Local Ca2+ release transients, or """"""""Ca2+ sparks"""""""", originated from ryanodine receptors on sarcoplasmic receptors have been identified in cardiac, skeletal, and smooth muscles. In systemic vascular smooth muscles, they have been implicated as a feedback modulator of membrane potential by increasing local [Ca2+], activating nearby Ca2+ -activated K+ (KCa) channels, leading to membrane hyperpolarization and vasodilation. Recently, we have identified and characterized Ca2+ sparks in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). In contrast to systemic vascular smooth muscle, activation of Ca2+ spark in PASMCs causes membrane depolarization. Endothelin-1 (ET-1), a major mediator of acute and chronic hypoxia induced pulmonary vasoconstriction, causes dramatic increase in Ca2+ spark frequency, which can be blocked by ET-A receptor antagonist, and non-selective cation channels. Inhibition of Ca2+ sparks with ryanodine significantly attenuated the ET-1 induced contraction of pulmonary arteries. Enhancement of Ca2+ spark by ET-1 is evident even at a threshold concentration (10-10 M), which at the best cause minimal contraction but is capable of potentiating hypoxic pulmonary vasoconstriction and contraction induced by other vasoconstrictor. Moreover, norepinephrine, which activates the PLC/PKC pathway similar to ET-1, causes the opposite effect of inhibiting Ca2+ sparks in PASMCs. Based on these novel findings, we propose that Ca2+ spark in PASMCs promote vasoconstriction, and vasoconstrictors modulate Ca2+ sparks through specific receptor dependent mechanisms. To test this hypotheses, we will apply a combination of state-of-the-art techniques including whole-cell patch clamp, laser-scanning confocal microscopy, and UV-pulse laser flash photolysis, RT-PCR, and gene-knockout with antisense oligonucleotide to examine if Ca2+ spark of PASMCs promotes vasoconstriction by (i) inducing membrane depolarization, (ii) providing Ca2+ for direct myofilament activation, and/or (iii) potentiating the contractile effects of other physiological stimuli. We will also characterize and identify signaling pathways for agonist induced Ca2+ sparks activation, and determine the molecular identity and functional role of non-selective cation channels in Ca2+ spark activation. This project will provide important information on the unique control of pulmonary circulation by subcellular local Ca2+ signaling.
Showing the most recent 10 out of 27 publications
