Intestinal Phosphate Transport Research Articles

Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.

We used a ribozyme loss-of-function approach to demonstrate that the protein product of a cDNA encoding a multifunctional membrane-associated protein binds the seco-steroid 1,25(OH)(2)D(3) and transduces its stimulatory effects on phosphate uptake. These results are paralleled by studies in which the ability of the hormone to stimulate phosphate uptake in isolated chick intestinal epithelial cells is abolished by preincubation with Ab099 directed against the amino terminus of the protein. We now report the complete sequence of the cloned chicken cDNA for the 1,25D(3)-MARRS (membrane-associated, rapid-response steroid-binding) protein and reveal it to be identical to the multifunctional protein ERp57. Functional studies showed that active ribozyme, but not a scrambled control, decreased specific membrane-associated 1,25(OH)(2)D(3) binding, but did not affect binding to the nuclear receptor for 1,25(OH)(2)D(3). Seco-steroid-dependent stimulation of protein kinase C activity was diminished as 1,25D(3)-MARRS protein levels were reduced in the presence of the ribozyme, as judged by Western blot analyses. Phosphate uptake in isolated cells is an index of intestinal phosphate transport that occurs during growth and maturation. Whereas cells and perfused duodena robustly responded to 1,25(OH)(2)D(3) in preparations from young birds, older animals no longer responded with stimulated phosphate uptake or transport. The age-related decline was accompanied by a decrease in 1,25D(3)-MARRS mRNA that was apparent up to 1 year of age. Together, these studies functionally link phosphate transport in the chick duodenum with the 1,25D(3)-MARRS protein and point to a previously uncharacterized role for this multifunctional protein class.

Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4-2 and by serum- and glucocorticoid-dependent kinase 1.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture.

Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40-70% of total effluent P production by trout. Particulate P concentrations accounted for 25-50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.

The sodium phosphate cotransporter family SLC34.

This review summarizes the characteristics of the solute carrier family SLC34 that is represented by the type ll Na/P(i)-cotransporters NaPi-lla (SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3). Other Na/P(i)-cotransporters are described within the SLC17 and SLC20 families. Type ll Na/P(i)-cotransporters are expressed in several tissues and play a major role in the homeostasis of inorganic phosphate. In kidney and small intestine, type ll Na/P(i)-cotransporters are located at the apical sites of epithelial cells and represent the rate limiting steps for transepithelial movement of phosphate. Physiological and pathophysiological regulation of renal and small intestinal epithelial transport of phosphate occurs through alterations in the abundance of type ll Na/P(i)-cotransporters.

Differential effects of 19-nor-1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 on intestinal calcium and phosphate transport

19-Nor-1,25-dihydroxyvitamin D2 (19-norD2) a less calcemic and phosphatemic analog of 1,25-dihydroxyvitamin D (1,25[OH]2D3), is approved for the treatment of secondary hyperparathyroidism in patients with kidney failure. We have previously demonstrated that 19-norD2 is less active than 1,25(OH)2D3 in stimulating bone resorption. In this study, we compared the potencies of 19-norD2 and 1,25(OH)2D3 in stimulating net calcium and phosphate absorption in the intestine. Mineral balance was assessed in normal rats during the last 4 days of a 14-day treatment with various daily doses of 19-norD2 or 1,25(OH)2D3. Calcium absorption increased from 16.5% ± 7.8% in vehicle-treated rats to 27.5% ± 7.2% in rats given 10 ng/day 1,25(OH)2D3 and to 21.6% ± 3.9%, 26.2% ± 5.5%, and 27.4% ± 5.1% in rats treated with 10, 50, and 100 ng/day 19-norD2, respectively. Thus comparable stimulation of calcium transport was attained with 10 ng 1,25(OH)2D3 and 100 ng 19-norD2. Similar results were obtained for phosphate absorption, with an increase from 28.2% ± 5.5% in vehicle-treated rats to 40.2% ± 4.7% in rats given 10 ng/day 1,25(OH)2D3 and to 32.9% ± 2.2%, 36.2% ± 4.5%, and 36.8% ± 3.8% in rats given 10, 50, and 100 ng/day 19-norD2, respectively. Vitamin D compounds are believed to increase calcium absorption by inducing a calcium channel (epithelial calcium transporter or calcium transporter-1 [CaT1]) on the luminal membrane, a calcium-binding protein (Calbindin D9k) in the cytosol, and a calcium pump (plasma membrane calcium adenosine triphosphatase-1 [PMCA1]) on the basolateral membrane. Northern-blot analysis of intestinal ribonucleic acid of vitamin D-deficient rats given seven daily injections of vehicle or 100 ng 1,25(OH)2D3 or 19-norD2 revealed that 19-norD2 was less potent than 1,25(OH)2D3 in stimulating expression of CaT1, Calbindin D9k and PMCA1. In summary, the reduced calcemic and phosphatemic activities of 19-norD2 can be attributed to lower potency in stimulating intestinal calcium and phosphate absorption. (J Lab Clin Med 2002;139:279-84)

