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

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.