Estrogen blocks 3-nitropropionic acid-induced [Ca 2+] i increase and cell damage in cultured rat cerebral endothelial cells

Abstract

Systemic administration of 3-nitropropionic acid (3-NPA, a mycotoxin) induces brain damage accompanied by disturbance in the blood–brain barrier (BBB). Since the endothelial cells are important components of the BBB and the first target of a systemic intoxication, in the present study, the effect of 3-NPA on primary cultured rat brain endothelial cells (rBECs) was examined by studying intracellular Ca 2+ ([Ca 2+] i) response using imaging techniques with fura-2. rBECs were prepared using a method of Kis et al. [Eur. J. Pharmacol. 368 (1999) 35–42] and Szabo et al. [Neurobiology 5 (1997) 1–16]. Almost all cells were immunoreactive to antibody against the factor VIII-related antigen (von-Willebrand factor). They showed a typical dose-dependent increase of [Ca 2+] i in response to ATP or bradykinin. Low concentrations of 3-NPA (1.7 mM, 3.4 mM) caused no changes, and a medium concentration (6.8 mM) increased the [Ca 2+] i gradually and progressively, and the increase was reversed incompletely back to the resting level after washing. A high concentration (13.6 mM) increased the [Ca 2+] i irreversibly. These elevations of [Ca 2+] i were absent in a Ca 2+-free medium. In endothelial cells treated with 17β-estradiol (above 10 −5 M) or with a selective estrogen receptor modulator, tamoxifen (5×10 −7 M), no elevation of [Ca 2+] i was observed with 3-NPA treatment. The response to ATP was impaired after application of 3-NPA, but it was preserved by cotreatment with 17β-estradiol or tamoxifen. An estrogen receptor antagonist ICI 182,780 inhibited these effects by 17β-estradiol or tamoxifen. Lysosomal neutral red uptake and TUNEL experiments revealed the necrotic but not apoptotic cell death at least in this acute stage. Data indicate that a medium to high concentration of 3-NPA induces damage on rBECs as revealed by an accumulation of [Ca 2+] i, but the damage was protected by cotreatment with 17β-estradiol or tamoxifen, suggesting that estrogen may be protective for the brain vascular damage via estrogen receptor.