear Corporation) was diluted with appropriate amounts of carrier
`NaCl. The radioactivity of 22Na was determined in a scintillation well counter peaked
`to the 1.3 meV signal. The radioactive compartment of the experiments in vitro with
`22Na contained initially approx. 8o 000 counts/min per ml. In some flux experiments,
`it was necessary to measure both 45Ca and "Na in the same sample by determining
`the radioactivity of the 22Na in the scintillation well counter and measuring the 45Ca
`by liquid scintillation techniques. Appropriate corrections were made for the ex-
`traneous counts introduced in the 45Ca determination by the 22Na which was in all
`cases less than 3% of the 45Ca activity. Pi was obtained as H332PO4 from New England
`Nuclear Corporation, specific activity ioo mC/ml. It was freed of pyrophosphate by
`acid reflux prior to use. Activity was determined by liquid scintillation techniques.
`
`Techniques in vitro for two-way flux determinations
`A modification of the Ussing short-circuit apparatus was designed and built as
`shown in Fig. 1. The shape of the opening was an elongated oval to comply with
`the physical limitations of the tissue. The total area of the orifice was 0.87 cm2. The
`electrical potential difference was monitored using a Keithley electrometer (Model
`600-A) via a pair of calomel electrodes and agar—saline bridges on each side of the
`preparation. Before each experiment, electrical potential between the bridges was
`measured without the intestinal preparation and a correction was applied for any
`deviation from zero. At the same time the resistance of the Ringers between the
`bridges was measured and a correction was subsequently applied to the short-circuit
`experiments on the intestinal mucosa. The short-circuit current was supplied from
`a 1.5-V dry cell using Ag—AgC1 electrodes and agar—saline bridges. Circulation and
`aeration was achieved using Ioo % O2 or 95% O,-5 % CO,. Where anaerobiosis was
`
`mV
`
`Na CI agar
`bridges
`
`NaCI agar
`bridge
`
`NaCI agar
`bridge
`Ag AgCI
`electrode
`
`Gas
`
`Membrane
`
`NA
`
`Fig. 1. Schematic representation of short-circuit membrane system in vitro.
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`

`

`84
`
`H. J. HELBOCK, J. G. FORTE, P. SALTMAN
`
`desired, 95%
`CO, was substituted. At the times indicated, aliquots were
`withdrawn from both compartments and the radioactivity determined as indicated
`above.
`All experiments reported in this paper were carried out using Sprague—Dawley
`male rats, approx. 40o g, which had been deprived of food 24 h prior to killing.
`Animals were killed with a blow on the head and the small intestine immediately
`extirpated. A 5-cm section of the small intestine lying distal to the pyloric muscu-
`lature was utilized. It was washed several times in cold 0.9% saline solution, opened
`along the mesentery and laid out as a flat sheet. This membrane was then mounted
`in the apparatus which was immediately filled with Ringers solution; the isotope
`was added and the experiments begun. Throughout the course of the experiments,
`short-circuit current was recorded. Periodically, the current was removed in order
`to monitor the transmucosal potential difference.
`
`Large-scale apparatus for measurements in vitro
`An alternate technique for flux measurements in vitro was developed which
`permits a large number of experiments to be carried out simultaneously with great
`ease. The apparatus is shown in Fig. 2. A segment of intestine extending from about
`2 cm to 14 cm below the pyloric valve was taken, washed with cold saline, everted,
`and mounted as indicated. Ringer solution was added to both compartments. Circu-
`
`MUCOSAL
`
`SEROSAL
`
`<- GAS
`
`GAS
`
`INTESTINE
`
`Fig. z. Apparatus used for flux measurements in vitro where no short circuit was applied.
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`

