title: Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells.
abstract: Intracellular calcium (Ca(2+)) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca(2+) signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca(2+) is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca(2+) is crucial for excitation-transcription coupling. However, information on Ca(2+) signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca(2+) signals, genetically encoded Ca(2+) indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca(2+) signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca(2+) of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca(2+) concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca(2+) signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca(2+) signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca(2+) signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca(2+) signals in PASMCs.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Krishna P
authors.initials: KP
authors.lastname: Subedi
authors.forename: Omkar
authors.initials: O
authors.lastname: Paudel
authors.forename: James S K
authors.initials: JS
authors.lastname: Sham
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D015374:Biosensing Techniques; D020013:Calcium Signaling / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology; D056966:Excitation Contraction Coupling / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D056489:Nucleoplasmins / Q000235:genetics / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012508:Sarcolemma / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D002135:Calcium-Binding Proteins; D019332:Endothelin-1; D056489:Nucleoplasmins; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D015544:Inositol 1,4,5-Trisphosphate
keywords: Ca2+ signaling; FRET; angiotensin; cameleon; endothelin