Dietary phosphorus regulates intestinal transport and plasma concentrations of phosphate in rainbow trout

Intestinal inorganic phosphate transport and its regulation have not been studied in fish. In this study, we initially characterized the mechanisms of intestinal inorganic phosphate transport in rainbow trout (Oncorhynchus mykiss) then determined the effects of dietary phosphorus concentrations on intestinal inorganic phosphate uptake, plasma inorganic phosphate, and intestinal luminal inorganic phosphate concentrations. In 11-g trout, the saturable mechanism of brushborder inorganic phosphate uptake had a Kt= 1.2 mmol l(-1) and a Vmax = 0.22 nmol mg(-1) min(-1), while the diffusive component had a Kd = 0.012 min(-1). Similar kinetic constants were obtained from 51-g trout, suggesting that development or size had little effect on transport. Tracer inorganic phosphate (1.18 mmol l(-1)) uptake was almost completely inhibited (>95%) by 20 mmol l(-1) unlabeled inorganic phosphate. Inorganic phosphate uptake (0.2 mmol l(-1)) was strongly inhibited (approximately 75% inhibition) by phosphonoformic acid, a competitive inhibitor of mammalian inorganic phosphate transport, as well as by the absence of Na+ (approximately 90% inhibition). Northern blot and reverse transcription-polymerase chain reaction indicated that the intestinal inorganic phosphate transporter in trout is not related to the cloned Na+ inorganic phosphate-II transporter of winter flounder. Intestinal luminal and plasma inorganic phosphate concentrations each increased with dietary P concentrations. Intestinal inorganic phosphate, but not proline, absorption rates decreased with dietary phosphorus concentrations. As in mammals and birds, a Na-dependent inorganic phosphate carrier that is tightly regulated by diet is present in trout small intestine.

Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine.

Stanniocalcin (STC) is an anti-hypercalcemic glycoprotein hormone previously identified in the corpuscles of Stannius in bony fish and recently in the human genome. This study undertook to express human STC in Chinese hamster ovary (CHO) cells and to determine its effects on calcium and phosphate absorption in swine and rat intestine. Unidirectional mucosal-to-serosal (Jm-->s) and serosal-to-mucosal (Js-->m) 45Ca and 32P fluxes were measured in vitro in duodenal tissue in voltage-clamped Ussing chambers. Addition of STC (10-100 ng/ml) to the serosal surface of the duodenum resulted in a simultaneous increase in calcium Jm-->s and Js-->m fluxes, with a subsequent reduction in net calcium absorption. This was coupled with an STC-stimulated increase in phosphate absorption. Intestinal conductance was increased at the highest dose of STC (100 ng/ml) in swine tissue. The addition of STC to the mucosal surface had no effect on calcium and phosphate fluxes. STC at doses of 10-1,000 ng/ml had no effect on short-circuit current in any region of the rat intestine. In conclusion, human recombinant STC decreases the absorption of calcium and stimulates the absorption of phosphate in both swine and rat duodenum. STC is a novel regulatory protein that regulates mammalian intestinal calcium and phosphate transport.

Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3.

The steroid hormone 1,25-(OH)2D3 modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH)2D3 rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH)2D3 significantly stimulated the appearance of 33P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 33P in the venous effluent. Vascular perfusion with a range of 1,25-(OH)2D3 concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH)2D3 and 25(OH)D3, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 nM 24,25(OH)2D3 nor 100 nM 25(OH)D3 stimulated 33P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25-(OH)2D3 and either 6.5 nM 24,25(OH)2D3 or 100 nM 25(OH)D3, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH)2D3, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 +/- 0.2% of controls). For 25(OH)D3, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 +/- 0.14% of controls. The presence or absence of lumenal Ca2+ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)2D3, or 6.5 nM 24,25(OH)2D3. In contrast, vascular perfusion with 100 nM 25(OH)D3 stimulated phosphate transport when lumenal Ca2+ was present but not when it was absent. The influence of lumenal P(i) on transcaltachia was then studied. Although 65 pM 1,25-(OH)2D3, 6.5 nM 24,25(OH)2D3, and 100 nM 25(OH)D3, initiate transcaltachia in the presence of lumenal P(i), the absence of the anion abolished the stimulatory effects of 1,25-(OH)2D3 and 24,25(OH)2D3 on calcium transport but not those induced by 25(OH)D3. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen.

Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25- hydroxyvitamin D3

The steroid hormone 1,25-(OH)2D3 modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH)2D3 rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH)2D3 significantly stimulated the appearance of 33P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 33P in the venous effluent. Vascular perfusion with a range of 1,25-(OH)2D3 concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH)2D3 and 25(OH)D3, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 nM 24,25(OH)2D3 nor 100 nM 25(OH)D3 stimulated 33P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25-(OH)2D3 and either 6.5 nM 24,25(OH)2D3 or 100 nM 25(OH)D3, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH)2D3, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 +/- 0.2% of controls). For 25(OH)D3, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 +/- 0.14% of controls. The presence or absence of lumenal Ca2+ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)2D3, or 6.5 nM 24,25(OH)2D3. In contrast, vascular perfusion with 100 nM 25(OH)D3 stimulated phosphate transport when lumenal Ca2+ was present but not when it was absent. The influence of lumenal P(i) on transcaltachia was then studied. Although 65 pM 1,25-(OH)2D3, 6.5 nM 24,25(OH)2D3, and 100 nM 25(OH)D3, initiate transcaltachia in the presence of lumenal P(i), the absence of the anion abolished the stimulatory effects of 1,25-(OH)2D3 and 24,25(OH)2D3 on calcium transport but not those induced by 25(OH)D3. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen.

Mechanisms of intestinal phosphate transport in small ruminants.

In order to study the localization and mechanisms of intestinal phosphate transport in sheep and goats, unidirectional inorganic phosphate (Pi) flux rates across isolated stripped epithelial tissues were measured in vitro by applying the Ussing-chamber technique. In the first experiment the tissues were obtained from animals which had been kept on an adequate dietary P supply. In the second experiment the animals had either been kept on an adequate Ca and P supply or were Ca- and/or P-depleted. Significant net Pi absorption was measured in all segments of the small intestine and in the proximal colon of sheep and in the duodenum and jejunum of goats. Since the experiments were carried out in the absence of any electrochemical gradient, this clearly indicates the presence of active mechanisms for Pi transport in the intestinal tract of small ruminants. In sheep jejunum, reduction of mucosal Na concentration to 1.8 mM or serosal application of ouabain (0.1 mM) resulted in significant decreases of net Pi absorption of the same order of magnitude, indicating that about 65% of active Pi transport in sheep jejunum is mediated by a Na-dependent active transport mechanism. The mechanism for the remaining Na(+)-independent active Pi transport has not yet been identified. Dietary P depletion caused hypophosphataemia and induced a significant stimulation of net Pi absorption in goat duodenum and jejunum. This increase was independent of dietary Ca supply and was not associated with increased plasma calcitriol concentrations. This suggests substantial differences in hormonal regulation of Pi transport in small ruminants in comparison with single-stomached species.

