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(Circulation Research. 2002;91:1168.)
© 2002 American Heart Association, Inc.

Cellular Biology

Nicotinic Acid Adenine Dinucleotide Phosphate Mediates Ca²⁺ Signals and Contraction in Arterial Smooth Muscle via a Two-Pool Mechanism

François-Xavier Boittin, Antony Galione, A. Mark Evans

From the Department of Biomedical Sciences (F.-X.B., A.M.E.), School of Biology, University of St Andrews, Fife, and University Department of Pharmacology (A.G.), University of Oxford, UK.

Correspondence to Dr A. Mark Evans, University of St Andrews, Division of Biomedical Sciences, School of Biology, Bute Building, St Andrews KY16 9TS, UK. E-mail ame3{at}st-and.ac.uk

	Abstract

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Previous studies of arterial smooth muscle have shown that inositol 1,4,5-trisphosphate (IP₃) and cyclic ADP-ribose mobilize Ca²⁺ from the sarcoplasmic reticulum. In contrast, little is known about Ca²⁺ mobilization by nicotinic acid adenine dinucleotide phosphate, a pyridine nucleotide derived from ß-NADP⁺. We show here that intracellular dialysis of nicotinic acid adenine dinucleotide phosphate (NAADP) induces spatially restricted "bursts" of Ca²⁺ release that initiate a global Ca²⁺ wave and contraction in pulmonary artery smooth muscle cells. Depletion of sarcoplasmic reticulum Ca²⁺ stores with thapsigargin and inhibition of ryanodine receptors with ryanodine, respectively, block the global Ca²⁺ waves by NAADP. Under these conditions, however, localized Ca²⁺ bursts are still observed. In contrast, xestospongin C, an IP₃ receptor antagonist, had no effect on Ca²⁺ signals by NAADP. We propose that NAADP mobilizes Ca²⁺ via a 2-pool mechanism, and that initial Ca²⁺ bursts are amplified by subsequent sarcoplasmic reticulum Ca²⁺ release via ryanodine receptors but not via IP₃ receptors.

Key Words: NAADP • calcium • smooth muscle • sarcoplasmic reticulum • ryanodine receptors

	Introduction

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It is generally accepted that Ca²⁺ release from the sarcoplasmic reticulum (SR) can be triggered by the ubiquitous Ca²⁺ mobilizing messenger IP₃ via the activation of one or more of the known inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) subtypes.^1,2 However, a consideration of the wide variety of cellular processes regulated by changes in intracellular Ca²⁺ concentration [Ca²⁺]_i, from cell differentiation and gene expression to muscle contraction, underscores the need for a versatile Ca²⁺ signaling system that would necessarily require multiple Ca²⁺ mobilizing second messengers. It is not surprising, therefore, that recent studies have demonstrated that intracellular Ca²⁺ release may also be triggered by Ca²⁺ mobilizing pyridine nucleotides, namely cyclic ADP-ribose (cADPR)^3,4 and nicotinic acid adenine dinucleotide phosphate (NAADP).^3,5

The enzymes for the synthesis and metabolism of NAADP and cADPR are present in mammalian cells,^3,6 and both messengers have been shown to release Ca²⁺ from microsomes derived from a variety of cell types including vascular smooth muscle.^6–12

It is generally accepted that cADPR mobilizes Ca²⁺ from SR/endoplasmic reticulum stores by activating ryanodine receptors (RyRs).^3,13–17 As yet, however, the Ca²⁺ release pathway through which NAADP mobilizes Ca²⁺ remains poorly characterized.

Recent studies have suggested that NAADP triggers Ca²⁺ release via a mechanism that is fundamentally different from those controlled by IP₃ or by cADPR, and that this may involve the activation of putative NAADP receptors.^8,18–20 Indeed, investigations in sea urchin eggs suggest that NAADP mobilizes Ca²⁺ from a store that is pharmacologically and physically distinct from those accessed by IP₃ and cADPR, respectively.^5,21,22 Despite the proposed physical segregation of the Ca²⁺ stores, however, all three Ca²⁺ release pathways may act in concert to initiate complex Ca²⁺ signals. Thus, NAADP can trigger Ca²⁺ release that is amplified by subsequent Ca²⁺ release via RyRs and via IP₃Rs.^12,19,22,23 Likewise, IP₃ can trigger Ca²⁺ release via IP₃Rs that is amplified by subsequent Ca²⁺ release via RyRs.^24,25

To date, however, there have been no investigations of Ca²⁺ mobilization by NAADP in intact vascular smooth muscle cells. We show here that NAADP is a powerful Ca²⁺ mobilizing second messenger in pulmonary artery smooth muscle cells, and propose that NAADP mobilizes Ca²⁺ via a 2-pool mechanism.

