Localization of Ryanodine Receptors in Smooth Muscle

From the Departments of Molecular Physiology and Biological Physics (R.E.L., A.P.S., A.V.S.), Anesthesiology (R.E.L.), Internal Medicine (A.P.S.), and Pathology (A.V.S.), University of Virginia Health Sciences Center, Charlottesville; the Department of Biomedical Sciences (G.F.N.), Institute of Medical Science, University of Aberdeen (Scotland), Foresterhill; the Department of Molecular Biology (S.F.), Vanderbilt University Medical Center, Nashville, Tenn; and the Department of Pharmacology (J.A.A.), University of Nevada School of Medicine, Reno.

Correspondence to Ryan E. Lesh, MD, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, PO Box 10011, Charlottesville, VA 22906-0011. E-mail rel4u{at}elvis.med.virginia.edu

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 ferrocyanide–stained 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–Ca²⁺-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 Ca²⁺ 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 Ca²⁺-release channels and origins of Ca²⁺ sparks, Ca²⁺ oscillations, and Ca²⁺ waves.

Key Words: smooth muscle • ryanodine receptor • electron microscopy • confocal microscopy immunofluorescence

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