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J. Biol. Chem., Vol. 281, Issue 45, 34312-34323, November 10, 2006

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Integrin Ligands Mobilize Ca²⁺ from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary Arterial Smooth Muscle Cells^*

Anita Umesh, Michael A. Thompson, Eduardo N. Chini, Kay-Pong Yip^¶, and James S. K. Sham¹

From the Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, the Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota 55905, and the ^¶Department of Physiology and Biophysics, University of South Florida, Tampa, Florida 33612

Received for publication, July 17, 2006 , and in revised form, September 8, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca²⁺ levels ([Ca²⁺]_i). Information on the Ca²⁺ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca²⁺]_i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins ₁, ₂, ₃, ₄, ₅, ₇, ₈, _v, ₁, ₃, and ₄, and immunoblots of ₅, _v, ₁, and ₃ confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca²⁺]_i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca²⁺ response was unrelated to Ca²⁺ influx and the inositol triphosphate receptor-gated Ca²⁺ store, but partially blocked by ryanodine or inhibition of lysosomerelated acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca²⁺ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca²⁺ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca²⁺ signaling in regulating PASMC function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular matrix (ECM)² protein receptors, or integrins, comprise a superfamily of structurally related heterodimeric transmembrane receptors that mediate cell-cell and cell-ECM interactions. Integrins physically bridge the ECM and cytoskeleton, and act as transducers of "outside-in" and "inside-out" signaling (1). Extracellular integrin ligation changes [Ca²⁺]_i in a variety of cell types, including platelets, neutrophils, monocytes, lymphocytes, fibroblasts, endothelial cells, osteoclasts, neurons, glomerulosa cells, epithelial and smooth muscle cells (2-5). Integrin ligation also activates other intracellular signaling molecules, including H⁺ and a plethora of protein kinases such as the focal adhesion kinase, Rac, and extracellular signal regulated kinases (1, 6). As a result, a myriad of cellular functions such as differentiation, proliferation, migration, apoptosis, and mechanosensing for shear and tension are affected (6, 7).

Integrins participate in controlling systemic vascular tone by invoking changes in smooth muscle [Ca²⁺]_i, resulting in relaxation or contraction. Synthetic ligands carrying the prototypic integrin-binding tripeptide motif, arginine-glycine-aspartate (RGD), decrease [Ca²⁺]_i causing vasodilation in rat cremaster muscle arterioles (8, 9). On the other hand, RGD-containing peptides constrict rat renal afferent arterioles through increased [Ca²⁺]_i (10, 11). Other integrin binding sequences also exist, such as leucine-aspartate-valine (LDV), which elevates [Ca²⁺]_i and causes vasoconstriction of rat cremaster muscle arterioles (12). Integrins ₅₁ and _v3 are, furthermore, directly involved in the vascular myogenic behavior, where blockade of either integrin inhibits myogenic constriction of skeletal muscle arterioles (13).

Apart from regulating tone, integrins play an important role in vascular pathogenesis. This is evidenced by the remodeling and deposition of ECM components seen in various vascular diseases such as atherosclerosis, hypertension, and restenosis (5). In the pulmonary vasculature, remodeling occurs in chronic obstructive pulmonary disease, asthma, scleroderma, and pulmonary hypertension (14). Interestingly, pulmonary vascular remodeling has been linked to integrin activation in rat models recapitulating pulmonary hypertension. Previous studies in monocrotaline-induced pulmonary hypertension show that degraded ECM components activate vascular smooth muscle cell integrin ₃, causing focal adhesion formation and initiation of the extracellular signal-regulated kinase signal transduction cascade (15). Incidentally, the increased PASMC proliferation and medial hypertrophy related to pulmonary hypertension also involves altered [Ca²⁺]_i homeostasis in PASMCs (16).

The influence of integrins on [Ca²⁺]_i homeostasis in governing systemic vascular tone, and the participation of integrins in pulmonary vascular remodeling suggest that integrin ligation would affect [Ca²⁺]_i homeostasis in PASMCs. However, information to this end is lacking. In this article, we therefore test this hypothesis by monitoring [Ca²⁺]_i levels in rat PASMCs upon exposure to soluble integrin ligands. We subsequently characterize the intracellular Ca²⁺ signaling cascade initiated by GRGDSP, and identify the ryanodine-sensitive Ca²⁺ stores and lysosome-related acidic organelles as the key mediators of this response.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Isolation and Culture of PASMCs—PASMCs were enzymatically isolated and transiently cultured as previously described (17). Briefly, lungs were removed from male Wistar rats (150-200 g) anesthetized with sodium pentobarbital (130 mg/kg intraperitoneal), upon which intrapulmonary arteries were dissected in HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 10 HEPES, 10 glucose (pH 7.4). Pulmonary arteries were carefully cleaned of connective tissue, and the endothelial layer removed by gently rubbing the luminal surface with a cotton swab. Arteries were incubated in ice-cold HBSS (30 min), and then in reduced Ca²⁺ (20 µM) HBSS (20 min, room temperature), upon which they were digested in reduced Ca²⁺ HBSS containing collagenase (Type I, 1750 units/ml), papain (9.5 units/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM), at 37 °C for 18 min. After washing with Ca²⁺-free HBSS, single smooth muscle cells were gently dispersed from the tissues by trituration in Ca²⁺-free HBSS, and cultured (16-24 h, 37 °C, 5% CO₂) on 25-mm glass coverslips in Ham's F-12 medium (with L-glutamine) supplemented with 0.5% fetal calf serum, 100 units/ml streptomycin, and 0.1 mg/ml penicillin.

RNA Isolation and RT-PCR—Intralobar pulmonary arteries and aorta were removed, cleaned of connective tissue, denuded of endothelium as above, frozen in liquid nitrogen, and kept at -80 °C until use. Tissues were homogenized in TRIzol reagent (Invitrogen) using a PowerGen homogenizer (Fisher Scientific), and total RNA was isolated according to the protocol supplied with the TRIzol reagent. Ethanol-precipitated RNA samples were dissolved in diethyl pyrocarbonate-treated water and quantified using a BioPhotometer spectrophotometer (Eppendorf, Hamburg, Germany).

Total RNA samples were subsequently used for cDNA synthesis with SuperScript II (Invitrogen). Primers for PCR (Table 1) were carefully designed to regions specific for each integrin subtype, based on sequence alignments generated by a Clustal W algorithm. At least one of each primer pair was designed to exon-exon junctions, to minimize the possibility of amplifying genomic DNA. Where it was not feasible to design primers to exon-exon junctions, primers were designed to span intronic regions, to differentiate between genomic DNA and cDNA. PCR was carried out for 30 cycles using PlatinumTaq DNA polymerase (Invitrogen), which involved denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. The resulting RT-PCR products were analyzed by 1.5% agarose gel electrophoresis.

Protein samples were analyzed by SDS-PAGE and Western blot. Samples were treated with Laemmli sample buffer with or without -mercaptoethanol (100 °C, 5 min), separated by an 8% polyacrylamide gel at a concentration of 20 µg/lane, and then electrotransferred onto Immobilon P membranes (0.45 mm, Millipore) using a tank transfer system (80 V, 3 h, 4 °C). Upon blocking (1 h, room temperature) with 5% skim milk in PBS containing 0.05% Tween 20 (PBST), membranes were incubated with primary antibodies diluted in PBST containing 1% bovine serum albumin (bovine serum albumin/PBST) at 4 °C overnight. The following primary antibodies were used: ₅ (1:1000, catalog number AB1949, Chemicon International, Temecula, CA); _v (1:250, catalog number 611012, BD Biosciences, San Diego, CA); ₁ (1:2000, catolog number AB1952, Chemicon International); and ₃ (1:500, catalog number 4702, Cell Signaling, Beverly, MA). After washing in PBST, membranes were incubated with horseradish peroxidase-coupled donkey anti-rabbit or sheep anti-mouse secondary antibodies (Amersham Biosciences) diluted in 1% bovine serum albumin/PBST (1 h, room temperature), again washed extensively, and ultimately detected using enhanced chemiluminescence (Amersham Biosciences). The anti-CD38 immunoblot was performed essentially as described above, except that for the negative control, the antibody (1:200, catalog number sc-7049, Santa Cruz Biotechnology, Santa Cruz, CA) was preadsorbed with the antigenic peptide (5 times excess by weight, sc-7049P, Santa Cruz) prior to use (overnight, 4 °C).