	Materials and Methods

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Cell Isolation
Single smooth muscle cells were isolated from second-order branches of the pulmonary artery of male Wistar rats (150 to 200 g). Briefly, an artery was dissected out and placed in a low-Ca²⁺ solution of the following composition (in mmol/L): 124 NaCl, 5 KCl, 1 MgCl₂, 0.5 NaH₂PO₄, 0.5 KH₂PO₄, 15 NaHCO₃, 0.16 CaCl₂, 0.5 EDTA, 10 glucose, 10 taurine, and 10 HEPES (pH 7.4). After 20 minutes, the artery was placed in the same solution containing 0.5 mg/mL papain (Fluka) and 1 mg/mL BSA (Sigma) for 1 hour at room temperature. Then, 0.25 mg/mL 1,4-DTT (Sigma) was added to the solution, followed by a further 30-minute incubation at room temperature. The tissue was then washed three times in fresh low-Ca²⁺ solution without enzymes, and single smooth muscle cells were isolated by gentle trituration with a fire-polished Pasteur pipette. Samples of the cell suspension were placed as required onto a glass coverslip in the experimental chamber and allowed to settle for 10 minutes.

Ca²⁺ Imaging
Cells were washed with physiological salt solution of the following composition (in mmol/L): 130 NaCl, 5.2 KCl, 1 MgCl₂, 1.7 CaCl₂, 10 glucose, and 10 HEPES (pH 7.45). Cells were then incubated for 30 minutes in the same solution with 5 µmol/L fura-2-acetoxymethyl ester (fura-2), washed, and allowed to equilibrate for 20 minutes. The experimental chamber was then placed on a Leica DMIRBE inverted microscope. Changes in [Ca²⁺]_i were monitored by assessing fura-2 fluorescence, using excitation wavelengths of 340 nm (F340) and 380 nm (F380), respectively, and an emission wavelength of 510 nm. Emitted fluorescence was monitored using a Hamamatsu 4880 image-intensifying charge-coupled device camera and recorded and analyzed using Openlab imaging software (Improvision) on an Apple Macintosh G4 personal computer. Fluorescence intensity was measured at 0.25 to 5 Hz, with background subtraction being carried out online. Changes in fura-2 fluorescence are reported as the F340/F380 ratio and as the estimated [Ca²⁺]_i.

Intracellular Dialysis of NAADP and IP₃
Ca²⁺ mobilizing second messengers were applied intracellularly in the whole-cell configuration of the patch-clamp technique and in current-clamp mode (I=0), as described previously.¹⁷ The pipette solution contained the following (in mmol/L): 140 KCl, 10 HEPES, 1 MgCl₂, and 0.005 fura-2 (free acid) (pH 7.4). Throughout each experiment the seal resistance was 3 G, whereas the series resistance and pipette resistance were 10 M and 2 to 3 M, respectively, as measured using an Axopatch 200B amplifier (Axon Instruments).

Experiments in the Absence of Extracellular Ca²⁺
To limit the impact on intracellular Ca²⁺ stores, cells were superfused with Ca²⁺-free physiological salt solution containing 1 mmol/L BAPTA using a flow pipe positioned close to the cell, as described previously.²⁶ By this method complete exchange of the extracellular solution is ensured within 5 seconds, as determined previously from the shift in the potassium equilibrium potential.²⁶ Superfusion with Ca²⁺-free solution began after first establishing the cell-attached patch configuration and was continued for 30 seconds before entering the whole-cell configuration and throughout the subsequent period of intracellular dialysis. All experiments were carried out at room temperature (22°C).

