Evidence that low plasma 1,25-dihydroxyvitamin D causes intestinal malabsorption of calcium and phosphate in juvenile X-linked hypophosphatemic mice.

X-linked hypophosphatemic (Hyp) mice are a model for human sex-linked vitamin D-resistant rickets. We have reported intestinal malabsorption of calcium in young Hyp mice, and in this report we have explored the mechanism for it. To test for resistance of the intestine to 1,25(OH)2 vitamin D3, this hormone was continually infused via osmotic minipumps into 4-week-old normal and Hyp mice at 0, 17, 50 or 150 ng/kg/day. After 3 days, 45Ca and inorganic 32P were administered by gavage, and the mice were sacrificed on the fifth day. The Hyp mice showed responses to the hormone equivalent to the normal mice in terms of increased intestinal absorption of both 45Ca and 32P, increased plasma isotope levels, increased femoral isotope content, and increased duodenal and renal 9 kD vitamin D-dependent calcium-binding protein (calbindin-D9K; CaBP). Plasma 1,25(OH)2D was measured in these mice. There were significant correlations of plasma 1,25(OH)2D to the intestinal absorption of 45Ca and 32P and to duodenal and renal CaBP. Plasma 1,25(OH)2D was also measured in stock normal and Hyp mice and was found to be lower in 4-week-old Hyp mice than in 4-week-old normal mice (113 +/- 10 pM (n = 18) vs. 67 +/- 10 (n = 20), normal vs. Hyp, p less than .01), but unchanged at 13 weeks of age (77 +/- 13 (n = 13) vs. 70 +/- 15 (n = 15), NS). This observed difference in plasma 1,25(OH)2D between normal and Hyp mice at 4 weeks of age was sufficient to explain the observed normal-to-Hyp differences in intestinal absorption of 45Ca and duodenal and renal CaBP. It also explained 72 +/- 18% of the observed difference in 32P absorption. We conclude that Hyp mouse intestine is not resistant to 1,25(OH)2D and that the lower plasma 1,25(OH)2D of 4-week-old Hyp mice causes intestinal malabsorption of calcium and phosphate.

Effect of 1,25,28-trihydroxyvitamin D2 and 1,24,25-trihydroxyvitamin D3 on intestinal calbindin-D9K mRNA and protein: is there a correlation with intestinal calcium transport?

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by dietary calcium of vitamin D-dependent calcium-binding protein and active calcium transport in the small intestine of lactating rats.

To test the hypothesis that vitamin D-dependent calcium-binding protein (CaBP) and active calcium (Ca) transport in the small intestine of vitamin D-replete lactating rats are regulated by dietary Ca intake, pregnant rats were given a high Ca (1.6% Ca and 1.4% phosphorus) or low Ca (0.1% Ca and 0.4% phosphorus) diet starting 3 days before delivery. Toward the end of lactation (days 16-23) the rats were killed, and active Ca transport (using everted gut sacs) and CaBP were determined in duodenum, jejunum, and ileum. The right tibiae were used for bone weight and ash determinations. The Ca transport ratios and CaBP concentrations in jejunum and ileum were significantly increased only in the low Ca group. In contrast, in the duodenum both parameters were equally high regardless of the diet. Nonlactating rats given the two diets for the same length of time had the expected increase in both parameters in the duodenum when fed the low Ca diet. Nonlactating rats, in contrast to lactating rats, had undetectable CaBP in jejunum and ileum regardless of diet. Lactating rats fed the high Ca diet had no net loss of bone at the end of lactation compared with rats on day 1 of lactation. In contrast, lactating rats fed the low Ca diet had a net loss of 44% of bone weight. Plasma 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] concentrations on the 21st day of lactation were (mean +/- SE) 538 +/- 96 and 46 +/- 18 pg/ml in rats consuming the low and high Ca diets, respectively. The comparable values for the nonlactating rats were 140 +/- 4 and 26 +/- 8 pg/ml. In conclusion, dietary Ca restriction during lactation can stimulate CaBP and active Ca transport in both jejunum and ileum, and both parameters appear to be modulated by dietary Ca via the circulating concentration of 1,25-(OH)2D3. In contrast, in the duodenum neither parameter appears to be related to dietary Ca, plasma 1,25-(OH)2D3 concentration, or lactation-associated bone loss.

Presence and localization of two vitamin D-dependent calcium binding proteins in kidneys of higher vertebrates.

