H. James Armbrecht

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.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 or 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 ofthe mRNA for the cytochrome P450. They also demonstrate that this regulation may change with age.

Ethanol intoxication and withdrawal among three age groups of c57bl/6nnia mice.

Data have not been forthcoming on the effects of chronic ethanol administration on intoxication and severity of withdrawal using animals representative of the life-span of a particular species. The purpose of this study was to examine ethanol intoxication and withdrawal among three age groups (3, 14, 25 months) of C57BL/6NNIA male mice. Ethanol was administered in a liquid diet for 14 days. Pair-fed control groups and laboratory chow groups were also employed. Blood ethanol levels, signs of intoxication and withdrawal, liquid diet consumption, and body weight were measured. Old mice were significantly more intoxicated than younger mice. However, young mice consumed more ethanol as compared to the older mice. Blood ethanol levels did not differ among the three age groups, although variability was high within each age group. The ethanol liquid diet groups did not show a decrease in body weight. Withdrawal was more severe for old animals than younger animals. The greater effects of ethanol observed in the old animals do not appear to be attributable to age differences in blood ethanol levels, amount of ethanol consumed, or body weight loss.

Changes in intestinal glucose transport over the lifespan of the rat

Age-related changes in intestinal glucose absorption were studied using everted intestinal sacs and brush border membrane vesicles prepared from male F344 rats. Glucose uptake by everted intestinal sacs was greatest in young (2-3-month-old) as compared with adult (12-14-month-old) and old (24-month-old) rats. The greatest decrease in glucose uptake occurred between 2 and 12 months. The addition of phloridzin reduced glucose uptake to similar levels in all age groups, suggesting that the age-related change was in the carrier-mediated component of glucose transport. In order to localize the site of decreased carrier-mediated glucose transport, experiments were performed using brush border membrane vesicles. Vesicular glucose uptake in the presence of Na was significantly greater in vesicles prepared from 2-month-old rats (133 +/- 18 pmol/mg/s), compared with those prepared from 12-month-old rats (82 +/- 13 pmol/mg/s). Kinetic studies performed under non-equilibrium conditions demonstrated that the major effect of age was on the Na-dependent component of the brush border transport system. There was a reduction in the Vmax from 335 +/- 37 pmol/mg/s in the young to 217 +/- 22 pmol/mg/s in the adult, but there was no change in the Km. Isotope exchange studies performed under equilibrium conditions confirmed a decrease in the activity of the glucose transporter with age. No age-related changes in Na uptake by brush border membrane vesicles were observed. These findings suggest that a decrease in the number and/or activity of Na-linked glucose carriers may account for the decrease in intestinal glucose transport with age.

Characterization and regulation of the vitamin D hydroxylases

The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases-the hepatic 25-hydroxylase, the renal 1α-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1α- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1α and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1α-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1α.

Effect of dietary calcium and phosphorus restriction on calcium and phosphorus balance in young and old rats.

Abstract Previous studies have shown that the serum levels of the primary regulators of calcium (Ca) and phosphorus (P) metabolism, 1,25-dihydroxyvitamin D and parathyroid hormone, may change with age. Therefore, the effect of age on the ability of the rat to maintain a positive Ca and P balance was determined. Young (1.5 months) and old (18 months) rats were divided into three groups and fed either a low-Ca, high-P diet; a high-Ca, low-P diet; or a high-Ca, high-P diet. After 14 days, the young rats were in positive Ca and P balance regardless of diet. The old rats on the low-Ca, high-P diet were in negative Ca balance and positive P balance. The old rats on the other diets were in positive Ca and P balance. The negative Ca balance of the old rats was due to decreased intestinal absorption of Ca. Intestinal absorption was assessed by determining the percentage of dietary Ca absorbed in vivo and by measuring the active transport of Ca using the everted gut sac in vitro . Intestinal P absorption showed little change with age, except for a decrease in old rats on the high-Ca, low-P diet. Renal adaptation to dietary Ca and P restriction was similar in both young and old animals. Plasma Ca levels were unchanged with age, but plasma P levels decreased with age regardless of diet. These changes in Ca balance with age may reflect the reported decrease in serum 1,25-dihydroxyvitamin D 3 levels and the slight increase in PTH levels with age. The inability of old rats to maintain a positive Ca balance in the face of Ca deprivation is consistent with a general characteristic of the aging process—the decreased ability of an organism to adapt to changes in the external environment.