Data Analysis
Data are expressed as mean±SEM for n experiments. Statistical significance was determined using a Student t test.

Drugs
All compounds were from Sigma, except papain (Fluka) and fura-2 (Molecular Probes). Ryanodine and thapsigargin were dissolved in DMSO. The minimum dilution of DMSO was 1:1000, which had no effect on the smooth muscle cells. All other stock solutions were in H₂O.

	Results

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NAADP Induces Global Ca²⁺ Oscillations in Pulmonary Artery Smooth Muscle Cells
The role of NAADP as a Ca²⁺-mobilizing second messenger in pulmonary artery smooth muscle cells was investigated by applying fixed concentrations intracellularly by dialysis from a patch pipette, in the whole-cell configuration and under current-clamp conditions (I=0). At 10 nmol/L, NAADP induced a global increase in the fura-2 fluorescence ratio (F340/F380), from 0.7±0.1 to 1.9±0.1, in isolated pulmonary artery smooth muscle cells. This occurred within 180 seconds of entering the whole-cell configuration (n="17; Figure 1A). In paired cells, intracellular dialysis of NAADP-free pipette solution had little or no effect over time periods (up to 10 minutes) that far exceeded those within which intracellular dialysis of NAADP triggered an increase in F340/F380 fluorescence ratio (n="17; Figures 1A and 1D). Moreover, NAADP (10 nmol/L) failed to induce an increase in the F340/F380 fluorescence ratio when applied intracellularly in combination with the fast calcium chelator BAPTA (1 mmol/L; n=4; Figures 1B and 1D). In marked contrast, superfusion with Ca²⁺-free extracellular solution containing BAPTA (1 mmol/L) had little or no effect on Ca²⁺ signals induced by intracellular perfusion of NAADP (10 nmol/L). In the absence of extracellular Ca²⁺, NAADP increased the fura-2 fluorescence ratio from 0.54±0.1 to 1.84±0.2 (n=6; Figures 1C and 1D). NAADP, therefore, triggers an increase in cytoplasmic free Ca²⁺ concentration in pulmonary artery smooth muscle cells by mobilizing intracellular stores.

View larger version (36K): [in this window] [in a new window]   Figure 1. NAADP induces global Ca2+ waves and contraction in isolated pulmonary artery smooth muscle cells. A, Top, Series of pseudocolor images of fura-2 fluorescence ratio (F340/F380) recorded in a pulmonary artery smooth muscle cell during the intracellular dialysis of 10 nmol/L NAADP. Bottom, Corresponding record of fura-2 fluorescence ratio against time and record of fura-2 fluorescence ratio in a different cell during intracellular dialysis of NAADP-free solution (control), respectively. In this and all subsequent figures, vertical lines at the start of the record indicate the point at which intracellular dialysis began. B, Record of fura-2 fluorescence ratio against time during intracellular dialysis of 10 nmol/L NAADP with 1 mmol/L BAPTA. C, Record of fura-2 fluorescence ratio against time during intracellular dialysis of 10 nmol/L NAADP in the absence of extracellular Ca2+ and in the presence of extracellular BAPTA (1 mmol/L). D, Bar chart showing maximal percentage change (mean±SEM) in fura-2 fluorescence ratio measured in pulmonary artery smooth muscle cells during intracellular dialysis of NAADP-free pipette solution (control), 10 nmol/L NAADP, and 10 nmol/L NAADP in the presence of 1 mmol/L intracellular and extracellular BAPTA, respectively. For this and all subsequent figures, intracellular dialysis was carried out under current-clamp conditions (I=0). Images were acquired at 3-second intervals.

In 80% of cells studied, the global Ca²⁺ wave triggered a pronounced contraction of the smooth muscle cell (Figure 1A). Furthermore, in 3 of 17 cells, NAADP (10 nmol/L) induced global, regenerative Ca²⁺ waves. These oscillations in [Ca²⁺]_i occurred with a frequency of 1.1±0.1 minute^-1 (see Figure 2A). The events leading up to the initiation of a global Ca²⁺ wave appeared complex, in that there was an initial increase in intracellular Ca²⁺ at the perimeter of the cell (Figures 1A and 1B). This initial "Ca²⁺ burst" was observed in 10 of 17 cells and appeared as (1) a uniform increase in fura-2 fluorescence ratio around the entire perimeter of the cell (Figure 1A) or (2) a spatially restricted "focal" Ca²⁺ burst covering between 2 and 10 µm of the perimeter of the cell (Figure 3C). The Ca²⁺ burst initiated by NAADP either declined back to basal levels without initiating a global Ca²⁺ wave, or preceded and then triggered a global Ca²⁺ wave (Figures 1A and 3C). Significantly, the global Ca²⁺ wave, but not the Ca²⁺ burst, induced contraction.

