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Altered Airway Responsiveness in CD38-Deficient Mice

Departments of Veterinary and Biomedical Sciences and Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul; Departments of Pediatrics and Pharmacology, College of Medicine, University of Minnesota, Minneapolis, Minnesota; and Trudeau Institute, Saranac Lake, New York

Correspondence and requests for reprints should be addressed to Mathur S. Kannan, BVSc, Ph.D., Professor of Pharmacology, Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, 1971 Commonwealth Avenue, Saint Paul, MN 55108. E-mail: kanna001{at}umn.edu

	Abstract

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Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38^–/–) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P 0.05) lower in cd38^–/– mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38^–/– mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38^–/– mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38^–/– mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.

Key Words: CD38 • cADPR • airway • smooth muscle • responsiveness

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Cyclic ADP-ribose (cADPR) has been shown to play a role in the regulation of intracellular calcium (Ca²⁺) in smooth muscles of the airways, intestine, seminiferous tubule, and blood vessels (1–6). It has been proposed that cADPR acts as a second messenger similar to that of inositol 1,4,5-trisphosphate (IP₃) (7–10). This hypothesis stems from several lines of evidence provided by recent investigations. In airway smooth muscle (ASM), a single protein, CD38, can both synthesize and degrade cADPR via its ADP-ribosyl cyclase and cADPR hydrolase activities, respectively (11). cADPR activates Ca²⁺ release from the intracellular stores in permeabilized ASM cells and cADPR antagonists attenuate intracellular Ca²⁺ responses to agonists (4, 12, 13). These results indicate a role for the CD38-dependent cADPR signaling pathway in ASM Ca²⁺ regulation.

The functional consequences of cADPR-mediated Ca²⁺ elevation have been investigated in a number of different cell types and model systems using cells isolated from CD38 knockout (CD38 KO) mice or treating cells with cADPR agonists or antagonists. For example, using CD38 deficient pancreatic ß-cells, it has been demonstrated that glucose-induced insulin secretion is dependent on CD38 and cADPR-mediated Ca²⁺ release (14). Likewise, acetylcholine (ACh)-induced Ca²⁺ release in pancreatic acinar cells is dependent on CD38 and cADPR (15). In studies using CD38-deficient neutrophils, monocytes, and dendritic cells, we have shown that CD38 and cADPR are necessary for Ca²⁺ responses to a number of different chemokines (16–18). With the use of a cADPR antagonist, it was demonstrated that cADPR mediates contraction of the smooth muscles of the seminiferous tubule and coronary artery (5). However, the role of CD38/cADPR signaling in ASM function has yet to be described.

The process of respiration is dependent on many factors, including airway caliber, which is regulated by a variety of contractile agonists such as ACh and endothelin-1 (ET-1). Although these agonists have been demonstrated to use cADPR-mediated Ca²⁺ release in ASM cells (12, 13), the contribution of CD38 in regulating the contractility and function of airway smooth muscle in vivo has not been investigated. In the present study, we characterized the role of CD38 and CD38-dependent cADPR signaling in airway responsiveness using the CD38 KO mice.

As a major contractile component in the airways, smooth muscle should determine airway tone and responsiveness to agonists. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in ASM is required for initiation of contraction and maintenance of airway tone. Therefore, we investigated the intracellular Ca²⁺ responses to contractile agonists in ASM cells isolated from airways of wild-type and CD38 KO mice.

Finally, even though CD38 is an important source for the synthesis of cADPR in mammalian cells, other ADP-ribosyl cyclases have been identified, including the GPI-anchored protein CD157 (BST-1) (19–21). A previous study from our laboratory suggested that the membrane bound CD38 was the source of ADP-ribosyl cyclase activity in ASM cells (11). Therefore, we determined the contribution of non-CD38 sources of ADP-ribosyl cyclase to the synthesis of cADPR in the airways using lung homogenates from CD38 KO mice.

