RETRACTED: Accumulation of glycerophosphocholine (GPC) by renal cells: osmotic regulation of GPC:choline phosphodiesterase.

Abstract

Although GPC has long been recognized as a degradation product of phosphatidylcholine, only recently is there wide appreciation of its role as a compatible and counteracting osmolyte that protects cells from osmotic stress. GPC is osmotically regulated in renal cells. Its level varies directly with extracellular osmolality. Cells in the kidney medulla in vivo and in renal epithelial cell cultures (MDCK) accumulate large amounts of GPC when exposed to high concentrations of NaCI and urea. Osmotic regulation of GPC requires choline in the medium, presumably as a precursor for synthesis of GPC. Choline transport into the cells, however, is not osmoregulated. The purpose of the present studies was to use MDCK cell cultures as a defined model to distin h whether osmotically induced accumulation of GPC results from increased GPC synthesis or decreased GPC disappearance. The rate of incorporation of 14C from [14C]choline into GPC, the steady-state GPC synthesis rate, and the activity ofphospholipase A2 (which can catalyze a step in the synthesis of GPC from phosphatidylcholine) are not increased by high NaCl and urea. In fact all are decreased by approximately one-third. Therefore, we find no evidence that high NaCI and urea increases the GPC synthesis rate. On the other hand, the rate coefficient for cellular GPC disappearance and the activity of GPC:choline phosphodiesterase (EC 3.1.4.2), which catalyzes degradation of GPC, are decreased by approximately two-thirds by high NaCl and urea. We conclude that high NaCl and urea increase the level of GPC by inhibiting its enzymatic degradation.Cells in the renal medullas of mammals contain large amounts of organic osmolytes [namely, GPC (1, 2), sorbitol, inositol, and glycine betaine (for review, see ref.3)]. When renal medullary osmolality rises (as, for example, during antidiuresis), the concentration of these organic osmolytes in medullary cells increases slowly over several days (4). The renal medullary organic osmolytes accumulate in response to the high extracellular concentrations of NaCl and urea in this region of kidney and vary with the NaCl and urea concentrations. The organic osmolytes help balance the high osmotic pressure of the extracellular NaCl. The utilization of these compounds, rather than inorganic salts, for this purpose is attributed to the organic osmolytes being compatible solutes (5, 6) that can accumulate to high levels in cells without perturbing intracellular macromolecules, whereas high concentrations of inorganic ions are perturbing. GPC is thought also to be a counteracting solute (5-7) that protects intracellular macromolecules from being denatured by the high concentration of urea in the renal medulla.

Caffeine causes glycerophosphorylcholine accumulation through ryanodine-inhibitable increase of cellular calcium and activation of phospholipase A2 in cultured MDCK cells

“…GPC was assayed by the combined chemical and enzymatic method previously described (Zablocki et al, 1991) that uses GPC:choline PDE to catalyze production of choline from GPC, then measurs the choline in a mixture of choline oxidase, peroxidase, phenol, and 4-aminoantipyrine that produces a "red dye". The absorbance was read at 500 nm wavelength in a spectrophotometer (Lambda 3B, Perkin-Elmer) against color reagent blank.…”

“…Incorporation of [ 14 C]choline into GPC was measured to estimate the rate of synthesis of GPC from choline, as previously described (Zablocki et al, 1991). In brief, confluent MDCK monolayers were first incubated for 3 h with or without 10 mM caffeine in the regular medium that contains 65 mM choline, were washed 4 times with choline-free medium, then were incubated with or without 10 mM caffeine in medium containing [ 1 4 C]choline (65 mM, 3.6 mCi/ml).…”

“…GPC:choline PDE activity was measured as described previously (Zablocki et al, 1991). In brief, confluent MDCK cells were rinsed 3 times with ice-cold PBS, scraped into 0.25 M sucrose solution containing 0.38% deoxycholic acid, then homogenized as described above.…”

“…As initially demonstrated in hepatocytes (Dawson, 1955), GPC is generated from choline via phosphatidylcholine which is a predominant phospholipid in mammalian renal cells (Morgan et al , 1963) and synthesized through the Kennedy pathway ( Wilgram and Kennedy, 1963). GPC synthesis entails the removal of fatty acid in part by the phospholipase A 2 ( P L A 2 ) activity (Zablocki et al, 1991). The GPC produced by enzymatic deacylations is degraded by hydrolysis to glycero 3-phosphate and choline in a reaction catalyzed by GPC:choline phosphodiesterase (GPC:choline PDE).…”

Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena

“…3.1.4.2) has been foand, both of which are active at alkaline pH, values (9). The latter activity plays an important role in osmoregulation (10). The high activity of these kidney and brain enzymes at alkaline pH values suggests that they do not belong to the lysosomal hydrolase pool.…”