Nirandon Wongsurawat

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.

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 parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.

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).