Calcium Imaging—PASMCs were loaded with Fluo-3 AM (10 µM) dissolved in Me₂SO containing 20% pluronic acid for 45 min at room temperature (Molecular Probes, Eugene, OR). Upon washing thoroughly with Tyrode solution containing (in mM) 137 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 D-glucose, and 10 NaHEPES (pH 7.4), the cytosolic dye was allowed to de-esterify for 20 min at room temperature.

Laser scanning confocal microscopy was used to monitor the fluorescence changes upon treatment of PASMCs with the respective agents. Images were acquired with a Zeiss LSM-510 inverted confocal microscope (Carl Zeiss Inc., Germany) using a Zeiss Plan-Neofluor x40 oil immersion objective (NA = 1.3) and an excitation wavelength of 488 nm. Fluorescence, measured at wavelengths >505 nm, was acquired in a framescan mode at either 5-(Figs. 3, 4, 5 and 6) or 10-s (Fig. 7) intervals. Photobleaching and laser damage to cells were minimized by attenuating the laser to 1% of its maximum power (25 milliwatts). In all cases during fluorescence recordings, reagents were applied manually but with care, so as not to disturb the cells or cause an artificial change in fluorescence.

View larger version (100K): [in this window] [in a new window]   FIGURE 1. RT-PCR shows the presence of multiple integrin subtypes in pulmonary artery (A) and aortic (B) smooth muscle. Table 1 gives the specifics of the primers used, as well as the expected sizes, which correspond with each of the products obtained in this figure. All primers were tested in other tissue types to verify their capability in efficiently and robustly amplifying the product of the expected size. This figure is a representative image of an RT-PCR, which was repeated a total of 3 times.

Changes in fluorescence intensity were used to calculate the cellular concentrations of Ca²⁺, using the following equation: [Ca²⁺]_i = (K_D·R)/[(K_D/[Ca²⁺]_rest) + 1 - R], where R is F/F₀, K_D of Fluo-3 is 1.1 µM, and the resting [Ca²⁺] ([Ca²⁺]_rest) is 100 nM. Changes in [Ca²⁺]_i (i.e. [Ca²⁺]_i) were the differences between the calculated values and the initial [Ca²⁺]_i at rest (i.e. 100 nM) (18). Concentrations of Ca²⁺ were similarly calculated for data obtained with the epifluorescence microscope, upon obtaining a background fluorescence value in an area devoid of cells by using the Ca²⁺ ionophone 4-Br-A23187 (EMD Biosciences) followed by Mn²⁺ quenching.

Imaging Lysosomes—Acidic organelles were labeled with the acidotropic dye Lysotracker Red DND-99 (Molecular Probes) diluted in Tyrode's solution (50 nM) at room temperature for 30-45 min. Labeled PASMCs were imaged with a Zeiss LSM-510 inverted microscope as described above, but with excitation and emission wavelengths of 543 and >560 nm, respectively. Images were acquired in framescan mode at 2-min intervals, and cellular changes in fluorescence intensity were represented as F/F₀, with F₀ being the fluorescence level immediately preceding treatment with the respective agent.