Low Phosphate Diet Upregulates the Renal and Intestinal Sodium-Dependent Phosphate Transporter in Vitamin D-Resistant Hypophosphatemic Mice

Renal and jejunal absorption of inorganic phosphate (P(i)) increases with dietary P(i) restriction in the rat. The defect in Na(+)-dependent phosphate transporter has been localized to the kidney of the Hyp mice; however, the adaptation to low-P(i) diet in both kidney and jejunum of the Hyp mice has not been well characterized. Therefore, the current studies were designed to characterize the adaptation of renal and jejunal Na(+)-dependent phosphate transport in the Hyp mice and compare it with normal mice. Low-P(i) diet significantly increased the slope of the initial rate of renal brush border membrane (BBM) phosphate uptake compared with corresponding values in mice raised on control-P(i) diet (0.035 vs 0.017) (P < 0.01). Kinetics of renal Na(+)-dependent phosphate uptake in Hyp mice showed a Vmax of 1.00 +/- 0.01 and 0.46 +/- 0.02 nmoles/mg protein/15 sec in low- and control-P(i) diets, respectively (P < 0.01), whereas, Km values were 0.07 +/- 0.04 and 0.02 +/- 0.01, respectively. Similar kinetic analysis in renal BBM of normal mice showed a Vmax of 2.4 +/- 0.17 and 1.18 +/- 0.09 (P < 0.01) and Km of 0.07 +/- 0.03 and 0.08 +/- 0.03 on low and control P(i) diets, respectively. Similarly, low-P(i) diet significantly increased the slope of the initial rate of intestinal phosphate uptake (0.013 and 0.007) (P < 0.01). Kinetics of jejunal Na(+)-dependent phosphate uptake in Hyp mice showed a Vmax of 0.36 +/- 0.01 and 0.2 +/- 0.02 nmoles/mg protein/15 sec, (P < 0.01) and Km of 0.13 +/- 0.06 and 0.06 +/- 0.01 mM in low- and in control-P(i) diet, respectively. Kinetic analysis in jejunal BBM of normal mice showed a Vmax of 0.47 +/- 0.04 and 0.18 +/- 0.01 nmoles/mg protein/15 sec (P < 0.01) and Km of 0.16 +/- 0.04 and 0.11 +/- 0.01 in low- and control-P(i) diets, respectively. The data indicates that low-phosphate diet upregulates the Vmax of the renal and jejunal Na(+)-dependent phosphate cotransporter in the hypophosphatemic mice.

L-thyroxine inhibits intestinal phosphate absorption in developing rabbits

Little is known about effects of l-thyroxine on phosphate absorption. To characterize effects of l-thyroxine on intestinal phosphate transport, four week old suckling rabbits were injected with l-thyroxine. 24 hours following injection, phosphate transport was studied in brush border membrane vesicles. Vesicles from l-thyroxine-treated and control animals demonstrated sodium-phosphate cotransport however uptake was significantly greater in controls than in treated animals. Controls demonstrated an “overshoot” in that peak phosphate uptake was nearly three-fold the equilibrium value whereas minimal overshoot was observed in treated animals. V max and K m for sodium-dependent phosphate transport were 230.4±6.4 pmol·mg protein −1·10s −1 and 0.034±0.05 mmol/L respectively for controls, and 97.7±4.4 pmol·mg protein −1·10s −1 and 0.023±0.006 mmol/L for treated animals. While phosphate uptake was greater in controls than in treated animals, maximal stimulation of phosphate uptake occurred between 75 and 100 mmol/L sodium in both groups. Uptake was half-maximally stimulated by 36.3±13.0 mmol/L sodium in controls, and 41.4±10.0 mmol/L sodium in treated animals. These results indicate that within 24 hours, l-thyroxine suppresses intestinal sodium-coupled phosphate transport in four week old suckling rabbits. This is due to decreased maximal transport capacity without changes in transporter affinity for phosphate or sodium.