	MATERIALS AND METHODS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Materials
All routinely used chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Hanks' balanced salt solution (HBSS) was purchased from Gibco BRL (Grand Island, NY). Goat polyclonal anti-rat CD38 antibody, donkey anti-goat IgG, and horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gradient gels and the Bio-Rad protein assay kit were purchased from Bio-Rad Laboratories (Hercules, CA). Protease inhibitor cocktail was obtained from Calbiochem (La Jolla, CA). Fura-2/AM was purchased from Molecular Probes (Eugene, OR). 8-Br-cADPR was synthesized as described previously (22). Methacholine hydrochloride solution was purchased from MethaPharm (Brantford, ON, Canada).

Experimental Animals
Mice 8–12 wk old were used in these studies. CD38-deficient mice were generated from C57BL/6J strain, as described previously (16). The wild-type (C57BL/6J) and the cd38^–/– (CD38 KO) mice were obtained from Trudeau Institute (Saranac Lake, NY) and maintained at the University of Minnesota animal facilities for use in the experiments. All studies were approved by The Animal Care and Use Committee of the University of Minnesota.

Assessment of Airway Responsiveness to Methacholine
Airway responsiveness to methacholine was measured using whole-body plethysmography, as described previously (23, 24). Mice were anesthetized with pentobarbital (90 mg/kg), tracheostomized, and intubated with an appropriately sized metal cannula that was secured in place with 3.0 silk. A polyethylene catheter was inserted orally into the lower third of the esophagus to estimate intrapleural pressure. The animal was then placed into a plethysmograph chamber (Buxco Electronics Inc., Sharon, CT) and connected to a ventilator (Harvard Apparatus, March-Hugstetten, Germany) set at a respiratory rate of 160 breaths per minute and a tidal volume of 150 µl. Respiratory flow signal was measured through a flow transducer (Sen Sym SCXL004; Buxco Electronics) connected to the plethysmograph. Lung volume was obtained by integration of the flow signal. Intraesophageal and airway pressure were measured with a pressure transducer (Validyne DP45; Buxco Electronics) directly connected to their respective ports. These data were fed into a computer through a pre-amplifier (MaxII; Buxco Electronics) and the data analyzed with the Biosystem XA software (Buxco Electronics). When the signal was stable, the basal lung resistance (RL) and dynamic compliance (C_dyn) were determined over a 1-min period. Body temperature was maintained at 37°C throughout the experiment.

The mice were challenged with normal saline followed by cumulative doses of methacholine (0, 12.5, 25, 50, and 100 mg/ml of saline) using an ultrasonic nebulizer. At the time of challenge, the respiratory rate was set at 100 breaths per minute at a tidal volume of 400 µl and nebulized for 10 breaths. The RL and C_dyn values were determined continuously following each dose of methacholine. To measure the degree of airway responsiveness, the peak responses following each dose of methacholine were obtained offline and calculated as the % changes from values following saline challenge.

Airway Smooth Muscle Cell Preparation
Tracheas from three mice were pooled for isolation of smooth muscle cells. The tracheas were excised and transferred immediately to ice-cold HBSS containing 10 mM HEPES, 11 mM glucose, 2.5 mM CaCl₂, and 1.2 mM MgCl₂ (HBSS; pH 7.4). The trachea was cleaned of connective tissue and finely minced in ice-cold HBSS. The HBSS was removed and the minced tissue was first incubated for 2 h in Earle's Balanced Salt Solution containing 20 U/ml papain and 0.005% DNase (Worthington Biochemical, Freehold, NJ) at 37°C with the subsequent addition of 0.4 mg/ml type IV collagenase and 0.3 U/ml elastase (Worthington Biochemical) and continued incubation at 37°C until cells were completely dispersed (10–15 min). Dispersion of cells was aided by gentle trituration with a siliconized glass pipette. Cells were pelleted by centrifugation, resuspended in HBSS, placed at 4°C overnight, and plated on coverslips the following day. The smooth muscle phenotype was confirmed by staining the cells with a specific -actin antibody and visualized using a fluorescence microscope.