`

`CALCIUM TRANSPORT BY RAT INTESTINE
`
`85
`
`lation was maintained and aeration kept constant using the bubbling devices as
`illustrated. The serosal compartment contained 20 ml of buffer, the mucosal compart-
`ment contained 6o ml. Aliquots were removed at desired time intervals from both
`sides to determine radioactivity. The temperature was maintained by emersing the
`apparatus in a circulating water bath at 37°
`
`Calcium flux in vivo
`The animals were anaesthetized with 15 mg sodium pentobarbital, the abdomen
`incised, and a segment of intestine 25 cm distal to the pyloric valve isolated by two
`ligatures. At all times, care was taken to maintain unimpaired blood supply to the
`ligated segment. The experimental solution, 2.0 ml, containing 47Ca at the desired
`concentration and in the desired medium was injected with a 27-gauge needle just
`below the proximal ligation. Immediately following removal of the needle, a third
`ligature was placed distal to the point of injection to avoid back leakage of the radio-
`active solution. Precautions were taken to prevent leakage from the site of injection.
`The segment of intestine was returned to the abdominal cavity and the rat maintained
`for i h under a warming lamp. At the conclusion of this period, the whole animal
`was counted in the animal counter. The isolated intestinal segment was then excised,
`and the animal was again counted. The amount of radioactivity within the animal
`with the intestinal segment removed divided by the total radioactivity (i.e. the animal
`plus intestinal segment) represented the fractional uptake of calcium during the r-h
`period.
`
`RESULTS
`
`Flux of 45Ca under short-circuit conditions
`In experiments using short-circuit conditions, the initial concentration of calci-
`um on both sides of the membrane was ro-5 M. The membrane potential measured
`in a series of over 20 intestinal segments ranged from —3.o to —4.5 mV (mucosa
`negative with respect to serosa in an external circuit). The current required to reduce
`
`0.049
`
`0.046
`
`0.041
`
`0050
`
`0.051
`
`0.044
`
`M -'S
`
`0038
`
`0033
`
`0033
`
`0.032
`
`0.016
`aots
`0060.016
`
`0.2
`
`0.1
`
`E
`U
`U
`
`a
`S
`
`0
`0
`
`30
`
`90
`
`60
`Time (min)
`Fig. 3. Bi-directional calcium flux measurenients for short-circuited system. The points represent
`time markers. The slope of the curve is the flux for that interval. The average numerical values
`for so experiments in each direction are presented for the S M direction above the curve,
`and the M S below. Initial calcium concentration, ro-5 M; no Pi present.
`
`Biochim. Biophys. Ada, 126 (1966) 81-93
`
`

`

`tih
`
`H. J. HELBOCK, J. 0. FORTE, P. SALTMAN
`
`the potential to o mV was 35-5o µA. The average transport of calcium in each
`direction for a series of ro experiments without P1 is given in Fig. 3. Two important
`features are to be noted in the graph. The most striking result is that the flux ratio
`(M S'S M) is essentially unity during all periods measured throughout 2 h.
`Another observation is that the permeability to calcium seems to increase following
`45 min of incubation. We believe that this lag period is an indication of the time
`necessary for the membrane to equilibrate with the bathing solutions since similar
`flux patterns were noted in both M S and S M measurements.
`
`Simultaneous determinations 4 22Na and 45Ca flux -under short-circuit conditions
`t is now well documented that the transport of sodium by the intestinal mucosa
`is an active process". The simultaneous measurement of this actively transported
`ion and calcium would provide an internal control in order to monitor membrane
`integrity. In Table I we report the averages of 5 experiments carried out in each
`direction M S and S M for both "Na and "Ca. The flux ratio for sodium is 1.34
`and represents a net M S movement equal to 2.02 µmoles Na/cm2 per h. The average
`short-circuit current for the same time period was "or µequiv/cm'2 per h, which is
`in reasonable agreement with the measured net flux of sodium. In the same table it
`is seen that there was essentially no net transport of calcium. The small values of
`S> M net flux are within the experimental errors of the procedure (P > 0. to).
`
`T.\ BEE I
`SIMULTANEOUS MEASUREMENT OF THE UN IDIRECTIONAL FLUXES OF 45Ca AND 22Na
`crircurer CON DiTIONS
`measurements
`Initial concentration of sodium, 144 mM: calcium, i 0 -5 M. NO VI was present. The
`were made for a i-h period following a 45-min equilibration of the membrane w ith the isotope
`
`in the apparatus. The results are the average of 5 experiments in each direction . The standard
`
`deviations are presented in parentheses.
`
`UNDER snoicr-
`
`lriii
`
`Iota
`22Na
`
`M S
`(mptnolcsico per h)
`
`(mymoleslcin2 per h)
`
`Flux ratio
`
`0.192 (
`(
`8.0o
`
`0.028)
`- 1.02)
`
`1).203 (
`5.98 (
`
`0.01. 7)
`0-30
`
`0-9-1
`1 .3-1
`
`The effect of anaerobiosis on 45Ca flux
`Using the apparatus described in Fig. 2 for large intestinal segments, we meas-
`ured the M > S transport of '"Ca under O, and then under N,. The results are shown
`in Fig. 4, with a comparable curve for a similar preparation maintained under O,.
`The slight increase in calcium transport seen under nitrogen could have been due to
`an enhanced permeability of the membrane caused by anoxia. It is quite clear that
`there was no inhibition of calcium flux under anaerobic conditions.
`
`The rate of calcium transport as a function of its concentration in vitro
`S fluxes of calcium
`Using the apparatus shown in Fig. r, unidirectional M
`were measured at concentrations from ro-6 to ro-3 M. This range was chosen because
`it encompasses the limits where the active or facilitated mechanism should be opera-
`tive as claimed by other investigators7,9. A plot of the log of the rate of transport
`
`Biochim. Biophys. Acta, 326 (1966) 81-93
`
`

