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Am J Physiol Heart Circ Physiol 291: H274-H282, 2006. First published February 10, 2006; doi:10.1152/ajpheart.01064.2005
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Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

Fan Zhang,1 Guo Zhang,1 Andrew Y. Zhang,1 Matthew J. Koeberl,2 Eryn Wallander,2 and Pin-Lan Li1

1Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia; and 2Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 7 October 2005 ; accepted in final form 27 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 ± 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 ± 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 ± 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 µM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 ± 4.42% to 51.80 ± 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.

beta-nicotinamide adenine dinucleotide phosphate; adenosine 5'-diphosphate-ribosyl cyclase; coronary artery; vascular smooth muscle

NICOTINIC ACID (NA) adenine dinucleotide phosphate (NAADP) is produced from NADP+ by a replacement of its nicotinamide (Nicot) group with NA via ADP-ribosyl cyclase or its mammalian homologue CD38 (1). NAADP was first identified to mobilize Ca2+ in sea urchin eggs (40), and recently this signaling nucleotide has been shown to act as an endogenous regulator of intracellular Ca2+ in a wide variety of cell types from plants to animals, thereby participating in the regulation of cell functions, such as fertilization, cell proliferation and differentiation, insulin secretion, and nitric oxide signaling, as well as muscle constriction (33, 37, 42, 50). In contrast to the well-characterized D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and cyclic ADP ribose (cADPR)/Ca2+ signaling pathways (38, 52), the mechanisms mediating NAADP/Ca2+ signaling still remain enigmatic. Recent studies (3, 7) from different tissues or cells have suggested that NAADP is a novel secondary messenger to mobilize intracellular Ca2+ via a mechanism completely different from Ins(1,4,5)P3 and cADPR. In sea urchin egg homogenates, for example, NAADP-induced Ca2+ mobilization undergoes homologous desensitization but does not affect subsequent Ca2+ release by Ins(1,4,5)P3 or cADPR, the inhibitors of Ins(1,4,5)P3 or cADPR, and the Ca2+ pump blocker of the Ins(1,4,5)P3/cADPR stores have no effects on the NAADP-induced Ca2+ release (15).

Although this NAADP-mediated Ca2+ signaling pathway has distinctive characteristics from that of other intracellular Ca2+ messengers by targeting different Ca2+ storing organelles, there is accumulating evidence that the coordination of action of NAADP with other Ca2+ mobilizing messengers in the Ca2+ cascade events is importantly involved in the regulation of intracellular Ca2+ levels (7, 9). For example, in pancreatic acinar cells, NAADP evoked a local Ca2+ release in the apical granular pole, and this local Ca2+ spike could be propagated to a global Ca2+ wave under the concerted action of cADPR and Ins(1,4,5)P3 (9). In other studies (15, 34, 57) using pulmonary arterial smooth muscle cells, it has been revealed that local Ca2+ could be sequestered to produce a global Ca2+ wave by Ca2+ release from the sarcoplasmic reticulum (SR) via Ca2+-induced Ca2+ release (CICR) mechanism, which is related to the activation of Ins(1,4,5)P3 receptors (IP3Rs) and ryanodine (Rya) receptors on the SR. It seems that NAADP serves as a trigger of global Ca2+ response to different agonists, which was named in some reports as a two-pool mechanism for the regulation of intracellular Ca2+ levels (5, 13). With a consideration of a wide variety of cellular processes are regulated by changes of intracellular Ca2+ concentrations, from fertilization to cell death, it is possible that this spatiotemporal Ca2+ pattern related to NAADP may importantly contribute to the regulation of different cell functions, either through its action as second messenger or via its activity to synchronize the actions of other second messengers.

Previous studies from our laboratory and by others have demonstrated that cADPR is an important Ca2+-signaling second messenger, which importantly contributes to the regulation of coronary vascular tone and vasomotor response. It has been reported that an ADP-ribosyl cyclase-mediated enzymatic pathway is present in coronary arterial smooth muscle, which is responsible for the production of cADPR under resting conditions or in response to different stimuli (22, 43, 59). Given that ADP-ribosyl cyclase is also responsible for the conversion of NADP+ into NAADP, we wondered whether NAADP may also be produced in coronary arterial smooth muscle and involved in the regulation of coronary arterial tone or vasomotor response and how this new signaling nucleotide participates in the regulation of intracellular Ca2+ concentration ([Ca2+]i) in coronary arterial smooth muscle cells (CASMCs). In addition, it is also imperative to know whether NAADP can interact with other intracellular second messengers to regulate cellular Ca2+ responses. To answer these questions, the present study first characterized the production of NAADP in coronary arterial smooth muscle preparation. Using HPLC analysis, we determined the conversion of NADP+ into NAADP by coronary homogenates, microsomes, and cytosols. We then addressed whether NAADP can induce Ca2+ release response in coronary arterial muscle cells using fluorescence imaging techniques and ultrasound microbubble intracellular delivery method. In reconstituted lipid bilayer with purified SR proteins, we examined whether NAADP changed Rya receptor (RyR) activity. To determine the role of endogenously produced NAADP in mediating the response to agonists, we tested the effects of endothelin-1 (ET-1) on the production of NAADP and analyzed the Ca2+ release and vasomotor responses to ET-1 before and after inhibition of lysosomal function, which has been reported to serve as a target for the signaling action of NAADP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture of CASMCs. The bovine CASMCs were cultured as described previously (44, 46). Briefly, the vessels were first rinsed with 5% FBS in medium 199 containing 25 mM HEPES with 1% penicillin, 0.3% gentamycin, and 0.3% nystatin and then cut into segments, and the lumen was filled with 0.4% collagenase in medium 199. After 30 min of incubation at 37°C, the vessels were flushed with medium 199. The strips of denuded arteries were placed into gelatin-coated flasks with medium 199 containing 10% FBS with 1% L-glutamine, 0.1% tylosin, and 1% penicillin-streptomycin. CASMCs migrated to the flasks within 3–5 days. Once growth was established, the vessels were removed and cells were grown in medium 199 containing 20% FBS. The identification of CASMCs was based on positive staining by an anti-{alpha}-actin antibody.

