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Biomedical subjects

M A Boltz

Publications and source records attributed to M A Boltz.

17 recordsLinked to original sources
PubMed2003

Hormonal regulation of 25-hydroxyvitamin D3-1alpha-hydroxylase and 24-hydroxylase gene transcription in opossum kidney cells.

In the kidney, 25-hydroxyvitamin D(3) (25(OH)D) is converted to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D) by the 25(OH)D(3)-1alpha-hydroxylase enzyme, which contains a terminal cytochrome P450 (CYP1alpha) (systematic name: CYP27B1). Likewise, the kidney also produces 24,25-dihydroxyvitamin D(3) and 1,24,25-trihydroxyvitamin D(3) via a 24-hydroxylase whose terminal cytochrome P450 is CYP24. The purpose of this study was to characterize the transcriptional regulation of the CYP1alpha and CYP24 genes by parathyroid hormone (PTH) and 1,25(OH)(2)D in the kidney. Promoter-reporter gene constructs were transfected into opossum kidney (OK) cells, a renal proximal tubular cell line with endogenous PTH and 1,25(OH)(2)D receptors. PTH and forskolin stimulated CYP1alpha promoter activity via a cAMP-dependent pathway acting through the phosphorylation of CREB (cAMP-dependent response element-binding protein). This stimulation did not require new protein synthesis but may be modulated by short-lived proteins. 1,25(OH)(2)D modestly inhibited basal and forskolin-stimulated CYP1alpha promoter activity. The stimulation of CYP1alpha promoter activity by PTH and forskolin can account for the effect of these hormones on renal CYP1alpha mRNA levels. CYP24 promoter activity in transfected cells was increased by both 1,25(OH)(2)D and PTH, but there was no interaction between the two. The modest effects of 1,25(OH)(2)D and PTH on promoter activity and their lack of interaction do not account for the effects of these hormones on renal CYP24 mRNA levels. This suggests that there may be important posttranscriptional regulation of CYP24 mRNA in the kidney.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2003

PTH increases renal 25(OH)D3-1alpha -hydroxylase (CYP1alpha) mRNA but not renal 1,25(OH)2D3 production in adult rats.

The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] production declines with age in the rat. The purpose of these studies was to determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1alpha, the cytochrome P-450 component of the 25(OH)D(3)-1alpha-hydroxylase. Young (2 mo) and adult (12 mo) male Fischer 344 rats were parathyroidectomized (PTX). After 72 h, PTX rats were injected with PTH or vehicle at 24, 6, and 3 h before death, and renal CYP1alpha mRNA levels were measured by ribonuclease protection assay. In young rats, PTH markedly increased plasma 1,25(OH)(2)D(3) and renal 1,25(OH)(2)D(3) production. However, in adult rats, the response to PTH was less than 30% of that seen in young rats. Renal CYP1alpha mRNA levels, on the other hand, were increased over fivefold by PTH in both young and adult rats. In in vitro studies, PTH/forskolin increased CYP1alpha mRNA levels over twofold in renal slices from both young and adult PTX rats. These studies demonstrate that the decreased capacity of PTH to increase 1,25(OH)(2)D(3) production in adult rats is not due to decreased induction of CYP1alpha mRNA.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2002

Development of a vitamin D-responsive organ culture system for adult and old rat intestine.

