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From the earliest times, cells had to struggle to keep calcium out in order to prevent the precipitation of phosphate as calcium phosphate. Utilization of phosphate bond energy and of phosphorylation reactions had been an early metabolic decision by the ur-cells from which life evolved (26). Three general strategies to minimize the rise in cytosolic calcium are to restrict entry, to develop ways to extrude the ion from the cell, and to store it by binding. Indeed all three of these strategies have been used--calcium channels restrict calcium entry, calcium pumps extrude calcium, and organelles bind and store calcium.

This study assembles information on the mechanisms involved in intracellular calcium regulation and their actual or potential relationship to cellular calcium transport. Topics discussed in detail are calcium channels, cellular calcium extrusion, sodium/calcium exchange, calcium-binding proteins, with special reference to the vitamin D-induced calbindin, calcium transport and disorders. Each topic is introduced by an overview followed by research papers dealing with relevant topics in each category. New information deals with calcium channels which are not voltage-sensitive, the structure and function of the plasma membrane Ca ATPase, the role of the Na/Ca exchanger in intracellular Na and proton regulation, a comprehensive overview of calcium transport with quantitative analysis of the role of the intestinal and renal calcium-binding proteins, description of the structure and function of the calbindin genes, and identification of calcium transport defects in diabetes and hypertension.

Soluble cytoplasmic binding proteins have been associated with a wide range of metabolites, including inorganic and organic anions, lipids and numerous xenobiotics. They occur in most tissues of the body. The function of these molecules has been debated since their discovery. Newer experimental methods have permitted direct demonstration of their role in catalyzing the intracellular transport of small molecules. They accomplish this by increasing the concentration of the diffusing species in the aqueous cytosolic phase. Thus, they are most important for molecules that would otherwise have very low concentrations in the cytosol. Certain binding proteins also interact directly with membranes to catalyze transfer of their ligands and possibly to target them to intracellular sites. This symposium summarizes current data and thinking on the mechanisms of cytoplasmic transport, with emphasis on the role of soluble carrier proteins in the overall uptake and metabolism of small molecules.

Kinetic analysis of transmural calcium transport, as evaluated by in situ intestinal loops, has confirmed the existence of two transport processes, a saturable, transcellular one that is regulated by vitamin D and predominates in the proximal intestine and a nonsaturable process similar in intensity throughout the intestine. Transport data obtained from everted sac experiments are kinetically consistent with events in the in situ loop. Analysis of the three component steps making up the saturable process, i.e., entry across the brush-border membrane, intracellular diffusion, and extrusion across the basolateral membrane, indicates that intracellular diffusion is likely to be the limiting step. Active calcium transport varies directly and proportionately with the content of calcium-binding protein (CaBP), a specific molecular expression of the action of vitamin D. Since CaBP is a cytosolic protein, it may act to facilitate calcium diffusion, a proposition advanced by Kretsinger, Mann, and Simmons and supported here quantitatively. We calculate that the rate of intracellular calcium diffusion in the absence of CaBP is only approximately 1/70 of what is found in the vitamin D-replete cell. Similar considerations have led to the proposal that calcium moved by the nonsaturable process travels largely via the paracellular route. The kinetic parameters derived here, i.e., Vm = 22 mumol X h-1 X g (wt wt-1, Km = 3.9 mM, and a nonsaturable rate of 0.16/h, can be used to predict calcium absorption data as determined in previously published balance experiments.

Calcium absorption in the mammal results from two types of movements: The segmenting and peristaltic contractions of the small intestine which move calcium along the intestinal tract, and transmural movement which carries calcium across the intestinal epithelium. Regulation of calcium absorption could, therefore, involve alterations in intestinal contractions or events that affect transmural movement. In what follows we shall first describe studies that deal with the effect of intestinal movement on calcium absorption. We then deal with regulation of transmural movement, emphasizing the feedback regulation of calcium itself.

