Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

Krishna P. Subedi, Omkar Paudel, James S.K. Sham

Research output: Contribution to journalArticle

Abstract

Intracellular calcium (Ca2++ plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca2+ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca2+ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca2+ is crucial for excitation-transcription coupling. However, information on Ca2+ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca2+ signals, genetically encoded Ca2+ indicators (cameleon, D3cpv, targeting the plasma membrane (PM, cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs and Ca2+ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca2+ signals in PASMCs.

Original languageEnglish (US)
Pages (from-to)C659-C669
JournalAmerican Journal of Physiology - Cell Physiology
Volume306
Issue number7
DOIs
StatePublished - Apr 1 2014

Keywords

  • Angiotensin
  • Cameleon
  • Endothelin
  • FRET

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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