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Molecular Mechanisms of Intestinal Transport of Calcium, Phosphate, and Magnesium
59.1
Introduction
Calcium, phosphorus, and magnesium belong to a group of essential chemical elements required to support a variety of biochemical processes by serving structural and functional roles. Although sophisticated homeostatic mechanisms have evolved to maintain serum levels, intracellular levels, and optimal mineral content in bone, disorders of mineral metabolism, including abnormalities of calcium, phosphorus, and magnesium homeostasis, are not uncommon. The homeostatic mechanisms affect primarily the three major target organs, the intestine, kidney, and bone. With parathyroid hormone (PTH) and vitamin D remaining the principal regulators, the emerging regulatory network including membrane transporters, associated proteins, and soluble mediators becomes increasingly more complex. In this chapter, we will focus primarily on the transport processes and proteins responsible for the absorption of all three of these nutrient molecules across the gastrointestinal tract. The transporters identified for uptake of calcium and magnesium across the apical membrane of enterocytes are members of the transient receptor potential (TRP) family of cation channels, while intestinal P i absorption across the apical membrane is now known to be mediated by a member of the sodium-phosphate cotransporter gene family ( SLC34 ). Additionally, molecules responsible for movement of these molecules across the cellular cytoplasm (e.g., calbindins for Ca 2 + ) and exit across the basolateral membranes (e.g., plasma membrane calcium ATPase [PMCA1b] for Ca 2 + ) of intestinal epithelial cells have also been identified and in some instances their functional relevance has been addressed in targeted knockout models. With the discovery of these channels and transporters, we now have a much clearer picture of the molecular mechanisms involved in the transport of these crucial nutrient molecules. The purpose of this chapter is thus to provide updated information regarding the molecular mechanisms of intestinal transport of calcium, phosphate, and magnesium.
59.2
Recommended Nutritional Requirements for Ca 2 + , Mg 2 + , and P i
In 1997, the Institute of Medicine (IOM) issued a comprehensive report that presented dietary reference values for intake of nutrients by Americans and Canadians ( Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. National Academy Press, Washington, DC, 1997; http://books.nap.edu/catalog/5776.html ). This report was part of a larger project that was conducted with the involvement of Health Canada, and other US governmental agencies that also supported the project. Since the publication of this report, there has been increasing interest in the possibility of enhanced roles for vitamin D and calcium in human health, thus resulting in increased calcium and vitamin D supplementation by the food industry and in the emerging
controversies concerning the adequate and safe levels of intake. In response to these trends, the Institute of Medicine conducted a review of data pertaining to calcium and vitamin D in an effort to identify Dietary Reference Intakes (DRIs) based on current scientific evidence regarding the roles of calcium and vitamin D in human health. At the time of the writing this chapter, a new report was available as a preprint pdf at http://www.nap.edu/ catalog.php?record_id=13050 ( Dietary reference intakes for calcium and vitamin D . Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Institute of Medicine of the National Academies, National Academy Press, Washington, DC, 2010).
DRIs are reference values that can be used to formulate and assess diets for healthy populations. The DRIs replace the periodic revisions of the Recommended Dietary Allowances (RDAs), which have been published since 1941 by the National Academy of Sciences. DRIs encompass the Estimated Average Requirement (EAR), the RDA, Adequate Intake (AI), and the tolerable Upper intake Level (UL). As has been the practice with dietary recommendations in the past from the Food and Nutrition Board (within the IOM), the DRIs that are included in these two reports apply to the general healthy population. RDAs and AIs are nutrient intake target levels that should decrease the risk of developing pathophysiological conditions related to that particular nutrient. Importantly, consideration of dietary practices associated with Ca 2 + and other nutrients has been limited to observations made within the United States and Canada, and may not be directly applicable to all populations.
The EAR is a nutrient intake value that is estimated to meet the requirement by 50% of the individuals in a particular life stage and gender group. At this intake level, the remaining 50% individuals would have either inadequate or excessive intake of a particular nutrient. In some cases, these recommendations have been extrapolated to estimate this value. The EAR is used in setting the RDA, and it may be one factor used for assessing and planning the adequacy of intake of various groups. The RDA is the average daily requirement that meets the nutritional needs of all individuals in a life stage and gender group (97%–98%) and is set two standard deviations above the EAR. It should be noted that the RDA applies to individuals, not groups. The EAR serves as the foundation for setting the RDA. Furthermore, the AI is set instead of an RDA if insufficient scientific evidence is available to calculate an EAR. The AI is based on observed or experimentally determined estimates of average nutrient intake by a group or groups of healthy people. The principle, intended use of the AI is as a goal for the nutrient intake of individuals. Additionally, the tolerable UL is the highest level of daily nutrient intake that will likely not pose any risk of adverse health effects to almost all individuals in the general population. This term is intended to set a level of intake that can, with high probability, be tolerated biologically. The UL is not intended to be a recommended level of intake.
The issue of calcium and vitamin D intake levels is still under debate. In the revised IOM report, the committee expanded the list of potential health indicators for both nutrients beyond positive calcium balance and optimal bone mineralization. Although their review took into consideration numerous emerging indications for calcium and vitamin D supplementation (i.e., cancer, cardiovascular diseases, diabetes, immunity, or neuropsychological functions), the conflicting nature of available evidence could not be used to establish health benefits with any level of confidence. Therefore, the evidence surrounding bone health continues to provide the most reasonable and supportable basis for DRI development. This is primarily relevant in the elderly, in whom intestinal calcium transport is declining, as are skeletal calcium reserves. In humans, intestinal absorption of calcium decreases with advancing age. Similarly, the physiological adaptation to low dietary calcium is also blunted in the elderly. These changes can be attributed to a progressive resistance to vitamin D that develops in the process of aging. In addition, renal production of 1,25(OH) 2 D 3 may also be impaired in the elderly. Consequently, the dietary intake of calcium required to maintain positive calcium balance progressively increases with advancing age.
In summary, the RDA or AI for an individual is the aim for the intake of a particular nutrient, while the UL should be used as a guide to limit intake. Chronic intake of amounts him excess of the UL may increase risk of adverse effects. The EAR is used to examine the possibility of inadequacy and to set the RDA. Evaluation of the true status of individuals however, requires clinical, biochemical, and/or anthropometric data. The findings of the two IOM reports and the current DRIs for dietary calcium, phosphorus, and magnesium for populations of various ages and genders are summarized in Table 59.1 .
