Thrombin receptor on rat primary hippocampal neurons: coupled calcium and cAMP responses

We have tested the hypothesis that hippocampal neurons respond to thrombin via a neuronal thrombin receptor. A human neuroblastoma cell line, SK-N-SH, known to be thrombin responsive morphologically, responded both to thrombin and thrombin receptor agonist peptide (TRAP 42-55) with elevation of intracellular calcium. In Western blots of membranes from SK-N-SH cells and cultured rat hippocampal neurons using an antibody against the N-terminal peptide of the human thrombin receptor, putative receptor proteins of 66 and 47 kDa were detected in both cells. Neurons were treated with thrombin and TRAP 42-55 (TRAP-14) to determine their effects on intracellular levels of calcium and cAMP. Only 10% of the neurons showed a rapid response to thrombin, but most responded rapidly to agonist peptide with a prolonged elevation of intracellular free calcium. Neuronal cAMP levels were decreased by 40% after 24 h thrombin treatment. This decrease in cAMP level could be blocked by both the Gi-protein inhibitor, pertussis toxin, and the thrombin inhibitor, hirudin, suggesting a possible involvement of Gi-protein-coupled receptor activation. Furthermore, rapid calcium and cAMP responses were apparently induced by pre-treatment of neurons with thrombin for 24 h and subsequent washout. In summary, these data indicate that rat primary hippocampal neurons have thrombin receptors whose responses to thrombin apparently are up-regulated by 24 h thrombin pre-treatment. These results may have implications for synaptic remodeling, learning and memory.