We looked for the presence of vitamin D-dependent intestinal calcium binding protein (CaBP), relative molecular mass (mol wt) about 10,000, in kidneys of mammals, birds, and reptiles, and compared the localization of this 10,000 mol wt CaBP with the localization of the 28,000 mol wt vitamin D-dependent CaBP. Antiserum directed against rat intestinal CaBP (RICaBP) was used with the unlabeled antibody peroxidase-antiperoxidase technique to localize the 10,000 mol wt CaBP. Kidneys of adult Swiss and DBA/2J mice were positive for the 10,000 mol wt CaBP, whereas kidneys of adult rat, chicken, and two species of lizard were negative. Extracts of adult rat kidney showed no detectable immunoreactivity with the antiserum to RICaBP by radial immunodiffusion assay. However, kidneys of postnatal rats (12 days old) exhibited limited immunocytochemical reactivity and relatively low immunoreactivity (about 1 micrograms/mg protein) for 10,000 mol wt CaBP. In both strains of mice, 10,000 mol wt CaBP was localized predominantly to the distal convoluted tubules, connecting tubules, and collecting tubules of the cortical labyrinth. In 12-day-old rats 10,000 mol wt CaBP was localized to the distal convoluted tubules and cortical ascending thick limbs. Absorption of the antiserum with electrophoretically pure RICaBP eliminated specific reactivity. By dual color staining of mouse kidneys, the population of cells reactive for the 10,000 mol wt CaBP was found to be similar, but not identical, to that population of cells reactive for the 28,000 mol wt CaBP. Absorption of the antiserum to RICaBP with either rat renal CaBP or chicken intestinal CaBP (both 28,000 mol wt CaBPs) had no affect on the positive reactivity of the distal mouse nephron for RICaBP. Conversely, absorption of the antiserum to rat renal CaBP with the 10,000 mol wt RICaBP had no affect on reactivity for the 28,000 mol wt CaBP. Thus, two immunologically distinct CaBPs, mol wt 10,000 and 28,000, are present in the adult mouse nephron; whereas, kidneys of adult rats, chickens, and saurian reptiles apparently contain significant levels of only 28,000 mol wt CaBP. Since the 12-day-old rat nephron contains both the 28,000 mol wt and the 10,000 mol wt CaBP it appears that in the kidney, ontogenetically, and perhaps evolutionarily as well, the 28,000 mol wt CaBP is more highly conserved.

Elevated levels of vitamin D-dependent calcium-binding protein (calbindin-D9k) in the osteosclerotic (oc) mouse.

The osteosclerotic (oc) mouse is an osteopetrotic mutation that has recently been identified as having rickets associated with its osteopetrosis. The presence of this rachitic lesion, unexplainable from a nutritional standpoint, prompted an investigation into the vitamin D endocrine system in these animals. The developmental appearance of vitamin D-dependent calcium-binding protein (calbindin-D9k) and alkaline phosphatase was studied in oc mutant and normal mice from birth to weaning, as were serum concentrations of 25-hydroxyvitamin D3 (25OHD3), 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium, and phosphorus. Intestinal and renal calbindin-D9k levels were markedly and precociously elevated (4- to 9-fold) in young suckling, but not newborn, mutant mice compared to values in normal controls. Serum 25OHD3 levels were very low to undetectable in 2-week-old mutant mice compared to normal values, while 1,25-(OH)2D3 levels were 6 times higher in mutants. The exact cause of this premature induction in mutants is unknown, but may be due to elevated circulating levels of 1,25-(OH)2D. Alkaline phosphatase activity was similar between phenotypes at all ages. These studies indicate that the rachitic lesion present in oc mutants may be the result of some inherited disorder in vitamin D metabolism in these animals. Alternatively, these data are also consistent with a normal appropriate response to hypocalcemia and hypophosphatemia resulting from decreased osteoclastic bone resorption.

Vitamin D-dependent calcium-binding protein of rat intestine: changes during postnatal development and sensitivity to 1,25-dihydroxycholecalciferol.