Calcium absorption from the intestine involves two sets of events. One, a saturable transcellular process is regulated by vitamin D via its molecular product, the calcium-binding protein (CaBP, MW = 8800). This transcellular movement is largely confined to the proximal portion of the intestine. The second process is nonsaturable, occurs throughout the length of the intestine and is paracellular. Evidence in support of these statements is discussed, with emphasis on kinetic considerations. It is proposed that CaBP acts as a ferry, amplifying the intracellular movement of calcium by a factor of about 60, thereby enabling transcellular calcium transport to reach the measured values of Vm = 22 mumol/h per gram (wet) duodenum, with Km = 3.9 mM. The transcellular process is subject to down-regulation and is influenced by functional or nutritional factors such as age or calcium intake. The nonsaturable process, on the other hand, is not directly influenced by these or related events. Vitamin D therapy alters active calcium transport, but may lead to undesirable effects at other target organs, e.g., kidney or bone. An increase in calcium intake is the simplest method for increasing the amount absorbed. Future research may show whether paracellular pathway alterations are a practical approach to changing the amount of calcium absorbed by the nonsaturable process.

Vitamin D-replete rats on high or low calcium diets received by intraperitoneal injection varying doses (62.5–750 ng/animal) of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 -D 3 ). The animals on the high calcium diet showed a progressive, dose-dependent response to the vitamin D metabolite; their duodenal levels of the cytosolic calcium-binding protein increased from about 50 nmol Ca bound /g mucosa to nearly 100 nmol Ca bound /g mucosa. The animals on the low calcium diet, whose calcium-binding protein base levels were about 100 nmol Ca bound /g mucosa, showed no response to the metabolite and their calcium-binding protein levels remained unchanged even with high doses of 1,25-(OH) 2 -D 3 . Vitamin D-deficient animals on a high calcium diet from weaning gave the same quantitative response to progressive doses of 1,25-(OH) 2 -D 3 as the replete animals, but the time and rate at which calcium-binding protein reached its maximum level in the intestine differed in the two groups. In the replete animals the calcium-binding protein response to 1,25-(OH) 2 -D 3 was completed by 1 h after treatment, whereas in the deficient rats the maximum response was not attained until 16–20 h. Plasma calcium responses to exogenous 1,25-(OH) 2 -D 3 occurred before the calcium-binding protein response in D-deficient animals and after in replete animals. The plasma calcium response therefore is not an index of the molecular response to vitamin D. It is concluded that the extent of the calcium-binding protein response to exogenous 1,25-(OH) 2 -D 3 is a function of the calcium status of the D-replete animal and that there exists an upper limit of this response. The time and rate of the calcium-binding protein response, on the other hand, depend on prior vitamin D status and may reflect a post-transcriptional as well as a transcriptional role of the metabolite in the intestinal cell.

Classical experimental rickets in the rat is a dual deficiency, resulting from both phosphate and vitamin D deficiency, with many of the features of rickets reproducible by simple phosphorus deficiency. Simple vitamin D deficiency differs markedly from experimental rickets, with only the absence of the vitamin D-dependent calcium-binding proteins common to both situations. The expression at the bone level of vitamin D deficiency differs in the two conditions, with rickets leading to profound structural and metabolic changes, whereas simple vitamin deficiency primarily compromises the regulatory function of bone, without obvious structural alterations. It is proposed that human nutritional rickets is the result of a nutritional vitamin D deficiency that aggravates the expression of a pre-existing metabolic defect in phosphate transport. Simple nutritional vitamin D deficiency, unaccompanied by rickets, may occur, but probably has always been rare.

Theophylline, when added to the incubation medium of everted duodenal sacs prepared from rats on a low-calcium diet, was found to inhibit transcellular Ca transport in a concentration-dependent manner, with an inhibitor constant (Ki) of 10.8 mM theophylline. Neither the rate of cellular Ca entry, as evaluated with the aid of brush-border membrane vesicles, nor the rate of cellular Ca extrusion, assessed by measuring ATP-dependent Ca uptake of basolateral membrane vesicles, was significantly altered by the addition of theophylline to the uptake media. However, Ca-binding by calcium-binding protein (CaBP; calbindin D9k), Mr approximately 8,800) was depressed by theophylline in a concentration-dependent manner, with Ki = 3.2 mM theophylline. Theophylline had no effect on Ca binding by calmodulin and the theophylline-induced inhibition of transcellular calcium transport was independent of adenosine 3',5'-cyclic monophosphate levels. Theophylline also had no effect on paracellular Ca movement. Since the theophylline-induced inhibition of Ca-binding by CaBP paralleled the inhibition of transcellular Ca transport, it is concluded that CaBP functions in transcellular Ca transport via its ability to bind Ca.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.