Table 59.1
Dietary Reference Intakes for Calcium, Phosphorus, and Magnesium
(Adapted from Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. Washington, DC: National Academy Press; 1997. http://books.nap.edu/catalog/5776.html and Dietary reference intakes for calcium and vitamin D . Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Institute of Medicine of the National Academies. Washington, DC: National Academy Press; 2010. http://www.nap.edu/catalog.php?record_id=13050 .)
| Life Stage Group a | Calcium | Phosphorus | Magnesium | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| EAR b | RDA c | UL d | AI e | EAR b | RDA c | AI e | EAR b | RDA c | AI e | ||||||
| Male | Female | Male | Female | Male | Female | Male | Female | Male | Female | ||||||
| 0–6 months | – | – | – | – | 1000 | 210 | – | – | 100 | – | – | – | – | 30 | 30 |
| 7–12 months | – | – | – | – | 1500 | 270 | – | – | 275 | – | – | – | – | 75 | 75 |
| 1–3 years | 500 | 500 | 700 | 700 | 2500 | 500 | 380 | 460 | – | 65 | 65 | 80 | 80 | – | – |
| 4–8 years | 800 | 800 | 1000 | 1000 | 2500 | 800 | 405 | 500 | – | 110 | 110 | 130 | 130 | – | – |
| 9–13 years | 1100 | 1100 | 1300 | 1300 | 3000 | 1300 | 1055 | 1250 | – | 200 | 200 | 240 | 240 | – | – |
| 14–18 years | 1100 | 1100 | 1300 | 1300 | 3000 | 1300 | 1055 | 1250 | – | 340 | 300 | 410 | 360 | – | – |
| 19–30 years | 800 | 800 | 1000 | 1000 | 2500 | 1000 | 580 | 700 | – | 330 | 255 | 400 | 310 | – | – |
| 31–50 years | 800 | 800 | 1000 | 1000 | 2500 | 1000 | 580 | 700 | – | 350 | 265 | 420 | 320 | – | – |
| 51–70 years | 800 | 1000 | 1000 | 1200 | 2000 | 1200 | 580 | 700 | – | 350 | 265 | 420 | 320 | – | – |
| > 70 years | 1000 | 1200 | 1200 | 1200 | 2000 | 1200 | 580 | 700 | – | 350 | 265 | 420 | 320 | – | – |
| Pregnancy | |||||||||||||||
| < 18 years | 1100 | 1300 | 3000 | 1300 | 1055 | 1250 | – | – | 335 | – | 400 | – | – | ||
| 19–30 years | 800 | 1000 | 2500 | 1000 | 580 | 700 | – | – | 290 | – | 350 | – | – | ||
| 31–50 years | 1000 | 580 | 700 | – | 300 | – | 360 | – | – | ||||||
| Lactation | |||||||||||||||
| < 18 years | 1100 | 1300 | 3000 | 1300 | 1055 | 1250 | – | – | 300 | – | 360 | – | – | ||
| 19–30 years | 800 | 1000 | 2500 | 1000 | 580 | 700 | – | – | 255 | – | 310 | – | – | ||
| 31–50 years | 1000 | 580 | 700 | – | – | 265 | – | 320 | – | – | |||||
a All groups not specified are males and females, except pregnancy and lactation.
b EAR , Estimated Average Requirement (in mg/day). The intake that meets the estimated nutrient needs of 50% of the individuals in a group.
c RDA , Recommended Dietary Allowance (in mg/day). The intake that meets the nutrient needs of 97%–98% of individuals in a group.
d UL , Upper Intake Level (in mg/day). The highest level of daily nutrient intake that will likely not pose any risk of adverse health effects to almost all individuals in the general population.
e AI , Adequate Intake (in mg/day). For healthy infants fed human milk, AI is the estimated mean intake.
59.3
Intestinal Calcium transport
Calcium is recognized as a critical element for normal homeostasis and is a major constituent of bone. An average 70 kg adult human has 1 kg of body calcium, with more than 99% of calcium found in bones and teeth in the form of calcium hydroxyapatite (Ca 10 [PO 4 ] 6 [OH] 2 ). Bone tissue serves as a reservoir and a source of calcium for critical metabolic needs provided by Ca 2 + in the circulatory system, extracellular fluid, muscle, and other tissues where it is necessary for mediating vascular tension, muscle function, nerve transmission, intracellular signaling, and hormone secretion. All the calcium in the body is acquired from maternal and dietary sources; therefore, a thorough understanding of the molecular mechanisms responsible for intestinal calcium absorption is critical ( Fig. 59.1 ). Calcium deposition rates to the skeleton vary with age, with highest rates of deposition being found in the neonatal period, followed by minimal deposition once growth ceases. In general, bone calcium deposition parallels removal of calcium from bone, with the difference between the two processes constituting the bone calcium mass. Bone calcium mass is very low in the newborn period; it increases until 35–45 years of age, and then it gradually decreases with increasing age in men and it abruptly decreases in women several years after menopause. Calcium from the diet is absorbed throughout the small and large intestines, with an active transport mechanism being predominant in the proximal small intestine under conditions of low calcium intake. When calcium intake levels are high, calcium is absorbed along the length of the small and large intestines via a paracellular absorptive mechanism. Several excellent reviews have been published on this subject.
59.3.1
Calcium Supply and Bioavailability
Current estimates indicate that the median total calcium intake from all sources (food and dietary supplements) for persons > 1 year of age range from 918 to 1296 mg/day, depending upon life stage. Dairy products including milk, yogurt, and cheese are rich sources of calcium, providing the majority of calcium from foods in the general diet. In the United States, an estimated 72% of calcium comes from such dietary sources. The remaining calcium comes from vegetables (7%), grains (5%), legumes (4%), fruit (3%), meat, poultry, and fish (3%), eggs (2%), and miscellaneous foods (3%) (U.S. Department of Agriculture/Economic Research Service Nutrient Availability Data (2009); http://www.ers.usda.gov/Data/FoodConsumption/NutrientAvailIndex.htm ). Calcium supplementation of a number of foods that do not naturally contribute this mineral (e.g., orange juice, cereals) is becoming widespread and makes it difficult for the national food composition databases (such as those maintained by USDA) to remain current and likely leads to some underestimation of actual calcium intake from food sources. Moreover, about 43% of all people in the United States and almost 70% of older women reported calcium intake from supplements. When calcium supplement use is taken into account based on these survey data, the average intake increases by about 7% for males and 14% for females.
Controlled metabolic studies undertaken across a wide age range by the USDA and described by Hunt and Johnson determined that fractional calcium absorption (percentage of a given dose of calcium that is absorbed) in men and nonpregnant women is approximately 25% of calcium intake. The fractional calcium absorption is influenced by age, physiological state (e.g., pregnancy), and the level of Ca 2 + intake. It is also influenced by the chemical form of calcium and modulated by dietary constituents. Calcium must be in a soluble form in order to be absorbed by the gastrointestinal tract. Even in the alkaline conditions of the ileum where calcium salts may be formed, some calcium ions remain in solution. Total calcium absorption is dependent upon three factors: (1) local solubility, (2) the rate of transepithelial transport of calcium across the intestinal epithelium, and (3) the transit time of chyme movement through a particular gut segment. Furthermore, calcium in vegetables and other solid foods must be digested before going into solution and being available for transport. Thus, the calcium present in foods with high fiber content is likely to be poorly absorbed compared to a more easily digestible food source with identical calcium contents, although true digestibility is difficult to predict and actual calcium transport rates may be higher than expected.