To study the role of the vitamin D-endocrine system during the perinatal period, we monitored vitamin D-dependent calcium-binding protein (CaBP) in rat intestine by radial immunodiffusion and polyacrylamide disc gel electrophoresis. Small amounts of CaBp were present 2 days before birth; these levels increased 74-fold by day 38 after birth. Approximately 80% of the increase in CaBP concentration occurred in a 5-day period at the time of weaning (days 17--22 after birth). Before this period, the concentration of CaBP was comparable to that found in rachitic (adult) rats. Administration of 1,25-dihydroxycholecalciferol to suckling rats on days 15 and/or 16 was followed by a premature increase in the amount of intestinal CaBP. These data demonstrate that although vitamin D-dependent CaBP is low in preweaned rats, the rat intestine is responsive to exogeneous 1,25-dihydroxyvitamin D3 at least as early as day 15 after birth. The close temporal correspondence between the increases in CaBP and previously reported changes in calcium transport and vitamin D metabolism suggest that the vitamin D-endocrine system plays a role in postnatal intestinal maturation and adaptation during the weaning period.

Low levels of intestinal vitamin D-dependent calcium-binding protein in juvenile X-linked hypophosphatemic mice.

Hyp mice are a model for human X-linked hypophosphatemia, the most common form of vitamin D-resistant rickets. The developmental appearance of intestinal vitamin D-dependent calcium-binding protein (CaBP) and alkaline phosphatase was studied in Hyp mice and normal mice from the perinatal period to adulthood. Both intestinal proteins were increased in the duodenum during weeks 2 and 3 of age, with values rising 10-fold or more above values measured in intestines of 1-week-old mice. During this developmental period and at most other ages, Hyp mice had levels of alkaline phosphatase and total intestinal protein comparable to those in control mice. On the other hand, the concentration of intestinal CaBP was decreased in juvenile Hyp mice during the weaning period at 2-3 weeks of age (35-65% of normal) and further depressed in the rapid growth phase at 4-6 weeks of age (15-45% of normal). During adulthood (7-35 weeks of age) Hyp mice maintained a CaBP concentration that averaged 71% of the level of control mice. These maturational defects in the Hyp intestine may play a contributory role in the bone disease in young Hyp mice.

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol.

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.

Expression of calbindin-D decreases with age in intestine and kidney.

The calbindins are Ca-binding proteins whose expression is regulated by 1,25-dihydroxyvitamin D3, the active metabolite of vitamin D3. The calbindins are found in high amounts in the proximal intestine (calbindin-D-9k) and the kidney (calbindin-D-28k), and they are thought to play a role in Ca transport by these tissues. Ca absorption by the intestine and perhaps the kidney declines with age, and this could be due to decreased expression of calbindin. Therefore, the expression of calbindins-D-9k and -D-28k was measured in F344 rats aged 2, 6, 13, and 24 months. mRNA levels were measured by dot blot hybridization to synthetic cDNA oligonucleotide probes, and protein levels were measured by enzyme-linked immunosorbent assay using specific antisera. Intestinal calbindin-D-9k mRNA decreased markedly between 2 and 6 months of age, but it then increased significantly between 13 and 24 months. Calbindin-D-9k protein paralleled the decrease in mRNA between 2 and 6 months, but continued to decline at 13 and 24 months despite the rise in mRNA. In the kidney, calbindin-D-28k mRNA declined between 2 and 13 months and then plateaued. Calbindin-D-28k protein followed a similar pattern. In the same studies expression of calmodulin by the intestine and kidney did not change with age. Plasma 1,25-dihydroxyvitamin-D3 correlated well with the expression of calbindin-D-9k in the intestine at 2 and 6 months of age and with the expression of calbindin-D-28k in the kidney at all ages. Decreased expression of calbindin-D with age may contribute to the age-related decrease in Ca transport in intestine and kidney.

Human growth hormone increases intestinal vitamin D-dependent calcium-binding protein in hypophysectomized rats.

