C. Tony Liang

Sodium Gradient- and Sodium Plus Potassium Gradient-Dependent L-Glutamate Uptake in Renal Basolateral Membrane Vesicles

SummaryA membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake ofl-glutamate by the basolateral membrane vesicles was studied. A Na+ gradient ([Na+]o>[Na+]i) stimulated the uptake ofl-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na+-l-glutamate cotransport system in the basolateral membrane. A K+ gradient ([K+]i>[K+]o) increased the uptake additionally. This effect was specific for K+ (Rb+). The action of the K+ gradient in enhancing the uptake ofl-glutamate had an absolute requirement for Na+. In the presence of Na+, but in the absence of a Na+ gradient. i.e., [Na+]o=[Na+]i, the K+ gradient also energized the concentrative uptake ofl-glutamate. This effect of the K+ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system forl-glutamate energized by both Na+ ([Na+]o>[Na+]i and K+ ([K+]i>[K+]o) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by whichl-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.

Bicarbonate-stimulated ATPase in the renal proximal tubule luminal (brush border) membrane

Abstract Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 m m HCO 3 − . The K a for HCO 3 − was 36 m m . Kinetic studies of the “HCO 3 − -ATPase” indicate that HCO 3 − had no effect on the K m for ATP and ATP did not alter the K a for HCO 3 − . Several anions, notably SO 3 2− , also accelerated the rate of dephosphorylation of ATP. The V for “SO 3 2− -ATPase” was fivefold greater than that for “HCO 3 − -ATPase.” The K a for SO 3 2− was 0.78 m m . Other anions including Cl − and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO 3 − stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCN − nor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p -trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H + flux. Carbonic anhydrase significantly increased the HCO 3 − -stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H + from tubular cell to lumen and support suggested interrelationships between HCO 3 − -stimulated ATPase, H + secretion, and bicarbonate transport in the kidney.

Actions of bFGF on mitogenic activity and lineage expression in rat osteoprogenitor cells: effect of age.

Rat osteoprogenitor cells were used to examine the effects of bFGF on DNA synthesis and the expression of osteoblast (OB)-related genes. bFGF, as low as 0.1 ng/ml, stimulated DNA synthesis. bFGF also increased the mRNA level of osteopontin (OP) and decreased that of type I collagen (COL I). When cultures were grown in dexamethasone (DEX) to induce OB lineage commitment, the expression of COL I, alkaline phosphatase (AP) and OP was greatly enhanced. Subsequent incubation with bFGF partially negated the stimulatory effect of DEX on AP and COL I mRNAs. bFGF also inhibited the expression of osteocalcin mRNA in cells grown in 1,25(OH)2D3 and DEX. Combined effects of bFGF with IGF-I or PDGF on DNA synthesis and OP expression were examined. bFGF + IGF-I, but not bFGF + PDGF, was more effective than PDGF alone. By comparing cells from adult and old animals, we found that bFGF-induced mitogenic activity was reduced significantly with age. In contrast, the effect of bFGF on the expression of OB genes was not significantly altered by age. These findings suggest that bFGF plays a dual role as a local positive and negative regulator on proliferation and osteogenic lineage expression, respectively, in osteoprogenitor cells, and that the mitogenic activity in response to bFGF was impaired in aging.

Effect of Locally Infused IGF‐I on Femoral Gene Expression and Bone Turnover Activity in Old Rats

Previously, we showed that the age‐dependent deficit in bone formation activity can be attributed in part to a decline in local expression of insulin‐like growth factor I (IGF‐I) and altered mitogenic response of old osteoprogenitor cells to IGF‐I. To establish the cellular basis for using IGF‐I as a possible therapeutic agent for osteoporosis, we examined the effect of locally infused (50 ng/day for 14 days) on the expression of osteoblast‐related genes in femurs of old rats. Northern and dot blot analyses showed that the expression of procollagen (I), osteopontin, alkaline phosphatase, and osteocalcin was increased 0.4‐ to 1.5‐fold in IGF‐1–treated femurs as compared with control femurs. Histomorphometric analyses were carried out in parallel experiments to assess the changes in bone remodeling activity. Trabecular bone volume, trabecular number, and trabecular thickness were increased 56%, 29%, and 23%, respectively, whereas trabecular separation was reduced 26% by IGF‐1 treatment. IGF‐I treatment increased significantly the osteoid volume, osteoid surface, osteoblast number, and osteoblast surface. Mineralizing surface and mineral apposition rate, kinetic indices of bone formation, were also stimulated by IGF‐I treatment. The bone formation rate was stimulated 81% in IGF‐I–treated femurs as compared with control femurs. In contrast, eroded surface and osteoclast surface, parameters associated with bone resorption, were not affected by IGF‐I treatment. These findings suggest that local administration of IGF‐I into femurs of old rats can stimulate the expression of matrix proteins and improve trabecular bone status by stimulating bone formation without any appreciable effect on bone resorption.

