Thrombin receptors on the cells of the blood–brain barrier

Abstract number: P0158

Bartha K., Vajda S., Kollár A., Machovich R., Adam V.

Semmelweis University, Hungary

Astrocytes induce blood–brain barrier (BBB) properties in brain capillary cells. Thrombin is rapidly produced in early moments following trauma to the CNS or other conditions that alter the BBB, and we reported previously the expression of thrombin receptors PAR-1, PAR-3 in monoculture of rat brain capillary endothelial cells (RBCE) and in astrocytes (Bartha et al. J. of Cerebral Blood Flow and Metab. 2000). In this study the kinetics of thrombin receptor signaling was examined in response to the rat PAR-1 agonist peptide (SFRLRN) and the PAR-4 agonist peptide (GYPGKF) by measuring [Ca2 + ]i transients. Experiments were performed in monoculture of RBCE cells, of astrocytes and in cocultures, to investigate if coculturing with astrocytes alters thrombin receptor signaling in RBCE cells. To monitor changes in [Ca2 + ]i, cells grown on extracellular matrix coated coverslips were loaded with Fura-2. Coverslips were transferred to a perfusion chamber fitted to a stage of an inverted microscope. Fluorescence emission and ratio were followed by digital imaging. Data analysis was carried out using a self developed image analysis software (Image Analyst v1.0) and fluorescence ratios were converted to [Ca²⁺]i. As a quantity taking into account both the magnitude and the length (t_1/2) of the [Ca²⁺]i response, the C²⁺ mobilizing potency (CMP) of the agonist was defined as the integrated rise in [Ca²⁺]i during the time of the Ca²⁺ signal. The maximal CMP of thrombin proved to be at least two times higher than that of SFRLRN, although the magnitudes of the [Ca²⁺]i transients were similar. The SFRLRN peptide elicited Ca²⁺ responses of much shorter duration (t_1/2:15 s) than that of thrombin, accordingly resulting in low CMP values. The magnitude of the [Ca²⁺]i response to GYPGKF was found to be lower than that of the thrombin, but its prolonged decay (t_1/2:54 s) resulted in a high CMP for GYPGKF. The data show, that PAR-4 activation in RBCE cells and in astrocytes triggered a more persistent elevation of [Ca²⁺]i, with a higher CMP compared to PAR-1, resulting in a total calcium response similar to that of activation with thrombin. In RBCE cell and astrocyte cocultures thrombin, SFRLRN and GYPGKF all elicited a [Ca2 + ]i transient with a similar activation kinetics as in a monoculture, indicating that thrombin receptor functions were retained.