HPLC analysis of NAADP production in coronary arterial muscle. Bovine coronary arterial homogenates were prepared as we described previously (44). Briefly, bovine hearts were obtained from a local slaughterhouse. The left anterior descending artery was dissected and homogenized in ice-cold HEPES buffer (pH 7.4), which contained (in mM) 20 HEPES, 1 EDTA, 255 sucrose, 1 PMSF, 1 Na3VO4, and 1 mg/ml leupeptin, and to 40 ml of HEPES buffer were added 1 tablet of complete proteinase inhibitor cocktail (Roche Diagnostics). After centrifugation of the homogenate at 6,000 g for 5 min at 4°C, the supernatant containing the membrane protein and cytosolic components was aliquoted and frozen in liquid N2 and termed the homogenate. Microsomes and cytosols were prepared by a differential centrifugation of the homogenate at 10,000 g for 20 min and at 100,000 g for 90 min, respectively. The pellet was named the microsomal fraction, whereas the supernatant was named the cytosolic fraction. To determine the production of NAADP, different amounts of homogenates (10, 50, 100, and 200 µg) were incubated for 30 min with 1 mM NADP+ and 30 mM NA at 37°C in HEPES buffer containing (in mM) 20 HEPES, 1 EDTA, and 255 sucrose (pH = 4.5), with a total reaction volume of 0.1 ml. Before HPLC analysis, the reaction mixtures were centrifuged at 4°C with the use of an Amicon microultrafilter at 13,000 rpm for 15 min to remove the proteins. The reaction products in the ultrafiltrate were analyzed on a Supelcosil LC-18 (3 µm, 4.6 x 150 mm) with a Supelcosil LC-18 guard column (5 µm, 4.6 x 20 mm; Supelco, Bellefonte, PA) by the use of a Hewlett-Packard 1090L solvent delivery system and a 1040A photodiode array detector (Hewlett-Packard, Avondale, PA). Data were collected and analyzed with a Hewlett-Packard Chemstation. Mobile phase consisted of 5 mM potassium dihydrogen phosphate (pH 5.5) containing 5 mM tetrabutylammonium dihydrogen sulfate (solvent A) and acetonitrile (solvent B). The nucleotides were separated in solvent A with a gradient of 0–25% solvent B over 12 min and then to 100% solvent B over the next 4 min. Solvent B was held constant for 4 min and then returned to 100% solvent A for the last 10 min. The flow rate was 0.8 ml/min. The column eluate was monitored at 254 nm. Peak identities were confirmed by comigration and absorbance spectra compared with the known standards. Quantitative measurements were performed by comparison of known concentration of standards.

To determine which part of the CASMCs had higher enzymatic activity regarding NAADP synthesis, the microsome and cytosol of coronary arterial preparations were made for the NAADP conversion experiments by HPLC analysis.

Fluorescence measurement of [Ca2+]i in CASMCs. A fluorescence image analysis system was used to determine [Ca2+]i in the primary cultures of CASMCs with fura-2 acetoxymethyl ester (fura-2) as an indicator (60). Having been loaded with 10 µM fura-2 at room temperature for 30 min, the cells were washed three times with Ca2+-free Hanks’ buffer. A fluorescence ratio of excitation at 340 nm to that at 380 nm (F340/F380) was determined after background subtraction, and [Ca2+]i was calculated by using the following equation: [Ca2+]i = Kdbeta[(R – Rmin)/(Rmax – R)], where Kd for the fura-2-Ca2+ complex is 224 nM; R is the fluorescence ratio (F340/F380); Rmax and Rmin are the maximal and minimal fluorescence ratios measured by addition of 10 µM of Ca2+ ionophore ionomycin to Ca2+-replete solution (2.5 mM CaCl2) and Ca2+-free solution (5 mM EGTA), respectively; and beta is the fluorescence ratio at 380-nm excitation determined at Rmin and Rmax, respectively (27, 58).

Ultrasound (Rich-Mar Sonitron 2000) and Optison (Perflutren protein type A microspheres) (51, 55) were employed to deliver NAADP into CASMCs for determination of its effect on [Ca2+]i. To enwrap NAADP in Optison, NAADP solution was mixed well with the Optison reagent in a volume ratio of 1:10 and incubated for 1 min (51, 55). After this mixture was added to the Ca2+-free Hanks’ buffer (pH 7.4) for a final concentration of NAADP at 100 nM, CASMCs were treated with ultrasound for 1 min (1 W/cm2) (51, 55). Before and after the ultrasound treatment, Ca2+ fluorescence assay was performed. The untreated CASMCs were also applied to ultrasound microbubble treatment for each group as a control. To determine the effects of bafilomycin A1 (Baf) or Rya on ET-1- or NAADP-induced Ca2+ response, CASMCs were pretreated with 100 nM Baf or 50 µM Rya for 30 min.

The ratio of fura-2 emissions, when excited at the wavelengths of 340 and 380 nm, was recorded with a digital camera (Nikon Diaphoto TMD Inverted Microscope). Metafluor imaging and analysis software were used to acquire, digitize, and store the images for off-line processing and statistical analysis (Universal Imaging).

Planar lipid bilayer analysis of NAADP effect on SR RyR Ca2+ release channels. The preparation of SR-enriched microsomes from bovine coronary arteries and reconstitution of these SR membranes into a planar lipid bilayer was performed by the protocol described previously (45, 54), with cesium used as the charge carrier. The Ca2+ release channel activity was detected in a symmetrical 300 mM cesium methanesulfonate and 10 mM MOPS solution (pH 7.2). An Integrating Bilayer Clamp Amplifier (Model BC-525C, Warner Instrument) was used to record single-channel currents. The amplifier output signals were filtered at 1 kHz with an eight-pole Bessel Filter (Frequency Devices). Currents were digitized at a sampling rate of 10 kHz. Data acquisition and analysis were performed with pClamp software (version 8, Axon Instruments). Channel open probability (NPo) in the lipid bilayer was determined from recordings of 3–5 min as described previously in our patch-clamp studies (45, 54). All lipid bilayer experiments were performed at room temperature (~20°C), because the bilayer system will be unstable at 37°C, which plagues observations of the effects of any pharmacological interventions on channel activity (45, 54).

The effects of NAADP (100 nM) and NADP+ (100 nM) on RyR Ca2+ (RyR/Ca2+) release channels of SR were determined, and the positive controls cADPR (100 nM) and Rya (2 µM) were used to validate the experimental condition. All doses of these compounds were based on previous studies from our laboratory or others (49, 54), and all compounds were added into the cis solution, with currents recorded at a holding potential of –40 mV.

ET-1-induced production of NAADP. Previous studies (22) have shown that oxotremorine (Oxo) activates ADP-ribosyl cyclase via M1 muscarinic receptors, leading to Ca2+ release from the SR in coronary arteries. Furthermore, Kinnear and associates (34) demonstrated that ET-1 evoked lysosomal Ca2+ release by NAADP and then caused a global Ca2+ wave. In the present study, we compared the effects of these two possible ADP-ribosyl cyclase-activating agonists. First, we examined their effects on the production of two different ADP-ribosyl cyclase products (22). In these experiments, the primary cultured CASMCs were treated with 100 nM ET-1 or 80 µM Oxo in the presence or absence of 6 mM Nicot. After the incubation at 37°C for 15 min, the cells were washed three times with Krebs buffer, and homogenates were prepared. Cell homogenates (100 µg) were used for the subsequent conversion assay. For the NAADP assay, the homogenates were incubated with 1 mM NADP+ and 30 mM NA in HEPES buffer (pH 4.5) at 37°C for 60 min, whereas 1 mM Nicot guanine dinucleotide was used as the substrate for cADPR conversion rate [cyclic GDP ribose (cGDPR) conversion rate] assay in HEPES buffer (pH 7.0).

The HPLC assay of NAADP was performed according to the protocol described above. As for the cGDPR analysis, the HPLC mobile was changed to 150 mM ammonium acetate (pH 5.5) containing 5% methanol (solvent A) and 50% methanol (solvent B). The solvent system was a linear gradient of 5% solvent B in A to 30% solvent B in A over 1 min, held for 25 min, and then increased to 50% solvent B over 1 min with the flow rate of 0.8 ml/min. The fluorescence detector was used with excitation wavelength of 300 nm and emission wavelength at 410 nm.

Coronary artery reactivity. The vascular reactivity of bovine coronary arteries was determined as described previously (23, 61). Small intramural coronary arteries from the left anterior descending artery were carefully dissected and placed in a Krebs bicarbonate solution containing (in mM) 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 1.18 NaH2PO4, 24 NaHCO3, 0.026 EDTA, and 5.5 glucose (pH 7.4). The small coronary arteries were then passed through its lumen by a segment of hair, and the endothelium was removed by gently rotating the hair segment (22). The denuded small artery (100–200 µm inner diameter) was transferred to a water-jacketed perfusion chamber and cannulated with two glass micropipettes at their in situ length. After a 90-min equilibration period under transmural pressure of 60 mmHg, the artery was activated by 80 mM KCl until reproducible constriction was obtained. The internal diameter of the arteries was measured with a video system composed of a stereomicroscope (Leica MZ8), a charge-coupled device camera (KP-MI AU, Hitachi), a video monitor (VM-1220U, Hitachi), a video measuring apparatus (VIA-170, Boeckeler Instrument), and a video printer (UP890 MD, Sony). The arterial images were recorded continuously with a videocassette recorder (M-674, Toshiba). The internal diameter of the arteries under 80 mM KCl was used as 100% constriction, and the subsequent artery constrictive responses to different agents were expressed as a percentage of the response to 80 mM KCl.

To determine ET-1 and Oxo-induced vasoconstriction, cumulative dose-response curves of ET-1 (10–10-10–7 M) and Oxo (10–8-10–4 M) were conducted by measuring changes in the internal diameter, respectively. The ET-1 and Oxo were then washed out with Krebs buffer until the contractility returned to the baseline level. To further determine the effects of Baf and Nicot on the ET-1- and Oxo-induced constriction of coronary arteries, 100 nM Baf or 6 mM Nicot were incubated for 15 min, and ET-1 and Oxo cumulative dose responses were redetermined. Throughout the experiments, Krebs buffer in the bath was continuously bubbled with a gas mixture of 95% O2-5% CO2 and maintained at 37 ± 0.1°C.

Statistical analysis. Data were presented as means ± SE. Significance of differences in mean values within and between multiple groups was examined using an ANOVA for repeated measures, followed by Duncan’s multiple-range test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Production of NAADP in coronary arteries. Figure 1 shows a representative reverse-phase HPLC chromatogram depicting a profile of NADP+ metabolites produced by coronary arterial homogenates. As shown in Fig. 1A, NAADP has a retention time of 18.3 min, and the unreacted NADP+ has a retention time of 16.3 min. Figure 1B presents a concentration-dependent conversion of NADP+ to NAADP by arterial homogenates. As the homogenate protein increased from 10 to 200 µg, the NADP+ conversion rate was increased from 77.9 ± 5.2 to 169.9 ± 5.9 nmol/min. When compared with the activity of purified ADP-ribosyl cyclase, the amount of NAADP produced by 1 mg of coronary arterial homogenates was equal to that produced by 5 x 10–4 ng of ADP-ribosyl cyclase. These results suggest that an enzyme responsible for the production of NAADP is present in bovine coronary arteries.


Figure 1
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  		Fig. 1. HPLC analysis of nicotinic acid adenine dinucleotide phosphate (NAADP) production. A: typical chromatogram of NADP+ and NAADP. B: conversion rate of NADP+ into NAADP at different protein concentrations of bovine coronary arterial homogenate. *P < 0.05 vs. 10 µg homogenate protein group (n = 6 experiments). mAU, milliabsorbance unit.

 
Figure 2 summarizes the maximal conversion rates of NADP+ into NAADP in the homogenates, microsomes, and cytosols prepared from coronary arteries in the presence of 1 mM NADP+. The conversion rates were 13.07 ± 1.17, 9.31 ± 0.81, and 18.30 ± 2.56 nmol·min–1·mg protein–1, respectively, in the coronary arterial homogenates, microsomes, and cytosols. It was obvious that the cytosols exhibited the highest conversion rate among three fractions of coronary preparations.


Figure 2
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  		Fig. 2. Location of NAADP production. Conversion rate of NAADP in homogenate (Homo), microsome (Mico), and cytosol (Cyt) prepared from bovine coronary arteries. *P < 0.05 compared with Homo group; #P < 0.05 compared with Micro group (n = 6 experiments).

 
Effects of NAADP on activity of reconstituted RyR/Ca2+ release channels from coronary arterial muscle SR. Figure 3 shows the effects of NAADP, NADP+, cADPR, and Rya on the activity of the reconstituted RyR/Ca2+ release channels in the planar lipid bilayer. Figure 3A shows representative recordings depicting the effects of NAADP, NADP+, cADPR, and Rya on the activity of these Ca2+ release channels. Figure 3B summarized results showing that both NAADP and NADP+ have no effects on the NPo of the reconstituted RyR/Ca2+ release channels, whereas cADPR and a low concentration of Rya (2 µM) can significantly increase the NPo of these channels. These results indicate that NAADP has no direct effect to activate RyR on the SR, as do cADPR and low doses of Rya.


Figure 3
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  		Fig. 3. Effects of NAADP (100 nM), NADP+ (100 nM), cyclic ADP ribose (cADPR; 100 nM) and ryanodine (Rya; 2 µM) on activity of reconstituted Rya receptor Ca2+ (RyR/Ca2+) release channels in planar lipid bilayer. A: representative recordings of channel currents trace under control condition and after addition of NAADP, NADP+, cADPR, or Rya into cis solution with "-c" indicating closed state of channel. B: summarized channel open probability (NPo) of RyR/Ca2+ release channels in the presence of NAADP, NADP+, cADPR, and Rya, respectively. *P < 0.05 compared with control, NAADP, and NADP+ group (n = 8 bilayers from 5 hearts).

