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 (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.
Calhau
C
C
Department of Biochemistry, Faculty of Medicine (U38-FCT), 4200-319 Porto, Portugal. ccalhau@med.up.pt
Martel
F
F
Pinheiro-Silva
S
S
Pinheiro
H
H
Soares-da-Silva
P
P
Hipólito-Reis
C
C
Azevedo
I
I
D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000098:blood supply* / Q000201:enzymology; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism
D007328:Insulin; D007457:Iodine Radioisotopes; D010749:Phosphoprotein Phosphatases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase