Identification of thrombin receptors in rat brain capillary endothelial cells

Both thrombin and plasmin induce contraction of brain endothelial cells, wh ich may increase capillary permeability thereby leading to disruption of th e blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells a nd astrocytes are therefore essential for understanding injury-related acti ons of thrombin in the brain. Using the reverse transcriptase-polymerase ch ain reaction method, the present study shows that primary cultures of rat b rain capillary endothelial (RBCE) cells and astrocytes derived from rat bra in express two different thrombin receptors. The first is proteolytically a ctivated receptor (PAR)-1, the receptor responsible for the vast majority o f the thrombin's cellular activation functions; the second is PAR-3, a rece ptor described to be essential for normal responsiveness to thrombin in mou se platelets. in addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functiona l significance of thrombin receptors was indicated by changes in [Ca2+](i) in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+](i) whereas , upon addition of active site-blocked thrombin or plasmin, [Ca2+](i) remai ned unchanged. The [Ca2+](i) signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the e xtracellular medium makes a contribution to the overall [Ca2+](i) rise. The amplitude of the transient [Ca2+](i) signal was dependent on the concentra tion of thrombin, and repeated application of the enzyme caused an essentia lly complete and long-term desensitization of the receptor. The PAR-1 agoni st peptide SFLLRN also elicited a transient increase in [Ca2+](i). After ac tivation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the gen eration of the [Ca2+](i) signal. Preheatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+](i) rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors fo r thrombin, PAR-1 and PAR-3, and probably both receptors are involved in th rombin-induced [Ca2+](i) signals. Plasmin itself does not elevate [Ca2+](i) but prevents the activation of receptors by thrombin.