View larger version (24K): [in this window] [in a new window]   Figure 2. Concentration-response relationship for NAADP in isolated pulmonary artery smooth muscle cells. A, Record of fura-2 fluorescence ratio (F340/F380) against time recorded in a pulmonary artery smooth muscle cell during intracellular dialysis of 10 nmol/L NAADP. B, Record of fura-2 fluorescence ratio against time recorded in a paired pulmonary artery smooth muscle cell during intracellular dialysis of 100 µmol/L NAADP and effect of subsequent extracellular application of 2.5 mmol/L caffeine. C, Bar chart showing maximal percentage change (mean±SEM) in fura-2 fluorescence ratio in paired pulmonary artery smooth muscle cells during intracellular dialysis of 10 nmol/L NAADP, 100 µmol/L NAADP, and during extracellular application of 2.5 mmol/L caffeine in absence and presence of intracellular NAADP (100 µmol/L), respectively.

View larger version (27K): [in this window] [in a new window]   Figure 3. NAADP induces spatially restricted Ca2+ bursts but not global Ca2+ waves or contraction after depletion of SR Ca2+ stores or block of RyRs. A, Record of fura-2 fluorescence ratio against time recorded in a pulmonary artery smooth muscle cell during the intracellular dialysis of 10 nmol/L NAADP. B, Record of fura-2 fluorescence ratio against time recorded during intracellular dialysis of 10 nmol/L NAADP after depletion of SR Ca2+ stores with 1 µmol/L thapsigargin. C, Top, Series of pseudocolor images of fura-2 fluorescence ratio recorded in a pulmonary artery smooth muscle cell during intracellular dialysis of 10 nmol/L NAADP after preincubation with 20 µmol/L ryanodine. Bottom, Record of fura-2 fluorescence ratio against time. D, Bar chart showing maximal percentage change (mean±SEM) in fura-2 fluorescence ratio in pulmonary artery smooth muscle cells during intracellular dialysis of 10 nmol/L NAADP under control conditions and in the presence of 1 µmol/L thapsigargin and 20 µmol/L ryanodine, respectively.

Our findings suggest that a global Ca²⁺ wave is only initiated by NAADP when the initial increase in cytoplasmic Ca²⁺ concentration breaches a given threshold (Figure 1A). Further analysis revealed that a global Ca²⁺ wave was initiated when the fura-2 fluorescence ratio within a given "region of interest" at the perimeter of the cell rose from 0.6±0.1 to 0.9±0.1 (n=10). This corresponded to a local increase in [Ca²⁺]_i from 100 to 400 nmol/L, as quantified by interpolation with a standard in vitro Ca²⁺ calibration curve.

Concentration Dependence of NAADP-Induced Ca²⁺ Signals
Low concentrations of NAADP (2 nmol/L) failed to initiate a global Ca²⁺ wave or contraction (not shown). The threshold concentration at which NAADP induced a global Ca²⁺ wave was 10 nmol/L (Figure 2A; n=17). As the concentration of NAADP was increased between 10 nmol/L and 10 µmol/L, no increase in the magnitude or frequency of the Ca²⁺ waves was observed (not shown). Thus, between 10 nmol/L and 10 µmol/L, NAADP initiates a global Ca²⁺ wave in an all-or-none manner. Surprisingly, however, intracellular dialysis of 100 µmol/L NAADP was less effective. In 3 of 6 cells paired with those infused with 10 nmol/L NAADP, 100 µmol/L NAADP had little or no effect on the [Ca²⁺]_i (Figure 2B). In the remaining 3 cells global Ca²⁺ signals of varying amplitude were observed. On average, 100 µmol/L NAADP increased the fura-2 fluorescence ratio to a peak of 41±19% (n=6), compared with an increase to a peak of 192±20% in cells dialyzed with 10 nmol/L NAADP (n=4, Figure 2C). The inability of 100 µmol/L NAADP to consistently induce a global Ca²⁺ wave in these cells did not, however, result from the presence of dysfunctional SR stores, because the increase in fura-2 fluorescence ratio induced by 2.5 mmol/L caffeine remained unaffected (Figures 2B and 2C).

