Identification of functionally segregated sarcoplasmic reticulum calcium stores in pulmonary arterial smooth muscle

In pulmonary arterial smooth muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 microm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 microm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca(2+) waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca(2+) stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca(2+) stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca(2+) via SERCA2b, and likely releases Ca(2+) via RyR1 to mediate vasodilation. The other is located centrally, receives Ca(2+) via SERCA2a, and likely releases Ca(2+) via RyR3 and RyR2 to initiate vasoconstriction.