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Ultrasound Microbubble Delivery of Ca 2+ Signaling Second Messengers into Bovine Coronary Arterial Smooth Muscle Cells
Author(s) -
ZHANG GUO,
LI PINLAN
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1110-b
Subject(s) - second messenger system , intracellular , chemistry , biophysics , inositol phosphate , inositol , inositol trisphosphate , biochemistry , receptor , biology
Ultrasound microbubble technique has been widely used for gene transfer and drug delivery into cells. So far little is known whether this delivery strategy can be used for studies of intracellular signaling molecules in the regulation of cell function. The present study was designed to test the applicability of this ultrasound microbubble mediated delivery method to the studies of Ca 2+ signaling second messengers. Freshly isolated bovine coronary arterial smooth muscle cells (CASMCs) were used to monitor Ca 2+ release response and Ca 2+ ‐dependent superoxide production. These cells were loaded with Fura‐2 Am and dihydroethidium (DHE), and Ca 2+ transient and conversion of DHE to ethidium (Eth) were monitored simultaneously. Ca 2+ mobilizing second messengers inositol‐1,4,5‐triphosphate (IP 3 ), cyclic ADP‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) were mixed with 40 μl microbubble solution, respectively before added into the reaction bath solution at 37°C. Ultrasound with a frequency of 1MHZ was then applied to drive microbubbles into cells. We found that IP 3 (5 μM), cADPR (10 μM) or NAADP (40 nM) induced a rapid increase in fura 2 F340/F380 fluorescent ratio accompanied by a relatively slow increase in DHE/Eth ratio within CASMCs. Maximal Δ[Ca 2+ ] i reached 729 ± 31, 691 ± 24, 592 ± 47 nM when we introduced IP 3 , cADPR or NAADP, respectively. These increases in intracellular Ca 2+ are accompanied by increases in superoxide production by 60–80%. These results suggest that this ultrasound microbubble delivery method is suitable for effective introduction of intracellular second messenger compounds to activate Ca 2+ release and Ca 2+ ‐dependent NAD(P)H oxidase. (Supported by NIH Grants HL57244, HL70726, and HL075316)