TY - JOUR
T1 - Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells
AU - Subedi, Krishna P.
AU - Paudel, Omkar
AU - Sham, James S.K.
PY - 2014/4/1
Y1 - 2014/4/1
N2 - 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.
AB - 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.
KW - Angiotensin
KW - Cameleon
KW - Endothelin
KW - FRET
UR - http://www.scopus.com/inward/record.url?scp=84900542307&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84900542307&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00341.2013
DO - 10.1152/ajpcell.00341.2013
M3 - Article
C2 - 24352334
AN - SCOPUS:84900542307
SN - 0363-6143
VL - 306
SP - C659-C669
JO - American Journal of Physiology
JF - American Journal of Physiology
IS - 7
ER -