The most common forms of supplemental calcium are calcium carbonate and calcium citrate. Calcium carbonate provides 40% of elemental calcium, compared with 21% for calcium citrate. This helps with compliance among the persons taking calcium carbonate supplements (fewer tablets needed and lower cost). However, compared with calcium citrate, calcium carbonate is more often associated with gastrointestinal side effects, such as constipation, flatulence, and bloating. Calcium citrate is also less dependent on low stomach pH for absorption and can be taken without food, by patients with achlorhydria, inflammatory bowel disease, or by patients with gastroesophageal reflux disease taking histamine-2 receptor blockers or proton-pump inhibitors. Several studies have reported on attempts to improve bioavailability of calcium by adding casein phosphopeptides to foods, or by the use of highly soluble salts such as calcium gluconate or calcium gluconate-glycerophosphate. Other studies have addressed the question of whether manipulation of certain diet components, such as adding rice cereal to infant formula or by increasing dietary fiber in weaning cereals, adversely affects calcium bioavailability or absorption. From many of these studies, it is now apparent that when calcium intake is adequate or high, differences in bioavailability, such as from increased solubilization, have little effect on intestinal calcium transport rates and calcium deposition to the skeletal system. However, under conditions of low dietary calcium intake and when calcium is present in a more insoluble form (such as in oxalate-rich spinach), the decrease in calcium transport becomes nutritionally significant. Several studies have sought to understand the high-absorbance rate of calcium from milk and suggested that lactose, phosphopeptides, and amino acids may play a role in this phenomenon. Interestingly, calcium bioavailability in some milk substitutes is decreased as compared to milk, despite the fact that nutritional quality is similar. Another area of interest is related to the effect of fat intake and the formation of calcium soaps on calcium bioavailability and intestinal transport rates. One study concluded that the addition of synthetic triglycerides to infant formula significantly improved calcium absorption in preterm neonates. However, the effect of fat intake on intestinal calcium homeostasis in older children and adults is not known. Furthermore, Sanyal et al. demonstrated that premicellar taurocholate, a component of bile, enhances calcium absorption from all regions of the rat small intestine.
A number of inorganic molecules and dietary components have also been recently reported to perturb intestinal calcium absorption. Phosphate may interfere with calcium absorption as a result of a formation of poorly soluble calcium-phosphate salts, but increasing dietary phosphate by a factor of 2.5 has no reported influence on calcium absorption, regardless of the amount of calcium consumed. However, indigestible organic phosphates, such as phytates of hexaphosphoinositol which are found in wheat bran, can form insoluble calcium salts. High concentrations of dietary magnesium can decrease calcium absorption presumably by competing for the calcium transport site, although we now know that calcium and magnesium are absorbed by different channel proteins. This effect could be due to interaction of magnesium ions with the specific calcium channels in the gut (TRPV6), as extracellular Mg 2 + has been shown to block the monovalent cation currents induced by TRPV6 in a heterologous expression system.
Certain antibiotics and cationic amino acids such as lysine stimulate intestinal calcium absorption. Similarly, lactose, other complex sugars, and glucose polymers promote calcium absorption. Although gastric acidity or achlorhydria was initially thought to have limited effects on intestinal calcium transport, there is an increasing concern that hypochlorhydria resulting from atrophic gastritis, bariatric surgery, or high-dose chronic use of proton pump inhibitors may create a risk for malabsorption of dietary and supplementary calcium and may increase the risk of osteopenia and osteoporosis in the long term.
Calcium is also known to be absorbed in the mammalian colon, with the cecum and the ascending colon having the highest rates; however, the transverse colon does not transport calcium. It has recently been demonstrated that acidic fermentation in the colon can enhance intestinal absorption of calcium and magnesium. Younes et al. concluded that the large intestine may represent a major site for calcium transport when acidic fermentation takes place. Likewise, another group demonstrated that fructooligosaccharides significantly enhanced calcium and magnesium absorption in the rat colon. These authors concluded that indigestible and fermentable carbohydrates facilitate colorectal absorption of these ions. Mineo et al. demonstrated that indigestible disaccharides directly affect the small intestinal and colonic epithelium by opening tight junctions between epithelial cells and thereby promoting Ca 2 + and Mg 2 + absorption. Additionally, another study showed that two fermentable carbohydrates (inulin and resistant starch) showed synergistic effects on intestinal calcium absorption and calcium balance in rats. Calcium absorption may also be enhanced by acetate and propionate in the human rectum and distal colon, by a nonsaturable, diffusive process.
The net epithelial transfer of calcium represents the sum of two opposing processes: transepithelial absorption and the loss of calcium in shed mucosal cells and in secretory fluids (saliva, gastric and pancreatic juices, and bile). Thus, fecal calcium contains unabsorbed calcium from dietary sources as well as endogenously excreted calcium. Abrams et al. estimated the endogenous fecal calcium losses at approximately 2.1 mg/kg/day in adults and 1.4 mg/kg/day in children. In contrast to urinary excretion, endogenous fecal Ca 2 + excretion is not significantly influenced by aging.
59.3.2
Molecular Mechanisms of Intestinal Calcium Transport
Depending on the segment and the luminal Ca 2 + concentration, absorption occurs via a weakly regulated paracellular route, or by a highly regulated and 1,25(OH) 2 vitamin D 3 -sensitive transcellular pathway. When dietary Ca 2 + intake and luminal concentration is low (< 20 mM), active transcellular transport in the duodenum predominates and accounts for approx. 80% of the total Ca 2 + absorption. At higher Ca 2 + supply (> 50 mM), the contribution of active transport diminishes to > 10%, largely due to the short duodenal transit time and downregulation of the key molecular components of the transcellular Ca 2 + transport pathway. In addition to calcium content in the diet, the amount of Ca 2 + transferred paracellularly is directly related to its solubility (decreasing in the distal small intestine), the sojourn of the chyme in a given intestinal segment (highest in the ileum), and the rate of paracellular diffusion from lumen to blood. Age is also an important factor determining the primary Ca 2 + absorption route. In newborns, it is largely passive and facilitated by the lactose content of breast milk. Ghishan et al. and Halloran and DeLuca demonstrated that as the neonate grows, passive absorption declines and calcitriol-stimulated active intestinal calcium absorption becomes more important. Three complementary models of transcellular Ca 2 + absorption have been proposed: a facilitated diffusion model, a vesicular transport model, and tunneling through the endoplasmic reticulum. All four modes of intestinal Ca 2 + transport are systematically discussed below.
59.3.2.1
The Paracellular Pathway of Dietary Calcium Absorption
Although paracellular Ca 2 + transport is the major route of absorption in the intestine with high-calcium diets, its importance or regulation has not been investigated in great detail. It is generally believed that contrary to the transcellular pathways, paracellular Ca 2 + transport is not tightly regulated. However, recent years have brought an increasing appreciation of the complexity and plasticity and dynamics of tight junction (TJ) assembly, and of the role of TJ components in paracellular Ca 2 + permeability. Biochemical and physiological aspects of tight junctions are reviewed in more detail elsewhere in this book (T. Ma “Gut Barrier: Tight Junctions”). Kutuzova and Deluca identified a number of 1,25-(OH) 2 D 3 target genes whose protein products are localized in the vicinity of intestinal tight junctions and which could influence TJ integrity and permeability. These included not only transporters and channels but also several intra- and intercellular matrix-related proteins and G-proteins. There were also a number of 1,25-(OH) 2 D 3 downregulated genes encoding proteins that are involved in the regulation of tight junction permeability, including sodium-potassium ATPase, claudin 3, aquaporin 8, cadherin-17, and RhoA. The conclusion was drawn that 1,25-(OH) 2 D 3 may increase intestinal epithelial tight junction permeability or modulate their selectivity toward Ca 2 + and other cations by regulating expression of proteins involved in tight junction formation. The increased tight junction permeability and/or selectivity, regulated by 1,25-(OH) 2 D 3 , could direct Ca 2 + absorption through the tight junction-regulated paracellular pathway in the intestinal epithelium. This suggestion is in agreement with a recent study that demonstrated that 1,25-(OH) 2 D 3 stimulated an increase in tight junction conductance, and increased paracellular Ca 2 + , Na + , Rb + , and mannitol transport in an enterocyte-like cell line (Caco-2). Under these conditions, no significant contribution of the Ca 2 + -ATPase-mediated transcellular pathway to overall transepithelial Ca 2 + transport was detected. More recently, Fujita et al. showed that 1,25-(OH) 2 D 3 upregulates claudin-2 and claudin-12 in intestinal epithelial cells via a VDR-dependent mechanism and that both claudins contribute to the vitamin D-induced increase in paracellular Ca 2 + permeability. Claudin-16 (paracellin 1) has also been implicated in playing a significant role in paracellular permeability of Ca 2 + and Mg 2 +, and mutations within the claudin-16 gene have been linked with familial hypomagnesaemia with hypercalciurea and nephrocalcinosis (FHHNC). However, due to a limited pattern of expression (selective expression at tight junctions of renal epithelial cells of the thick ascending limb of the Henle loop), the role of claudin-16 in intestinal Ca 2 + permeability is unlikely.