`

`CALCIUM TRANSPORT BY RAT INTESTINE
`
`87
`
`versus the log of the concentration of calcium is shown in Fig. 5. Over the i000-fold
`range of concentration studied, there was a linear increase in the rate of calcium
`transport. If there had been a significant fraction of the transport that was facilitated
`either by an active or a passive carrier, then we would have expected deviations
`from linearity. No such effect was observed and there was no evidence for any mecha-
`nism operative other than simple diffusion.
`
`40
`
`30
`
`ZS 20
`
`10
`
`02 <
`
`N0
`
`30
`
`60
`
`-5
`
`-4
`
`-3
`
`Time (min)
`log mole/I Cot `
`Fig. 4. Calcium flux, M -a S, in non-short-circuited system under aerobic (lower line) and anaerobic
`conditions (upper line). Initial concentration of calcium, 3 mM.
`
`Fig. 5. Short circuit, M —> S flux of calcium as a function of calcium concentration in Pi-free
`Ringer in vitro.
`
`2
`
`0
`
`-
`
`-5
`
`-4
`
`—3
`
`log mumoles Ca2*/cm2 per h
`
`log mole/I Ca 2+
`Fig. 6. Uptake of calcium from intestinal segments of rat small intestine in vivo. Time of uptake, 1 h.
`
`In order to test the possibility that exchange diffusion might be operative in
`this system, we raised the concentration of non-radioactive calcium in the serosal
`compartment to 2.6 io-3 M and measured the transport M —> S of 45Ca over the same
`concentration range as had been studied in Fig. 5. Identical rates of transport were
`observed under these conditions as had been observed previously. There was no
`evidence that exchange diffusion enhances the rate of carrier movement in the mem-
`brane in order to facilitate transport of calcium.
`
`The effect of concentration of calcium on transport in vivo
`Studies were carried out in vivo to determine whether calcium concentration
`had a significant effect on rate of calcium uptake. Fig. 6 shows the results of transport
`in vivo over a ioo-f old concentration range between bo-5 M and bo-3 M. Our results
`confirmed the earlier experiments of WASSERMAN12 which indicated that calcium
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`