After overnight recovery at 4°C, cells were pelleted by low-speed centrifugation and resuspended in HBSS. The cell suspension (200 µl) was then "beaded" onto glass coverslips and allowed to attach at 37°C in 95% O₂/5% CO₂ for 30 min. Attached cells were then maintained at 37°C in 95% O₂/5% CO₂ until loading with Fura-2/AM. Coverslips with attached cells were placed in HBSS containing 5 µM Fura-2/AM and incubated at 37°C for 30 min. Coverslips were washed in HBSS, treated as described below, and mounted on a 150-µl open slide chamber (Warner Instruments, Hamden, CT), placed on the stage of a Nikon Diaphot inverted microscope for measurement of intracellular calcium response during perfusion with acetylcholine or ET-1, as described below.

Digital Video Fluorescence Imaging
Fluorescence excitation, image acquisition, and real-time data analyses were controlled using a video fluorescence imaging system (Metafluor; Universal Imaging Corporation, Downington, PA). A Nikon Fluor x40 oil immersion objective lens was used for imaging. Fura 2/AM-loaded cells were excited alternately at 340 and 380 nm using a Lambda DG-4 high-speed filter changer (Sutter Instrument Co., Novato, CA). Fluorescence emissions were collected separately for each wavelength using a 510-nm barrier filter. Images were acquired using a Photometric Cool Snap 12-bit digital camera (Roper Scientific, Tucson, AZ). The ratio of intensities of Fura-2 emissions at excitation wavelengths 340 and 380 nm was calculated every 0.75 s. Intracellular calcium concentration was calculated from the ratio of intensities at 340 nm and 380 nm by extrapolation from a calibration curve, as described previously (12, 13).

Agonist-Induced Intracellular Ca²⁺ Responses
Cells were perfused with HBSS and basal [Ca²⁺]_i was measured for 1–2 min. Cells were then exposed for 4–5 min to one of the following agonists: ACh (100 nM or 1 µM) or endothelin-1 (ET-1, 200 nM), and [Ca²⁺]_i was measured. The cells were then washed with HBSS. The net [Ca²⁺]_i was calculated by subtracting the basal from the peak [Ca²⁺]_i and is represented as the intracellular Ca²⁺ response to the agonist. The Ca²⁺ responses to ACh and ET-1 were also integrated from baseline over the duration of exposure (240 s) to the agonists and represented as total Ca²⁺ response in nM.

Effect of 8-Br-cADPR on Agonist-Induced Intracellular Ca²⁺ Response
Fura-2/AM–loaded cells were incubated with 100 µM 8-Br-cADPR in HBSS for 15 min. The concentration and duration of exposure to 8-Br-cADPR were determined previously (12). The intracellular Ca²⁺ response to 200 nM ET-1 was subsequently determined in these cells.

Western Blot Analysis of CD38 Expression
Lungs obtained from wild-type and CD38 KO mice were homogenized in Tris-HCl buffer (pH 7.2) containing protease inhibitors. The homogenate was subjected to low-speed centrifugation to remove cell debris, nuclei, and mitochondria. Microsomal membranes were prepared by centrifuging at 100,000 x g for 60 min. The microsomal pellet was resuspended in the same buffer and protein content was determined using the BioRad protein assay kit.

CD38 protein in HASM microsomes was detected by Western blot analysis, as described earlier (13, 25). Microsomal protein (100 µg) was subjected to electrophoresis on 4–15% gradient polyacrylamide gels, and the proteins in the gels were transferred to a polyvinylidene difluoride (PVDF) membrane. The proteins on the membrane were blocked in PBS containing 1% milk concentrate and 0.025% Tween-20. The PVDF membranes were incubated with a polyclonal goat anti-rat CD38 antibody for 1 h. This antibody was raised against a CD38 peptide that is conserved between mice and rats, polyclonal in nature and cross-reacts with mouse CD38. A horseradish peroxidase–conjugated donkey anti-goat IgG was used as a secondary antibody. The blots were developed using chemiluminescence substrate before exposure to X-ray film. Standard molecular weight markers were electrophoresed simultaneously for comparing the molecular weights of the visualized proteins in the membrane.