The purpose of this study was to develop an organ culture system for adult and old rat small intestine that is responsive to vitamin D. Explants from F344 rats were cultured on Millipore tissue culture inserts placed in 6-well dishes at a temperature of 28 degrees C and in the presence of 95% oxygen. Explants from young (2 months old), adult (12 months old), and old (22 months old) rats were viable for up to 12 hr as determined by constant rates of DNA and protein synthesis. Hormonal responsiveness was characterized by measuring the capacity of 1,25-dihydroxyvitamin D [1,25(OH)2D], the hormonal form of vitamin D, to increase mRNA levels of the intestinal 24-hydroxylase cytochrome P-450 (CYP24). Jejunal explants from young rats increased CYP24 mRNA levels in a linear fashion with an EC50 of 3 nM in response to 1,25(OH)2D. There was no change with age in the magnitude of the jejunal response with regard to time (0-12 hr) or dose (0.1-100 nM). However, in the duodenum, 1,25(OH)2D increased CYP24 mRNA to significantly higher levels in the adult compared to the young. Since the 24-hydroxylase is the first step in the degradative pathway for 1,25(OH)2D in the intestine, increased duodenal expression of the 24-hydroxylase may contribute to the decreased action of 1,25(OH)2D on the adult duodenum.

Animals↗
PubMed2002

Differences in intestinal calcium and phosphate transport between low and high bone density mice.

The purpose of this study was to determine whether there are differences in intestinal Ca and phosphate transport in mice having different peak bone densities. Intestinal transport was measured in C57BL/6 (C57, low bone density) and C3H/He (C3H, high bone density) female mice. Unidirectional (mucosal to serosal) transport of Ca was 58% higher in C3H compared with C57 mice, as measured by everted duodenal sacs. The capacity of the duodenal mucosa to take up Ca was also higher in the C3H mice. This uptake highly correlated with Ca transport across the intestine. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], which stimulates intestinal Ca absorption, markedly stimulated unidirectional Ca transport and uptake to similar levels in both strains of mice. On the other hand, unidirectional phosphate transport in C3H mice was only 36% that of C57 mice. mRNA levels of the plasma membrane Ca pump were 90% higher in the duodenum of C3H mice. There was no difference between strains in duodenal calbindin or 24-hydroxylase mRNA levels. Regarding vitamin D metabolism, there was no difference in serum 1,25(OH)(2)D(3) levels or in renal 1alpha-hydroxylase mRNA levels. The combination of high intestinal Ca transport and low phosphate transport may contribute to the high peak bone density seen in the C3H relative to the C57 mouse.

Animals↗
PubMed2001

Differential responsiveness of intestinal epithelial cells to 1,25-dihydroxyvitamin D3--role of protein kinase C.

Non-transformed rat intestinal epithelial cell (IEC) lines were used to study the action of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D) in the intestine. The capacity of 1,25(OH)2D to increase the expression of the cytochrome P450 component of the vitamin D 24-hydroxylase (CYP24) was determined in IEC-6 and IEC-18 cell lines. In IEC-6 cells, which are derived from crypt cells isolated from the whole small intestine, 1,25(OH)2D markedly increased expression of CYP24 protein and mRNA within 12 h. In contrast, in IEC-18 cells, which are derived from crypt cells from the ileum only, 1,25(OH)2D did not increase expression of CYP24 until 24-48 h. The maximal levels of CYP24 mRNA seen in the IEC-18 cells were only 31% of the maximal levels seen in the IEC-6 cells. In the presence of 1,25(OH)2D, phorbol esters rapidly increased CYP24 mRNA levels in IEC-18 cells from almost undetectable to levels seen in IEC-6 cells. Protein kinase inhibitors abolished the stimulation by 1,25(OH)2D and by phorbol esters in both cell lines. Stimulation of mRNA levels by phorbol esters required new protein synthesis but stimulation by 1,25(OH)2D did not. These studies demonstrated that the rapid action of 1,25(OH)2D in IEC-6 cells is related to the activation of protein kinase C, an event which is missing in the IEC-18 cells. This differential response to 1,25(OH)2D probably takes place at a post-receptor site, since the number of vitamin D receptors in each cell line was found to be similar.

Animals↗
PubMed1999

Effect of age on calcium-dependent proteins in hippocampus of senescence-accelerated mice.