Global Ca²⁺ Waves by NAADP Require the Release of Ca²⁺ From Ryanodine-Sensitive SR Stores
To determine the role of ryanodine-sensitive SR stores in the generation of Ca²⁺ waves by NAADP, we studied the effect of blocking RyR function with ryanodine and of depleting SR Ca²⁺ stores with thapsigargin, respectively. When cells were preincubated (15 minutes) with thapsigargin (1 µmol/L), 10 nmol/L NAADP failed to induce a global Ca²⁺ wave and contraction (n=4; Figure 3B, bottom panel). However, low-magnitude and spatially restricted Ca²⁺ bursts were still observed (Figure 3B). On average the maximum increase in the F340/F380 ratio measured only 27±3% (n=8, Figure 3D). After preincubation (20 minutes) of cells with 20 µmol/L ryanodine, intracellular application of NAADP (10 nmol/L) again failed to induce a global Ca²⁺ wave, and no cell contraction was observed (n=9, Figure 3C). Once more, however, low-magnitude and spatially restricted Ca²⁺ bursts were observed (Figure 3C). On average the maximum increase in the F340/F380 ratio under these conditions measured only 27±3% (n=8, Figure 3D). In marked contrast, no Ca²⁺ bursts were observed in cells exposed to intracellular dialysis of NAADP-free pipette solution after preincubation with thapsigargin (n=3) and ryanodine (n=3), respectively.

Global Ca²⁺ Waves by NAADP Do Not Require the Activation of IP₃Rs
In pulmonary artery smooth muscle cells, intracellular dialysis of 1 µmol/L IP₃ triggered a global Ca²⁺ wave (n=5, Figure 4A). The Ca²⁺ wave was triggered 180 seconds after entering the whole-cell configuration, as was the case with NAADP, as indicated by an increase in the fura-2 fluorescence ratio from 0.6±0.01 to a peak of 2.5±0.1 (n=5). The global Ca²⁺ wave by IP₃ remained in the presence of ryanodine and peaked at a level not significantly different from that of control (Figure 4B). Thus, after preincubation (20 minutes) of cells with 20 µmol/L ryanodine, intracellular dialysis of 1 µmol/L IP₃ increased the fura-2 fluorescence ratio from 0.8±0.1 to a peak of 1.8±0.2 (n=5).

View larger version (25K): [in this window] [in a new window]   Figure 4. IP3R antagonist xestospongin C blocks Ca2+ release by IP3 but not by NAADP. A, Record of fura-2 fluorescence ratio (F340/F380) against time recorded in a pulmonary artery smooth muscle cell during intracellular dialysis of 1 µmol/L IP3. B, Record of fura-2 fluorescence ratio against time in a pulmonary artery smooth muscle cell during intracellular dialysis of 1 µmol/L IP3 after preincubation with 20 µmol/L ryanodine. C, Record of fura-2 fluorescence ratio against time in a pulmonary artery smooth muscle cell during intracellular dialysis of 1 µmol/L IP3 after preincubation with 0.1 µmol/L xestospongin C. D, Record of fura-2 fluorescence ratio against time in a pulmonary artery smooth muscle cell during intracellular dialysis of 100 nmol/L NAADP in presence of 0.1 µmol/L xestospongin C. D, Bar chart showing maximal percentage change (mean±SEM) in fura-2 fluorescence ratio for experiments described in panels A through C.

