Lysosome-dependent Ca(2+) release response to Fas activation in coronary arterial myocytes through NAADP: evidence from CD38 gene knockouts

Abstract

Activation of the death receptor Fas has been implicated in the development of vascular injury or disease, but most studies have focused on its role in the regulation of cell apoptosis and growth. The present study was designed to examine the early response of coronary artery to Fas activation by its ligand, FasL. The hypothesis being tested is that CD38 signaling pathway mediates FasL-induced intracellular Ca(2+) release through nicotinic acid adenine dinucleotide phosphate (NAADP) in mouse coronary arterial myocytes (CAMs) and thereby produces vasoconstriction in coronary arteries. HPLC analysis demonstrated that FasL markedly increased NAADP production in CAMs from wild-type mice (CD38(+/+)) but not in cells from CD38 knockout (CD38(-/-)) mice. Using fluorescent Ca(2+) imaging analysis, we found that FasL (10 ng/ml) significantly increased Ca(2+) release from 142.5 +/- 22.5 nM at the basal level to 509.4 +/- 64.3 nM in CD38(+/+) CAMs but not in CD38(-/-) CAMs. However, direct delivery of NAADP, the CD38 metabolite, into CD38(-/-) CAMs still markedly increased Ca(2+) release, which could be significantly attenuated by a lysosomal function inhibitor, bafilomycin A1 (Baf), or a NAADP antagonist, pyridoxalphosphate-6-azophenyl-2-disulfonic acid. Confocal microscopy further demonstrated that FasL produced a typical two-phase Ca(2+) release with a local Ca(2+) burst from lysosomes, followed by a global Ca(2+) response in CD38(+/+) CAMs. In isolated perfused septal coronary arteries from CD38(+/+) mice, FasL was found to significantly increase U-46619-induced vasoconstriction from 29.2 +/- 7.3 to 63.2 +/- 10.3%, which was abolished by Baf (100 nM). These results strongly indicate that the early response of CAMs to FasL is to increase intracellular Ca(2+) levels and enhance the vascular reactivity through stimulation of NAADP production and lysosome-associated two-phase Ca(2+) release in coronary arteries.

Fig. 1.

Genotyping confirmation of wild-type (W) and CD38 knockout (KO) mice and identity determination of isolated coronary arterial myocytes (CAMs). A: a PCR product gel document from genotyping. See text for detailed description of lanes. B: CAM identity was confirmed by positive staining with anti-α-smooth muscle actin primary antibody and Alexa-Fluor 488-conjugated anti-mouse IgG secondary antibody (green stain), and negative control staining was shown in endothelial cells (ECs).

Fig. 2.

HPLC analysis of nicotinic acid adenine dinucleotide phosphate (NAADP) conversion rate in CAMs. A: typical chromatogram of NADP⁺ and NAADP. B: conversion rate of NADP⁺ to NAADP in CD38^+/+ and CD38^−/− CAMs under Fas ligand (FasL; 10 ng/ml) in the presence or absence of the CD38 inhibitor nicotinamide (Nicot; 6 mM). Values are means ± SE (n = 6 experiments). *P < 0.05 vs. vehicle only. #P < 0.05 vs. NADP⁺ group.

Fig. 3.

FasL induced Ca²⁺ release in mouse CAMs as shown by fluorescent Ca²⁺ imaging analysis. A: intracellular Ca²⁺ responses to FasL (10 ng/ml) in the absence or presence of bafilomycin A1 (Baf; 100 nM), ryanodine (Rya; 50 μM), or 2-aminoethoxydiphenyl borate (2-APB; 100 μM) in CAMs from wild-type (CD38^+/+) mice. B: intracellular Ca²⁺ response to FasL (10 ng/ml) or NAADP (1 μM) with or without pretreatment with Baf (100 nM) or pyridoxalphosphate-6-azophenyl-2-disulfonic acid (PPADS; 50 μM) in CAMs from CD38^−/− mice. Values are means ± SE (n = 6 experiments). *P < 0.05 vs. FasL group. #P < 0.05 vs. vehicle group.

Fig. 4.

CD38 plasmid transfection rescued FasL-induced Ca²⁺ release in CAMs of CD38^−/− mice. A: confirmation of CD38/green fluorescent protein (GFP) transfection in CAMs. SMCs, smooth muscle cells. B: FasL-induced Ca²⁺ response in CD38-rescued CAMs was restored to the level in that of CD38^+/+ CAMs. Values are means ± SE (n = 6 experiments). *P < 0.05 vs. CD38^−/− group.

Fig. 5.

Confocal microscopic analysis of FasL-induced 2-phase Ca²⁺ release in CAMs of CD38^+/+. A: sequential images of intracellular Ca²⁺ response to FasL (10 ng/ml) with the fluorescence indicator fluo-4. The occurrence of color from green to red represents Ca²⁺ release. B: FasL-induced 2-phase Ca²⁺ release in CAMs of CD38^+/+ in the presence or absence of Baf (100 nM) or Rya (50 μM). The ratio of Ca²⁺-dependent fluorescence intensity to basal level was quantified to present the intracellular Ca²⁺ release response. Values are means ± SE (n = 6 experiments). *P < 0.05 vs. first phase in control group. #P < 0.05 vs. second phase in control group.

Fig. 6.

Confocal microscopic confirmation of first-phase Ca²⁺ release from lysosomes in CAMs of CD38^+/+. A: sequential images show that the Ca²⁺ release in the first phase (4.5 min) appeared as an unevenly Ca²⁺-sparking image (green images in Ca²⁺/fluo-4), and the highest Ca²⁺ release regions were colocalized well with lysosomes/rhodamine (red images in Lyso/Rho), which resulted in strong yellow spots (overlay). In contrast, the subsequent global Ca²⁺ release in the second phase (10 min) was evenly distributed within the whole cell, and no specific regional yellow spots were observed. B: summarized results showed colocalization coefficiency of Ca²⁺/fluo-4 and Lyso/Rho at the basal level, first-phase (4.5 min), and second-phase (10 min) Ca²⁺ releases, respectively. Values are means ± SE (n = 6 experiments). *P < 0.05 vs. other groups.

Fig. 7.

FasL increased the coronary artery vasoconstriction response to U-46619 in CD38^+/+ but not CD38^−/− mice. A: effects of FasL on U-46619-induced vasoconstriction in coronary arteries from CD38^+/+ (+/+) and CD38^−/− (−/−) mice under FasL (10 ng/ml) treatment. B–D: this FasL-sensitized vasoconstrictor effect was almost blocked by pretreatment with the lysosomal function inhibitor Baf (100 nM; B) or the SR Rya/Ca²⁺ antagonist Rya (50 μM; C), but not by the IP₃ receptor antagonist 2-APB (100 μM; D). Values are means ± SE (n = 6 experiments). *P < 0.05 vs. CD38^−/− group.