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

✍ Scribed by C. Calhau; F. Martel; S. Pinheiro-Silva; H. Pinheiro; P. Soares-da-Silva; C. Hipólito-Reis; I. Azevedo

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 215 KB
• Volume: 84
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.10027

✦ Synopsis

Abstract

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 μM), 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 μM) 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 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 ^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. J. Cell. Biochem. 84: 389–400, 2002. © 2001 Wiley‐Liss, Inc.