`

`SS
`
`H. J. HELBOCK, J. G. FORTE, P. SALTMAN
`
`uptake is proportional to concentration. Calculation of the amount of calcium (io-5 M
`in 2.0 ml of injected volume) removed from a 25-cm-long section in vivo (using 5o cm2
`as an approximate surface area) gives a value of 0.2 mµmole/cm2 per h. This value
`is comparable to the calcium flux found in vitro (see Table I).
`
`The effect of Pi concentration on calcium mobility
`Utilizing the techniques in vitro of Fig. 2, we measured the unidirectional M> S
`flux of calcium at two concentrations as a function of Pi at pH 7.4. Data from these
`experiments are presented in Table II. The ability of Pi to enhance uptake of calcium
`was dependent upon the initial concentrations of each of these two ions. At the lower
`concentration of calcium, to-fold excess of Pi was required for maximal stimulation.
`
`TABLE II
`
`CALCIUM FLUX in vitro,
`
`S, AS A FUNCTION OF Pi CONCENTRATION NON-SHORT-CIRCUITED SYSTEM
`
`Values are given in mµmoles calcium/cm2 per h and represent the average of at least 5 experiments
`for each condition.
`
`Calcium
`concentration
`(mM)
`
`Calcium flux at Pi concentration (nal)
`
`1.2
`
`5.7
`
`10.7
`
`0.4
`3.0
`
`45.7
`
`13.0
`102.0
`
`14.0
`10.5
`
`TABLE 111.
`
`UPTAKE in vivo OF CALCIUM, 1O-5 M, FROM AN INTESTINAL SEGMENT WITH AND WITHOUT Pi
`
`Period of uptake, T h. Values given are averages of 6 experiments at each condition and are
`percentages of original 47Ca taken up by the animal. Volume of solution injected, 2.0
`
`P i (mill)
`
`Uptake of
`calcium (%)
`
`23
`54
`
`Further increases in Pi caused no inhibition. However at 3 mM calcium, maximum
`stimulation was reached at approx. a 2:1 ratio of Pi to calcium; at higher concen-
`trations, Pi caused a marked inhibition. The inhibition observed at high concen-
`trations of calcium and Pi was due to visible precipitation and the resulting removal
`from the transport pool of calcium ions. Similar stimulation of calcium transport by
`Pi is seen in vivo where the presence of Pi plays an important regulator role in trans-
`port. Table III presents the results from a series of experiments with and without Pi.
`In the presence of excess Pi, there was better than a 2-fold increase in the uptake
`of calcium, 1 o-5 M.
`These experiments led us to examine the movement of P1 in the short-circuited
`system. It was possible that although calcium per se was not actively transported,
`a net active flux of Pi might have carried with it calcium as a counter ion. That Pi
`was indeed actively pumped M S is shown in Table IV. When we measured 45Ca
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`

`

`CALCIUM TRANSPORT BY RAT INTESTINE
`
`89
`
`trans-mucosal movement in the presence of the same concentration (3.7 mM) of P1,
`we found that the flux ratio of calcium was now 1.56. These results indicate the net
`serosal movement of both Pi and calcium with the possibility that calcium is moving
`as the counter ion in the transport process. When Pi was removed from the system,
`we observed the same results as seen in other experiments presented in this paper;
`the flux ratio for calcium returned to unity.
`
`TABLE IV
`
`SHORT-CIRCUIT FLUX RATIOS (M
`
`S/S M) OF CALCIUM, P1, AND CALCIUM + P1
`Initial concentrations of calcium, ro-5 M; Pi, 3.7 mM, where tested. Flux values are given as
`mumolesicm2 per h. The results are the average of 5 experiments in each direction.
`
`Condition
`
`M
`
`S M
`
`Flux ratio
`
`Calcium P i
`
`Calcium
`PI
`Calcium +
`
`Pi
`
`0.185
`70.1
`0.195
`81.7
`
`0.191
`45.5
`0.125
`58.7
`
`4-
`
`2
`
`1.54
`
`1.39
`
`0.97
`
`1.50
`
`EDTA
`
`NTA
`
`citrate
`
`fructose
`
`Coe' only
`
`0
`
`15
`
`30
`
`45
`
`60
`
`Time (min)
`Fig. 7. Effects of various chelating agents on the rate of calcium transport, M a S, in the non-
`short-circuited system. Initial calcium, 2.67 mM; P1, 10.7 mM. Chelate concentration; EDTA,
`8 mM; nitrilotriacetate (NTA), 8 mM; citrate, 8 mM; fructose, 27 mM.
`
`TABLE V
`EFFECT in Vivo OF CITRATE AND FRUCTOSE ON THE UPTAKE OF CALCIUM, 3 mM, IN THE PRESENCE
`OF Pi AT TWO CONCENTRATIONS
`Period of uptake, 1 h. Values are average of 6 animals at each condition, and are given as per cent
`of original calcium present.
`
`Condition
`
`Calcium uptake
`at Pi concentration
`
`1.2 mM 10.7 mM
`
`Calcium
`Calcium + fructose (3o mM)
`Calcium + citrate
`(3 mM)
`
`12.7
`
`12.1
`
`6.3
`8.4
`11.3
`
`Biochim. Biophys. Acta, 126 (1966) 81-93
`
`