Preincubation (15 minutes) of cells with the IP₃R antagonist xestospongin C (0.1 µmol/L)²⁷ abolished the increase in F340/F380 ratio induced by 1 µmol/L IP₃ (n=5, Figures 4A and 4B). In marked contrast, Ca²⁺ waves by 10 nmol/L NAADP remained unaffected in paired cells preincubated (15 minutes) with 0.1 µmol/L xestospongin C (Figure 4C). The peak increase in F340/F380 ratio induced by NAADP was 208±42% (n=5) in the absence and 211±54% in the presence of xestospongin C (n=5; Figure 4D).

	Discussion

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Recent studies have suggested that NAADP triggers Ca²⁺ release via a mechanism that is fundamentally different from those controlled by IP₃ or by cADPR, and that this may involve the activation of putative NAADP receptors.^8,11,19,20 Indeed, It has been proposed that NAADP mobilizes Ca²⁺ from a store that is pharmacologically and physically distinct from those accessed by IP₃ and cADPR, respectively.^5,21,22 Furthermore, all three Ca²⁺ release pathways may act in concert to initiate complex Ca²⁺ signals. Thus, NAADP can trigger Ca²⁺ release that is amplified by subsequent Ca²⁺ release via RyRs and via IP₃Rs.^12,19,22,23 We have, therefore, investigated the role of NAADP as a Ca²⁺-mobilizing second messenger in pulmonary artery smooth muscle.

NAADP Induces Global Ca²⁺ Oscillations in Pulmonary Artery Smooth Muscle Cells
Intracellular dialysis of NAADP into acutely isolated pulmonary artery smooth muscle cells induced global, regenerative Ca²⁺ waves that were abolished when NAADP was coinfused with the fast Ca²⁺ chelator BAPTA. In contrast, superfusion of cells with Ca²⁺-free extracellular solution containing BAPTA had little or no effect on Ca²⁺ signals by NAADP. Thus, intracellular dialysis of NAADP mobilizes Ca²⁺ from intracellular stores in pulmonary artery smooth muscle cells, as would be expected given previous investigations.^{5,8,11,19–22}

The Ca²⁺ signals induced by NAADP were, however, complex. Spatially restricted Ca²⁺ bursts were observed before the initiation of a global Ca²⁺ wave. The Ca²⁺ bursts took two clearly identifiable forms. In one subset of cells, a uniform increase in Ca²⁺ around the entire perimeter of the cell was observed, indicative of an increase in Ca²⁺ proximal to the plasma membrane as has been reported previously in sea urchin eggs.²² In the second subset of cells, a focal increase in Ca²⁺ was triggered at the perimeter of the cells, between 2 and 10 µm in diameter. The Ca²⁺ burst either decayed or preceded and then triggered a global Ca²⁺ wave and contraction in the smooth muscle cell. Significantly, only the global Ca²⁺ wave and not the initial Ca²⁺ burst triggered contraction. These data suggested that the initial Ca²⁺ burst had to breach a threshold before it was able to initiate a global Ca²⁺ wave, consistent with the proposal that NAADP induces global waves via a 2-pool mechanism by priming IP₃- and cADPR-sensitive SR stores.²²

NAADP Triggers Ca²⁺ Release From a Discrete Intracellular Store That Is Amplified by Subsequent Ca²⁺ Release From Thapsigargin-Sensitive SR Stores
Previous investigations have demonstrated that NAADP mobilizes Ca²⁺ from a thapsigargin-insensitive store.^8,9,22 It was surprising, therefore, that prior depletion of SR Ca²⁺ stores with thapsigargin resulted in marked attenuation of NAADP-induced Ca²⁺ signals in pulmonary artery smooth muscle cells. Importantly, however, thapsigargin did not block the initiation of the spatially localized Ca²⁺ bursts by NAADP. Thus, NAADP initially mobilizes Ca²⁺ from a discrete, thapsigargin-insensitive store, which then triggers a global Ca²⁺ wave by subsequent Ca²⁺ release from the SR. Further support for this proposal may be derived from the fact that high concentrations of NAADP can induce self-inactivation without affecting Ca²⁺ release from the SR by RyR activation.

