0169 : Segregated calcium stores are important for stretch-induced calcium signaling in smooth muscle cells: Implication in pulmonary hypertension

Pulmonary arterial smooth muscle cells (PASMC) are submitted to stretch forces exerted by the blood pressure. They can transduce a mechanical stimulus of stretch into a biological response of contraction, a mechanism called myogenic tone which involves Ca2+ influx through stretch-activated channels (SAC). We investigate how the subcellular organization of Ca2+ stores in PASMC (sarcoplasmic reticulum (SR), lysosomes and mitochondria) is important for the Ca2+ response to stretch during PH.Studies were performed in freshly isolated PASMC from control rats and rats with a pulmonary hypertension induced by an injection of monocrotaline (MCT). Inward currents from SAC were recorded after a negative pressure applied by a patch-clamp pipette. Cytosolic Ca2+ was also measured with the fluo4 probe and mitochondrial Ca2+ with the rhod2 probe after an osmotic shock. A pharmacological approach coupled with different coimmunolabelings of ryanodine receptors (RyR) and SERCA2 pumps was used to investigate which RyR subtype is involved in Ca2+ response to stretch.PASMC exhibit segregated Ca2+ stores: a subplasmalemnal SR store with RyR1 and SERCA2b, associated to mitochondria and a perinuclear SR store with RyR3 and SERCA2a. We showed that a stretch of PASMC induces an inward Ca2+ influx through SAC that is amplified by a Ca2+ release by RyR1, which is buffered by mitochondria. In MCT rats, the subcellular organization of RyR subtypes is modified: RyR3 and SERCA2a are not only expressed in a perinuclear area but also in a subplasmalemnal level. This is correlated with a different organization of mitochondria. In MCT rats, this new organisation leads to a greater amplification by all RyR subtypes and to a higher Ca2+ increase induced by stretch. Furthermore, the Ca2+ response to stretch is enhanced by a mechanism independent on extracellular Ca2+, involving caveolae.The spatial organization of Ca2+ stores in PASMC is important for cell signaling and plays a causal role in PH