Measurement of ADP Ribosyl Cyclase Activity
ADP-ribosyl cyclase activity was assayed by measuring the conversion of nicotinamide guanine dinucleotide (NGD), an analog of nicotinamide adenine dinucleotide (NAD), to the fluorescent product cGDPR. The resultant fluorescence was measured at an excitation wavelength of 305 nm and an emission wavelength of 410 nm using a spectrofluorometer (26).

ADP-ribosyl cyclase activity in the lung homogenates was also determined using 3-deaza-NAD as the substrate and measuring the amount 3-deaza-cADPR formed. Lung homogenates from wild-type and CD38 KO mice were incubated with 100 µM 3-deaza-NAD in buffer at pH 7.2 at 37°C in a total volume of 50 µl. After incubation for different time periods (10 and 30 min, and 2, 10, and 24 h), 5 µl was removed and mixed with 5 µl of 100 mM HCl to stop the reaction. The amount of 3-deaza-cADPR formed was determined using a sea urchin egg homogenate calcium release bioassay (22). The reaction mixture (1.5 µl) was mixed with 150 µl of fluo-3 loaded sea urchin egg homogenate and calcium release was measured using a BMG FluoStar 96-well fluorescence plate reader. The preparation of the egg homogenate has been previously described (22). Samples containing increasing concentrations of 3-deaza-cADPR (0–1,000 nM) were used in the assay to obtain a standard curve. Calcium release by the samples was measured over a period of 300 s and the slope of the curve at the peak release was determined. The slopes obtained for all the samples collected were converted to amount of 3-deaza-cADPR formed using the standard curve. Recombinant ADP-ribosyl cyclase was used as a positive control. In this study we chose 3-deaza-NAD as the substrate because the product 3-deaza-cADPR is 70 times more potent than cADPR in the calcium release bioassay and it is metabolically stable to enzymatic degradation (27).

Measurement of cADPR Levels
cADPR levels in lung tissues were measured as described previously (28). Tissue samples were homogenized in 10 ml of ice-cold 0.6 M perchloric acid, and cell debris were separated by centrifugation at 17,500 x g for 10 min. The supernatant was treated with an organic solvent mixture containing three parts of 1,1,2-trichlorotrifluoroethane and one part of tri-n-octylamine, and the aqueous layer containing cADPR was removed. The samples containing cADPR were treated with hydrolytic enzyme mixture (0.44 U/ml nucleotide pyrophosphatase, 12.5 U/ml alkaline phosphatase, 0.0625 U/ml NADase, 2.5 mM MgCl₂, and 20 mM sodium phosphate, pH 8.0) at 37°C to remove all the nucleotide contaminants. After this step, the enzymes were removed by filtration and the cADPR in the aqueous mixture was collected. Aliquots ( 100 µl) were taken and mixed with 0.3 µg/ml ADP-ribosyl cyclase, 30 mM nicotinamide, and 100 mM sodium phosphate, pH 8.0 to convert cADPR in the sample to NAD⁺. Cycling reaction was started by the addition of 100 µl cycling reagent containing 2% ethanol, 100 µg/ml alcohol dehydrogenase, 20 µM resazurin, 10 µg/ml diaphorase, 10 µM FMN, 10 mM nicotinamide, 0.1 mg/ml BSA, and 100 mM sodium phosphate, pH 8. The reaction was allowed to proceed for 2 h and the increase in the fluorescence of resorufin (544 nm excitation and 590 nm emission) was measured using a fluorescence plate reader. cADPR standards were used to determine the concentration of cADPR in the samples.

