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This Article Full Text Full Text (PDF) All Versions of this Article: 289/2/L338    most recent 00328.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Yang, X.-R. Articles by Sham, J. S. K. Search for Related Content PubMed PubMed Citation Articles by Yang, X.-R. Articles by Sham, J. S. K.

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca²⁺ stores in pulmonary arterial smooth muscle cells

¹Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland; ²Department of Physiology and Biophysics, University of South Florida, Tampa, Florida; and ³Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee

Submitted 1 September 2004 ; accepted in final form 17 April 2005

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca²⁺ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, 15–20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca²⁺ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca²⁺ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 ± 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca²⁺ sparks (FDHM = 44.6 ± 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca²⁺ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca²⁺ regulation of cellular functions in PASMCs.

calcium sparks; ryanodine receptors; calcium signaling; intralobar pulmonary arteries; perinuclear sarcoplasmic reticulum

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