Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K-Akt-endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.

We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL-8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that IL-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca 2؉ signaling as well as cell migration, which was completely blocked by bafilomycin A1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. A type II transmembrane protein, CD38, possesses ADP-ribosyl cyclase (ADPR cyclase) 3 and cyclic ADP-ribose hydrolase (cADPR hydrolase) activity (1, 2). These two enzyme activities are involved in the conversion of ␤-nicotinamide adenine dinucleotide (␤-NAD ϩ ) first to cADPR and then to ADPR (3-5). The metabolite cADPR is known to increase intracellular Ca 2ϩ concentration, [Ca 2ϩ ] i , by releasing Ca 2ϩ from intracellular stores or by Ca 2ϩ influx through plasma membrane Ca 2ϩ channels in a variety of cells (6 -10). It was shown that CD38 can also synthesize NAADP from NADP in the presence of nicotinic acid by a base exchange reaction in vitro (11). However, it still remains unclear whether the base exchange reaction occurs physiologically as intracellular nicotinic acid concentration is less than the millimolar concentration that is required for the enzymatic synthesis of nicotinic acid adenine dinucleotide phosphate (NAADP) in vitro (12).NAADP is a potent Ca 2ϩ -releasing messenger in a variety of cell types, including mammalian cells (13-15). Although D-myo-inositol 1,4,5-trisphosphate (IP 3 ) and cADPR are firmly established as secondary Ca 2ϩ messengers, receptor-mediated formation of NAADP has been shown in a limited number of cellular systems (16 -18). It has been demonstrated that NAADP triggers Ca 2ϩ release from thapsigargin-insensitive Ca 2ϩ stores through the activation of channels distinct from those sensitive to ryanodine and IP 3 (19). In sea urchin eggs, NAADP releases Ca 2ϩ from acidic Ca 2ϩ stores, lysosome-related organelles (20). However, NAADP can also release Ca 2ϩ from the endoplasmic reticulum (21-23).Previously, we have reported that IL-8 stimulated cADPR formation by activation of CD38 via cGMP/protein kinase G and induced an increase of [Ca 2ϩ ] i and migration of LAK cells (24). In this study we investigated whether NAADP is involved in IL-8-induced Ca 2ϩ signaling and migration of LAK cells. We showed that NAADP plays a key role in IL-8-stimul...

To investigate the mechanism by which an increase in pacing frequency or distension increases the secretion of atrial natriuretic peptide (ANP), the changes in atrial volume during contraction (atrial stroke volume), transmural transport of the extracellular fluid (ECF), and ANP secretion were quantified in the beating perfused rabbit atria. The atrium was stimulated by transmural field stimulation or by atrial distension induced by an increase in intraatrial pressure. Atrial stretch and incremental increases in pacing frequency up to 2 Hz activated the secretion of ANP coincident with an increase in atrial stroke volume and the transendocardial translocation of the ECF. These results showed positive relationships between changes in the secretion of ANP and the atrial stroke volume or the translocation of the ECF. The translocation of the ECF was also positively correlated with the change in atrial stroke volume. The accentuated secretion of ANP and translocation of the ECF waned at higher stimulating rates to show a peak value. Even under this condition, the secretion of ANP was a function of the translocation of the ECF. These data suggest that the increases in atrial stroke volume and translocation of ECF are fundamental factors in the ANP stimulation in response to atrial stretch and increases in atrial rate.

No abstract

Antimycin A (AMA), an inhibitor of electron transport in mitochondria, has been used as a reactive oxygen species (ROS) generator in biological systems. Here, we investigated the in vitro effect of AMA on apoptosis in HeLa cells. AMA inhibited the growth of HeLa cells with an IC(50) of about 50 microM. AMA efficiently induced apoptosis, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), Bcl-2 down-regulation, Bax up-regulation, and PARP degradation. All caspase inhibitors used in this experiment, especially pan-caspase inhibitor (Z-VAD), could rescue some HeLa cells from AMA-induced cell death. When we examined the changes of the ROS, H(2)O(2) or O(2) (.-), in AMA-treated cells, H(2)O(2) and O(2) (.-) were markedly increased. In addition, we detected the depletion of GSH content in AMA-treated cells. Pan-caspase inhibitor showing the efficient anti-apoptotic effect significantly reduced GSH depletion by AMA. Superoxide dismutase (SOD) and catalase did not reduce intracellular ROS, but these could strongly rescue the cells from apoptosis. However, these anti-apoptotic effects were not accompanied by the recovery of GSH depletion. Interestingly, catalase significantly decreased the CMF negative (GSH depletion) and propidium iodide (PI) positive cells, indicating that catalase strongly maintained the integrity of the cell membrane in CMF negative cells. Taken together, these results demonstrate that AMA potently generates ROS, induces the depletion of GSH content in HeLa cells, and strongly inhibits the growth of HeLa cells throughout apoptosis.