Intestinal calcium-binding protein (CaBP) levels of rats fed a high (1.5%) Ca diet were the same whether the animals were parathyroidectomized (PTX), sham-operated controls pair-fed with the PTX animals, or sham-operated controls fed ad libitum. Consequently, a given base level of CaBP seems to be parathyroid hormone independent and not closely related to feed intake. On the other hand, whereas the ad libitum fed controls more than doubled their intestinal CaBP in response to a 2-day low-calcium (0.02%) regimen, neither the parathyroidectomized animals nor the pair-fed sham-operated controls were able to do so. Since the latter two groups consumed less feed and therefore less vitamin D than the ad libitum fed animals, the inability to increase CaBP in response to a low-calcium diet may have been caused by a restricted vitamin D intake rather than by the absence of parathyroid hormone.

S35-sulfate was injected intraperitoneally- into 7-day-old rats and their long bones were removed after intervals of time. The epiphyses were separated from the diaphyses for analysis. From the diaphyses freed of bone marrow about 82 per cent of the S35 which they contained was extracted with a 2.5 N solution of sodium hydroxide. More, about 91 per cent of the S35, was thus extracted from the epiphyses. Dialysis of the extracts against water showed that the fraction of S35 which was dialyzable decreased rapidly with time. After 1 hour about 80 per cent and 50 per cent of the S35 in the extracts of diaphyses and epiphyses, respectively were found in the dialysates, after 24 hours about 20 per cent and 4 per cent, and after 120 hours 12 per cent and 1 per cent. Similar values for the S35 in inorganic sulfate were found when the extracts were chromatographed on an anion exchange resin, dowex-2. The S35, other than inorganic sulfate, was in the form of bound sulfate, which was released by acid hydrolysis. Uronic acid and hexosamines, primarily galactosamine, were associated with the S35. Indeed, on paper electrophoretograms and paper chromatograms the major S35-labelled component which was seen resembled chondroitin sulfate in its mobility. On the paper chromatograms, also a second S35-labelled component with a mobility lower than that of chondroitin sulfate was found. It is unlikely that the latter is a breakdown product of chondroitin sulfate, produced in the course of extraction with the sodium hydroxide solution. In fact, both components were also found in sodium versenate homogenates which had been dialyzed extensively against water. On the basis of these results it is suggested that the greatest part of the S35-labelled materials previously demonstrated by autoradiography to be progressively deposited in the metaphyses after 24 hours,—as the concentration of S35-sulfate concurrently decreased in the epiphyseal cartilage plates,—are akin to the chondroitin sulfate of the epiphyseal cartilage plates and are derived from the latter.

Weanling rats were given 2 mg. of 17-ß-estradiol benzoate at weekly intervals for 4 weeks. Twenty-four hours after each intraperitoneal injection of the estrogen 100 µc. of S35-sulfate or 11 µc. of Ca45 was similarly injected. The animals were sacrificed 24 hours after the last dose of isotopes. An effect of estradiol benzoate on calcium metabolism was deduced from the observation that the concentration of calcium in some tissues of the treated rats was higher than the concentration in the tissues of untreated rats. Alkaline extracts of the distal metaphyses of femurs from the estradiol-treated and from control rats, given S35-sulfate, were shown by chromatography on an anion exchange resin to contain from 9 to 22 per cent of the S35 as inorganic sulfate. From similar bone samples, 6 to 21 per cent of the S35 was removed by decalcification with sodium versenate. Most of the remaining S35 was associated with uronic acid and hexosamine; on paper chromatograms and paper electrophoretograms S35 was shown to be part of material which migrated and was metachromatic in the same way as purified chondroitin sulfate. Autoradiograms of the proximal ends of tibiae from the animals given estradiol benzoate showed that both the S35 and Ca45 were deposited in the metaphyses in strata. The arrangement of the strata of S35, however, was different from the arrangement of the strata of Ca45. This difference in arrangement is interpreted as indicating that most of the S35 in the metaphysis was derived from the chondroitin sulfate of the cartilage plate which the metaphysis had replaced.