We examined the effects of hypophysectomy and pituitary hormone replacement on vitamin D-dependent calcium-binding protein (CaBP) in rat small intestine. The concentration of immunoreactive CaBP per mg intestinal protein was decreased by at least 56% in hypophysectomized rats compared to that in intact pair-fed controls. Alkaline phosphatase and total protein also were reduced by hypophysectomy, but pair-feeding produced comparable decreases. Daily injections of 2, 10, or 50 micrograms human GH (hGH) for 9 days produced a dose-dependent increase in CaBP. At the highest hGH dose (50 micrograms), the content of CaBP was increased 2- to 4-fold to intact levels. By comparison, the increases in total protein and alkaline phosphatase were small (25% to 40% and 80% to 90%, respectively). The induction of CaBP preceded the other protein responses; half-maximal increases in CaBP occurred after 2 days of hGH (50 micrograms/day) treatment before statistically significant changes in total protein or alkaline phosphatase activity. hGH was the most potent pituitary hormone tested; ovine TSH (25 mU/day) had no effect on CaBP, and ovine PRL (10 or 50 micrograms/day) increased CaBP by only 25-27% (P = 0.014). These studies indicate that the vitamin D-dependent intestinal CaBP in hypophysectomized rats is regulated by GH and provide further evidence that the pituitary may be involved in regulating vitamin D-dependent intestinal adaptations.

Intestinal calcium absorption in the aged rat: evidence of intestinal resistance to 1,25(OH)2 vitamin D.

We investigated the role of circulating 1,25-dihydroxycholecalciferol (1,25(OH)2D) and intestinal resistance to 1,25(OH)2D in the diminished intestinal calcium absorption capacity of the senescent rat. We measured plasma 1,25(OH)2D, total and unoccupied duodenal vitamin D receptor, duodenal calbindin D9k protein (calbindin D), and net dietary calcium absorption in rats at several ages. As expected, circulating 1,25(OH)2D, calbindin D, and net calcium absorption decreased with age. However, no age-related changes were evident in intestinal vitamin D receptor levels. We then measured duodenal calcium absorption from in situ intestinal loops after continuous s.c. infusion of 1,25(OH)2D for up to 6 days and found that despite a marked elevation of plasma 1,25(OH)2D duodenal calcium absorption was significantly lower in old compared with young rats. To assess calcium absorption over a wide physiological range of plasma 1,25(OH)2D, in a dose-response study we altered plasma 1,25(OH)2D by continuous infusion of 1,25(OH)2D (at 0, 4, or 14 ng/100 g BW/day) for 9 days. We found that the slope of the linear regression between plasma 1,25(OH)2D and duodenal Ca transport in old rats was only 46% of that observed in young rats, suggesting an age-related resistance of the duodenal calcium transport process to the hormonal action of 1,25(OH)2D. Collectively, our observations suggest a dual defect in vitamin D metabolism in old animals: one defect related to the low circulating levels of 1,25(OH)2D and a second defect related to a relative intestinal resistance to the action of 1,25(OH)2D, which is apparently not due to a reduction in intestinal vitamin D receptor levels.

Growth hormone and parathyroid hormone stimulate intestinal calcium absorption in aged female rats.

Aged (16-month-old) female rats (n = 8/treatment) were injected for 12 days with GH (100 micrograms/100 g x day), PTH (8 micrograms/100 g x day), GH plus PTH, or vehicle (V) in an experiment designed to determine the effects of these hormones on intestinal mineral absorption in senescent rats. PTH and GH increased fractional net calcium absorption to a similar extent (PTH, 1.6-fold; GH, 1.4-fold) even though PTH increased serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] 3.7-fold, and GH had no significant effect. GH plus PTH caused no further increase in serum 1,25-(OH)2D3 above that caused by PTH alone, but resulted in an additive effect on net calcium absorption (2.3-fold increase). PTH and GH also had statistically independent effects on phosphate absorption; magnesium absorption was elevated only by PTH. Duodenal calbindin-D9k levels were increased by GH (from 3.79 +/- 0.72 to 6.98 +/- 0.73 micrograms/mg protein) and PTH (from 3.23 +/- 0.46 to 7.55 +/- 0.75 micrograms/mg protein); PTH plus GH treatment resulted in an additive effect on calbindin-D9k levels. Additional in vitro transport studies in the human intestinal cell line Caco-2 showed that 72 h of pretreatment with the local mediator of GH action, insulin-like growth factor-I (at 10 and 100 ng/ml), stimulated transcellular calcium transport (22% and 44%, respectively) regardless of concomitant 1 nM 1,25-(OH)2D3 pretreatment (80% increase). Our findings suggest a 1,25-(OH)2D3-mediated mechanism for PTH-induced changes in calcium and phosphorus absorption. In contrast, the effects of GH in the senescent rat are independent of changes in circulating 1,25-(OH)2D3 and our data suggest that these effects may be mediated by insulin-like growth factor-I.