`

`90
`
`H. J. HELBOCK, J. G. FORTE, P. SALTMAN
`
`The e cc!, of c.!-,clatcs on calcium transport in vitro and vivo
`As indicated in Tables II and V, at a concentration of 3.o mM calcium, the.
`addition of 10.7 mM P; caused a marked decrease in the M
`S movement of calcium
`both in vivo and in vitro. In order to overcome this inhibition, we added a series of
`chelating agents to the calcium solution. The results in vitro are shown in Fig. 7.
`These experiments were carried out on the non-short-circuited membrane using the
`apparatus in Fig. 2. It was evident that in the presence of excess I'; both citrate
`and fructose enhance calcium movement. EDTA and nitrilotriacetate were also tried.
`However, at the concentrations tested, these chelates appear to have increased the
`permeability of the membrane by chelating the endogenous calcium of the tissue.
`The effects of citrate and fructose were not seen if the Pi concentrations were in-
`sufficient to inhibit calcium movement. Fructose and citrate were tested in vivo using
`3 mM calcium and 10.7 mM Pi. Five animals were utilized for each experimental
`condition. It is clear from Table V that both fructose and citrate manifested similar
`activity in vivo as in vitro.
`
`DISCUSSION
`
`The experimental evidence above indicates that calcium ion per se, is not
`actively transported from mucosal to serosal surfaces of the rat small intestine. Only
`when Pi, or perhaps other suitable anions, are actively transported is there any evi-
`dence for a flux ratio of calcium different from unity. Further, at concentrations
`between ro-6 and ro-3 M, there is no evidence that calcium transport is facilitated
`by a membrane-bound carrier. These findings are not in agreement with other in-
`vestigations where a specific "calcium pump" has been proposeds,12. It is pertinent
`to examine some of the reasons for the discrepancies in results and interpretations
`and, in turn, to consider some physiological phenomena in light of our findings.
`We believe that the fundamental differences in the interpretation of the mecha-
`nisms of calcium transport can be traced to the experimental conditions used. In the
`experiments using everted intestinal loops, isotopes were injected on either one or
`both sides of the sacs, and sampling carried out at a fixed time intervals. Each sac
`could only be sampled once. It should be noted that the everted sacs contained a
`total volume of serosal fluid of about 0.5 ml, while the mucosal compartment was
`about 2.5 ml. Absorption and adsorption by the membrane became significant sources
`for error. The two techniques utilized by us permit continuous sampling of both sides
`of a single membrane and the detection of any changes in the flux as a function of
`time. The short-circuit technique permits continuous monitoring of membrane activity
`and eliminates the net movement of ions due to an electrical field. Perhaps the most
`important difference in technique was that in our experiments the large fluid volumes
`in both compartments relative to the small surface area of the tissue reduced errors
`introduced by membrane adsorption and absorption of the ion. Our methods insure
`continuous aeration of both sides of the membrane, rapid mixing of the liquid com-
`partments, and eliminate errors introduced by differences in hydrostatic pressure.
`What are the physiological and biological implications of our findings? That
`calcium may interact with a wide variety of ligands in the intestinal lumen to either
`enhance or impair its accumulation has long been knownls. We have been able to
`show quite clearly that under conditions where excess of Pi leads to the precipitation
`
`Biochinz. Biophys. Ada, 126 (1966) 81-93
`
`

