The endoplasmic-sarcoplasmic reticulum of smooth muscle: immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for the control of Ca2+ homeostasis.

Abstract

Abstract. Cryosection immunofluorescence and immunogold labeling with antibodies against specific markers were used in rat vas deferens smooth muscle fibers to reveal the molecular arrangement of the endomembrane system (referred to variously in the text as ER or sarcoplasmic reticulum [SR]; S-ER or ER/SR) known to participate in the control of Ca 2+ homeostasis. The lumenal ER chaperon, immunoglobulin binding protein (BiP), as well as protein disulfide isomerase, and calreticulin, a Ca 2+ binding protein expressed by most eukaryotic cells, appeared to be evenly distributed throughout the entire system (i.e., within [a] the nuclear envelope and the few rough-surfaced cisternae clustered near the nucleus; [b] single elements scattered around in the contractile cytoplasm; and [c] numerous, heterogeneous, mainly smooth-surfaced elements concentrated in the peripheral cytoplasm, part of which is in close apposition to the plasmalemma). All other structures, including nuclei, mitochondria, Golgi complex, and surface caveolae were unlabeled. An even distribution throughout the endomembrane system appeared also for the proteins recognized by anti-ER membrane antibodies. In contrast, calsequestrin (the protein that in striated muscles is believed to be the main actor of the rapidly exchanging Ca 2+ storage within the lumen of the sarcoplasmic reticulum) was found preferentially clustered at discrete lumenal sites, most often within peripheral smoothsurfaced elements of moderate electron density. Within these elements dual labeling revealed intermixing of calsequestrin with the other lumenal ER proteins. Moreover, the calsequestrin-rich elements were enriched also in the receptor for inositol 1,4,5-trisphosphate, the second messenger that induces Ca 2+ release from intracellular stores. These results document the previously hypothesized molecular heterogeneity of the smooth muscle endomembrane system, particularly in relation to the rapid storage and release of Ca 2+.

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

“…Although not resolved as clearly as in chemically fixed specimens, the organelles retained part of their dense appearance and were therefore easily recognized. The polyclonal antibodies used were addressed to components of two of the investigated organelles, the Ca2+ binding protein calreticulin (CRT), specific of the ER lumen (Villa et al, 1993;Nash et al, 1994), and mannosidase II (MA-NII), a GC membrane enzyme (Velasco et al, 1993). With the first, the immunogold labeling was strong, uniformly and strictly confined to the lumen of ER cisternae ( Figure 2A) including the nuclear envelope ; short and often slightly swollen ER cisternae, most often covered with discrete, dense polysomes; a clearly visible GC and various dense granules (arrows).…”

“…Distinction among individual ER cisternae had therefore not been possible. Immunocytochemistry and subcellular fractionation studies, carried out in many nonmuscle cell types Volpe et al, 1991;Sharp et al, 1992;Villa et al, 1993;Lievremont et al, 1994;Caspersen and Treiman, 1995) including PC12 (Rooney and Meldolesi, 1996), documented heterogeneities in the distribution of various ER proteins participating in Ca2+ homeostasis, including pumps and channels (see Pozzan et al, 1994). Whether such molecular heterogeneities give rise to heterogeneous distributions of calcium within the ER had however not been established.…”

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

“…glutaraldehyde and 2% (vol./vol.) OsO 4 [33]. For immunoelectron microscopy, INS-1E cell pellets were fixed at room temperature in a mixture of 4% (wt/vol.)…”

Annexin VI Participates in the Formation of a Reversible, Membrane-Cytoskeleton Complex in Smooth Muscle Cells

“…The "non-junctional" regions are comprised of caveolae which occur in close proximity to Ca 2ϩ -storage sites in the sarcoplasmic reticulum (4,5). During contraction, the sarcolemma displays a sequential arrangement of firm, inwardcaving anchoring regions and flexible, outward bulging, hingelike domains, an organization which is reminiscent of a barrel when viewed in three-dimensions (6,7).…”