Detection of Cyclic ADP-ribose (cADPR)—cADPR levels were measured in cultured PASMCs at passage 3-4. Cells were isolated and cultured in Ham's F-12 media overnight as described above, and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum thereafter. Prior to treatment with GRGDSP (1.25 mM), cells were washed three times with PBS, and equilibrated in Tyrode's solution for at least 30 min. Upon GRGDSP treatment, nucleotides were extracted from cells with 10% (w/v) trichloroacetic acid at 4 °C, followed by removal with watersaturated diethyl ether. The aqueous layer containing cADPR was adjusted to pH 8 with 1 M Tris and contaminating nucleotides other than cADPR were removed with a mixture containing hydrolytic enzymes with the following final concentrations: 0.44 unit/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline phosphatase, 0.0625 unit/ml NADase, 2.5 mM MgCl₂. Incubation proceeded at 37 °C for 2 h. Detection of cADPR was performed with some modifications to the cycling method described recently (19). Briefly, 0.1 ml of cADPR standard or nucleotides extracted from cell samples were incubated with 100 µl of cycling reagent containing (final concentrations) 0.3 µg/ml ADP-ribosyl cyclase, 4 mM nicotinamide, 100 mM sodium phosphate (pH 8), 0.8% (v/v) ethanol, 40 µg/ml alcohol dehydrogenase, 8 µM resazurin, 0.04 units/ml diaphorase, and 4 µM flavin mononucleotide. Following a 2-h incubation at room temperature, the increase in resorufin fluorescence was measured using a Spectramax XPS fluorescent plate reader with excitation and emission wavelengths of 544 and 590 nm, respectively (Molecular Devices, Sunnyvale, CA). Results are the averages of three to four independent experiments and are normalized to the value at time 0.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Representative image of immunoblot analysis of pulmonary artery and aortic smooth muscle. Total protein (20 µg/lane) treated with Laemmli sample buffer either with (+) or without (-) -mercaptoethanol, were separated by 8% SDS-PAGE and subsequently analyzed for integrin immunoreactivity. Antibodies to integrin 5, v, 1, and 3 were used as indicated under "Experimental Procedures."

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Order of potency of RGD peptides in eliciting PASMC [Ca2+]i transients. A, four different RGD peptides (GRGDSP, GRGDNP, GRGDTP, and cyclo-RGD) and 2 control peptides (GRGESP and GRADSP) were applied (0.5 mM) at time = 50 s and changes in fluorescence were recorded and expressed as changes in [Ca2+]i. B, calculated changes in peak levels of [Ca2+]i are summarized. Only GRGDNP and GRGDSP significantly elevated [Ca2+]i levels (asterisks, p < 0.05). Data acquired from the following numbers of cells from 6 to 8 separate dishes were averaged to obtain the values in the figure: 82, 89, 61, 21, 76, and 62 for GRGDNP, GRGDNP, GRGDTP, cyclo-RGD, GRGESP, and GRADSP, respectively. Cells were prepared from individual animals, and were repeated in cells obtained from two to four replicate animals.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Integrin Expression in PASMCs—To determine the repertoire of integrins expressed in endothelium-denuded rat pulmonary arteries, RT-PCR was performed using primers designed to integrins ₁, ₂, ₃, ₄, ₅, ₇, ₈, _v, ₁, ₃, and ₄ (Table 1). Fig. 1 shows a representative image of an RT-PCR, in which positive mRNA expression was observed for all integrin subtypes examined (Fig. 1a). We further compared the integrin expression profile to that in endothelium-denuded aorta (Fig. 1b). As illustrated in Fig. 1, the pattern of integrin expression was similar in both tissue types, except that integrin ₄ consistently could not be detected in the aortic smooth muscle. Sizes of the PCR products obtained in all cases corresponded to that expected (see Table 1). All PCR primers were tested prior to use in other tissues (e.g. heart, small intestine, and brain) and shown to efficiently amplify products of the expected size (data not shown). Negative controls, in which the reverse transcriptase was omitted, yielded no PCR products (data not shown).