Additional evidence has accumulated demonstrating that 1,25-(OH) 2 D 3 enhanced both cell-mediated active and passive, paracellular Ca 2 + and P i transport in a Ussing chamber study with rat small intestine and for Ca 2 + transport in Caco-2 cell monolayers. More recently, Tudpor et al. demonstrated nontranscriptionally mediated, short-term increase in paracellular Ca 2 + transport across rat duodenum treated in vitro with 1,25-(OH) 2 D 3 . This solvent drag-induced increase in Ca 2 + transport was dependent on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein (MAP) or extracellular signal-regulated (Erk) kinase (MEK) activity. Unlike the passive, Ca 2 + gradient-directed transport, solvent drag-induced paracellular Ca 2 + absorption resembles a secondary active transport in that it is dependent on the activity of Na + /K + -ATPase that creates a paracellular hyperosmotic gradient resulting from a sodium concentration ~ 15 mM higher than the surrounding milieu and is stimulated by high glucose level.
59.3.2.2
Facilitated Diffusion: Apical Ca 2 + Entry
Early studies indicated that over a wide range of transepithelial Ca 2 + transport rates, Ca 2 + influx at the apical membrane is correlated in a 1:1 fashion with the apical to basolateral Ca 2 + flux. Ca 2 + entry was postulated to occur via Ca 2 + -selective channels at the luminal membrane, under the influence of a steep inwardly directed electrochemical gradient. Initial studies demonstrated that 1,25(OH) 2 D 3 affected Ca 2 + influx in enterocyte-like cells. This calciotropic hormone significantly increased radiolabled Ca 2 + uptake within several minutes in a dose-dependent manner in rat, intestinal epithelial cells. These and other studies suggested that a Ca 2 + channel was activated by 1,25(OH) 2 D 3 through a cAMP/PKA-dependent pathway in the mammalian intestine.
Further studies utilized functional expression cloning in an attempt to identify the apical calcium influx transporter/channel. The first such experiments were done by Hoenderop et al., who utilized a cDNA library constructed from poly(A) + RNA isolated from primary cultures of rabbit kidney cells. The cDNA clones were copied into cRNA, which was microinjected into Xenopus laevis oocytes, followed by screening for Ca 2 + uptake activity in the presence of a cocktail of known voltage-gated Ca 2 + channel inhibitors (the renal and intestinal Ca 2 + channels were known to NOT be voltage-gated). After extensive screening procedures, a single transcript was isolated which encoded a novel epithelial Ca 2 + channel (first called ECaC1 and recently renamed as transient receptor potential channel TRPV5; also known as ECaC and CaT2). When the TRPV5 channel was expressed in Xenopus oocytes and in HEK293 cells (human, embryonic kidney cells), calcium transport was inhibited by (in order of potency) La 3 + > Cd 2 + > Mn 2 + , whereas Ba 2 + and Sr 2 + had no effect. Permeability to Na + was negligible in the presence of Ca 2 + . Moreover, the TRPV5 channel was shown to be induced by 1,25(OH) 2 D 3. Overall, these observations demonstrated that TRPV5 possesses all the expected characteristics of the calcium influx in Ca 2 + -transporting epithelia. The TRPV5 transporter was subsequently determined to be expressed predominantly in the kidney and was conclusively shown to be responsible for renal Ca 2 + reabsorption.
Subsequent studies by Hediger and coworkers applied the functional expression cloning technique to the search for the intestinal calcium entry channel, and they were able to identify an intestinal Ca 2 + transporter, which they called CaT1. CaT1 shares 80% amino acid sequence identity to TRPV5. CaT1 has subsequently been renamed TRPV6 and is also known in the literature as ECaC2 and CaT-like. Electrophysiological studies, as described in detail below, demonstrated that the characteristics of TRPV6 are comparable to those measured for TRPV5, but the expression pattern of TRPV6 is more ubiquitous. Moreover, functional properties of both TRPV5 and TRPV6 are in congruence with the known properties of the putative epithelial calcium channels, which are responsible for intestinal and renal calcium (re)absorption. TRPV6 is currently thought to be the major player in active, intestinal calcium transport.
59.3.2.2.1
The Transient Receptor Potential (TRP) Family of Channel Proteins
TRP channel proteins form a large and diverse, but related family of proteins that are expressed in many tissues and cells types. The large functional diversity of this family of proteins is reflected in their variable permeability to ions, activation mechanisms, and their involvement in a wide range of physiological processes. The TRP channels can be divided by sequence homology into at least six subfamilies, designated TRPC (canonical or classical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystins, PKD-type), TRPA (for ankyrin), TRPML (for mucolipin), and the more distantly related subfamily TRPN (N for “nomp,” no mechanoreceptor potential). An intriguing subfamily within the TRP superfamily is the TRPV family, consisting of six members. This group of channels includes TRPV1-4, which respond to heat, osmolarity, odorants, and mechanical stimuli, whereas TRPV5 and 6 are epithelium Ca 2 + channels and have been implicated in maintaining body-Ca 2 + balance by facilitating Ca 2 + (re)absorption in the kidney and small intestine.
Extensive scientific evidence supports the concept of paracellular transport of calcium with high dietary intake levels, as described earlier in this chapter. However, Morgan et al. reported the discovery of another intestinal calcium channel (Ca v 1.3; CACNA1D), homologous to the neuroendocrine L-type Ca v 1.3 calcium channel. This group’s immunocytochemical studies demonstrated its expression on the apical membrane of enterocytes from the proximal jejunum to the mid-ileum, and our own observations further determined Ca v 1.3 expression in the human and mouse colon. Perfusion studies with 1.25 mM luminal calcium revealed L-type calcium channel activity; this activity was inhibited by phloridizin, nifedipine and Mg 2 + , and activated by Bay K 8644. These findings demonstrated channel-mediated rather than paracellular calcium transport. Furthermore, none of these compounds affected the expression of the active calcium transport channels (e.g., TRPV5 and TRPV6; discussed below). These authors thus conclude that intestinal Ca v 1.3 may mediate a significant route of calcium transport during times of dietary calcium sufficiency. In contrast to other channels such as TRPV6 that work under a hyperpolarized setting, Ca v 1.3 is hypothesized to contribute to an alternative, TRPV6-independent, route of intestinal Ca 2 + absorption. While TRPV6 plays a more dominant role under the polarizing conditions between meals, Ca v 1.3 plays a dominant role under postprandial depolarizing conditions, such as during digestion, when nutrients and luminal Ca 2 + are abundant. In in vitro studies with Caco-2 cells, Nakkrasae et al. showed that Ca v 1.3 was the sole apical channel responsible for the prolactin-stimulated transcellular calcium transport. The recently reported phenotype of Ca v 1.3-null-knockout mice includes a smaller skeleton, lower body weight, and lower bone mineral content. However, intestinal Ca 2 + absorption has not been measured in these mice and since Ca v 1.3 is expressed in multiple tissues, including osteoblasts, the role of this Ca 2 + channel in intestinal epithelial cells would have to be more precisely elucidated in a tissue-specific knockout model, especially that the contribution of Ca v 1.3 to either basal or vitamin D-regulated intestinal Ca 2 + absorption in vivo has been recently questioned by Reyez-Fernandez and Fleet.