 
Role of exogenous NAADP in inducing Ca2+ release response. The above results have shown that NADP+ could be converted to NAADP in the coronary myocytes and that NAADP could not activate RyR/Ca2+ channels on the SR. Therefore, we further explored the mechanism by which NAADP increases [Ca2+]i and the lysosome-related actions. As shown in Fig. 4A, the F340/F380 ratio was increased from 0.85 to 1.32 when 100 nM NAADP was introduced into CASMCs by an ultrasound-microbubble delivery method. In the presence of Baf, NAADP-induced Ca2+ release was substantially blocked. Figure 4B summarizes NAADP-induced alterations of [Ca2+]i in the absence or presence of 100 nM Baf (n = 6 experiments). When 100 nM NAADP was introduced into CASMCs, [Ca2+]i was significantly elevated from 142 ± 12 to 711 ± 47 nM. NAADP-induced Ca2+ release effects were reduced to 259 ± 18 nM when cells were pretreated by 100 nM Baf. However, there were no changes in basal [Ca2+]i in CASMCs treated with vehicle through ultrasound and microbubble introduction.


Figure 4
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  		Fig. 4. Exogenous NAADP (100 nM)-induced Ca2+ release in primary cultured coronary arterial smooth muscle cells (CASMCs) in the absence or presence of bafilomycin A1 (Baf; 100 nM). A: an online recording of fura-2 fluorescence ratio of F340 vs. F380 (F340/F380) against time after treatment of NAADP or Baf plus NAADP with ultrasound and microbubble optison. B: effects of NAADP and Baf plus NAADP on intracellular Ca2+ concentration ([Ca2+]i). *P < 0.05 vs. control and Baf + NAADP group (n = 6 experiments).

 
ET-1-induced production of NAADP. By HPLC analysis, we examined whether ET-1 can enhance NAADP production in bovine coronary arteries by determining the NAADP conversion rate in CASMCs. Figure 5A demonstrates that ET-1 could significantly increase NAADP conversion rate in these cells from 3.69 to 5.05 nmol·min–1·mg protein–1, whereas Nicot, an inhibitor of ADP-ribosyl cyclase, almost completely blocked this effect. Oxo had no effect on the NAADP conversion rate compared with that of the control. In contrast, as shown in Fig. 5B, ET-1 had no effect on the cGDPR conversion, whereas Oxo could markedly enhance the cGDPR production. Nicot also exerted profound inhibitory effects on the Oxo-induced cGDPR conversion.


Figure 5
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  		Fig. 5. HPLC assay of NAADP and cyclic GDP ribose (cGDPR) conversion rate in primary cultured CASMCs. A: NAADP conversion rate under endothelin-1 (ET-1; 100 nM) and oxotremorine (Oxo; 80 µM) treatment in the absence or presence of nicotinamide (Nicot; 6 mM). B: cGDPR conversion rate under ET-1 (100 nM) and Oxo (80 µM) treatment in the absence or presence of Nicot (6 mM). *P < 0.05, ET-1 group vs. other group (A), or Oxo group vs. other group (B) (n = 6 experiments).

 
Role of endogenous NAADP in mediating ET-1-induced Ca2+ release response. Figure 6A shows several images of the fura-2 fluorescence ratio F340/F380 recorded in CASMCs treated by ET-1 (100 nM) with or without pretreatment of Baf (100 nM) or Rya (50 µM). A spatially localized Ca2+ burst around cell boundary precedes the global Ca2+ increase, as indicated by the change of red color within the cell in the ET-1-treated group. In the Baf-pretreated group, both the Ca2+ burst in the area under the cell membrane and global Ca2+ increases were significantly decreased, whereas in the Rya-pretreated group, only the global increase in intracellular Ca2+ was selectively blocked. Figure 6B shows a digitally converted recording of the fura-2 fluorescence ratio F340/F380 against time for [Ca2+]i. Consistent with fluorescence images shown above, there were two peaks when CASMCs were treated with ET-1, with a small peak at 1 min corresponding to a local [Ca2+]i burst and a big peak around 3 min representing the global [Ca2+]i increase or release of Ca2+ from the SR. When the cells were pretreated with Baf, both peaks were almost completely abolished. In the presence of a high concentration of Rya (50 µM), a RyR/Ca2+ release inhibitor, only the large peak of Ca2+ increase was substantially blocked, whereas early small peak remained. Figure 6C summarized ET-1-induced [Ca2+]i fluctuations in the absence or presence of Baf or Rya. It is clear that there were no differences among the basal level of [Ca2+]i in all the three groups, namely ET-1, Baf, and Rya treatment groups. ET-1 produced significant increases in [Ca2+]i from 151 ± 43 nM of the basal level to 529 ± 32 nM at the first small Ca2+ transient peak, followed by a large increase of 1,815 ± 84 nM 3 min after the addition of ET-1. In the presence of 100 nM Baf, this ET-1-induced two-phase Ca2+ response was significantly attenuated. In contrast, Rya only significantly blocked the ET-1-induced large global Ca2+ increase, but it had no effect on the early small Ca2+ response to ET-1.