It is interesting to note, however, that depletion of SR Ca²⁺ stores with thapsigargin resulted in such marked attenuation of Ca²⁺ signals by NAADP, such that cell contraction was no longer observed. This suggests that Ca²⁺ signals by NAADP are more heavily dependent on the SR in pulmonary artery smooth muscle than is the case in, for example, pancreatic acinar cells.¹⁹

Ca²⁺ Signals by NAADP Require Subsequent Ca²⁺ Release via RyRs but not via IP₃Rs
Consistent with previous investigations,^12,19,22,23 we found that inhibition of RyRs with ryanodine resulted in marked attenuation of Ca²⁺ signals by NAADP. Under these conditions, only spatially restricted Ca²⁺ bursts were observed, as was the case after depletion of SR stores with thapsigargin. The ability of ryanodine to block global Ca²⁺ waves by NAADP did not, however, result from slow depletion of SR Ca²⁺ stores, as demonstrated by the finding that ryanodine had little effect on Ca²⁺ signals triggered by IP₃. Thus, global Ca²⁺ waves by NAADP, but not local Ca²⁺ bursts, require subsequent SR Ca²⁺ release via RyRs.

This raises the possibility that Ca²⁺ signals by NAADP in pulmonary artery smooth muscle may be modulated by cADPR, which may release SR Ca²⁺ via RyRs and/or sensitize RyR to Ca²⁺-induced Ca²⁺ release,¹³ as has been shown to be the case in pancreatic acinar cells.²⁸

In marked contrast to previous reports,^12,19,22,23 we found no evidence to support a role for IP₃Rs in the generation of NAADP-induced Ca²⁺ waves in pulmonary artery smooth muscle cells. Thus, the IP₃R antagonist xestospongin C blocked Ca²⁺ signals induced by IP₃, but not those induced by NAADP. It would appear, therefore, that NAADP first induces local Ca²⁺ bursts via a mechanism independent of RyRs, IP₃Rs, and SR Ca²⁺ stores, which then initiates a global Ca²⁺ wave and contraction in pulmonary artery smooth muscle cells by further Ca²⁺ release from SR stores via RyRs, but not via IP₃Rs.

Clearly, our finding that initial Ca²⁺ bursts by NAADP do not induce further Ca²⁺ release via IP₃Rs is contrary to previous reports.^12,19,22,23 This discrepancy may be explained if the initiation of a global Ca²⁺ wave relies on Ca²⁺-induced Ca²⁺ release and if IP₃Rs are expressed in a Ca²⁺-insensitive form²⁹ in pulmonary artery smooth muscle. Alternatively, in pulmonary artery smooth muscle, NAADP receptors may colocalize with SR "compartments" with a high density of RyRs, and not with SR compartments where IP₃Rs are concentrated.³⁰

It is also interesting to note that recent investigations in veinous smooth muscle have shown that IP₃ induces a global Ca²⁺ wave by first activating IP₃Rs, which in turn trigger further Ca²⁺ release via RyRs.^24,25,31 This is clearly not consistent with our finding that ryanodine has little effect on the magnitude of global signals by IP₃ in arterial smooth muscle. These contrary findings may result from differences in the spatial localization of RyRs and IP₃Rs in arterial versus venous smooth muscle. Thus, IP₃Rs may colocalize with clusters of RyRs in venous smooth muscle, thereby allowing for the threshold for Ca²⁺-induced Ca²⁺ release via RyRs to be breached with ease. In contrast, IP₃Rs in pulmonary artery smooth muscle cells may be localized in sections of the SR that are spatially segregated from sections of the SR within which RyRs are clustered, leading to spatially segregated Ca²⁺ signals via these two families of SR Ca²⁺ release channel.