59.3.2.2.2
TRPV5 and TRPV6
The epithelial Ca 2 + channel family is restricted to two members, and genomic cloning demonstrated that TRPV5 and TRPV6 channels are transcribed from distinct genes. Interestingly, TRPV5 and 6 are juxtaposed on human chromosome 7q35, with a distance of only 22 kb separating them, which suggests that a single ancestral gene was duplicated over evolutionary time. An analogous situation was observed in the mouse genome, in which the two genes are located close to one another on chromosome 6, in a region that is syntemic to human chromosome 7q33-35. To date, TRPV5 and TRPV6 have been cloned from many species, including rabbit, rat, mouse, and human. The putative proteins exhibit an overall amino acid sequence homology of 75%–80%. Interestingly, several domains within these proteins are completely conserved between species, including the membrane topology of the protein with six putative transmembrane segments and the postulated pore region. Furthermore, detailed sequence analysis of these proteins identified several putative phosphorylation sites, for PKC-, PKA-, and cGMP-dependent kinase. Phosphorylation of Thr 709 by cAMP-dependent protein kinase A has recently been reported to mediate PTH-induced channel’s opening probability and Ca 2 + reabsorption in the kidney. Another example of phosphorylation-dependent regulation of TRPV5 was provided by Topala et al., who showed that in renal epithelial cells, activation of the extracellular calcium sensing receptor (CaSR) stimulates TRPV5-mediated Ca 2 + influx via a PMA-insensitive PKC-mediated phosphorylation of Ser 299 and Ser 654 . While TRPV6 was not affected by CaSR activation, phosphorylation may also play a role in regulating this primarily intestinal Ca 2 + channel. Inhibition of tyrosine phosphatases increases TRPV6 activity—an effect abolished by mutations Tyr 161 and Tyr 162 . Moreover, ATP stabilizes TRPV6 Ca 2 + current, a phenomenon that can be reversed by PKC stimulation with phorbol ester (PMA). Two residues at both the N- and C-termini, Ser 144 and Thr 688 , synergistically contribute to mediate the effect of PMA; these two amino acids have been postulated to form ATP binding domains, allowing to form a bridge between the termini and modulating TRPV6 channel properties.
Additionally, both the TRPV5 and six proteins contain PDZ motifs and ankyrin repeats in the NH 2 -terminal region, which are conserved in a diverse range of receptors and ion channels, including the TRP superfamily. PDZ motifs are recognized by proteins containing PDZ interacting domains, and these protein-protein interactions may be involved in protein targeting and multiprotein complex assembly. PDZ domain interactions serve not only scaffolding and cytoskeletal attachment roles but there is also evidence that they can regulate the functions of their ligands. PDZ domain-containing proteins typically interact with COOH-terminal PDZ motifs in target proteins, but interactions could also occur with the N-terminal motifs that are present in TRPV5 and 6. Ankyrin repeats have similar roles as PDZ domain/motif interactions, in that they can link transporters and cell adhesion molecules to spectrin-based cytoskeletal elements in specialized membrane domains. Kim et al. proposed that PDZK2 is an essential TRPV6-interacting protein and a physiological modulator of TRPV6 activity. A direct interaction between the TRPV6 C-terminal PDZ-binding motif and PDZK2 was identified by GST pull-down assay. Although heterologous overexpression of both TRPV6 and PDZK2 did not affect the activation of TRPV6, mutation of the four critical residues in TRPV6 (EYQI) decreased peak current amplitude of the channel and RNAi-mediated knockdown of endogenous PDZK2 in HEK293 cells significantly decreased current density in divalent-free media (DVF).
Several studies addressed the expression of TRPV5 and TRPV6 in the gastrointestinal tract. Initially, Northern blot analysis showed expression of rabbit TRPV5 in duodenum and jejunum, whereas ileum was negative. However, these hybridizations were performed prior to the identification of the TRPV6 isoform, using full-length cDNA probes that did not discriminate between the highly homologous TRPV5 and TRPV6 transcripts. Subsequent experimental approaches using isoform-specific probes, quantitative PCR analysis, and immunohistochemical studies found expression of both channels in the intestine. However, these studies demonstrated that TRPV6 transcript levels in the gut are at least three orders of magnitude higher than TRPV5 transcript levels. Another study addressed the issue of whether TRPV5 has an important physiological role in the intestine, by creating TRPV5-knockout mice. These animals exhibit intestinal Ca 2 + hyperabsorption, most likely mediated by increased TRPV6 and calbindin-D 9k expression levels, which suggests a predominant role for TRPV6 in intestinal calcium transport. Consistent with this observation, targeted deletion of the TRPV6 gene resulted in perturbed Ca 2 + homeostasis, including a 60% decrease in intestinal Ca 2 + absorption, deficient body weight gain, decreased bone mineral density, and reduced fertility. Changes in bone density were later associated with increased bone resorption driven by increased osteoclasts differentiation and activity in TRPV6 −/− mice. Similarly, homozygous knock-in of a D541A mutation in the TRPV6 pore region into mouse TRPV6 gene resulted in significantly impaired intestinal Ca 2 + uptake despite an increase in duodenal TRPV5 expression during feeding with low Ca 2 + diet.
In the mammalian intestine, TRPV6 expression is found in the duodenum, jejunum, cecum, and colon where it is colocalized in epithelial cells along with other molecular components of intestinal calcium absorption, calbindin D 9k , and PMCA1b. One study conducted by Hediger and coworkers demonstrated expression of TRPV6 throughout the entire digestive tract from esophagus to colon. Additional studies estimated TRPV6 and TRPV5 mRNA expression levels in the mouse by quantitative PCR analysis, and resulting data were normalized for the amount cDNA used for the amplification. This study demonstrated that TRPV6 mRNA expression was highest in duodenum and cecum, lower in the colon, and even lower in the ileum. This investigation also demonstrated that TRPV5 mRNA was expressed at much higher levels in the kidney as compared to the duodenum and cecum, whereas ileum and colon did not express TRPV5 mRNA. Immunohistochemical techniques have also been used to determine the distribution of TRPV6, calbindin D 9k , and PMCA1b proteins in the small intestinal epithelium. TRPV6 was localized along the brush-border membrane, whereas calbindin D 9k was found in the cellular cytoplasm and PMCA1b was expressed at the basolateral membrane. Further detailed immunolocalization studies demonstrated expression of TRPV6 on the apical membrane of enterocytes in the entire small intestine and colon.
When considered in their entirety, the current data strongly suggest that the epithelial Ca 2 + channel TRPV6 is the major transcellular mediator of Ca 2 + uptake from the intestinal lumen. Thus, the remainder of this section will focus on this intestinal calcium channel.