Figure 6
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  		Fig. 6. ET-1 induced Ca2+ release response in the absence or presence of Baf and Rya. A: serial images of fura-2 fluorescence ratio F340/F380 recorded in primary cultured CASMCs with treatment of ET-1 (100 nM), Rya (50 µM), or Baf (100 nM). Spatially localized Ca2+ burst (First) around the cell boundary precedes global Ca2+ wave (Second) in ET-1-treated group with a significantly blocking effect in both Baf- and Rya-pretreated group. B: fura-2 fluorescence ratio against time. C: summary of results. #P < 0.05 vs. basal; *P < 0.05 vs. Baf- or Rya-pretreated group (n = 6 experiments).

 
Effects of Baf and Nicot on the ET-1-induced constriction of bovine coronary artery. Figure 7A presents some typical microscopic photomicrographs showing coronary artery reactivity to ET-1 in the absence and presence of Baf or Nicot. ET-1 was found to induce coronary vasoconstriction, which was attenuated by either Baf or Nicot. As summarized in Fig. 7B, ET-1 (10–11-10–7 M) produced a dose-dependent constriction in the freshly isolated and pressurized small coronary arteries, which was significantly attenuated by 6 mM Nicot or 100 nM Baf, respectively. As a comparison, Oxo-induced vasoconstriction was significantly blocked by Nicot but not by Baf (Fig. 7C). However, neither Nicot nor Baf itself had any vasoconstrictor effects on the prepressurized coronary artery (data not shown).


Figure 7
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  		Fig. 7. ET-1-induced bovine coronary artery constriction in the absence or presence of Baf or Nicot. A: typical microscopic recording of ET-1-induced coronary artery constriction under different treatment. B and C: summarized results showing effects of Nicot (6 mM) or Baf (100 nM) on ET-1 (10–10-10–7 M) and Oxo (10–8-10–4 M)-induced vasoconstriction. *P < 0.05 vs. ET-1 alone-treated group (B) or Oxo alone-treated group (C) (n = 6 arterial preparations from different hearts).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that 1) an enzymatic pathway for NAADP production is present in coronary arteries, 2) NAADP had no direct effect on RyR because it did not alter the activity of the reconstituted channels from coronary arterial smooth muscle SR, 3) NAADP-stimulated Ca2+ release was blocked by a lysosome function inhibitor, and 4) ET-1 increased the conversion of NADP+ into NAADP in CASMCs and induced an increase in [Ca2+]i and vasoconstrictor response through a NAADP-lysosome dependent mechanism.

Production of NAADP in coronary arterial myocytes. In 1987, Lee and his associates (16) reported that the metabolites of NADP+ had Ca2+-releasing effects from sea urchin egg microsomes. This NADP+ metabolite was later identified as NAADP (6, 10, 12, 31). It has been demonstrated that a soluble protein, ADP-ribosyl cyclase, and its membrane-bound homologous CD38 are involved in the production of NAADP (1). Both of the enzymes can exchange the terminal Nicot group of the NADP+ with NA to produce NAADP under the reaction of transglycosylation, which has been demonstrated in a variety of cells and tissues, including sea urchin eggs, pancreatic acinar cells, human T lymphocytes, rat brain, and rat smooth muscle cells (21, 22, 41, 45). In addition to the production of NAADP, ADP-ribosyl cyclase and CD38 are also responsible for the synthesis of cADPR with the substrate of NAD+ by a cyclizing reaction. The enzymatic production of cADPR and the cADPR signaling pathway have been well characterized in a variety of tissues and cells (2, 19, 26, 28, 29). Recent works in our laboratory have demonstrated that this cADPR-mediated Ca2+ signaling is present in cardiovascular tissues, such as the myocardium, renal microvessel, and coronary arteries (21–23, 43, 59). However, so far little is known about the production and action of NAADP in coronary circulation. Given the peculiar properties of ADP-ribosyl cyclase and its ability to produce both cADPR and NAADP, we wondered whether NAADP could also be synthesized in the coronary artery and participate in the regulation of coronary arteral vascular tone or vasomotor response.