Administration of parathyroid extract to immature rats that had been given S35 24 hours earlier resulted on the average in a 30% increase in the level of S35 in the plasma, a 25% higher output of S35 in the urine, a 17% increase in the pelt content of S35, and a 14% increase in the ratio (S35 in humerus ends:S35 in humerus shafts), as compared with litter mates treated with an inactive control solution. The findings are interpreted as indicating that calcium mobilization induced by the action of parathyroid hormone proceeds by removal of both organic and inorganic portions of bone. Concomitant histological observations confirmed the inference drawn from the chemical analyses, namely, that administration of parathyroid extract induced a higher than normal metabolic activity in the ends of the long bones and resulted in increased bone formation and resorption.

Osteomalacia and rickets are disorders of bone mineralization. There are many causes, but all may be controlled by suitable treatment. Except in vitamin-D deficiency, treatment often must be continued for years with careful chemical and clinical control. Relatively simple investigations enable one to distinguish between the various causes.

Treatment of duodenal tissue from rats deficient in vitamin D with 1,25-dihydroxy-vitamin D3 [1,25-(OH)2-D3] led to more than a doubling of calcium uptake by the isolated cells and the appearacne in those cells of previously undetectable calcium-binding protein (CaBP). Treatment with the precursor, 25-hydroxy-vitamin D3, was without effect on calcium uptake or CaBP. Cells from vitamin D-replete animals took up three and a half times more calcium than cells from deficient animals. This rapid (90-minute) effect of in vitro treatment with a physiological dose (4.7 X 10(-8)M) of 1,25-(OH)2-D is the first such report and is in accord with the regulatory role of the hormone-like sterol.

Ca-dependent and Mg-dependent ATPase activity was found to be depressed significantly in renal membrane preparations from vitamin D-deficient as compared to normal rats. Administratiin of 2000 i.u. vitamin D2 to deficient rats 30 hours before sacrifice restored the membrane enzyme activity to normal. ATP binding by enzymes was also reduced in vitamin D deficiency and raised by repletion. Vitamin D may therefore be required for normal Ca-and Mg-dependent ATPase activity.

Duodenal calcium transport was resolved into a saturable and a nonsaturable process by means of an in situ ligated loop procedure applied to Wistar rats at 3, 12, 19, 24, 30, 40, 60, 110, and 150 days of age. All postweaning animals were males that had been placed on a 1.5% calcium, 1.5% phosphorus semisynthetic diet. Duodenal calcium-binding protein (CaBP) levels were determined at all ages. The newborn rat had no saturable transport component and no CaBP. Its nonsaturable component was very high. With increasing age the saturable component and CaBP varied biphasically, increasing steeply until the animals were about 35 days old; thereafter, each decreased to low but detectable values. The nonsaturable component, on the other hand, decreased in near-linear fashion in the first 35 days; in animals beyond that age it remained invariant. The difference in age dependence between the saturable and nonsaturable components may be considered to constitute additional evidence for the existence of the two transport processes. CaBP and the saturable transport process were highly correlated, further proof that both are vitamin D dependent. Histological studies have revealed the presence of many vacuoles in the intestinal cells of the very young rats; these vacuoles were absent in rats older than 35 days. It is suggested that these vacuoles may be implicated in a pinocytosislike nonsaturable transport that is superimposed on the nonsaturable, non-vitamin D-dependent calcium transport found in all enterocytes.

Publisher Summary Homeostasis is a term introduced by Cannon to designate the maintenance of the milieu interne, a concept originally developed by Claude Bernard. The term homeostasis is used to refer to the steady-state level of plasma constituents and their maintenance. The chapter describes extracellular calcium homeostasis, its mechanisms, and the way it may be altered in disease. The calcium in the blood is found principally in the plasma, only about 3% of the total blood calcium occurring in the red blood cells. Of the calcium in plasma, about 45% is bound to protein, mostly albumin. The remainder is diffusible largely in the form of ionized calcium with small amounts of diffusible calcium also occurring as complexes of citrate, phosphate, and other organic or inorganic complexes. Plasma calcium regulation may be analyzed in terms of three characteristics: the set value around which the calcium level is regulated and has been termed [U] by Aubert and Bronner; the precision with which the set value is maintained and can conveniently be expressed as the coefficient of variation the set value; and the rate with which a positive or negative calcium load is overcome.

