P. Fournier

Effect of some alkaline phosphatase inhibitors on intestinal calcium transfer

1. There is a good correlation between the capacity of sugars to stimulate calcium transfer and their capacity to be phosphorylated by the intestinal alkaline phosphate with a part of the phosphate liberated from an ester phosphate. 2. On the sugar dependent and sugar independent calcium transfer, inhibitors of this enzyme act differently. 3. Phosphate, a competitive inhibitor suppresses both transfers. 4. Only the dependent sugar transfer was suppressed with phloridzin acting competitively at the sugar site, or with EDTA which could react close to the active site. 5. L-phenylalanine and phenobarbital, not competitive inhibitors does not act on either type of calcium transfer, the sugar dependent or the sugar independent.

Does the inhibition of microvillus protein phosphorylation by lysine explain the activity of the latter on calcium transfer

Abstract 1. 1. Is the activity of l -lysine on calcium absorption related to the fact that its phosphorylation is competitive with that of the microvilli proteins involved in the mineral transfer? 2. 2. The microvilli proteins phosphorylation is not cyclic GMP-dependent but is actually inhibited by l -lysine, used in general at a 100 mM concentration. 3. 3. The electrophoresis is followed by an autoradiograph which reveals the existence of a phosphorylated protein with a molecular weight of 140,000 daltons. Another phosphorylable protein, clearly visible in some preparations but only detectable in others, has a molecular weight close to 70,000 daltons. 4. 4. The inhibition by lysine of the microvilli proteins phosphorylation is not specific to a given protein, but is also observed for phosphorylable cytosolic proteins. 5. 5. A scheme for calcium transfer is proposed. It involves a protein whose phosphorylation should reduce the membrane permeability to calcium. 6. 6. The following three attributes of the phosphorylable membrane protein—its molecular weight; the fact that another protein (probably its monomer) is also phosphorylable; its well known capacity for phosphorylation—suggest that this protein might actually be alkaline phosphatase whose correlations in calcium metabolism are well known.

The influence of phosphorylable molecules on calcium transfer in the rat ileum

Abstract Amino or hydroxylated molecules have been tested from both points of view of their effect on calcium absorption in ileal loops, and of their ability to be phosphorylated. Some molecules, very effective in enhancing calcium absorption, are also highly phosphorylable: aspartic and glutamic acids and creatine. Other molecules, virtually ineffective, are not phosphorylable: L-alanine, L-valine, asparagine and glutamine. Ileal mucosa extract induces transphosphorylation from ATP to lysine and arginine, the amino acids the most potent in increasing calcium absorption. The observation that both isomers of the same ose (D- and L-xylose) or that a non absorbable polyoside (raffinose) increase calcium absorption may be linked to the fact that all are phosphorylated in vitro by a purified ileal phosphatase. Injection of either sorbitol or creatine into an ileal loop induces formation of their phosphorylated derivatives, thereby providing circumstantial evidence for a direct relationship between the phosphorylation ability of these molecules and their involvement in calcium transfer mechanisms.

Homodimer and heterodimer forms of adult rat intestinal alkaline phosphatase

Three forms of alkaline phosphatase have been isolated from different sections of the small intestine: F3 180 kDa from the duodenum; F2 150 kDa principally jejunal; F1 120 kDa the only ileal form. Their catalytic properties have been compared as well as the electrophoretic properties the dimer and monomer of their phosphorylated intermediates. Pi was a competitive inhibitor of F1 and F3, whereas glycerophosphate was competitive inhibitor only of F3. Pi was a non competitive inhibitor of F2 and of a mixture F1 plus F3. Heating the phosphorylated enzyme preparations led to their dissociation into the phosphorylated monomers: F1 and F3 appear to be homodimers 65 kDa and 90 kDa peptides respectively whilst F2 seems to be a dimer formed from one of each monomer. F1 was phosphorylated faster but less intensively than F3. F2 was strongly phosphorylated over a long time-course and its 65 kDa monomer fraction was phosphorylated more strongly for longer than that from F1.

Stimulation of ileal calcium absorption by sorbitol, L-xylose, or creatine via a decrease in luminal sodium concentration: Relation with concomitant changes in enterocyte energy metabolism

Ligated ileal loops, 30 cm in length, of 4-month-old male Wistar rats were instilled with 3 ml of a 10 mM CaCl2 solution (added with 0.25 μCi 45Ca) in the absence (control) or presence of 100 mM sorbitol, L-xylose, or creatine. Ileal calcium (Ca) transport, measured by plasma 45Ca appearance, was found to be similar 30 minutes after fluid instillation in all four instances. However, thereafter, 45Ca appearance in plasma did not increase further in control animals whereas it increased twice as much during the subsequent 30 minutes in the presence of sorbitol, L-xylose, or creatine. However, when loops of similar length were instilled with only 1.0 ml of such solutions, the sorbitol effect was already observed during the first 30 minutes. The stimulation of ileal Ca absorption induced by the presence of sorbitol appeared to be due to a cellular effect, associated with a decreased flux across the paracellular pathway, as indicated by 3H-mannitol absorption. The presence of sorbitol in instilled ileal solution induced a significant decrease in luminal Na, K, bicarbonate, and Cl concentrations at each time point studied (30, 60, 120, or 240 minutes after instillation). Thirty minutes after instillation, no difference in soluble Ca concentration was observed between control and experimental rats. After 60 minutes, Ca concentration was dramatically decreased in control rats but it remained nearly constant in experimental animals. Thus, the presence of substances enhancing ileal Ca transport favored the maintenance of soluble Ca in ileal solution during longer time peroids than their absence. In the ileal enterocyte, these substances induced a twofold increase of ATP content compared with controls. Furthermore, the size of mitochondria and the number of cristae and dense granules was increased 60 minutes after the presence of sorbitol in ileal solution. In face of the concomitant decrease of luminal Na concentration and increase of enterocyte ATP content, we propose that carbohydrates may act to stimulate Ca transport by decreasing Na, K-ATPase activity, thereby favoring an increase in activity of Ca ATPase. Interestingly, enhanced ileal Ca transport was associated with an apparent enhancement of mitochondrial activity of the ileal mucosa, as reflected by an increase in mitochondrial size and the number of cristae and dense granules.