Protease-activated receptor 4-mediated Ca2+ signaling in mouse lung alveolar epithelial cells

Protease-activated receptor (PAR)-4 is a recently identified low-affinity thrombin receptor that plays a pathophysiological role in many types of tissues including the lung. Here, we showed for the first time that PAR4 mRNA and protein are expressed on primary cultured mouse lung alveolar epithelial cells by reverse transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemical analyses. In a fura 2-AM-loaded single epithelial cell, stimulation with thrombin (1 U/ml) and a PAR4 agonist peptide (AYPGKF-NH2, 1–100 μM) increased intracellular Ca2+ concentration ([Ca2+]i), which consisted of an initial peak phase followed by a slowly decaying delayed phase, while a PAR1 agonist peptide, TFLLR-NH2 (1–100 μM), induced a transient increase in [Ca2+]i. AYPGKF-NH2 (10 μM)-induced [Ca2+]i response was attenuated by a PAR4 antagonist peptide (tcY-NH2), a phospholipase C inhibitor, U-73122 (1–10 μM) or a Ca2+-ATPase inhibitor, thapsigargin (1 μM). Removal of extracellular Ca2+ or an inhibitor of store-operated Ca2+ entry, trans-resveratrol (1 μM) shortened the time to shut off the Ca2+ response without any significant effects on the magnitude of the peak [Ca2+]i. Thus, stimulation of PAR4 appeared to mobilize Ca2+ from intracellular stores in the initial peak response and to enhance Ca2+ entry through the store depletion-operated pathway in the delayed phase. The latter mechanism probably contributed to the longer responsiveness of PAR4 stimulation.