Expression and regulation of the thrombin receptor (PAR-1) in the central nervous system

Cell culture studies demonstrated that thrombin can induce neural cell process retraction, alter gene expression and modulate cell viability. These cellular responses to thrombin are mediated by activation of the thrombin receptor (PAR-1). To better understanding the role of thrombin in the CNS, in situ hybridization and immunohistochemical techniques were used to analyze the cellular localization of PAR-1 mRNA in rat brain. PAR-1 mRNA was broadly distributed across the neuraxis, although expression was very focal and often anatomically limited within specific neural structures. The highest density of hybridization occurred in individual neurons in neocortex, cingulate/retrosplenial cortex and subiculum, subsets of nuclei in hypothalamus, thalamus, pretectum and ventral mesencephalon and discrete cell layers in the hippocampus, cerebellum and olfactory bulb. Patterns of hybridization included neuronal, glial and ependymal cells, although white matter was uniformly negative, as were most cerebrovascular endothelial cells. Expression of PAR-1 mRNA by hippocampal astroglia and nigral dopaminergic neurons was confirmed by combined in situ hybridization/immunohistochemistry. Comparison between PAR-1 and prothrombin cRNA hybridization demonstrated distinct but overlapping brain distributions.;Mechanisms of PAR-1 regulation have been shown to be novel and cell type-specific, however, little is known about PAR-1 regulation in neural cells. Thus, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies. Scatchard analysis revealed high affinity, specific binding to a single affinity class of receptors. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide. Differential antibody binding was used to demonstrate rapid and near-complete thrombin-induced PAR-1 cleavage and internalization with protein synthesis-dependent replacement of intact receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), phospholipid inflammatory mediator, lysophosphatidic acid (LPA), and injury-related condition, hypoglycemia. The effect of hypoglycemia was shown by RNase protection to be at least partially pre-translational. Thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.