The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane-bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco-2 cells. ALP activity expressed on the apical surface of blood-brain barrier cells, and its relationship with (125)I-insulin internalization were investigated under physiological conditions using p-nitrophenylphosphate (p-NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and (125)I-insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto-oriented active site which was functional at physiological pH. Orthovanadate (100 microM), an inhibitor of phosphatase activities, decreased both RBE4-ALP activity and (125)I-insulin internalization. In the presence of L-arginine (1 mM) or adenosine (100 microM) RBE4-ALP activity and (125)I-insulin, internalization were significantly reduced. However, D-arginine (1 mM) had no significant effect. Additionally, RBE4-ALP activity and (125)I-insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 microM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 microM and 100 microM), beta-estradiol (100 microM), iron (100 microM) or in the presence of all-trans retinoic acid (RA) (10 microM). The ALP inhibitor levamisole (500 microM) was able to reduce (125)I-insulin internalization to 69.1 +/- 7.1% of control. Our data showed a positive correlation between ecto-ALP activity and (125)I-insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood-brain barrier may be modulated through ALP.

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The liver has an important role in the detoxification of organic cations from the circulation. [3H]‐1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+), a low molecular weight organic cation, is efficiently taken up and accumulated by rat hepatocytes through mechanisms partially unknown. The aim of the present work was to characterize further the uptake of MPP+ by rat isolated hepatocytes. The putative interactions of a wide range of drugs, including inhibitors/substrates of P‐glycoprotein, were studied. The uptake of MPP+ was investigated in rat freshly isolated hepatocytes (incubated in Krebs‐Henseleit medium with 200 nM [3H]‐MPP+ for 5 min) and in the rat liver in situ (perfused with Krebs‐Henseleit/BSA medium with 200 nM [3H]‐MPP+ for 30 min). [3H]‐MPP+ accumulation in the cells and in tissue was determined by liquid scintillation counting. Verapamil (100 μm), quinidine (100 μm), amiloride (1 mM), (+)‐tubocurarine (100 μm), vecuronium (45 μm), bilirubin (200 μm), progesterone (200 μm), daunomycin (100 μm), vinblastine (100 μm), cyclosporin A (100 μm) and cimetidine (100 μm) had a significant inhibitory effect on the accumulation of [3H]‐MPP+ in isolated hepatocytes. Tetraethylammonium (100 μm) had no effect. In the rat perfused liver, both cyclosporin A (100 μm) and verapamil (100 μm) had much less marked inhibitory effects as compared to their effects on isolated hepatocytes (0% against 35% and 45% against 96% of inhibition, respectively). Inhibition of alkaline phosphatase activity by increasing or decreasing the pH of the incubation medium or by the presence of vanadate (1 mM) or homoarginine (500 μm) led to a significant increase in the accumulation of [3H]‐MPP+ in isolated hepatocytes. It was concluded that, in addition to the type I organic cation hepatic transporter, [3H]‐MPP+ is taken up by rat isolated hepatocytes through P‐glycoprotein, a canalicular transport system that usually excretes endobiotics and xenobiotics. We proposed that the reversal of transport through P‐glycoprotein may be related to the loss of efficiency of alkaline phosphatase in isolated hepatocytes.

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