Abstract-The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanidestained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca 2ϩ -release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca 2ϩ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca 2ϩ -release channels and origins of Ca 2ϩ sparks, Ca 2ϩ oscillations, and Ca 2ϩ waves. (Circ Res. 1998;82:175-185.) Key Words: smooth muscle Ⅲ ryanodine receptor Ⅲ electron microscopy Ⅲ confocal microscopy immunofluorescence

The ryanodine receptor (RyR) functions as the calcium release channel of the sarcoplasmic reticulum activated by electromechanical coupling in skeletal and cardiac muscles. In smooth muscle, inositol trisphosphate releases calcium from internal stores during pharmacomechanical coupling, but these cells also contain ryanodine-sensitive calcium stores. In this study, we establish the presence of anti-RyR antibody binding sites in vascular and endocardial endothelium. Both types of endothelia also contain messenger RNA, which hybridizes to a cardiac RyR isoform cDNA probe. Western blots of endothelial cell homogenates demonstrate the presence of a single, high molecular weight band of protein that corresponds to the cardiac RyR isoform. Confocal micrographs of endothelial cells labeled with a specific anti-RyR antibody reveal an intense fluorescent signal surrounding the nucleus and distributed in a nonhomogeneous pattern throughout the cytoplasm. This pattern of fluorescence is consistent with the electron microscopic distribution of the endoplasmic reticulum. The pattern of immunofluorescence seen with the anti-RyR antibody is distinctly different from that seen with the mitochondrial fluorophore rhodamine 123. Our findings suggest that the RyR plays a role in endothelial signaling.

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric Gi protein (the Gγ subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.

Antisense oligodeoxynucleotides (ODNs) have been used to modify gene expression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN-mediated changes in protein expression of cultured cells, use of these compounds to achieve antisense regulation of specific proteins in intact tissue has been limited. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of force-generating capabilities and normal ultrastructure and (2) to develop a method for efficient introduction of antisense ODNs into intact tissue. Sheets of ODN-containing, reversibly permeabilized rat outer longitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membrane depolarization or carbachol stimulation; the G protein-coupled calcium sensitization pathway was also intact after 7 days. Reversible permeabilization, a method previously used to load smooth and cardiac muscle with aequorin and heparin, was effective for loading > 95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5'-AAGGGCCATTTTGTT-FITC-3'). Confocal microscopy of reversibly permeabilized smooth muscle loaded with fluorescent antisense ODNs revealed intense nuclear fluorescence and less intense, homogeneous, cytoplasmic fluorescence. Internally radiolabeled ODNs (homologous to the above sequence) showed complete degradation between 4 and 16 hours after introduction into the cells. In summary, we have demonstrated methods for long-term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential utility for investigating many questions in smooth muscle biology. At present, however, a major limitation of this approach is the short half-life of phosphorothioated ODNs.