Adaptation to phosphate deprivation in osteoblast-like cells.

The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolayer culture these cells transport inorganic phosphate and L-alanine via Na(+)-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60-70 per cent increase in Na(+)-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na(+)-dependent alanine uptake and Na(+)-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased Vmax and was blocked completely by pretreatment with cycloheximide (70 microM). In these cells recovery of intracellular pH after acidification with NH4Cl is due primarily to the Na+/H+ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na+/H+ exchange was inhibited during phosphate deprivation.

Hypertonic upregulation of amino acid transport system A in vascular smooth muscle cells.

The A10 line of vascular smooth muscle cells has Na+ dependent transport systems for alanine, proline, and Pi, whereas uptake of leucine, myo-inositol and D-glucose is Na+ independent. When A10 cells were incubated for 4 h in medium made hypertonic by addition of sucrose, there was a marked increase in Na(+)-dependent transport of alanine and proline but no change in Na(+)-dependent Pi uptake or Na(+)-independent uptake of leucine and inositol. Intracellular alanine content was increased 61% by the hypertonic treatment. Other nonpenetrating solutes, such as cellobiose and mannitol, reproduced the effect of sucrose, but urea, a penetrating solute, did not. Studies with 2-(methylamino)-isobutyric acid revealed that the upregulation by hypertonicity involved only system A. Increases in alanine and proline uptake also occurred after incubating the cells in isotonic medium containing 0.1 mM ouabain, suggesting that an increase in intracellular Na+ may be part of the intracellular signal for upregulation of system A. Hypertonic upregulation of Na(+)-dependent alanine transport occurred also in primary cultures of vascular smooth muscle cells. The response was blocked by actinomycin D and cycloheximide, indicating that gene transcription and protein synthesis play important roles in the mechanism leading to increased alanine uptake. We conclude that vascular smooth muscle cells, during prolonged hypertonic stress, activate system A and accumulate specific neutral amino acids which may act as organic osmolytes to help maintain normal cell volume.

Vanadate action on renal phosphate transport.

Insulin stimulates reabsorption of phosphate (Pi) in the renal proximal tubule. Previous studies have shown that vanadate can mimic the action of insulin on various tissues. In the present study, we tested the action of vanadate on renal Pi transport both in control rats and in rats made diabetic by injection of streptozotocin. Vanadate was administered orally for 4 days by inclusion in drinking water (0.7 mg/ml). By the 4th day, vanadate treatment of control rats did not change acid-base status, plasma glucose or the filtered load of Pi, but the urinary excretion of Pi was reduced to 2.5 +/- 0.9 compared with 17.6 +/- 3.5 mumol/mg creatine (P < 0.02) in untreated control rats. However, Na+/Pi cotransport by isolated brush border membrane vesicles was not different between the two groups. Findings in parathyroidectomized rats were similar. By the 4th day of vanadate treatment of diabetic rats, there was reversal of polyuria, polydipsia and hyperglycemia with no change in acid-base status. The filtered load of Pi was decreased by vanadate, and urinary Pi excretion also tended to decrease but not significantly. The values for Pi excretion were 21.4 +/- 7.6 in vanadate treated diabetics and 36.1 +/- 4.5 mumol/mg creatinine in untreated diabetics. In contrast to vanadate, daily injections of insulin did not change the filtered load of Pi but reduced urinary Pi excretion in diabetic rats to 15.6 +/- 2.2 mumol/mg creatinine (P < 0.02). These findings suggest that vanadate stimulated tubular Pi reabsorption in control rats but not in diabetic rats. Vanadate treatment of diabetic rats may tend to decrease tubular Pi reabsorption in contrast to the action of insulin.