We focused our attention on a few integrin subtypes that have been characterized most extensively in other smooth muscles and examined the protein expression in endothelium-denuded pulmonary arteries and aorta. Western analysis of integrins ₅, _v, ₁, and ₃ confirmed the presence of these proteins in both tissue subtypes (Fig. 2). Immunoblots were performed using both reduced and nonreduced protein samples, as certain antibodies were generated against reduced or non-reduced antigens. Some integrin subunits including ₅ and _v contain a disulfide-linked cleavage site at the C terminus of the protein, yielding 2 polypeptides in the presence of reducing agents (20). Subunits have internally disulfide-bonded cysteine-rich domains in the C termini that influence mobility on SDS-PAGE gels depending on the presence of reducing agents (21). A number of antibodies from various companies were tested for each of the integrin subtypes, and those giving the most specific products of the expected size were chosen. The anti-₅ antibody recognized a broad band of 150 kDa under non-reducing conditions in both PA and aorta, but was not reactive with the reduced protein as indicated by the manufacturer. Two bands of about 120 and 150 kDa were immunoreactive to the anti-_v antibody, presumably representing the reduced and non-reduced forms, respectively, because the 150-kDa band was more prominent under nonreducing conditions. A single band of about 120 kDa was detected by anti-₁ in both tissues regardless of the presence of reducing agents, and a single 100-kDa band was recognized by the anti-₃ antibody only under reducing conditions.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Concentration dependence of [Ca2+]i response to GRGDSP. A, time course of the change in [Ca2+]i evoked by the application of 3 different (0.5, 1.25, and 2.5 mM) GRGDSP concentrations. GRGDSP was applied at time = 50 s. B, comparison of [Ca2+]i levels induced upon exposure to 0.5, 1.25, and 2.5 mM GRGDSP at 7.5 min post-application. Values are the average of data acquired from individual cells in multiple dishes for experiments (i.e. 0.5 mM, n = 89 cells from 8 dishes; 1.25 mM, n = 71 cells from 8 dishes; 2.5 mM, n = 102 cells from 8 dishes). Cells were obtained from individual rats, and experiments were repeated in cells prepared from at least 3 replicate rats.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Ca2+ influx is not involved in the GRGDSP-elicited [Ca2+]i response. The GRGDSP-induced (1.25 mM) Ca2+ response was monitored upon pretreating PASMCs with: A, nifedipine (1 µM, 20 min) to block L-type Ca2+ channels; B, SKF 96365 (30 µM, 30 min) to block non-selective Ca2+ channels; and C, 0 mM Ca2+ Tyrode buffer containing 2 mM EGTA to create conditions of no external Ca2+, to eliminate Ca2+ entry. Arrow represents the time of GRGDSP application. D, peak [Ca2+]i elicited by GRGDSP, showing that Ca2+ influx does not contribute to the GRGDSP-induced response. Values in the figure reflect the average of data acquired from individual cells in multiple dishes. A, control, n = 74 cells from 7 dishes; nifedipine, n = 65 cells from 7 dishes; B, control, n = 46 cells, 7 dishes; SKF, n = 34 cells, 5 dishes; C, control, n = 67 cells, 8 dishes; 0 Ca, n = 60 cells, 7 dishes. Cells were obtained from individual animals, and experiments were repeated using cells prepared from at least 2 different rats.

After establishing that the active motif of integrin ligands indeed elicited [Ca²⁺]_i changes in rat PASMCs, we further characterized the [Ca²⁺]_i effects of GRGDSP, because this is the more commonly occurring sequence in endogenous integrin ligands. The [Ca²⁺]_i response evoked by GRGDSP was concentration-dependent (Fig. 4). A concentration of 1.25 mM GRGDSP was used in all subsequent experiments, as this concentration elicited a consistently robust response.

