C. Hipólito-Reis

Inward transport of [3H]-1-methyl-4-phenylpyridinium in rat isolated hepatocytes: putative involvement of a P-glycoprotein transporter.

1 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. 2 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. 3 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. 4 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. 5 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). 6 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. 7 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.

Alkaline phosphatase from rat liver and kidney is differentially modulated

OBJECTIVE To investigate the effect of inhibitors of alkaline phosphatase (ALP) and modulators of P-glycoprotein (Pgp), multidrug resistance protein (MRP) and hepatic taurocholate uptake on the activity of tissue-nonspecific ALP (TNALP) in liver and kidney. DESIGN AND RESULTS ALP activity was determined in rat liver and kidney homogenates. Levamisole had a stronger inhibitory effect on renal TNALP than on the hepatic isoform. 1,3-dimethylxanthine (theophylline) almost abolished renal TNALP activity whereas its effect on hepatic TNALP was less intense. 3-isobutyl-1-methylxanthine (IBMX) and lidocaine produced opposite effects, activating hepatic TNALP and inhibiting the kidney isoform. Quinidine significantly inhibited renal TNALP without affecting hepatic TNALP. Kaempferol activated both liver and kidney isoforms, the effect being more pronounced on hepatic TNALP. CONCLUSIONS a) renal TNALP seems to be more sensitive to inhibition than hepatic TNALP, b) TNALP activity studies should take into account the source of ALP isoform and c) ALP pharmacological manipulation in vivo may produce different and even opposite effects in different tissues/organs.

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto‐phosphatase activity

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 125I‐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 125I‐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 μM), an inhibitor of phosphatase activities, decreased both RBE4‐ALP activity and 125I‐insulin internalization. In the presence of l‐arginine (1 mM) or adenosine (100 μM) RBE4‐ALP activity and 125I‐insulin, internalization were significantly reduced. However, d‐arginine (1 mM) had no significant effect. Additionally, RBE4‐ALP activity and 125I‐insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 μM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 μM and 100 μM), β‐estradiol (100 μM), iron (100 μM) or in the presence of all‐trans retinoic acid (RA) (10 μM). The ALP inhibitor levamisole (500 μM) was able to reduce 125I‐insulin internalization to 69.1 ± 7.1% of control. Our data showed a positive correlation between ecto‐ALP activity and 125I‐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. J. Cell. Biochem. 84: 389–400, 2002.

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto‐ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco‐2 cells, a putative modulation of 1‐methyl‐4‐phenylpyridinium (MPP+) apical uptake by an ecto‐ALP activity. Ecto‐ALP activity and 3H‐MPP+ uptake were evaluated in intact Caco‐2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane‐bound ecto‐ALP expressed on the apical surface of Caco‐2 cells was studied at physiological pH using p‐nitrophenylphosphate as substrate. The results showed that Caco‐2 cells express ALP activity, characterized by an ecto‐oriented active site functional at physiological pH. Genistein (250 µM), 3‐isobutyl‐1‐methylxanthine (1 mM), verapamil (100 µM), and ascorbic acid (1 mM) significantly increased ecto‐ALP activity and decreased 3H‐MPP+ apical transport in this cell line. Orthovanadate (100 µM) showed no effect on 3H‐MPP+ transport and on ecto‐ALP activity. On the other hand, okadaic acid (310 nM) and all trans‐retinoic acid (1 µM) significantly increased 3H‐MPP+ uptake and inhibited ecto‐ALP activity. There is a negative correlation between the effect of drugs upon ecto‐ALP activity and 3H‐MPP+ apical transport (r = −0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco‐2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto‐ALP activity may be involved in this process. J. Cell. Biochem. 87: 408–416, 2002.

Rat serum alkaline phosphatase electrophoretic fractions: variations with feeding, starvation and cellulose fibre ingestion

The effect of feeding, starvation and fibre ingestion on alkaline phosphatase (ALP) activity (E.C. 3.1.3.1) was studied in Wistar rat serum. Using identical assay conditions for total ALP activity determination and for electrophoretic ALP isoenzymes/fractions activity calculation, alpha- and beta-naphthyl phosphates and p-nitrophenyl phosphate were used as substrates and 2-amino-2-methyl-1-propanol/HCI was used as buffer, respectively. Total activity with beta-naphthyl phosphate was significantly higher than with alpha-naphthyl phosphate and p-nitrophenyl phosphate; with alpha-naphthyl phosphate it was significantly higher than with p-nitrophenyl phosphate. With all substrates, fed animals had significantly higher total activity than starving ones. Electrophoresis allowed the separation of two fractions. The second fraction activity was significantly higher in the fed group than in the starving ones, irrespective of the substrate used. Starving animals with fibre showed higher values of this fraction than starving animals without fibre, the difference reaching statistical significance with alpha-naphthyl phosphate. The first fraction predominated in both starved groups and the second in the fed group. The second fraction was identified as intestinal ALP. We conclude that the mechanical stimulation of the digestive tract appears to influence the passage of intestinal ALP to serum. The experimental conditions used enable quantification of electrophoretic fractions based on total activity. Activity depends on the substrate used.

Importance of assay conditions in visualization and quantitation of serum alkaline phosphatase isoenzymes separated by electrophoresis

The importance of separation and identification of serum alkaline phosphatase (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. Each serum ALP fraction/isoenzyme quantitation has both practical and theoretical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and serum ALP electrophoretic fractions/isoenzymes activities were quantified. Different results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-methyl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions (9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were used. Higher total serum ALP activity was always observed with beta-naphthyl phosphate, independently of the buffer (or mixture of buffers) and pH used. Electrophoresis allowed the separation of two serum ALP fractions. Activity of both serum ALP electrophoretic fractions was always higher with beta-naphthyl phosphate, except with carbonate/bicarbonate pH 10.4. The effect of a change in pH was buffer- (or mixture of buffers) and substrate-dependent; the addition of a second buffer (to that previously used) was not always accompanied by an increase or decrease (of the same magnitude) in our results. The results obtained with different buffers (or mixture of buffers) were not identical with substrates and pH values. It is concluded that (i) from the same electrophoretic separation of serum ALP fractions/isoenzymes, different values for its activity can be obtained by changing the assay conditions used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoretic fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each case.

Effect of thiamine on 3H-MPP+ uptake by Caco-2 cells

Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP+) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP+ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP+ intestinal apical uptake and ecto-ALP activity by thiamine (T+) and thiamine pyrophosphate (TPP, a T+ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-3H]-4-phenylpyridinium acetate (3H-MPP+) uptake were evaluated in intact Caco-2 cells. T+ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease 3H-MPP+ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and 3H-MPP+ uptake were concentration-dependent. The results suggest that the effect of T+ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T+ may occur through hEMT, in Caco-2 cells.