59.3.2.2.3
TRPV6 Molecular Structure and Protein-Protein Interactions
Crystal structure of TRPV6 has been described by Saotome et al., and the structural elements and requirements for activity of TRPV5 and TRPV6 channels have been recently reviewed by van Goor et al. TRPV6 likely forms homotetramers in the plasma membrane of cells. This tetrameric organization closely resembles the structure of the Shaker potassium channel, which is composed of four tandemly associated homologous domains. The clustering of the four subunits is thought to create an aqueous pore centered at the fourfold symmetry axis. This proposed tetrameric architecture implies that aspartic acid residues D542 and D541 form a negatively charged ring structure that functions as a calcium-selectivity filter, analogous to voltage-gated calcium channels. Recently, Niemeyer et al. identified the third ankyrin repeat in TRPV6 as being critical for physical assembly of TRPV6 subunits into the tetrameric form. Deletion or mutation of amino acid residues within this ankyrin repeat renders the channel nonfunctional and abolishes tetrameric formation. It was suggested that the third ankyrin repeat initiates a molecular zippering process that proceeds past the fifth ankyrin repeat and creates an intracellular anchor that is necessary for assembly of the functional subunits. However, in a recent report by Phelps et al., which provided detailed mapping and crystal structure of the N-terminal ankyrin repeat domain (ARD) of TRPV6, TRPV6-ARD’s were monomeric in solution. Furthermore, the packing and symmetry of the TRPV6-ARD crystals seemed incompatible with tetrameric assembly of the ARD around a fourfold symmetry axis. While previous studies demonstrated that the integrity of the ARD is important in channel assembly, this study indicates that it is not mediated through self-tetramerization of the ARD. The authors hypothesized that TRPV6 assembly may be assisted by additional cellular factors, which require the ARD but are unable to bind an ARD destabilized by mutation or partial deletion.
In addition to forming homotetramers, TRPV5 and TRPV6 can form heterotetramers. This observation is based upon cross-linking studies, coimmunoprecipitations and molecular mass determination of TRPV5/6 complexes using sucrose gradient sedimentation. When these two isoforms are coexpressed in some tissues, they may oligomerize, and this hetero-oligomerization may influence the functional properties of the Ca 2 + channels formed. As both these proteins exhibit different channel kinetics with respect to Ca 2 + -dependent inactivation, Ba 2 + selectivity and sensitivity for inhibition by ruthenium red, the influence of the heterotetrameric composition on channel properties could be important in certain tissues and cell types. In the intestine, however, TRPV6 has been shown to be expressed at levels as much as 100–1000 times higher than TRPV5, and no experimental evidence has directly demonstrated that there is actually heterotetramerization of the two channels in the intestine.
A number of regulatory proteins have been described that modify the activity, and biophysical and pharmacological properties of ion channels and transporters by direct, physical interactions, including TRPV5 and TRPV6. An auxiliary protein of TRPV6 was identified by screening a mouse kidney cDNA library using the yeast two hybrid system . This study identified S100A10 as a protein that specifically associates with the carboxy-terminus of the TRPV6. S100A10 is a 97 amino acid protein, which is a member of the S100 superfamily, that is present in vertebrates, insects, nematodes, and plants. This protein is predominately present as a heterotetrameric complex with annexin 2, which has been implicated in numerous biological processes including endocytosis, exocytosis, and membrane-cytoskeletal interactions. A recent report suggested a regulatory role for the S100A10-annexin 2 heterotetramer in vitamin D-mediated, intestinal calcium transport and in TRPV6 function and/or regulation. The association of S100A10 with TRPV6 was restricted to a short-peptide sequence NH 2 -VATTV-COOH located in the carboxy-terminus of the channel, a region that is conserved across species. Intriguingly, the NH 2 -TTV-COOH sequence in the putative S100A10 binding motif of TRPV6 resembles an internal, type I PDZ consensus binding sequence (NH 2 -S/TXV-COOH). However, S100A10 does not contain PDZ domains, suggesting that the interaction with TRPV6 is distinct. The first threonine of the S100A10 interaction motif is crucial for binding to TRPV6. In fact, the activity of TRPV6 was abolished when this particular residue was mutated, demonstrating the necessity for calcium channel function. Furthermore, these mutant channels were mislocalized within cells, indicating that the S100A10-annexin 2 heterotetramer facilitates the translocation of TRPV6 channels to the plasma membrane. The importance of annexin 2 in the process was demonstrated by siRNA-mediated knockdown of annexin 2, which significantly inhibited the currents through TRPV6, exemplifying that annexin 2 in conjunction with S100A10 is necessary for normal TRPV6 activity. More recently, Borthwick et al. found that formation of the annexin 2-S100A10-TRPV6 complex was dependent on forskolin-induced and calcineurin (CnA)-dependent dephosphorylation of annexin 2 in lung and intestinal epithelial cells. This study demonstrated that cAMP/PKA/CnA signaling was important for annexin 2-S100A10 complex formation and interaction with target molecules in both absorptive and secretory epithelia.
Interestingly, similar to the epithelial calcium channels, S100A10 expression was found to be vitamin D sensitive. In addition, annexin 2 expression levels have been shown to increase with 1,25(OH) 2 D 3 treatment. Thus, physical interaction and coregulation of TRPV6 with S100A10 and annexin 2 could control trafficking of these channels to the plasma membrane. 1,25(OH) 2 D 3 has been shown to act via rapid nongenomic and slower genomic actions. The genomic effects are mediated by interaction with nuclear VDR/RXR heterodimers. Recently, it was reported that annexin 2 serves as a membrane receptor for 1,25(OH) 2 D 3 and that it mediates a rapid effect of the hormone on intracellular calcium homeostasis. It was shown that that 1,25(OH) 2 D 3 specifically bound to annexin 2 on the plasma membrane of rat osteoblast-like cells. Partially purified plasma membrane proteins and purified annexin 2 exhibited specific, saturable binding for tritiated 1,25(OH) 2 D 3 . These results suggest that annexin 2 may serve as a receptor for rapid actions of 1,25(OH) 2 D 3 ; however, this concept is still under debate. Taken together, these findings show that the S100A10-annexin 2 complex is required for the trafficking of TRPV6 to the plasma membrane and therefore is involved in overall calcium homeostasis. In addition to association with annexin 2, TRPV6 has also been found to associate with cyclophilin B (CypB) in the human placenta. When coexpressed in Xenopus oocytes, CypB increased TRPV6-mediated calcium uptake, a phenomenon that could be inhibited with CypB inhibitor, cyclosporin A. Although, as with most cyclophilins, CypB is ubiquitously distributed, its role in intestinal Ca 2 + transport has not been elucidated.
59.3.2.2.4
Physiology of TRPV6-Mediated Ca 2 + Transport
In the intestinal lumen, Ca 2 + concentration varies, but it is often in the millimolar range, while inside the absorptive cell, the Ca 2 + concentration is much lower (~ 100 nmol/L). This gives an approximate 10,000-fold concentration gradient across the apical membrane, and moreover, the membrane electric potential provides an additional driving force for calcium transport (with a relative negative charge on the cytoplasmic side of the membrane). Thus, transport of Ca 2 + into intestinal epithelial cells does not require the consumption of metabolic energy. Ca 2 + influx mediated by TRPV6 does not appear to be coupled with NaCl or protons. Transport activity is sensitive to pH, with Ca 2 + uptake activity increasing at alkaline pH. TRPV6 is permeable to Ba 2 + and Sr 2 + , but not Mg 2 + . Its apparent affinity for Ca 2 + ( K m ) is 0.44 mM.