`

`CALCIUM TRANSPORT BY RAT INTESTINE
`
`91
`
`of calcium, the presence of a suitable chelate solubilizes the ion and enhances its
`movement across the intestine. In part, we believe that this mechanism for transport
`explains the elegant work of DUPUIS AND FOURNIER16 and LENGEMANN et al.20 who
`were able to demonstrate that lactose and many other sugars and polyols enhance
`the nutritional availability and uptake of calcium.
`The interrelationship of calcium with inorganic phosphate is also of significance.
`As mentioned above, the solution chemistry of these two ions can result in the removal
`of calcium participation in any transport mechanism. On the other hand, calcium
`may interact with Pi and then be actively transported from the mucosa to the serosa.
`It should be pointed out that in the short-circuited intestinal preparation, the net
`uptake of calcium from M —> S induced by Pi appeared to be due entirely to a de-
`creased permeability of calcium from S -> M. Similar observations, whereby the back
`flux of a transporting system is under direct enzymic or metabolic control, has been
`observed in several other tissues, such as nerve21, muscle22, gastric mucosa23, and
`frog skin24. Even for the sodium-transport system of the intestine, SCHULTZ AND
`ZALUSKY25 reported that various methods of inhibition, such as glucose deprivation,
`2,4-dinitrophenol, and oubain, significantly increased the S —> M or backflux of
`sodium. For a formal treatment of this problem, the reader is referred to a paper
`by ROSENBERG AND WILBRANDT26.
`In the absence of short circuit for the system in vitro as well as in the intact
`animal, alteration of the net charge on the calcium ion or its complexes will have
`an appreciable effect on its transport. The dominant soluble form of calcium is un-
`charged in the presence of phosphate ion at pH 7.4. Thus the M S flux of the
`calcium will be enhanced because the influence of the membrane potential is di-
`minished, and this phenomenon may explain the increased calcium uptake observed
`in the presence of Pi both in vivo and in vitro. Although a significant fraction of
`calcium appears to move passively, it is possible that its movement with Pi is im-
`portant in meeting the nutritional requirements of the organism. In part, this inter-
`action of calcium with Pi might explain the activity of many other divalent ions
`that have been studied in biological systems. Thus interference of calcium transport
`by strontium that has been observed by some investigators27 could be explained on
`the competitive interaction of these divalent ions at the transport site which is
`activated by Pi.
`We have not, as yet, extensively investigated the role of various vitamins and
`hormones on this transport process. The literature regarding activities of vitamin D,
`parathyroid hormone, calcitonen, and other important mediators of calcium metabo-
`lism has been extensive. Alterations in calcium metabolism evoked by these com-
`ponents might well be due to either a change in the movement of Pi or to a funda-
`mental alteration in the permeability of the biological membranes to calcium18. If
`the former were true, alteration of the metabolic carrier mediating Pi movement could
`well influence the extent of the binding of metal ion. If the membrane permeability
`were changed, it would be possible to observe either an increase or decrease in the
`passive component of the calcium ion flux.
`During the last few years, there has been an intensive investigation of other
`organs and organelles with respect to calcium metabolism. In the systems studied
`including mitochondria and sarcoplasmic reticulum of muscle, it has been difficult
`to study these systems in the complete absence of phosphate at the ultimate site of
`
`Biochinz. Biophys. Acta, 126 (1966) 81-93
`
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`92
`
`H. J. HELBOCK, J. G. FORTE, P. SALTMAN
`
`the deposition of the calcium ion. Although the work of CHANCE29, CARAFOLI et al.30
`and others, carried out in the absence of Pi in the external medium suggests that
`calcium ion is actively pumped into the mitochondria, hydrolysis of endogenous phos-
`phate esters, such as ATP, would provide inorganic phosphate and would account
`for their observations with respect to electron transport. Recent studies of calcium
`in the sarcoplasmic reticulum indicate that the movement of calcium is very rapid
`and linked to ATP metabolism". Whether or not there is a system directly involved
`in calcium pumping in these tissues remains to be determined.
`It has been very fashionable to consider that all ions are actively pumped
`against concentration gradients by systems which require metabolic energy and are
`mediated by specific membrane carriers. Recent work' concerning the transport of
`trace metals, such as iron, zinc, copper and manganese has demonstrated that passive
`mechanisms can be important in the regulation and control of ion movement. In
`these passive systems, the solution chemistry of the particular ion being studied is
`of fundamental importance. In each case, the interaction of the divalent or trivalent
`metal ions with ligands and chelates can significantly influence the rate of transport.
`In the case of calcium, it has been possible to show that its movement across the
`gut wall is intimately related to the active transport of phosphate ion. It will be
`interesting to see whether or not other di- and trivalent metal ions manifest similar
`behavior.
`
`ACKNOWLEDGEMENTS
`
`This work was supported in part by grants from the John A. Hartford Foun-
`dation, U.S. Public Health Service Research Grant AM O5484, and L'.S. Public
`Health Service Research Career Development Award 2-K3-GM-3250 to Dr. PAUL
`SALTMAN. We wish to thank Mrs. FRANCES WILLIAMS BOOTH for her essential partici-
`pation as a research technician through the course of this work. Dr. ULRIK V. LAssEN-
`was of great help to us in the initial phases of this investigation. The critical and
`stimulating reviews of the manuscript by Drs. SAMUEL ALLERTON, PETER CuRRAN
`and ANTHONY NORMAN are appr