Concentration Dependence of Ca²⁺ Signals by NAADP
Previous studies in pancreatic acinar cells, T lymphocytes, and sea urchin eggs have demonstrated that high concentrations of NAADP prove ineffective as a trigger of intracellular Ca²⁺ release.^{19,23,32–34} It has been proposed, therefore, that NAADP receptors may self-inactivate in the presence of high concentrations of NAADP. Our findings provide some support for this proposal, because global Ca²⁺ signals by NAADP were consistently observed between 10 nmol/L and 10 µmol/L, whereas 100 µmol/L NAADP proved to be less effective. Thus, 100 µmol/L NAADP failed to induce a Ca²⁺ signal in 3 of 6 cells and induced a global Ca²⁺ signal somewhat smaller than control in the other 3 cells. The failure of 100 µmol/L NAADP to consistently induce Ca²⁺ signals in pulmonary artery smooth muscle cells was not, however, due to the presence of dysfunctional SR stores, because SR Ca²⁺ release triggered by caffeine remained unaffected after self-inactivation of NAADP signaling by intracellular dialysis of 100 µmol/L NAADP. The fact that high concentrations of NAADP conferred a variable degree of self-inactivation in pulmonary artery smooth muscle cells suggests that the susceptibility to self-inactivation is less than has been reported in other preparations.^{19,23,32–34} Ca²⁺ mobilization by NAADP may therefore be more robust in pulmonary artery smooth muscle cells than in other cell types,^{19,23,32–34} raising the possibility that the characteristics of the Ca²⁺ release process triggered by NAADP may be tailored to suit a particular function in a given cell type. Unfortunately, the robust nature of NAADP signaling in pulmonary artery smooth muscle cells mitigates against the use of self-inactivation as a tool for identifying vasoconstrictors that mediate Ca²⁺ signals via NAADP, as described in studies of secretagogue signaling in pancreatic acinar cells.¹⁹

Physiological Significance of Ca²⁺ Signaling by NAADP
Consideration of the wide variety of cellular processes regulated by changes in [Ca²⁺]_i in vascular smooth muscle, from cell differentiation and gene expression to muscle contraction and relaxation,^1,2,35 underscores the need for a versatile Ca²⁺ signaling system. Such versatility with respect to Ca²⁺ signaling would necessarily require equally versatile mechanisms of Ca²⁺ mobilization. When taken together with the fact that the enzymes for the synthesis and metabolism of NAADP are present in vascular smooth muscle,^6,36 the findings of the present investigation suggest that this requirement may be fulfilled, in part, by NAADP. Thus, relatively low concentrations of NAADP mobilize Ca²⁺ from intracellular stores in arterial smooth muscle cells and do so by a mechanism distinct from that of IP₃. The pronounced smooth muscle cell contraction triggered by the associated Ca²⁺ wave suggests that NAADP likely mediates intracellular Ca²⁺ release and, in part, contraction triggered by certain vasoconstrictors. However, development of selective NAADP antagonists is required before this can be proven.

Recent investigations have demonstrated that different Ca²⁺ signals may regulate differential gene expression^37,38 and trasncription,³⁹ respectively, in preparations including native vascular smooth muscle. It seems likely, therefore, that the required code for differential gene expression and transcription could be determined, at least in part, by the discrete mechanisms through which IP₃ and NAADP trigger Ca²⁺ release and by the spatiotemporal characteristics of the resultant Ca²⁺ signal. Thus, differential activation of these signaling pathways by vasoactive agents may trigger smooth muscle contraction and promote, over prolonged periods of stimulation, associated, differential gene expression required to maintain normal physiology, or promote the development of pathophysiologies associated with, for example, hypertension and atherogenesis.

In conclusion, we have demonstrated that NAADP acts as a potent Ca²⁺ mobilizing second messenger in arterial smooth muscle. Our data support previous proposals that NAADP induces Ca²⁺ signals via a 2-pool mechanism,^22,21 which requires initial Ca²⁺ release from a thapsigargin-insensitive Ca²⁺ store.^8,9 We propose, therefore, that NAADP induces Ca²⁺ bursts from a thapsigargin-insensitive store, which are then amplified by subsequent SR Ca²⁺ release via RyRs, but not via IP₃Rs. Given that certain agonists mediate Ca²⁺ signals via NAADP in pancreatic acinar cells whereas others mediate Ca²⁺ signals via IP₃, it seems likely that certain vasoconstrictors may mediate Ca²⁺ signals and contraction in arterial smooth muscle by NAADP, whereas others rely on IP₃. Further investigations of Ca²⁺ signaling by NAADP in arterial smooth muscle will, therefore, advance our understanding of the fundamental mechanisms that regulate arterial function in health and disease.

	Acknowledgments

 
This work was funded by a Wellcome Trust Project Grant (reference No. 056423).

Received July 12, 2002; revision received September 9, 2002; accepted November 4, 2002.

	References

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