Glucocorticoids Inhibit Intestinal Phosphate Absorption in Developing Rabbits

Children are apt to develop osteoporosis during chronic glucocorticoid therapy, in part due to suppression of intestinal calcium absorption. Little is known about effects of glucocorticoids on phosphate absorption. To characterize effects of glucocorticoids on intestinal phosphate transport, 4-wk-old suckling rabbits were injected with methylprednisolone. Twenty-four hours following injection, phosphate transport was studied in brush border membrane vesicles. Vesicles from steroid-treated and control animals demonstrated sodium-phosphate cotransport. Uptake was significantly greater in controls than in methylprednisolone-treated animals. Controls demonstrated an “overshoot” in that peak phosphate uptake exceeded equilibrium by nearly 100%, whereas minimal overshoot was observed in methylprednisolone-treated animals. Vmax and Km for sodium-dependent phosphate transport were 556.6 ± 13.2 pmol-mg protein-1·10 s-1 and 0.051 ± 0.01 mmol/L, respectively, for controls and 285.4 ± 3.5 pmol·mg protein-1·10 s-1 and 0.044 ± 0.01 mmol/L for methylprednisolone-treated animals. Although phosphate uptake was greater in controls than in methylprednisolone-treated animals, maximal stimulation of phosphate uptake occurred between 75 and 100 mmol/L sodium in both groups. Uptake was half-maximally stimulated by 32.5 ± 8.1 mmol/L sodium in controls and 41.4 ± 6.2 mmol/L sodium in treated animals. These results indicate that within 24 h methylprednisolone suppressed intestinal sodium-coupled phosphate transport. This is due to decreased maximal transport capacity without changes in transporter affinity for phosphate or sodium.

Ontogeny of intestinal phosphate absorption in rabbits.

The ontogeny of intestinal phosphate transport was examined in brush-border membrane vesicles prepared from 2-wk, 4-wk, 6-wk, and 3-mo-old rabbits. At all four ages, vesicles prepared from the duodenum demonstrated sodium-phosphate cotransport in that uptake in the presence of a sodium gradient was significantly greater than uptake in the presence of a potassium gradient. While sodium-independent phosphate uptake was comparable at all ages, sodium-phosphate cotransport was greatest in the youngest animals and declined with increasing age. Peak phosphate uptake was 10-fold the equilibrium value at 2 wk and declined to 3-fold in adults. Maximal transport capacity of sodium-phosphate cotransport fell from 2,292 +/- 161 at 2 wk to 286 +/- 12 pmol.mg-1.10 s-1 at 3 mo, whereas the Michaelis constant did not change with age, varying between 0.032 and 0.054 mM. At all ages, uptake was half-maximally stimulated between 30 and 50 mM sodium, and Hill coefficients were between 1.5 and 2. Generation of inside negative diffusion potentials did not significantly enhance phosphate uptake at any age. These data suggest that throughout development, the transport of each phosphate molecule across the rabbit duodenal brush-border membrane is coupled to the transport of two sodium molecules. There were distinct developmental changes in the distribution of the sodium-phosphate cotransport along the length of intestine. In 2-wk old animals, sodium-phosphate cotransport was present in the duodenum, proximal and distal jejunum, and proximal ileum. In 4-wk-old animals, sodium-phosphate cotransport was present throughout the duodenum and proximal and distal jejunum, and in 6-wk and 12-wk-old animals, sodium-phosphate cotransport was observed only in the duodenum and proximal jejunum.