The senescence-accelerated P8 mouse (SAMP8) is a well-characterized model for the age-related decline in acquisition and retention. Calcium-dependent protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase (CAM K) have been implicated in these processes in the hippocampus. Therefore, the expression of hippocampal PKC and CAM K was determined in SAMP8 mice aged 4, 8, and 12 months. As measured by Western blotting, total hippocampal PKC-gamma protein declined linearly with age. In addition, the distribution of the PKC-gamma also changed with age. The amount of PKC in the particulate fraction declined linearly with age relative to the soluble PKC. The decline in total PKC and particulate PKC correlated with the previously reported decline in retention but not with the decline in acquisition. Western blotting revealed no consistent change in CAM KII protein levels. In addition to protein levels, Ca-dependent protein kinase activity may also be affected by changes in intracellular Ca concentration. Therefore, the levels of calbindin and the plasma membrane Ca pump, two proteins involved in maintaining low levels of intracellular Ca, were measured in the hippocampus. Calbindin protein declined progressively with age, but there was no significant change in total plasma membrane Ca pump expression. These studies demonstrate a decrease in the amount and distribution of hippocampal PKC-gamma in the SAMP8 between 4 and 12 months that is associated with decreased retention.

Aging↗
PubMed1999

Capacity of a low calcium diet to induce the renal vitamin D 1a-hydroxylase is decreased in adult rats.

Young animals adapt to a low calcium diet by increasing renal production of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active metabolite of vitamin D. However, the capacity of adult animals to adapt is markedly diminished. With the recent cloning of the cytochrome P450 component (CYP1a) of the renal 1-hydroxylase enzyme complex, it is now possible to determine directly the effect of dietary calcium and maturation on the expression of renal 1-hydroxylase. Using a ribonuclease protection assay, it was found that feeding a low Ca diet markedly increased renal CYP1a mRNA levels in young rats. However, feeding this diet to adult rats produced an increase in CYP1a mRNA that was only 10% that of the young rats. These studies demonstrate that a low calcium diet increases renal 1,25-dihydroxyvitamin D production in young animals but not in adult animals by increasing CYP1a expression. Since the low calcium diet increased plasma parathyroid hormone levels to similar levels in both age groups, this suggests that in the adult there is a renal refractoriness to parathyroid hormone.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1999

Intestinal plasma membrane calcium pump protein and its induction by 1,25(OH)(2)D(3) decrease with age.

The plasma membrane Ca pump of intestinal absorptive cells has been proposed as a component in the vitamin D-dependent active transport of Ca. Because intestinal Ca transport declines with age, the purpose of this study was to determine if changes in Ca pump expression parallel this decline. Intestinal levels of the plasma membrane Ca pump protein were measured by Western blotting in Fischer 344 rats that were 2, 12, and 24 mo of age. Ca pump protein levels declined by 90% in the duodenum and 65% in the ileum between 2 and 12 mo of age, the time during which active Ca transport declines markedly. The effect of age on the induction of the Ca pump by 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the active metabolite of vitamin D, was determined. Rats were made deficient in 1,25(OH)(2)D(3) by feeding a high-strontium diet, and they were then dosed with 1,25(OH)(2)D(3) or vehicle at 48, 24, and 6 h. In 12-mo-old rats 1,25(OH)(2)D(3) induced duodenal Ca pump protein to only 39% and active Ca transport to 33% of that seen in 2-mo-old animals. These studies demonstrate that decreased expression of the plasma membrane Ca pump protein, along with calbindin protein, parallels the decline in intestinal Ca transport and its response to 1,25(OH)(2)D(3) with age.

Aging↗
PubMed1998

Capacity of 1,25-dihydroxyvitamin D to stimulate expression of calbindin D changes with age in the rat.