Vitamin D-dependent calcium binding protein in rat uterus: differential effects of estrogen, tamoxifen, progesterone, and pregnancy on accumulation and cellular localization.

The present studies were undertaken to characterize the expression of calcium binding protein (CaBP or calbindin-D9k) in uterine tissues. Using immunohistochemical techniques, calbindin-D9k was localized to the uterine (luminal) epithelium of pregnant rats, but not present in the uterine epithelium of nonpregnant rats. Calbindin was found also in the uterine smooth muscle and endometrial stromal cells of pregnant animals. These latter localizations were reproduced in uteri of 21-day-old nonpregnant rats by administration of tamoxifen or physiological doses of estrogens. Estrogen and tamoxifen produced half-maximal increases of uterine calbindin at daily doses of 0.1 and 10 micrograms, respectively, and maximal responses at 0.3 and 40 micrograms/day. Testosterone and progesterone, at doses which increased the growth of the uterus, did not induce calbindin-D, and both hormones blocked estradiol's effect on uterine calbindin-D appearance. The epithelial localization of calbindin in pregnant uteri was not reproduced in nonpregnant animals by either estradiol (3 micrograms/day) or progesterone (1 mg/day). The localization of calbindin in uterine epithelium during pregnancy appears to be dependent upon an as yet unknown factor. In view of the large surface area of the luminal epithelium in pregnant animals, and the pregnancy-related expression of calbindin in these cells, we propose that uterine epithelium plays an important role in transport of calcium during pregnancy.

Estrogen inhibits calbindin-D28k expression in mouse uterus.

The cellular localization and hormonal controls of calbindin-D9k expression in the rodent reproductive tract have suggested new functions for this protein. The present studies were undertaken to extend the earlier studies of calbindin-D9k to the related protein, calbindin-D28k. Immunohistochemical studies revealed that calbindin-D28k was absent from female rat reproductive tissues, but was abundantly expressed in immature mouse uterus and oviduct. Immunoreactivity was restricted to the endometrial and glandular epithelium of the uterus and the oviductal epithelium. Neither 1,25-dihydroxyvitamin D- nor strontium-containing diets (to blunt 1,25-dihydroxyvitamin D production) affected expression of calbindin-D28k. Uterine, but not oviductal, calbindin-D28k decreased markedly at sexual maturity; this pattern persisted in pregnant mice and was reproduced in immature mice by the administration of estradiol (3 micrograms/day for 3 days). RNA extraction and Northern analyses demonstrated that estrogen markedly decreased calbindin-D28k mRNA abundance in the uterus, but not in the oviduct. These findings suggest that estrogen affects mammalian calbindin-D28k expression and represent a rare example of estrogen-induced down-regulation of gene expression.

Epidermal growth factor increases intestinal calbindin-D9k and 1,25-dihydroxyvitamin D receptors in neonatal rats.

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.

The presence and estrogen control of immunoreactive calbindin-D9k in the fallopian tube of the rat.

The present study was undertaken to localize and investigate the endocrine control of immunoreactive 9K calbindin-D9k in the fallopian tube (oviduct) of the rat. Rat fallopian tubes were excised with the uterus, immediately fixed by freeze-substitution, and processed for immunoperoxidase staining. Staining employed a rabbit antiserum against purified rat intestinal calbindin-D9k and the streptavidin-biotin technique. Calbindin-D9k immunoreactivity was localized to luminal epithelial cells of the fallopian tube of mature rats, with no staining observed in other tissue layers of the tube. Epithelial cells in both the isthmus and the ampulla were positive for calbindin-D9k. In weanling rats, which have little ovarian function but high levels of 1,25-dihydroxyvitamin D, no immunoreactive calbindin-D9k was observed in any part of the tube. However, after daily injections of estradiol (6 micrograms/day) for 3 days, intense staining was observed in the epithelial cells of the immature rat fallopian tube. Progesterone treatment (1 mg/day for 3 days) of immature rats had no effect on calbindin-D9k in fallopian tube. The lumen of the fallopian tube (oviduct) is the key location for fertilization, a process that requires a narrowly defined concentration of extracellular calcium. By analogy to the intestine, calbindin-D9k may play a role in the transcellular movement of calcium across the fallopian tube epithelium in the fallopian tube lumen.

Intermediate filaments in eosinophilic cells of epithelioid sarcoma: a light-microscopic, ultrastructural, and electrophoretic study.

