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

Abstract

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.

FIGURE 1.

SERCA2a and SERCA2b, but not SERCA1 or SERCA3, are functionally expressed in rat pulmonary arterial smooth muscle. A, RT-PCR fragments of SERCA1, SERCA2a, SERCA2b, and SERCA3 amplified from total mRNA extracted from second and third order branches of the pulmonary arterial tree (PA), skeletal muscle (SM), heart (H), and brain (B) mRNAs. The predicted sizes of the PCR products were 340 bp for SERCA1, 694 bp for SERCA2a, 501 bp for SERCA2b, and 264 bp for SERCA3. B, Western blots for SERCA1, SERCA2a, SERCA2b, and SERCA3 in lysates of: smooth muscle from second order branches of the pulmonary arterial tree (PA), skeletal muscle (SM), heart (H), and brain (B). Each blot was also probed with an antibody selective for actin to ensure equal protein loading (not shown).

FIGURE 2.

SERCA2a and SERCA2b are differentially distributed within isolated pulmonary arterial smooth muscle cells. A and B, images show the distribution of labeling for SERCA2a (A) and SERCA2b (B). Panel a, transmitted light image of PASMC. Scale bar, 10 μm. Panel b, deconvolved Z-section taken through the cell with labeling for the given SERCA subtype (red) in relation to the nucleus (blue) and the plasma membrane (dotted line). Panel c, three-dimensional reconstruction of a series of Z-sections (Z step 0.2 μm) obtained through the cell fluorescently labeled as in panel b. Panel d, three-dimensional representation showing the distribution by color of individual volumes of labeling for the given SERCA subtype in the perinuclear (orange), extraperinuclear (pink), and subplasmalemmal (light blue) regions. C, bar chart shows the density of labeling (μm³ of labeling per μm³; mean ± S.E.) for SERCA2a and SERCA2b within the three designated regions of isolated PASMCs.

FIGURE 3.

RyR1, RyR2, and RyR3 are differentially distributed within isolated pulmonary arterial smooth muscle cells. A–C, images show the distribution of labeling for RyR1 (A), RyR2 (B), and RyR3 (C). Panel a, transmitted light image of PASMC. Scale bar, 10 μm. Panel b, three-dimensional reconstruction of a series of Z-sections (Z step 0.2 μm) obtained through the cell fluorescently labeled in relation to the nucleus (blue) and the plasma membrane (dotted line). Panel c, three-dimensional representation showing the distribution by color of individual volumes of labeling for the given RyR subtype in the perinuclear (orange), extraperinuclear (pink), and subplasmalemmal (light blue) regions. D, bar chart shows the density of labeling (μm³ of labeling per μm³; means ± S.E.) for each of the RyR subtypes within the three designated regions of isolated PASMCs.

FIGURE 4.

A cyclopiazonic acid-insensitive sarcoplasmic reticulum Ca²⁺ store underpins Ca²⁺ signaling in response to ET-1 in pulmonary arterial smooth muscle cells. A–C, the effect on the Fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) in the absence (A) and presence of cyclopiazonic acid (B) and thapsigargin (C). D, bar chart shows the means ± S.E. for the percentage change in Fura-2 fluorescence ratio induced by ET-1 in the absence and presence of cyclopiazonic acid (CPA) and thapsigargin (Thapsi), respectively.

FIGURE 5.

Schematic representation of the spatial and functional compartmentalization of the sarcoplasmic reticulum in a pulmonary arterial smooth muscle cell. NCX, sodium/calcium exchanger; PKA, cAMP-dependent protein kinase; ARC, ADP-ribosyl cyclase; cADPR, cyclic adenosine diphosphate-ribose; MLCK, myosin light chain kinase.