Vitamin D-replete (+D) and vitamin D-deficient (−D) rats received large doses of calcium (2–18 mg) by intraperitoneal injection and their response to the calcium load was analysed in terms of the instantaneous and time-dependent responses of the plasma calcium concentration, [Cas]. Following an initial expansion, [Cas] returned to the preinjection value in a strictly exponential manner, with t1/2 = 22.5 ± 2.0 (SE) min in + D and 51 ± 5.2 min in −D animals. In both groups of animals, these rates were independent of the calcium load. Extraprolation of [Cas] to t = 0, i.e., the time just after administration of the calcium, revealed that the amount of calcium circulating at that moment was only about one-fifth of the amount that would have been found if all of the injected calcium had remained in the plasma. Calculations suggest that in all animals about four-fifths of the injected calcium load became distributed virtually instantaneously in the extracellular water. In both + D and − D groups the fraction of the injected load that left the plasma instantaneously was independent of the calcium load, of [Cas] at t = 0 or of the animals' plasma volume. The ability of rats to disperse some 80% of the load to outside the plasma would seem to constitute a major mechanism of acute plasma calcium regulation. Dilution was insufficient, however, totally to reduce [Cas] to the preinjection level. That occurred exponentially, with most of the calcium presumed to enter the skeleton. This exponential rate was markedly and significantly slower in the vitamin D-deficient animals than in their controls.

Transepithelial calcium transport in the intestine involves an active and a passive route. The active route is totally vitamin D-dependent, transcellular, and is largely expressed in the proximal intestine. Of the three steps involved in transcellular transport--entry into the mucosal cell, intracellular movement, and extrusion at the basolateral pole of the cell--neither entry nor extrusion appears rate-limiting in the absence of vitamin D, even though both are enhanced as a result of the action of the vitamin D. However, intracellular calcium movement inside the mucosal cell can match the experimental Vm of transcellular transport only in the presence of the vitamin D-dependent calcium-binding protein (CaBP, Mr = 8.8kDa). CaBP is thought to act as the equivalent of a calcium ferry by amplifying the intracellular movement of calcium. Thus, the major action of vitamin D on cellular calcium transport is via its hormonal product, CaBP, which amplifies intracellular calcium movement by raising total and free calcium levels in the transporting cell.

Biosynthesis of the calcium-binding protein (CaBP) found in mucosal scrapings or isolated cells of rat intestinal mucosa (1) requires 1,25-dihydroxyvitamin D3 (1,25-(OH) 2-D3), the dihydroxylated metabolite of vitamin D (2). Studies of this process have suggested (3,4) that it is akin to steroid hormone induced protein synthesis, involving a sequence of two cytosolic and one nuclear receptor for 1,25-(OH) 2-D3, with CaBP synthesis the ultimate result. Cell-free translation of the chick CaBP, a larger molecule, and of the pig CaBP (MW ≃ 10,000, like that of the rat [5]) has been effected (6,7). In addition to an absolute requirement for 1,25-(OH)2-D3, CaBP biosynthesis is also regulated by calcium, inasmuch as animals on high calcium intakes have low levels of CaBP, while low calcium intakes are associated with much higher CaBP levels (5,8,9). Moreover, in vitro studies with isolated intestinal cells have suggested (10) that raising the calcium concentration of the medium depresses the amount of CaBP that can be synthesized. In addition, the calcium status of the whole animal affects the amount of circulating 1,25-(OH)2-D3, with larger amounts circulating and reaching the intestinal target cells in animals on low calcium intakes (11).

Vitamin D-replete (+D) and vitamin D-deficient (-D) rats received by intraperitoneal injection varying amounts of 1,25-dihydroxyvitamin D3, and 4 h (+D) or 9 h (-D) later everted duodenal sacs were prepared to evaluate active calcium transport, i.e., the amount of calcium found in the serosal fluid. At the same time, duodenal calcium-binding protein (CaBP) content was measured. Calcium transport was a close positive function of CaBP content. It was not detectable when CaBP content was zero and increased linearly without plateauing as CaBP content increased to 100 nmol calcium bound/g mucosa. Trifluoperazine (TFP) inhibited active calcium transport in a concentration-dependent manner. Experiments using vesicles prepared from brush-border or basolateral membranes indicated that TFP inhibited the calcium-extrusion process, with virtually no effect on calcium entry. It is concluded that vitamin D exerts its major regulation of active calcium transport in the rat duodenum via CaBP on transport steps beyond brush-border entry.