EFFECT OF AGE ON DUODENAL 1,25-DIHYDROXYVITAMIN D-3 RECEPTORS IN WISTAR RATS

We confirmed our previous observation that duodenal Ca2+ absorption and serum 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels declined concurrently in old (24 months old) rats as compared to young (6 months old) rats. It is well known that 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) expresses its action after binding to specific receptor molecules. In this paper, we compared certain properties of rat duodenal 1,25-(OH)2D3 receptors from old and young animals. Receptor preparations were incubated with [3H]1,25-(OH)2D3 to quantitate the number of unoccupied and total receptor sites and showed that total and unoccupied receptor sites decreased by 22 and 16%, respectively in old rats. Endogenously occupied sites were reduced by 43% in duodenum of the old rat and, consequently, the percentage of receptor occupancy also declined. Age did not affect the dissociation constant (KD) of 1,25-(OH)2D3 from the receptor; the sedimentation coefficient (3.3 S) of the tritiated 1,25-(OH)2D3-receptor complex in sucrose density centrifugation; or its affinity for DNA. The data are consistent with the hypothesis that the age-related decline in Ca2+ absorption in the intestine may be due, in part, to the decrement in the circulating level of 1,25-(OH)2D and a reduction of intestinal 1,25-(OH)2D3 receptor occupancy status.

Alterations in mRNA expression of duodenal 1,25-dihydroxyvitamin D3 receptor and vitamin D-dependent calcium binding protein in aged wistar rats

Previously, we reported that uptake of calcium into isolated duodenal cells and duodenal brush border membrane vesicles decreased in senescence. Decreases in duodenal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor number and 9k vitamin D-dependent calcium binding protein (CaBP) were also observed in aged rats. In this study, we examined the steady state mRNA levels of duodenal 1,25-(OH)2D3 receptor and CaBP in both adult (6-month-old) and old (24-month-old) rats. We identified one major band of 4.4 kb for 1,25-(OH)2D3 receptor mRNA. The size of the transcript was not affected by age. The content of 1,25-(OH)2D3 receptor mRNA (normalized with poly(A)+RNA) decreased 23% in the aged rat as compared to the adult rat. The expression of CaBP was also examined. A single band of 0.6 kb was observed for CaBP mRNA. The size of CaBP mRNA was not altered with age. However, the abundance of CaBP mRNA (normalized with poly(A)+RNA) was reduced 20% in the senescent rat. Thus, the results in the present study were consistent with our previous findings that the number of 1,25-(OH)2D3 receptors and the level of CaBP declined in the aged rat. However, the precise mechanism leading to the age-related deficit in mRNA expression remains to be elucidated.

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

SummaryThein vivo andin vitro effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)2D3 given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)2D3 with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10−15m, was maximal at 10−13m, and was diminished at higher (10−11m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D3 analogs was 1,25-(OH)2D3=1-(OH)D3>25-(OH)D3>1,24,25-(OH)3D3>24,25-(OH)2D3>D3. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)2D3 was five orders of magnitude greater than D3. Kinetically, 1,25-(OH)2D3 increased theVmax of calcium uptake; the affinity for calcium (Km=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)2D3,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)2D3. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)2D3 induction of calcium transport and specific biochemical correlates.

In Vitro Effects of Parathyroid Hormone on Kidney Cortical Slices: cAMP Responses and Concomitant Inhibition of the Na+ Gradient-Dependent Uptake of Phosphate by Brush Border Membrane Vesicles Isolated from the Renal Slices

Mouse renal cortical slices were incubated with parathyroid hormone (30 U/ml) for 2 min. Brush border membrane vesicles isolated from the treated slices had a decreased Na+ gradient-dependent uptake of phosphate. Concomitantly, the hormone elicited the activation of adenylate cyclase, the increase in tissue level of cAMP, and the enhancement of cAMP-dependent protein kinase.

Alpha-2-adrenergic modulation of the parathyroid hormone-inhibition of phosphate uptake in cultured renal (OK) cells.

Parathyroid hormone enhances the formation of cAMP and decreases the Na+-dependent uptake of phosphate in cultured renal cells derived from the American opossum (OK cells). Epinephrine, acting as an alpha 2-adrenergic agonist, inhibits the PTH-induced synthesis of cAMP by a pertussis toxin-sensitive mechanism and blunts the inhibition of phosphate transport by PTH. Na+-dependent alpha-methylglucoside and Na+ uptakes by the cells are unaffected by PTH and epinephrine. These findings suggest that alpha 2-adrenergic agonists may selectively modulate PTH-sensitive phosphate transport in the renal proximal tubule.

Effects of basic fibroblast growth factor on osteoblast-related gene expression in the process of medullary bone formation induced in rat femur

Abstract. The effects of basic fibroblast growth factor (bFGF) on osteogenic differentiation in-vivo were investigated using a rat bone marrow ablation model. bFGF was infused directly into rat femora for 6 days after bone marrow ablation. The contralateral femur was infused with vehicle only and used as control. Bone formation was induced in the rat femoral cavity, and the gene expression of osteoblast markers was examined. Treatment with bFGF at 50 and 100 ng/day significantly enhanced the mRNA levels of osteopontin compared with the levels in the control leg, with increases of 25% and 24%, respectively. In contrast, bFGF infusion at 50 ng/day provoked a significant (nearly 20%) inhibition of expression for type I collagen. Infusion of bFGF at a higher dose exhibited an inhibitory tendency for bFGF action on gene expression. There were no significant changes in alkaline phosphatase and osteocalcin mRNA levels in response to any dose of bFGF. The findings presented here suggest that bFGF modulates osteogenic differentiation in-vivo and may play an important role in the process of bone remodeling.