Studies in rats and humans have shown that there is an age-related decline in the stimulation of intestinal Ca transport by 1,25-dihydroxyvitamin D3 [1,25(OH)2D], the active metabolite of vitamin D. The calbindins are a family of vitamin D-dependent calcium-binding proteins found in the intestine (calbindin D-9k) and kidney (calbindin D-28k) and are thought to play a role in calcium transport and homeostasis. The purpose of this study was to determine if the capacity of 1,25(OH)2D to stimulate the expression of calbindin protein and mRNA changes with age. Young (2 months), adult (12 months), and old (22 months) male F344 rats were made deficient in 1,25(OH)2D by being fed a strontium-containing diet and then being given a single i.p. injection of 1,25(OH)2D. Calbindin protein levels were measured by immunological techniques using specific antisera, and mRNA levels were measured by Northern and dot blots. The maximal amount of calbindin D-9k protein induced by 1,25(OH)2D declined with age in the duodenum but not in the ileum. In time-course studies, there was a delay in calbindin D-9k induction in the duodenum but not in the ileum of adult rats compared to young rats. In contrast to protein induction, maximal calbindin D-9k mRNA levels in response to 1,25(OH)2D were greater in the adult animal and showed no time lag compared to those in the young animal. In the kidney, maximal levels of renal calbindin D-28k protein and mRNA did not change with age, but there was delayed induction in the adult. These studies demonstrate that there is an age-related decrease in the induction of calbindin protein in response to 1,25(OH)2D in the duodenum, but not in the ileum or kidney. This decline may be due to decreased translation of calbindin D-9k mRNA into protein in the duodenum with age.

Age Factors↗
PubMed1998

Induction of the vitamin D 24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 is regulated by parathyroid hormone in UMR106 osteoblastic cells.

The expression of the vitamin D 24-hydroxylase is highly regulated in target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D), where it may modulate the action of 1,25(OH)2D. In UMR106 osteoblastic cells, 1,25(OH)2D and PTH synergistically induce 24-hydroxylase expression. The purpose of these studies was to characterize the interaction between 1,25(OH)2D and PTH with regard to the messenger RNA (mRNA) levels of the cytochrome P450 component of the 24-hydroxylase (CYP24). PTH alone had no effect on CYP24 mRNA levels, and 1,25(OH)2D alone produced only a modest increase. However, 1,25(OH)2D and PTH together synergistically increased CYP24 mRNA levels 3-fold compared with 1,25(OH)2D alone. PTH also increased the sensitivity of UMR cells to 1,25(OH)2D from 10(-8) to 10(-10) M. PTH worked through the cAMP signaling pathway as evidenced by the lack of effect of PTH (3-34) and by the full activity of 8-bromo-cAMP. PTH in the presence of 1,25(OH)2D increased CYP24 gene transcription as shown by nuclear run-on studies and by activation of a CYP24 promoter-reporter construct after transfection. PTH also increased vitamin D receptor number in UMR cells, but this occurred at times later than the increase in transcription. These studies demonstrate that PTH in the presence of 1,25(OH)2D works through the cAMP-dependent signaling pathway to increase transcription of the CYP24 gene, to increase CYP24 protein levels, and to increase 24-hydroxylase activity.