In the present study, HPLC analysis showed that the homogenates from bovine coronary arterial smooth muscle converted NADP+ into NAADP in a concentration-dependent manner under a reaction condition of pH 4.5, which had similar efficiency to that observed for cADPR production under pH 7.4 (21, 22). These results provide the first direct evidence indicating that NAADP is an enzymatic product of NADP+ in coronary arterial smooth muscle. To determine the localization of NAADP production in coronary arterial smooth muscle, we further prepared microsomes and cytosols from the coronary arteries and determined NAADP production in different fractions. When compared with homogenates, NAADP conversion rate was higher in the cytosol but lower in microsomes. It seems that NAADP production may primarily be an event that occurs in cytosol of CASMCs. Although two membrane-bound ADP-ribosyl cyclases, CD38 and CD157, and a cytosolic soluble ADP-ribosyl cyclase isoform were reported (39, 53), our previous studies demonstrated that cytosolic ADP-ribosyl cyclase activity may be primarily derived from internalized CD38 in coronary arterial smooth muscle. In addition, other studies (11, 30, 62) also demonstrated that CD38 internalization is of importance in mediating cADP-ribose production in cell cystosols. Therefore, based on current understanding, a high level of ADP-ribosyl cyclase activity to convert NAAP+ to NAADP may be associated with the internalization of a membrane-bound enzyme. This internalized cytosolic ADP-ribosyl cyclase is responsible for catalyzing the exchange of the Nicot group of NADP+ with NA to produce NAADP under acidic conditions.

NAADP-induced Ca2+ release. Previous studies (25, 32, 35, 47) reported that NAADP possibly acts on RyRs on the SR to release Ca2+. These results were mostly based on pharmacological blockade or activation. There is no direct evidence that NAADP can activate RyRs. In previous studies, we demonstrated that cADPR directly activated RyR/Ca2+ release channels on the SR of coronary arterial smooth muscle by reconstituted lipid bilayer methods (54). This effect of cADPR on SR RyR/Ca2+ release channels is related to the dissociation of FKBP12.6 from RyRs (54). Therefore, we wondered whether NAADP also stimulates Ca2+ release in coronary arterial myocytes through its action on RyR as does cADPR. To explore this possiblity, we performed lipid bilayer reconstitution experiments using the isolated SR from coronary arterial smooth muscle. It was found that NAADP had no effect on the activity of reconstituted RyR/Ca2+ release channels from coronary arterial SR, suggesting that NAADP could not directly activate Ca2+ release from the SR in coronary arterial myocytes and that the SR RyR/Ca2+ release channels are not a target for the action of NAADP to produce Ca2+ release. Studies (34) from other laboratories demonstrated that NAADP evoked highly localized intracellular Ca2+ signals by mobilizing Ca2+ from thapsigargin (a SR Ca2+-ATPase inhibitor)-insensitive stores and that heparin and high-dose Rya [inhibitors of Ins(1,4,5)P3 and RyRs on the SR, respectively] had no inhibitory effects on NAADP-initiating local Ca2+ wave. Taken together, these results reveal an important fact that NAADP-induced Ca2+ release is independent of its direct effect activating RyR/Ca2+ channels in coronary arterial myocytes.

To further explore the mechanism mediating the action of NAADP and to determine the functional role of NAADP in the regulation of [Ca2+]i in coronary arterial myocytes, we performed several series of experiments to determine the actions of exogenous and endogenous NAADP in increasing [Ca2+]i. It was found that NAADP produced a rapid Ca2+ release response when introduced into CASMCs by ultrasound microbubble delivery technique. This NAADP-induced Ca2+ release response was blocked by inhibition of lysosomal Ca2+ release and vacuolar proton pump function with Baf. These results indicate that NAADP is able to mobilize intracellular Ca2+, which may be associated with lysosome function.

Although the present study demonstrates that a lysosome-associated mechanism may be importantly involved in NAADP-induced Ca2+ release, there are some other reports indicating that NAADP may induce Ca2+ release through other mechanisms. In this regard, it seems that the controversy is dependent on the cell or tissue types used in different experiments. For example, in early studies using the sea urchin eggs or pancreatic acinar cells, NAADP was demonstrated to mobilize Ca2+ from a store that is distinct pharmacologically and physically from the Ins(1,4,5)P3- and cADPR-sensitive Ca2+ stores (7, 14). However, recent studies (18) in T lymphocytes and other cells have demonstrated that the Ca2+ release channel sensistive to NAADP in these cells is the RyR. There is also evidence that NAADP could directly activate isolated RyRs reconstituted in lipid bilayers from rabbit skeletal muscle (RyR1) (32) and cardiac microsomes (RyR2) (48). In addition, many studies have found that NAADP-induced Ca2+ release rarely operates in isolation but rather in combination with other pathways or factors. It is often observed that the resultant Ca2+ signals evoked by NAADP are boosted by Ca2+ release via activation of RyRs, IP3Rs, or both (14, 15, 24, 36, 40). Now two models for the role of NAADP in inducing Ca2+ release have been proposed to interpret the results obtained from different cells (56). In the first model, the endoplasmic reticulum (ER) or SR expressing IP3Rs and RyRs as a single pool is responsible for the action of NAADP. To induce Ca2+ release, NAADP interacts either directly with RyRs or via a separate protein that may indirectly activate RyRs (18, 25). This model can be clearly used to explain the results obtained from T lymphocytes, where NAADP-evoked release can be abolished by either RyR blockers or thapsigargin. A second model describes a two-pool or trigger hypothesis, which is based on the assumption that a NAADP-sensitive Ca2+ store exists, which is possibly a thapsigargin-insensitive acidic store (15). This NAADP-sensitive Ca2+ store is responsible for a localized signal, which is amplified by CICR through IP3Rs and RyRs on the ER to produce a large global Ca2+ increase (7, 8, 13, 14). If NAADP induces Ca2+ release through this mechanism, a localized NAADP-induced signal would persist in the presence of IP3Rs and RyR antagonists or thapsigargin, but it could be abolished by agents that dissipate storage of some acidic organelles, such as the vacuolar H+ pump inhibitor, Baf, or a disruptor of lysosomal-related organelles, glycyl-phenylalanyl-naphthylamide (56). As discussed above, in CASMCs, NAADP may exert its Ca2+ mobilizing action through the mechanism described in model 2, because NAADP did not alter the SR RyRs/Ca2+ release channel activity and inhibition of lysosome function abolished the effect of NAADP.