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.

Fluidizing effects of centrophenoxine in vitro on brain and liver membranes from different age groups of mice

This study examined the effects of different concentrations of centrophenoxine on physical properties of synaptic plasma membranes and liver microsomes using electron spin resonance procedures. Membranes of different age groups of mice were labeled with the 5-doxyl stearic acid spin-label and membrane fluidity determined in the presence and absence of different concentrations of centrophenoxine. Centrophenoxine had a direct effect on membranes as shown by a significant increase in membrane fluidity. This effect was greatest in liver microsomes as compared to synaptic plasma membranes. Age differences were not observed in centrophenoxine-induced fluidization. Effects of centrophenoxine in vivo may be due in part to the drug acting directly on the physical properties of the membrane lipid environment.

Calcium transport by basal lateral membrane vesicles from rat small intestine decreases with age

There is a marked decrease in active Ca2+ transport by the rat small intestine with age, particularly between 2 and 12 months. Much evidence suggests that the active component of Ca2+ transport resides in the energy-dependent pumping of Ca2+ across the intestinal basal lateral membrane. Therefore, we have characterized Ca2+ uptake by basal lateral membrane vesicles isolated from young (2-3 month old) and adult (12-14 month old) rats. In vesicles from the proximal duodenum, ATP-dependent Ca2+ uptake was about 4-times greater in the young animal than in the adult. There were no age differences in Ca2+ uptake in the absence of ATP. In vesicles from the ileum, Ca2+ uptake was much less than in the duodenum. The age differences in the ileum were smaller, and ATP-dependent Ca2+ uptake in the young was only twice that seen in the adult. Osmotic lysis of duodenal vesicles reduced Ca2+ uptake to low levels in both age groups, indicating that most of the Ca2+ was being taken up into an osmotically active space. Kinetic studies of Ca2+ uptake showed that there was no change in the apparent affinity but a 5-fold decrease in the Vmax of the adult Ca2+ transport system compared to that of the young animal. This marked decrease in the capacity of basal lateral membrane vesicles to actively transport Ca2+ may contribute to the decline in intestinal Ca2+ absorption with age.

Regulation of Calcium Metabolism by the Vitamin D Hydroxylases

Serum Ca must be maintained at about 10 mg/100 ml for the proper function of nerve, muscle, and bone. Serum Ca is maintained through the action of vitamin D metabolites and parathyroid hormone (PTH) on intestine, kidney, and bone (DeLuca, 1988; Minghetti and Norman, 1988) (Figure 1). Vitamin D 3 (cholecalciferol) itself has very little biological activity. It must first be hydroxylated in the liver to from 25-hydroxyvitamin D 3 (25[OH]D), which has some biological activity. However, the major activation takes place in the kidnev. Where 25 (OH)D is hydroxulated to 1,25-dihydroxyvitamin D 3 (1,25[OH]) 2 D)the metabolite of vitamin D with the most biological activity. 1,25(OH) 2 D is then inactivated by the 24-(OHases) in various target tissues. The charactristics of these vitamin D hydroxylases (OHases) and their regulation in health and diseasse are the subject of this review.

Changes in the Production and Action of 1,25-Dihydroxyvitamin D and Parathyroid Hormone with Age

Serum calcium is closely maintained at about 10 mg/dl throughout the life-span of both humans and rodents. This regulation is necessary for the proper function of many organ systems including nerve, muscle, and bone. Serum Ca is maintained primarily through the action of 1,25-dihydroxyvitamin D, the hormonal form of vitamin D, and parathyroid hormone (PTH) on intestine, kidney, and bone. Although serum Ca does not change with age, there is increasing evidence from both human and animal studies that serum levels of 1,25-dihydroxyvitamin D and PTH change markedly with age. In addition, the action of 1,25-dihydroxy-vitamin D on the intestine and the action of PTH on the kidney may change with age. Age-related changes in the levels and actions of these hormones may result in altered Ca homeostasis and may contribute to the loss of bone that is seen in the elderly (see Chapter 6).