Statistical Analysis
To analyze the effect of different doses of methacholine on changes in lung resistance and dynamic compliance in the two groups of mice, we used two-way repeated measures ANOVA. Intracellular Ca²⁺ responses to agonists (net and integrated calcium responses) in cells obtained from wild-type and CD38 KO mice were compared using Student's t test. The effect of 8-Br-cADPR on intracellular Ca²⁺ responses was compared using one-way ANOVA. All values are represented as mean ± SEM. Two means were considered significantly different when P value was < 0.05.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
CD38 Is a Critical Source of cADPR in ASM Cells
To confirm the absence of CD38 in the lungs obtained from the CD38 KO mice, we prepared microsomal membranes from lung homogenates and performed Western blot analysis using a polyclonal goat anti-rat CD38 antibody. CD38 protein was detected in wild-type lungs, indicating that CD38 is expressed within the lung, but not in the CD38 KO lungs (Figure 1A). Next, to determine whether CD38 is the constitutively active cyclase expressed in the lung, we measured ADP-ribosyl cyclase activity and cADPR levels in lung homogenates isolated from wild-type and CD38 KO mice. ADP-ribosyl cyclase activity was first determined fluorometrically using NGD as the substrate. As shown in Figure 1B, incubation of lung microsomes from wild-type mice with NGD resulted in a time-dependent accumulation of the fluorescent product cyclic GDP-ribose. In contrast, no change in fluorescence was detected upon incubation of lung microsomes obtained from CD38 KO mice with NGD (Figure 1B), indicating that CD38 is present as a constitutively active ADP-ribosyl cyclase in the lung.

View larger version (20K): [in this window] [in a new window]   Figure 1. (A) Western immunoblot analysis for CD38 expression in lung microsomes. Microsomal proteins were separated by SDS-PAGE and transferred to PVDF membrane. CD38 protein was probed using polyclonal anti-rat CD38 antibody. Note no detectable CD38 immunoreactivity in lung microsomes from CD38 KO mice. (B) ADP-ribosyl cyclase activity in lung microsomes obtained from wild-type and CD38 KO mice using NGD as the substrate. The data is represented as fluorescence of cGDPR v.s. time (s). The ADP-ribosyl cyclase activity is undetectable in lung microsomes from CD38 KO mice. (C) ADP-ribosyl cyclase activity in lung homogenates obtained from wild-type (squares) and CD38 KO (triangles) mice using 3-deaza-NAD as the substrate. The data shown are the amount of 3-deaza-cADPR formed upon incubation of the lung homogenates (equal amount of total protein) with 3-deaza-NAD as determined by a calcium release bio-assay using sea urchin egg homogenates. Recombinant Aplysia cyclase (triangles) was used as a positive control. Note no detectable ADP-ribosyl cyclase activity in the lung homogenates obtained from CD38 KO mice. (D) Levels of cADPR in lung homogenates. cADPR levels were measured in lung homogenates obtained from wild-type and CD38 KO mice (n = 4–6 mice for each group). Note significantly (P 0.05) lower level of cADPR in lung homogenates obtained from CD38 KO mice compared with wild-type mice.

Finally, using a sensitive cycling assay, we measured cADPR levels in the lungs of wild-type and CD38 KO mice. The basal levels of cADPR measured in lung homogenates obtained from wild-type mice were 808.5 ± 186.2 fmol/mg (Figure 1D). cADPR levels were significantly lower (49.65 ± 10.78 fmol/mg) in lung homogenates obtained from CD38 KO mice (Figure 1D). Taken together, these data strongly suggest that cADPR production in the lungs is critically dependent on the presence of CD38.

