Abstract
In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.
Highlights
Nicotinic acid adenine dinucleotide phosphate (NAADP)2 is a newly discovered Ca2ϩ messenger with unique properties that has been extensively investigated in various tissues and cell lines [3,4,5,6,7,8]
Effects of NAADP were prevented by bafilomycin A1, by desensitizing concentrations of NAADP and by ryanodine and thapsigargin
Actions of NAADP Photoreleased from a Caged Analogue on Whole Cell Ca2ϩ Transients—In the first series of experiments presented here, NAADP was photoreleased from a caged analogue to test whether or not NAADP could exert any effect on whole cell Ca2ϩ transients accompanying action potentials in cardiac ventricular myocytes
Summary
Cell Isolation—Guinea pig ventricular myocytes were isolated using methods described previously. Measurement of Ca2ϩ Transients—The loading of ventricular myocytes with fluo-4 was achieved by either inclusion of the dye in the patch pipette solution (75 M) or incubation for 15 min with the acetoxymethyl ester of fluo-4 (fluo-4 AM, 5 M). Bafilomycin A (Calbiochem-Novabiochem Ltd., Nottingham, UK, stock solution dissolved in Me2SO) and NAADP-AM were applied to ventricular cells by a rapid switch local perfusion system (Warner Instrument Corp) to ensure rapid application. When ryanodine and thapsigargin (Calbiochem-Novabiochem Ltd., Nottingham, UK) were applied in the solution superfusing the cells, a tap close to the inflow of the bath was used to switch to the drug-containing solution. Control hearts were perfused for 10 min with a Ca2ϩ-containing solution, composition in mM: NaCl 137, KCl 5, NaHCO3 12, CaCl2 1.8, glucose 5, sodium pyruvate 1, NaH2PO4 0.4, MgCl2 1, NaOH 1, EGTA 0.1, pH 7.4, gassed with 95% O2/5% CO2. Data are quoted in the text as mean Ϯ S.E
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