Abstract
Protease‐activated receptor 2 (PAR2) is a G‐protein‐coupled receptor that contributes to prostate fibrosis and lower urinary tract symptoms (LUTS). In addition to fibrosis, aberrant smooth muscle tone in the prostate has been hypothesized to play a role. We therefore examined PAR2 expression in primary human prostate smooth muscle cells (PSMC) and studied the downstream signaling effects of PAR2 activation. Signaling pathways involved in the process were assessed using the PAR2 activating peptide SLIGKV‐NH2. We show that PAR2 is expressed in PSMC and that PAR2 activation mediates a biphasic elevation in intracellular Ca2+ and phosphorylation of myosin light chain 20 (MLC20), causing cellular contraction as assessed in a gel contraction assay. Intracellular Ca2+ flux was inhibited by a phosphoinositide hydrolysis inhibitor, U73122, showing a requirement for phospholipase C β (PLCβ) activation. PSMC expressed mRNA for L‐type voltage dependent Ca2+ channels (VDCC) as well as Ca2+ release activated channels (CRAC), a hitherto unreported finding. Secondary intracellular Ca2+ oscillations were abrogated only by BTP2, the CRAC channel inhibitor, but not by nifedipine, an inhibitor of VDCC. These data suggest that, PAR2 activation and subsequent Ca2+ entry through CRAC channels are important mechanisms in prostate smooth muscle contraction.
Highlights
PAR2 belongs to a unique family of ligand‐activated, seven transmembrane domain containing, G‐protein coupledAbbreviations: BTP2, [N‐{4‐[3,5‐bis(Trifluoromethyl)‐1H‐pyrazol‐1‐yl] phenyl}‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide]; CRAC, Ca2+ release activated channels; LUTS, lower urinary tract symptoms; MLC20, myosin light chain 20; MLCK, myosin light chain kinase; PAR2, Protease‐activated receptor 2; PLCβ, phospholipase C β; PSMC, prostate smooth muscle cells; VDCC, L‐type voltage dependent Ca2+ channels.receptors.[1]
Our results demonstrate that the level of phosphorylated MLC20 at 300 seconds after SLIGKV‐NH2 treatment is significantly increased in prostate smooth muscle cells (Figure 2C,D)
The key findings of our study are that PAR2 is expressed in smooth muscles of the prostate and upon stimulation, PAR2 causes contraction of these cells by activating surface CRAC channels (Figure 6)
Summary
PAR2 belongs to a unique family of ligand‐activated, seven transmembrane domain containing, G‐protein coupled. Several tissues express the PAR2 mRNA and physiological activators of this receptor include trypsin and the mast cell‐derived serine protease, tryptase. These proteases cleave the extracellular N‐terminal domain of PAR2 and lead to a tethered ligand that initiates downstream signaling.[2-4]. The increase in cytosolic free Ca2+ triggers calmodulin‐dependent activation of myosin light chain kinase (MLCK), subsequently phosphorylating MLC20 to form actin myosin crossbridges and leading to muscle contraction.[12,13]. Another mechanism of Ca2+ entry into smooth muscle cells is through VDCC and CRAC, both of which are activated by depletion of Ca2+ from intracellular stores.[14]. Our studies reveal a novel role of PAR2 in mediating contraction of prostatic smooth muscle through PLCβ that stimulates Ca2+ release, initially from internal stores and through surface CRAC channels
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