Characterization of the Source of [Ca²⁺]_i Mobilized by GRGDSP—We proceeded to examine pharmacologically the source of Ca²⁺ mobilized by GRGDSP in PASMCs (Figs. 5, 6 and 7). To determine whether the GRGDSP-induced rise in [Ca²⁺]_i resulted from extracellular Ca²⁺ influx through L-type Ca²⁺ channels, PASMCs were treated with nifedipine (1 µM) for 20 min prior to GRGDSP application (Fig. 5A). Compared with time-matched controls ([Ca²⁺]_i = 200.9 ± 12.9 nM, n = 74 cells), the GRGDSP elicited changes in [Ca²⁺]_i did not differ significantly with nifedipine treatment ([Ca²⁺]_i = 171.6 ± 15.7 nM, n = 65 cells, p > 0.05). Treatment of PASMCs with SKF96365 (30 µM, 20 min), a non-selective Ca²⁺ channel blocker inhibiting both L-type Ca²⁺ channels and capacitative Ca²⁺ entry, also had no significant effect on the GRGDSP-induced [Ca²⁺]_i increase (Fig. 5B). GRGDSP caused [Ca²⁺]_i elevations of 142.7 ± 16.6 nM in SKF 96365-treated cells (n = 34 cells), compared with 136.7 ± 14.9 nM (n = 46 cells; p > 0.05) in time-matched controls. To further confirm that GRGDSP was not causing Ca²⁺ entry, PASMCs were exposed to the peptide under nominally Ca²⁺-free conditions (Fig. 5C). Elimination of extracellular Ca²⁺ altogether did not affect the GRGDSP-induced Ca²⁺ response in PASMCs when compared with time-matched controls, where [Ca²⁺]_i was increased by 194.5 ± 13.6 and 223.1 ± 20.1 nM in control (n = 67 cells) versus EGTA-treated cells (n = 60 cells), respectively (p > 0.05).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. GRGDSP mobilizes [Ca2+]i from ryanodine and bafilomycin A1-sensitive stores, but not the IP3 receptor-gated store. PASMCs were exposed to GRGDSP (1.25 mM) upon pretreatment with: A, ryanodine (50 µM, 30 min); B, Xestospongin C (10 µM, 30 min); and C, bafilomycin A1 (3 µM, 30 min). Arrows in A and B indicate the time of GRGDSP application, but reflects the time of bafilomycin A1 treatment in C. GRGDSP treatment in C is illustrated by the horizontal bar. Inset shows averaged [Ca2+]i levels upon stimulation with the Ca2+ ionophore 4Br-A23187 in the presence of 10 mM Ca2+. Cells were challenged with the ionophore after stimulation with GRGDSP ± bafilomycin A1. Note that the y axis scale ([Ca2+]i) reaches the micromolar range, indicating that Fluo-3 fluorescence intensity was not saturated by treatment of PASMCs with GRGDSP ± bafilomycin A1. D, summary of peak [Ca2+]i response elicited by GRGDSP application (compared with [Ca2+]i immediately preceding GRGDSP application) as a percent of control. Values represent the average of data acquired from individual cells in multiple dishes for experiments in A and B, and the average of data acquired from individual dishes in C. A, control, n = 106 cells from 12 dishes; ryanodine: n = 115 cells from 12 dishes; B, control, n = 103 cells, 12 dishes; Xestospongin C, n = 105 cells, 12 dishes; C, control, n = 11 dishes; bafilomycin, n = 12 dishes. Experiments were repeated using cells prepared from three individual animals. Asterisks indicate p < 0.10 relative to the values without blocker treatment.