Airway Responsiveness to Methacholine Is Diminished in CD38 KO Mice
In this study, we have determined the contribution of CD38 to airway responsiveness after methacholine challenge. The lung resistance values after saline challenge in the wild-type (1.68 ± 0.11 cm H₂O/liter/s) and CD38 KO (1.79 ± 0.25 cm H₂O/liter/s) mice were not significantly different from each other, and are comparable to lung resistance values reported in this stain of mice by other investigators. Methacholine challenge resulted in a dose-dependent increase in lung resistance and decrease in dynamic compliance (Figure 2). Furthermore, in CD38 KO mice, the airway responsiveness (as measured by the change in lung resistance and dynamic compliance from baseline) to methacholine was significantly (P 0.05) attenuated as compared with the wild-type controls. The lung resistance was increased to 5.36 ± 0.37 cm H₂O/liter/s in wild-type mice, whereas it was increased to 3.42 ± 0.40 cm H₂O/liter/s in CD38 KO mice upon exposure to 100 mg/ml of methacholine. As compared with saline challenge, this represents a maximum increase in lung resistance of 220% in the wild-type and 90% in the CD38 KO mice (Figure 2A). The dynamic compliance values in wild-type and CD38 KO mice during saline challenge were not significantly different from each other, and exposure to cumulative doses of methacholine resulted in a dose-dependent decrease in dynamic compliance. As compared with saline, the change in dynamic compliance following methacholine challenge was significantly (P 0.05) lower in the CD38 KO mice (maximum decrease of 37%) than in the wild-type controls ( 64% decrease) (Figure 2B). These findings demonstrated the contribution of CD38 to airway responsiveness.

View larger version (14K): [in this window] [in a new window]   Figure 2. Changes in lung resistance (A) and dynamic compliance (B) in response to different doses of methacholine in naïve wild-type (WT) and CD38 knockout (KO) mice. Anesthetized, tracheostomized, and ventilated mice (n = 7 for WT and n = 6 for KO) were challenged with different doses of methacholine. Total pulmonary resistance responses are plotted to different methacholine doses and are expressed as % increase from pulmonary resistance values obtained following saline challenge. Dynamic compliance at different doses of methacholine is represented as percent decrease from compliance values following saline challenge. Values shown are mean ± SEM. * Denotes significant (P 0.05) difference from responses in KO mice.

View larger version (28K): [in this window] [in a new window]   Figure 3. Net intracellular Ca2+ responses to 100 nM ACh (A), 1 µM ACh (B), and 200 nM ET-1 (C) in ASM cells isolated from wild-type (WT) and CD38 KO (KO) mice. The net intracellular Ca2+ responses were calculated by subtracting the peak [Ca2+]i from the basal [Ca2+]i. Note attenuated net Ca2+ responses to 100 nM ACh and 200 nM ET-1 in cells isolated from CD38 KO mice compared with wild-type controls (n = 61 and 95 cells from WT mice, respectively, for responses to ACh and ET-1; 95 cells from KO mice for responses to ACh and ET-1). Integrated Ca2+ responses to 100 nM ACh (D), 1 µM ACh (E), and 200 nM ET-1 (F) in ASM cells isolated from wild-type (WT) and CD38 KO (KO) mice. The total Ca2+ responses are shown in nM Ca2+ over the period of integration. Note attenuated total Ca2+ response to agonists in cells isolated from CD38 KO mice compared with wild-type controls. Data obtained from at least four different cell isolations from tissues pooled from two to three mice for each isolation. *Denotes statistical significance (P 0.05); n = 54 and 26 cells from WT mice, respectively, for responses to ACh and ET-1; 74 and 32 cells from KO mice for responses to ACh and ET-1, respectively.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of 8-Br-cADPR on intracellular Ca2+ responses to ET-1 in cells isolated from wild-type and CD38 KO mice. Fura-2-AM-loaded cells were incubated with 100 µM 8-Br-cADPR for 15 min and then the net intracellular Ca2+ responses to 200 nM ET-1 were determined. Data obtained from cells isolated from at least six mice. Note a significant attenuation of the response in cells from wild-type but not in cells from CD38 KO mice (P 0.05).

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

The generation of CD38-deficient mice has provided a means to study the functional role of CD38 in many organ systems in vivo. In the pancreas, CD38/cADPR signaling mediates glucose-induced insulin secretion from the islet cells and CD38-deficient mice have decreased glucose tolerance (14, 30). Another study using the CD38 KO mice demonstrated the role of CD38/cADPR signaling in osteogenesis (31). Using the same mouse phenotype, we demonstrated in previous studies a role for CD38 in modulating innate and acquired immunity (16, 18, 32). cADPR, a nucleotide catalyzed by CD38, was shown to mediate ET-1–induced Ca²⁺ release and contractility of peritubular smooth muscle cells obtained from rat seminiferous tubules (5). Similarly, the role of cADPR in the contraction of coronary arterial smooth muscle has been recently demonstrated (6). The study reported here provides the first evidence of the contribution of CD38 and cADPR to ASM contractility and airway function.

