Asrar B. Malik

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Gαi prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Gαi in activating the downstream MAPK pathway. Expression of constitutively active Gαi2 mutant or Gβ1γ2 subunits increased reporter activity 3–4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.

Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells

Caveolae exist in most cell types (with certain exceptions, e.g., erythrocytes, lymphocytes, and neurons) and are particularly abundant in endothelial cells (ECs) and smooth muscle cells (SMCs) of blood vessels. It is clear that the major plasmalemma vesicle structure in ECs and SMCs is caveolae as opposed to clathrin-coated vesicles. The number of caveolae is high in continuous endothelium (e.g., 73 caveolae per square micrometer in ECs of intramuscular capillaries) and low in fenestrated or discontinuous endothelium. Caveolae have a lipid composition similar to that of membrane rafts, but in addition, they possess other proteins, including the organelle-specific structural protein caveolin and the more recently identified cavin. Caveolae appear to represent a specialized form of membrane raft domain, where caveolin-1, glycosphingolipids, and cholesterol are preferentially concentrated. Instead of assembling these structures in the plasma membrane, cells may in fact build caveolae in the Golgi apparatus and then send them to the plasma membrane proper, where they are incorporated, a process involving Na/K-ATPase. This chapter describes the structure and function of caveolae and caveolin proteins and their interaction with membrane receptors, transporters and signaling proteins that are related to the pulmonary vascular permeability and pulmonary hypertension.