Animals↗
PubMed1997

Induction of 24-hydroxylase cytochrome P450 mRNA by 1,25-dihydroxyvitamin D and phorbol esters in normal rat kidney (NRK-52E) cells.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), acts on intestinal, renal, and bone cells to regulate skeletal and mineral metabolism. 1,25(OH)2D also induces 24-hydroxylase activity in these target cells. The 24-hydroxylase hydroxylates 1,25(OH)2D to 1,24,25-trihydroxyvitamin D and 25(OH)D to 24,25-dihydroxyvitamin D. The production of 1,24,25-trihydroxyvitamin D is thought to be the first step in the inactivation of 1,25(OH)2D by its target tissues. Previous studies have characterized the induction of the 24-hydroxylase by 1,25(OH)2D in clonal cell lines from intestine and bone. The purpose of these studies was to characterize the induction of the 24-hydroxylase by 1,25(OH)2D in the kidney, using the clonal rat renal cell line NRK-52E. 1,25(OH)2D (10(-7)M) increased the mRNA levels for the cytochrome P450 component of the 24-hydroxylase (P450cc24) by sevenfold after 36 h in NRK-52E cells. 1,25(OH)2D increased P450cc24 mRNA levels in a dose-dependent manner with an EC50 of 10(-8) M. In parallel experiments, 1,25(OH)2D significantly increased 24-hydroxylase enzyme activity after 48-72 h. The increase in P450cc24 mRNA induced by 1,25(OH)2D required ongoing transcription and translation and was inhibited by H-7, a protein kinase C inhibitor. Tetradecanoyl phorbol acetate markedly increased the magnitude of the tissue responsiveness to 1,25(OH)2D by a protein kinase C-dependent pathway. These studies demonstrate that 1,25(OH)2D increases P450cc24 mRNA levels in NRK-52E cells by a mechanism requiring new protein synthesis and involving protein kinase C. This is in contrast to the action of 1,25(OH)2D in intestinal cells, which does not require new protein synthesis, and in osteoblastic cells, which does not involve protein kinase C.

Animals↗
PubMed1994

Expression of plasma membrane calcium pump mRNA in rat intestine: effect of age and 1,25-dihydroxyvitamin D.

The capacity of the small intestine to actively transport Ca declines markedly with increasing age in the rat. The basal-lateral plasma membrane Ca pump is thought to be an important component of the active transport mechanism. Therefore, the purpose of this study was to determine if there are changes in the expression of the intestinal Ca pump with age, mRNA levels were quantitated by Northern and dot blot analysis using a cDNA probe based on the sequence of the plasma membrane Ca pump expressed in the rat intestine (PMCA1). In the duodenum, Ca pump mRNA levels were 3-4 times higher in young (2 months) rats compared to adult (12 months) and old (27 months) rats. In the ileum, Ca pump mRNA levels were one third those of the duodenum, and ileal levels were higher in young rats compared to adult rats. These changes in mRNA levels with age and segment were significantly correlated with Ca pump activity as measured in basal-lateral membrane vesicles in vitro. To determine intestinal responsiveness to 1,25(OH)2D, rats were fed a strontium diet to induce vitamin D deficiency. In young animals, 1,25(OH)2D significantly increased Ca pump mRNA levels 4-fold in the duodenum. 1,25(OH)2D had a similar effect in the adult duodenum. These studies demonstrate that there are changes in Ca pump mRNA levels with age and intestinal segment. Since there was no change in the capacity of 1,25(OH)2D to increase Ca pump mRNA levels, the decline in Ca pump expression may be due to the age-related decrease in serum 1,25(OH)2D rather than to decrease responsiveness to 1,25(OH)2D.

Aging↗
PubMed1993

Phorbol ester markedly increases the sensitivity of intestinal epithelial cells to 1,25-dihydroxyvitamin D3.

We have used a clonal intestinal epithelial cell line (IEC-18) to study the mechanism of action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D) in vitro. 1,25(OH)2D(10(-7) M) elevated by over 10-fold the mRNA levels for the cytochrome P450 component (P450cc24) of the 1,25(OH)2D-24-hydroxylase. Increased P450cc24 mRNA levels were detectable at 6 h and peaked at 36 h. Below a concentration of 10(-7) M, 1,25(OH)2D had almost no effect. However, addition of phorbol ester for 2 h made the intestine responsive to 1,25(OH)2D concentrations as low as 10(-9) M.

Animals↗
PubMed1993

Effects of 1,25-dihydroxyvitamin D3 and phorbol ester on 25-hydroxyvitamin D3 24-hydroxylase cytochrome P450 messenger ribonucleic acid levels in primary cultures of rat renal cells.