A significant observation of the present study is that the in vivo responsiveness of airways to methacholine is attenuated in the CD38 KO mice as compared with wild-type controls. In most mammalian systems, CD38 is considered to be the primary ADP-ribosyl cyclase enzyme and the major source of cADPR (33–36). In the context of airways, ASM contractility through its effect on airway caliber influences pulmonary resistance. The reduced airway responsiveness to methacholine, as measured by changes in pulmonary resistance, in the CD38 KO mice suggests a role for CD38 in the maintenance of airway tone. The potential mechanisms involved in the diminished airway responsiveness to methacholine challenge in CD38 KO mice are many, but most likely stem from reduced cADPR-mediated Ca²⁺ release from the sarcoplasmic reticulum in the smooth muscle cells. This hypothesis is supported by our findings of reduced intracellular Ca²⁺ responses to agonists whose effects are known to involve CD38/cADPR signaling in ASM cells (12, 13). The finding that CD38 gene disruption results in significantly lower airway responsiveness to methacholine suggests that CD38 may have a role in airway hyperresponsiveness. This remains to be determined in a model of inflammatory airway disease such as asthma.

In earlier studies, we have demonstrated that stimulation of muscarinic or ET-1 receptors in ASM cells results in Ca²⁺ release from intracellular stores through activation of both inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor (RyR) channels (37, 38). The Ca²⁺ mobilizing role for cADPR in ASM cells is supported by the observation that a cADPR antagonist attenuates agonist-elicited Ca²⁺ responses (4, 12, 13). Furthermore, cADPR-mediated Ca²⁺ release during ET-1 and ACh stimulation of ASM cells involves activation of RyR channels (12, 37). The diminished Ca²⁺ responses to ACh and ET-1 in ASM cells reported in the present study in the CD38 KO mice may reflect reduced RyR channel activation. We also observed that the net intracellular Ca²⁺ responses in ASM cells obtained from the wild-type and the CD38 KO mice were similar when stimulated with a high concentration (1 µM) of ACh. It has been demonstrated that at high concentrations of muscarinic agonist, there is preferential recruitment of M₃ muscarinic receptors coupled to G_q type of G-proteins (39–42). Low concentrations of ACh preferentially stimulate M₂ muscarinic receptors, which are coupled to G_i type G-proteins (39, 42). In a previous study, we have shown that the CD38/cADPR pathway in ASM cells is coupled to M₂ muscarinic receptors (12). The finding that the net Ca²⁺ responses to a high ACh concentration (1 µM) in ASM cells isolated from wild-type and CD38 KO mice are similar suggests that other signaling pathways, such as IP₃, are involved in the regulation of intracellular Ca²⁺ and may compensate for the lack of CD38/cADPR signaling. When the area under the Ca²⁺ response to 1 µM ACh was analyzed, we found that the response in cells obtained from the CD38 KO mice was significantly lower than in cells from the wild-type controls. However, the integrated calcium responses to 1 µM ACh were higher (78% of response in the wild type) than to 100 nM ACh (68% of response in the wild type). Whether the calcium responses to higher ACh concentrations are similar in the cells obtained from the wild-type and the CD38 KO mice remains to be determined. These results indicate that the Ca²⁺ response to a higher concentration of ACh is not sustained and may have a functional consequence. Indeed, the results of lung function measurements did demonstrate clear attenuated responses to methacholine challenge in the CD38 KO mice compared with the wild-type controls.