The macroscopic properties of this channel expressed in X. laevis oocytes indicate that this protein works as a facilitative uniporter, that constitutively transports substrate down the concentration gradient with saturation kinetics, but without obvious gating mechanisms. Basic electrophysiological studies for TRPV6 have shown that outward currents are extremely small, indicating the channel is nearly completely inwardly rectifying. The current through TRPV6 is carried exclusively by Ca 2 + at extracellular Ca 2 + concentrations exceeding 10 μM. TRPV6 shows high selectivity for calcium, with Ca 2 + to Na + permeability ratios ( P Ca 2 + / P Na + ) of over 100. This channel is effectively blocked by trivalent and divalent cations, and is relatively insensitive to L-type voltage gated channel blockers (as discussed in detail below).
59.3.2.2.5
Pharmacology of TRPV6
Relatively little is known about effective pharmacological tools to modulate TRPV6 activity. Examination of Ca 2 + channel blockers revealed sensitivity toward ruthenium red, Gd 3 + , and La 3 + . The inhibition magnitude increased as follows: Gd 3 + , La 3 + > Pb 2 + , Cd 2 + > Co 2 + , Ni 2 + . These findings are consistent with studies on Ca 2 + current and flux in the small intestinal epithelium. Furthermore, TRPV6 is rather insensitive to the L-type voltage-gated calcium channel blockers nifedipine, diltiazem, and verapamil, with TRPV6 being inhibited only 10%–15% when these compounds were used at 100 μM. Econazole, miconazole, and SKF96365 were shown to inhibit Ca 2 + uptake in TRPV6 expressing oocytes, with econazole being the most effective inhibitor (~ 50% inhibition with 50 μM). The inorganic polycationic dye ruthenium red, which binds to phospholipids, inhibits TRPV6 in a voltage-dependent manner. Furthermore, xestospongin, a noncompetitive inositol 1,4,5-triphosphate receptor antagonist, seems to block TRPV6 activity as well. Additionally, capsaicin has been reported to block TRPV6. 2-APB (2-aminoethoxydiphenyl borate), a store-operated calcium channel (SOC) inhibitor also effectively inhibited human TRPV6 but not TRPV5. Searching for more specific inhibitors, Simonin et al. described cis -22a through an in silico method of ligand-based virtual screening. No direct activator of TRPV5 or TRPV6 has been identified to date. The pharmacology of TRP cation channels, including TRPV5 and TRPV5, was reviewed in detail by Vriens et al.
59.3.2.3
Facilitated Diffusion: Calcium Diffusion Across the Intestinal Epithelial Cell Cytoplasm
Epithelial cells involved in transcellular calcium transport are continuously challenged by substantial Ca 2 + moving through the cytosol, while the cells simultaneously need to maintain very low levels of intracellular Ca 2 + . Free ionized cytoplasmic Ca 2 + concentrations have to be tightly controlled due to cytotoxic effects, and due to potential inhibitory effects on TRPV5 and TRPV6-mediated Ca 2 + currents. Some of these effects are mediated by associated proteins like calmodulin and the protein kinase C substrate 80K-H. The facilitated Ca 2 + diffusion model proposes binding of calcium to an intracellular buffering protein, which delivers the calcium to the basolateral membrane for export. Mathematical modeling predicts that intracellular diffusion of this protein/Ca 2 + complex is the rate-limiting step in transepithelial calcium movement. Indeed, the 1,25(OH) 2 D 3 -dependent Ca 2 + -binding protein calbindin-D 9k (CBD 9k ) has been detected in intestinal epithelial cells and has been proposed to deliver calcium to the basolateral membrane for export into the interstitial space. The expression level of calbindin-D 9k in the intestinal epithelium closely correlates with the efficiency of Ca 2 + absorption, and therefore, this protein plays a central role in the facilitated diffusion model. Interestingly, calbindin D 9k may directly enhance plasma membrane Ca 2 + -ATPase (PMCA) activity, which is the principle mechanism of calcium export across the basolateral membrane.
Calbindin-D 9k belongs to a group of intracellular proteins that bind Ca 2 + with high affinity, which causes the protein to undergo structural changes due to electrostatic interactions. Another similar protein called calmodulin (CaM) is known to interact with and regulate the activity of voltage-gated Ca 2 + channels in a Ca 2 + -dependent fashion. The ubiquitously expressed CaM directly interacts with an “IQ motif” present in the carboxy-terminal of these channels, where it functions as a Ca 2 + sensor. This IQ motif, however, is not present in TRPV6. It is unknown whether calbindin-D 9k can perform a similar Ca 2 + sensor function for which a specific interaction with TRPV6 would be required. Furthermore, the striking colocalization of calbindin-D 9k with TRPV6 in the intestine suggests that a functional interaction between these two proteins may occur. Further experiments are needed to delineate whether the function of calbindin-D 9k is restricted to its buffering capacity to maintain low Ca 2 + concentrations in the cell in close vicinity to the channel opening, or whether physical interaction between calbindin-D 9k and TRPV6 is needed to exert a direct regulatory function.
Despite the large body of literature on the role of calbindins D 9k acting both as a Ca 2 + buffer and a ferry facilitating trans-cytoplasmic movement of Ca 2 + to the basolateral site in the intestinal epithelial cells, its precise role in both functions remains controversial. Interestingly, TRPV6 −/− , CBD 9k −/− , or TRPV6 −/− /CBD 9k −/− double-knockout mice had normal plasma Ca 2 + . In the same report by Benn et al., under low dietary calcium conditions, wild type, TRPV6 −/− and CBD 9k −/− mice showed a 4.1-, 2.9-, and 3.9-fold increase in duodenal calcium transport, respectively. A significant, 2.1-fold increase in duodenal calcium transport was also observed in TRPV6 −/− /CBD 9k −/− double-knockout mice fed a low-calcium diet. 1,25(OH) 2 D 3 administration to vitamin D-deficient null mutant and wild-type mice also resulted in a significant increase in duodenal calcium transport. These studies with genetically engineered mice challenge the dogma that TRPV6 and calbindin-D 9k are essential for vitamin D 3 -induced active intestinal calcium transport and imply that either naturally or through compensatory mechanisms, active intestinal Ca 2 + transport can occur in the absence of TRPV6, CBD 9k or both. Alternative transport models described below (vesicular transport model and Ca 2 + tunneling through ER) may partially explain the lack of a significant phenotype in CBD 9k -deficient mice.
59.3.2.4
Facilitated Diffusion: Calcium Extrusion Across the Basolateral Membrane
Two transport proteins have been implicated in the cellular exit of Ca 2 + . One of them is a bi-directional Na + /Ca 2 + exchanger NCX1, which under physiological electrochemical gradients, is responsible for basolateral Ca 2 + extrusion. It is widely expressed in absorptive epithelia, although it is considered to be of primary importance in the renal distal convoluted tubules, and of minor significance in the intestinal epithelium. The physiological importance of this exchanger is highlighted by the embryonic lethality of NCX1 null-knockout mice. In heterozygous mice, expression of NCX1 protein in the tubular epithelial cells and Ca 2 + influx via NCX1 in renal tubules are markedly attenuated. The second transporter involved in basolateral Ca 2 + extrusion is a P-type ATPase, a high-affinity Ca 2 + efflux pump PMCA1b, which is abundantly expressed in the intestine. Calcium is expelled through a channel-like opening formed by the transmembrane elements, and phosphorylation is believed to bring about the necessary conformational change such that Ca 2 + bound to the protein is propelled through the opening. Similar to NCX1, PMCA1b null knockout in mice is embryonically lethal.