The renal 25-hydroxyvitamin D3 24-hydroxylase enzyme, which may be the starting point in the catabolic pathway for vitamin D metabolism, is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonal form of vitamin D. The purpose of this study was to investigate the regulation of the cytochrome P450 component of this enzyme (P450cc24) by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate (TPA). P450cc24 messenger RNA (mRNA) levels were measured using the full-length rat complementary DNA probe (p108). In primary cultures of rat renal tubular cells, 1,25-(OH)2D3 produced a 26-fold increase in P450cc24 mRNA which was detectable at 4 h, maximal at 24 h, and returned almost to baseline by 48 h. The induction was inhibited by actinomycin D, 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB), and cycloheximide, and it was specific for vitamin D compounds containing a 1-hydroxyl group. TPA alone had no effect, but TPA in the presence of 1,25-(OH)2D3 produced an increase in P450cc24 mRNA within 30 min, and this increase peaked at 2 h. TPA also shifted the dose-response curve of 1,25-(OH)2D3 to the left, so that 1,25-(OH)2D3 was effective at a concentration as low as 1 nM. In the same experiments, TPA increased c-fos mRNA levels, and this increase was accelerated by 1,25-(OH)2D3. These studies suggest that the induction of P450cc24 mRNA by 1,25-(OH)2D3 is a receptor-mediated genomic event and that this induction may account for the stimulation of 24-hydroxylase enzyme activity by 1,25-(OH)2D3. In addition, TPA accelerates the effect of 1,25-(OH)2D3 by a mechanism which may involve protein kinase C.

Animals↗
PubMed1991

Expression of 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 in kidney and intestine. Effect of 1,25-dihydroxyvitamin D and age.

To study the mechanism of hormonal regulation of the 25-hydroxyvitamin D 24-hydroxylase, a DNA probe complementary to the published sequence of the recently cloned P450 component [(1991) FEBS Lett. 278, 195] was employed. Young (2 month) and adult (12 month) F344 rats, deficient in 1,25-dihydroxyvitamin D, were given a single dose of 1,25-dihydroxyvitamin D. In young rats, 1,25-dihydroxyvitamin D markedly increased P450 mRNA levels within 3 h in both kidney and intestine, and maximal levels were attained at 16 and 3 h, respectively. In adult animals, maximal induction of mRNA was diminished in the kidney, and the decline was slower in the intestine. Time of maximal induction did not change with age. These studies demonstrate for the first time regulation of the 24-hydroxylase enzyme by 1,25-dihydroxyvitamin D at the level of the mRNA for the cytochrome P450. They also demonstrate that this regulation may change with age.

Aging↗
PubMed1986

Effect of age on parathyroid hormone and forskolin stimulated adenylate cyclase and protein kinase activity in the renal cortex.

The capacity of parathyroid hormone (PTH) to stimulate the renal production of 1,25-dihydroxyvitamin D declines with age. Since the action of PTH in the kidney is mediated by cAMP, we have examined the effect of PTH and forskolin on renal cortical adenylate cyclase in young (3 months), adult (13-15 months), and old (25-27 months) F344 rats. PTH-dependent adenylate cyclase, measured as cAMP accumulation in cortical slices, was reduced in adult and old rats compared to young rats over a PTH concentration range of 0.015-15 units/ml. There was no difference in PTH-dependent adenylate cyclase activity between adult and old rats. Renal plasma membrane preparations demonstrated similar changes in PTH-stimulated adenylate cyclase activity. There was no difference in calcitonin, forskolin, or guanyl-5-ylimidodiphosphate (Gpp(NH)p) stimulation of adenylate cyclase in plasma membranes from each age group, suggesting that the defect lies in the membrane receptor for PTH. The decreased adult sensitivity to PTH could be reversed by thyroparathyroidectomy. PTH stimulation of cytosolic protein kinase activity did not change with age. These results suggest that the decrease in PTH-dependent adenylate cyclase is due to the alterations at the level of PTH receptor. These alterations may be in response to the increase in serum PTH seen in these animals with increasing age.

Adenylyl Cyclases↗