Intestinal phosphate transport in spontaneously hypertensive rats and genetically matched controls

Hypophosphatemia has been documented in patients with hypertension and in spontaneously hypertensive rats compared with genetically matched control Wistar-Kyoto rats. However, renal tubular reabsorption is increased in spontaneously hypertensive rats. Therefore, it was hypothesized that decreased serum phosphate levels in spontaneously hypertensive rats may be related to a decrease in the intestinal transport of phosphate. To test this hypothesis, sodium-dependent phosphate uptake by jejunal brush-border membrane vesicles of spontaneously hypertensive rats and genetically matched Wistar-Kyoto rats was determined. Phosphate uptake consisted of two components: sodium-independent passive diffusion across the brush border and sodium-dependent, carrier-mediated uptake. The initial rate of uptake in spontaneously hypertensive and Wistar-Kyoto rats was linear up to 20 seconds. The initial rate and time course of jejunal sodium-dependent phosphate uptake was decreased in adult spontaneously hypertensive rats compared with corresponding mean values in Wistar-Kyoto rats. This decrease was secondary to a decrease in Vmax rather than Km, suggesting that the number and/or the activity of the sodium-phosphate transporters is decreased. Sodium-dependent phosphate uptake was pH dependent, with greater uptake at pH 6.0 than at pH 7.4. However, uptake values were lower in spontaneously hypertensive rats than in Wistar-Kyoto rats at all pH levels tested. In contrast, sodium-dependent phosphate uptake in weanling rats (prehypertensive state) was not significantly different between spontaneously hypertensive and Wistar-Kyoto rats. Vitamin D deficiency in both spontaneously hypertensive and Wistar-Kyoto rats decreased Vmax and Km of sodium-dependent phosphate uptake, whereas 1,25(OH)2 vitamin D3 administration increased Vmax and Km in both spontaneously hypertensive and Wistar-Kyoto rats. These results suggest that the hypophosphatemia seen in adult spontaneously hypertensive rats is secondary to a decrease in sodium-dependent phosphate uptake compared with controls. The sodium-phosphate transporter in spontaneously hypertensive rats is responsive to vitamin D administration.

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3-modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.

Intestinal phosphate transport: Mechanistic and regulatory aspects

Intestinal phosphate transport: Mechanistic and regulatory aspects

Mechanisms and regulation of intestinal phosphate transport.

I will like to express my indebtedness to Dr. Marlin M. Walling (January 17, 1941 — November 30, 1979), a true scientist and scholar. He inspired and generated most of the data included in this presentation.

Phosphate fluxes in isolated enterocytes from vitamin D replete and vitamin D deficient rats--early effects of calcitriol.

In the present work we studied rapid in vitro effects of calcitriol (1,25(OH)2 vitamin D3) on the intestinal transport of inorganic phosphate (Pi). Enterocytes from vitamin D replete (D+) as well as vitamin D depleted (D-) rats were isolated mechanically from the duodeno-jejunum. In this model, Pi uptake was a temperature and Na+-dependent phenomenon. The in vitro-addition of calcitriol (1 pM) resulted in a significant enhancement of initial Pi uptake rate by enterocytes from D+ (P less than 0.01) and D- (P less than 0.05) rats. This effect which was Na+-dependent, was observed within the time of 20 min, but not before. A similar effect on Pi uptake rates of D+ or D- enterocytes could be elicited by the in vitro addition of the methyl ester of cis-vaccinic acid (MCVA) which is thought to increase membrane fluidity by modifying the lipid composition of the cell membrane. The stimulatory effect of calcitriol on Pi uptake rate was blunted in the presence of the methyl ester of transvaccinic acid (MTVA) thought to decrease membrane fluidity. Enterocyte Pi efflux rate constant (oKPi) remained unchanged in the presence of calcitriol (1 pM). In conclusion, the study demonstrates a rapid in vitro effect of calcitriol on Pi uptake by isolated enterocytes from D+ and D- rats. It suggests, but does not prove, that the hormone may act via an action independent of genomic nuclear activation.

Antirachitic Activity of 1α-Hydroxyergocalciferol and 1α-Hydroxycholecalciferol in Rats

Weanling vitamin D-deficient rats fed a high calcium, low phosphate diet were injected daily with 1α-hydroxyergocalciferol or 1α-hydroxycholecalciferol at doses of 2.5, 25 or 250 ng. Intestinal calcium and phosphate transport, serum phosphorus level, epiphyseal plate calcification, and percent femur ash increased as a function of the dose. Both compounds were equally effective in eliciting these responses demonstrating their equivalency as antirachitic compounds.