Four PMCA isoforms have been identified to date and alternatively spliced transcripts have been detected ; PMCA1b is however the predominant isoform in the gut, where it is abundantly expressed in the small intestine. Several studies indicated that PMCA1b is positively regulated by 1,25(OH) 2 D 3 in the intestine to increase Ca 2 + absorption. Northern blot analysis indicated that repletion of vitamin D-deficient chickens with 1,25(OH) 2 D 3 increases PMCA mRNA expression in the duodenum, jejunum, ileum, and colon. Additional studies by Johnson and Kumar demonstrated that 1,25(OH) 2 D 3 causes an increase in the abundance of PMCA protein and stimulates Ca 2 + extrusion. However, plasma 1,25(OH) 2 D 3 was not found to be correlated with PMCA1 expression in humans. PMCA activation is dependent upon CaM and inhibition of CaM is, in turn, known to prevent PMCA stimulation. Although some data suggest that extrusion is not the rate-limiting step for Ca 2 + absorption, the estimated V max of PMCA1b has been reported to be in the range of 20–30 nM Ca/min/mg protein , which should be adequate to extrude Ca 2 + even at the highest rates of Ca 2 + transport.
59.3.2.5
Vesicular Transport Model
According to this model, apical formation of Ca 2 + loaded vesicles is initiated upon TRPV-mediated Ca 2 + uptake. These vesicles can then be transported vectorially by microtubules, or they can fuse with endoplasmic reticulum followed by passive diffusion within the ER lumen. Ca 2 + is then released from the ER at the basolateral membrane through calcium release channels and extruded by NCX1 and PMCA1b (see below). Alternatively, these vesicles may fuse with lysosomes, which move laterally to eventually coalesce with the lateral membrane and release the content through exocytosis. This route is also stimulated by 1,25(OH) 2 D 3 . Net epithelial Ca 2 + is inhibited by chloroquine, suggesting that this may be the main route of transcellular Ca 2 + trafficking.
59.3.2.5.1
Vesicular Transport: Apical Entry
Secondarily to TRPV5/6-mediated Ca 2 + influx, the rapid increase in Ca 2 + concentration around the apical region can disrupt the actin filaments near the Ca 2 + channels and initiate the formation of Ca 2 + -enriched endocytic vesicles. It is also possible that Ca 2 + influx promotes transitory insertion of P-type calcium channels in the surface membrane, which is then followed by compensatory endocytosis and formation of vesicles containing Ca 2 + transporters. Indeed, both TRPV5 and TRPV6 have been found to colocalize with a marker of recycling endosomes Rab11a in renal epithelial cells. A third possibility is direct filling of a vesicle by the Ca 2 + channel. Ultimately, the newly formed vesicles are transported vectorially by microtubules.
59.3.2.5.2
Vesicular Transport: Transcellular Ca 2 + Movement and Basolateral Ca 2 + Exit
An earlier study by Warner and Coleman utilizing X-ray probe analysis in intestinal epithelial cells found discrete localizations of Ca 2 + under transport conditions rather than a diffuse cytoplasmic presence. Subsequently, Davis et al., working with chick duodenum, identified these vesicles as lysosomes. The same group later showed that in 1,25(OH) 2 D 3 -treated rachitic chicks there was an increased Ca 2 + concentration and lysosomal count in intestinal epithelial cells. These vesicular/lysosomal structures were membrane bound, enriched in the lysosomal marker acid phosphatase, had a high Ca 2 + content, moved laterally, and eventually coalesced with the lateral plasma membrane, leading to the exocytosis of their contents. The involvement of lysosomes in transcellular Ca 2 + transport in intestinal epithelia was confirmed in several other studies (for review see Ref. ). According to the vesicular transport model, exocytosis delivers Ca 2 + to the basolateral membrane of the polarized intestinal epithelium. Indeed, lysosomotropic agents such as chloquine, quinarine, or ammonium chloride inhibit the net duodenal transport of Ca 2 + . Moreover, Ca 2 + transport is also accompanied by secretion of lysosomal cathepsin B.
59.3.2.6
Tunneling Through the Endoplasmic Reticulum
This model of transcellular Ca 2 + transport was demonstrated in pancreatic acinar cells. Although not confirmed experimentally, this route may also contribute to vectorial Ca 2 + transport in intestinal epithelial cells. According to this model, Ca 2 + enters the cell through channels as in the preceding models and is transported to the basolateral membrane through passive diffusion in the endoplasmic reticulum. This process involves active Ca 2 + buffering rather than the passive buffering that has been described for facilitated diffusion. Ca 2 + is extruded to the interstitium via basolaterally expressed Ca 2 + -ATPases and Na + /Ca 2 + exchangers. It has been hypothesized that the endoplasmic reticulum buds off transport vesicles filled with calcium. Fig. 59.2 depicts the three models of transcellular Ca 2 + transport discussed above. Although still little is known about the relative contribution of each mechanism in the transepithelial Ca 2 + transport in the intestine, it is likely that all three models function in a coordinated fashion to provide the necessary redundancy aimed at meeting the short- and long-term systemic Ca 2 + requirements.
59.3.3
Regulation of Intestinal Calcium Absorption
59.3.3.1
Dietary Calcium
The importance of adequate calcium intake has been best illustrated by studies utilizing VDR and 1α-hydroxylase-knockout mice. The skeletal phenotype of VDR-null mice can be completely rescued by feeding the animals a diet high in calcium, phosphorus, and lactose. Additionally, the abnormal phenotype of 1α-hydroxylase-knockout mice can be corrected by high dietary Ca 2 + intake. The expression of calcium transport proteins was subsequently studied in these knockout models, so as to be able to distinguish the effects of hypocalcemia from those of vitamin D deficiency. Thus studies were initially performed in 1α-hydroxylase-ablated mice fed normal versus high Ca 2 + rescue diets. It is known that under normal circumstances, increased plasma calcium levels act via a negative feedback loop to stimulate PTH secretion, which eventually suppresses 1α-hydroxylase activity, which in turn decreases renal 1,25(OH) 2 D 3 production and calcium transport in the intestine. This seems to be mediated by the expression of the intestinal calcium transport proteins TRPV6, calbindin-D 9k , and PMCA1b, which was normalized by feeding the knockout mice the high calcium rescue diet. Comparable observations were made in VDR-knockout mice where duodenal TRPV6 mRNA levels were induced by dietary calcium. Studies with both of these animal models revealed that calcium supplementation can increase transcriptional initiation of genes encoding calcium transporter proteins in the absence of circulating 1,25(OH) 2 D 3 , but the molecular mechanism of this vitamin D-independent calcium sensitive pathway remains elusive. It is, however, likely that in addition to the 1,25(OH) 2 D 3 response elements in the promoter regions of the TRVP6 and the calbindin-D 9k genes, calcium responsive cis -acting elements are also present. Several promoter elements have been proposed to function as calcium-sensitive transcriptional modulators, including the serum-responsive element in the cAMP/calcium responsive element. However, detailed promoter studies are necessary to characterize fully any putative calcium-responsive elements in the genes responsible for transepithelial calcium transport in the small intestine.
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