Involvement of NAADP in ET-1-induced Ca2+ response in CASMCs and coronary vasoconstriction. The next questions we addressed are whether and how endogenous NAADP play a role in mediating physiological response of coronary arterial muscle. ET-1 is an important endothelium-derived vasoconstrictor. ET-1 exerts a wide spectrum of biological effects on smooth muscle cells via ETA and ETB receptors, including inhibition of voltage-gated K+ channels, activation of L-type Ca2+, as well as mobilization of intracellular Ca2+ (4, 17, 20). Recent studies (34) have indicated that ET-1 exerts its Ca2+ mobilizing action in pulmonary arteries through a lysosome-associated Ca2+ activation via NAADP. In the present study, we demonstrated that ET-1 significantly increased the conversion of NADP+ into NAADP in coronary arterial myocytes, indicating that NAADP may serve as a signaling messenger to produce ET-1-induced mobilization of intracellular Ca2+ in coronary arterial myocytes and vasoconstriction. Indeed, Ca2+ fluorescence imaging analysis demonstrated that ET-1 induced a sequential two-phase increase in [Ca2+]i in CASMCs. The first phase Ca2+ spike occurred 1 min after ET-1 was added into the bath solution, and the second phase Ca2+ transient occurred 3 min later, which is strong and represents a global increase in [Ca2+]i. In the presence of Baf, both phases of Ca2+ response were markedly attenuated. However, a relatively high concentration of Rya, which is reported to be a classic inhibitor of SR RyR/Ca2+ channels, only abolished the late Ca2+ increase that functionally was related to Ca2+ release from SR Ca2+ stores. These results suggest that ET-1 may first stimulate Ca2+ release from lysosomes through NAADP action and then activate Ca2+ release from the SR via RyRs-sensitive CICR mechanism. This further supports the view that NAADP serves as a trigger or initiating factor to mediate ET-1-induced Ca2+ release from the lysosome, whereby the subsequent large Ca2+ release from the SR is evoked via a CICR mechanism by activation of RyRs.

To further address the functional significance of this NAADP-lysosome Ca2+ signaling, bovine coronary vascular reactivity to ET-1 was examined in the absence and presence of lysosome H+-ATPase inhibitor Baf and ADP-ribosyl cyclase inhibitor Nicot. It was found that ET-1 produced dose-dependent vasoconstriction in isolated and perfused coronary arteries, which was significantly attenuated by inhibition of ADP-ribosyl cyclase or lysosomal Ca2+ release. However, Oxo-induced coronary arterial constriction was only attenuated by Nicot but not by Baf. This action of Oxo is consistent with those in our previous studies that cADPR was involved in the Oxo-induced coronary arterial constriction and that the cADPR-mediated Ca2+ release is associated with thapsigargin-sensitive Ca2+ pool in the SR. In addition, these results were also consistent with the findings discussed above that NAADP was an endogenous enzymatic product of ADP-ribosyl cyclase in response to ET-1 in coronary arterial preparation and that NAADP mobilized Ca2+ in arterial smooth muscle cells via lysosome-related mechanism. Taken together, these functional results indicate that the lysosomal Ca2+-mobilizing action of NAADP plays a crucial role in ET-1-induced vasoconstrictor response in coronary arteries.

In summary, the present study demonstrated that 1) ET-1 activated ADP-ribosyl cyclase, leading to the production of NAADP under acidic conditions or acidic organelles; 2) NAADP produced Ca2+ release response independently of RyRs/Ca2+ release channel activation; 3) the Ca2+ release action of NAADP was blocked by inhibition of lysosomal function; 4) NAADP-induced two-phase Ca2+ response to ET-1 was associated with lysosome Ca2+-triggering action and subsequent CICR, which results in a large global increase in [Ca2+]i; and 5) the lysosome-associated Ca2+ regulatory mechanism through NAADP participated in the coronary vasoconstrictor response to ET-1. We conclude that NAADP is important in the regulation of [Ca2+]i in CASMCs and in the mediation of coronary vasoconstriction response to ET-1.


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This study was supported by National Heart, Lung, and Blood Institute Grants HL-057244 and HL-075316.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P.-L. Li, Dept. of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth Univ., 410 N. 12th St., PO Box 980613, Richmond, VA 23298 (e-mail: pli{at}mail1.vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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