ET-1 is a potent constrictor of airway smooth muscle and implicated in the regulation of bronchomotor tone in both health and disease (43, 44). In this study we observed that the net as well as the integrated intracellular Ca²⁺ responses to ET-1 were significantly lower in myocytes obtained from CD38 KO mice than in myocytes obtained from wild-type mice. The results demonstrated that ET-1 also uses CD38-dependent cADPR to mediate the elevation of intracellular Ca²⁺ in ASM cells. Similar findings have been reported in smooth muscle cells lining the seminiferous tubules using a cADPR antagonist (5).

In this study, the intracellular Ca²⁺ elevation in response to contractile agonists were significantly lower in ASM cells from CD38 KO mice compared with cells from wild-type controls. Upregulation of CD38 expression in ASM cells, on the other hand, results in augmented intracellular Ca²⁺ responses to agonists (13). These findings support the interpretation that the level of CD38 expression in ASM cells affects the magnitude of intracellular Ca²⁺ responses to agonists. Similar correlation between CD38 expression and function has been demonstrated in other cell types. For example, CD38-deficient mice have reduced glucose tolerance resulting from impaired insulin secretion, whereas transgenic mice overexpressing CD38 in pancreatic ß-cells have increased insulin secretion induced by glucose compared with control mice (14, 30). These studies provide evidence that the level of CD38 expression correlates with physiologic function in different cell types.

Previous studies have reported no detectable cADPR levels in lymphoid or myeloid cells obtained from CD38 KO mice (18, 35). However, in the kidney, heart, and brain of CD38 KO mice, the levels of cADPR are comparable to those in the wild-type controls, indicating the presence of a non-CD38 novel ADP-ribosyl cyclase. In fact, it has been recently demonstrated that a novel, regulatable ADP-ribosyl cyclase is expressed in brain homogenates prepared from CD38 KO mice (29). Studies from our laboratory using smooth muscle membranes of airways and uterus have demonstrated that CD38 possesses ADP-ribosyl cyclase activity (11, 25). In whole lung homogenates obtained from CD38 KO mice, there is no measurable ADP-ribosyl cyclase activity, suggesting that CD38 is the primary constitutively active cyclase expressed in the lungs. In the present as well in our previous (18) studies, we have detected very low levels of cADPR in lung homogenates obtained from the CD38 KO mice, suggesting that an alternate producer of cADPR may be present at very low levels in the lung. However, other cADPR activating mechanisms are unlikely to be involved in the intracellular Ca²⁺ release in response to ET-1, because the cADPR antagonist does not inhibit the response to ET-1 in cells isolated from the CD38 KO mice.

In summary, airway responsiveness to methacholine in CD38 KO mice was significantly attenuated compared with the wild-type control mice. This finding demonstrates the functional importance of CD38/cADPR signaling in airways. Furthermore, the intracellular Ca²⁺ responses to contractile agonists were diminished in ASM cells from CD38 KO mice, confirming the Ca²⁺ mobilizing role of CD38-generated cADPR in ASM cells. Finally, the lack of inhibition of ET-1–dependent Ca²⁺ responses in cells isolated from CD38 KO, but not from wild-type mice by the cADPR antagonist demonstrates that CD38 is the primary source for cADPR production and no additional cADPR activating mechanisms are involved in ET-1–stimulated ASM cells.

	Acknowledgments

 
The authors thank Dr. Richard Graeff, Soner Dogan and Xin Ge for help.

	Footnotes

 
This study has been supported by grants from the National Institutes of Health (HL57498 to M.S.K., DA11806 to T.F.W., and AI43629 to F.E.L.), and by an Academic Health Center Faculty Development Grant to M.S.K. and T.F.W.

Conflict of Interest Statement: D.A.D. has no declared conflicts of interest; T.A.W. has no declared conflicts of interest; A.G.P.G. has no declared conflicts of interest; C.M. has no declared conflicts of interest; T.F.W. has no declared conflicts of interest; F.E.L. has no declared conflicts of interest; and M.S.K. has no declared conflicts of interest.

^* Contribution of these authors to the study was equal.

Received in original form July 29, 2004

Received in final form November 9, 2004

	References

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 

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