A 41-year-old woman had a mass in her thigh for 5 months. The neoplasm had areas histologically typical of epithelioid sarcoma, but there were also cells with abundant eosinophilic, vacuolated cytoplasm and peripheral, distorted nuclei. These cells resembled signet-ring cells but contained no intracellular mucin. Rather, their cytoplasm was filled with large aggregates of 7-10 nm diameter, nonperiodic filaments, and lipid droplets. Lipid droplets and filaments are common in epithelioid sarcomas, but their presence in sufficient volume to displace the nucleus in a signet-ring fashion has not been described. Electrophoretic analysis of a tumor extract showed that actin was present in insufficient concentration (1-3%) to account for the filamentous aggregates. However, two proteins of 51,000 and 63,000 daltons apparent molecular weight comprised 40-50% of the total protein, suggesting that one or both were the major subunits of the abundant cytoplasmic filaments. Filaments of identical ultrastructural size have been described in a variety of human neoplasms and may be related to a filament characterized in other species and termed vimentin.

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression.

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)2D3 concentrations decreased with age in rats of both genders. Abundance of calbindin-D28K and its mRNA in kidney and calbindin-D9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)2D3 may be responsible for low plasma 1,25-(OH)2D3 concentrations observed in older animals. Plasma PTH and 1,25-(OH)2D3 concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D9K and calbindin-D9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)2D3 concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)2D3-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.

Femoral abnormalities and vitamin D metabolism in X-linked hypophosphatemic (Hyp and Gy) mice.

X-linked hypophosphatemia is a genetic bone disease in humans and mice. Two closely linked mutations in mice, Hyp and Gy, cause low plasma phosphate and a rachitic and osteomalacic bone disease. Because of the controversy as to whether Gy is a good model for X-linked hypophosphatemia, the phenotypic severity of these two mutations was compared in both sexes and on two genetic backgrounds. The depression in plasma levels of phosphate was similar in all 10-week-old mutant mice. Male Hyp mice and heterozygous female Hyp mice were affected with similar severity in terms of reduced tail growth, shortened femora, reduced femoral mineral content, and abnormal mineral composition of the femoral matrix. In contrast, male Gy mice did not survive on the C57BL/6J background and were more severely affected than female Gy mice on the B6C3H background. The hybrid B6C3H background ameliorated the bone disease compared with the inbred C57BL/6J background for both mutant strains. There was no evidence of change in the plasma levels of 1,25-dihydroxyvitamin D, duodenal level of vitamin D-dependent calcium-binding protein, or urinary level of calcium in these adult mutant mice. In summary, Gy mice have a sexual dimorphism not present in Hyp mice. These two genes may indicate the presence of multiple gene loci in the human disease, with multiple proteins involved in the pathophysiology of the bone disease.

Parathyroid hormone-related protein mRNA in sheep endometrium and myometrium during late gestation and labor.

OBJECTIVE: To determine quantitative changes of parathyroid hormone-related protein (PTHrP) mRNA in sheep myometrium and endometrium during late gestation and labor. METHODS: Tissues were obtained from 22 pregnant ewes under halothane anesthesia: early controls at 131 days' gestational age (dGA) not in labor (ECNL; n = 6); during cortisol-induced premature labor (CPL at 131 dGA; n = 6); in term spontaneous labor (STL at 140-145 dGA; n = 5); or term control animals not in labor (TCNL at 140-145 dGA; n = 5). Total RNA was extracted and subjected to Northern blot analysis. Blots were probed with a human PTHrP cDNA probe, stripped, and rehybridized with an 18s rRNA nucleotide probe to normalize PTHrP mRNA levels. RESULTS: Endometrial PTHrP mRNA:18s was unaffected by gestational age or labor in tissues from the four groups. In contrast, myometrial PTHrP mRNA:18s ratio was decreased when TCNL and STL were compared with both ECNL and CPL groups (P < .05). CONCLUSIONS: Significant changes occur at the end of gestation in pregnant ovine myometrium PTHrP mRNA but not in the endometrium. In myometrium PTHrP mRNA levels were down-regulated at term regardless of whether labor was present. It seems that PTHrP mRNA levels in myometrium are related to gestational age rather than to labor per se in sheep. We hypothesize that PTHrP may play a role in maintaining uterine quiescence until term, at which time levels fall allowing myometrial contractile activity to increase unopposed by PTHrP.