It has recently been shown that in addition to ryanodine and IP₃ receptor-gated intracellular Ca²⁺ stores, that PASMCs contain an active intracellular Ca²⁺ store of lysosomal origin that is sensitive to nicotinic acid adenine dinucleotide phosphate (NAADP), but insensitive to thapsigargin treatment (24, 25). To test the possibility that GRGDSP was promoting Ca²⁺ release through a lysosome-related acidic organelle, we treated the PASMCs with bafilomycin A1 (Fig. 6C). Bafilomycin A1 is a macrolide antibiotic that selectively inhibits the vacuolar H⁺-ATPase (V-ATPase) at nanomolar to low micromolar concentrations, and the P-type ATPases at high micromolar concentrations (26, 27). It potently inhibits both ATPase and H⁺ pumping activities of the V-ATPase, causing dissipation of the proton gradient that energizes Ca²⁺ uptake into these compartments, thereby inhibiting the NAADP-sensitive Ca²⁺ activity (26-29). As reported by Kinnear et al. (24), bafilomycin A1 treatment alone elevated [Ca²⁺]_i in PASMCs, presumably arising from the lysosome-related acidic compartments as a result of inhibiting the lysosomal proton motive force. In addition, the GRGDSP-induced response in the bafilomycin A1-treated cells was significantly reduced to 50.4% of control values ([Ca²⁺]_i (control) = 79.0 ± 15.9 nM, n = 11 dishes versus [Ca²⁺]_i (bafilomycin) = 39.9 ± 11.6 nM, n = 11 dishes, p < 0.1). The GRGDSP-induced [Ca²⁺]_i response was taken to be the peak [Ca²⁺]_i elicited by GRGDSP relative to the [Ca²⁺]_i just prior to GRGDSP application. The inset to Fig. 6C shows the same dishes treated with the Ca²⁺ ionophore 4Br-A23187 upon completion of the experiment. Treatment with 4Br-A23187 caused [Ca²⁺]_i changes in the micromolar range in both the presence and absence of bafilomycin A1, demonstrating that Fluo-3 fluorescence intensity had not been saturated by GRGDSP treatment.

The fact that none of the above pharmacological treatments completely eliminated the response to GRGDSP suggested that multiple Ca²⁺ signaling pathways were involved. This was verified by depletion of both intracellular and extracellular Ca²⁺ sources (Fig. 7A). Upon washing the Fluo-3-loaded cells with 2 mM calcium-Tyrode, thapsigargin (20 µM) was applied under nominally Ca²⁺-free conditions in the presence or absence of bafilomycin A1. After an additional 30 min, caffeine (5 mM) was applied to ensure complete depletion of the ryanodine-sensitive Ca²⁺ store, upon which PASMCs were ultimately exposed to GRGDSP (1.25 mM). The initial peak [Ca²⁺]_i upon application of the 0 Ca/EGTA/thapsigargin/bafilomycin A1 mixture was higher in the presence of bafilomycin A1 ([Ca²⁺]_i = 251.3 ± 8.5 nM, n = 202 cells) compared with that in its absence ([Ca²⁺]_i = 180.6 ± 7.0 nM, n = 160 cells; p < 0.05), presumably due to the additional release of Ca²⁺ by bafilomycin A1 from the acidic organelles. [Ca²⁺]_i levels subsequently decreased below baseline in both populations, but remained higher in the bafilomycin-treated cells ([Ca²⁺]_i =-22.8 ± 0.1 nM, n = 202 cells) in contrast to controls ([Ca²⁺]_i =-43.0 ± 0.1 nM, n = 160 cells, p < 0.05). Comparison of the GRGDSP-elicited peak change in [Ca²⁺]_i to that immediately preceding peptide application (Fig. 7A) established that addition of bafilomycin A1 completely abolished the peak [Ca²⁺]_i from 130.3 ± 7.9 nM in controls (n = 160 cells) to 13.7 ± 6.6 nM (202 cells).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Simultaneous inhibition of ryanodine- and bafilomycin A1-sensitive intracellular Ca2+ stores abolishes the GRGDSP-induced Ca2+ response. A, GRGDSP-induced Ca2+ response in nominally Ca2+-free conditions in which ryanodine and IP3-gated intracellular Ca2+ stores were depleted with thapsigargin (black trace); and lysosome-related acidic organelle was additionally blocked with bafilomycin A1 (gray trace). B, treatment of PASMCs with ryanodine (50 µM) and bafilomycin A1 (3 µM) in the presence of 2 mM extracellular Ca2+ (applied at t = 3 min 20 s; arrow, for 30 min, gray trace). C, bar graph summarizing the peak [Ca2+]i of the GRGDSP-induced response, expressed as a percentage of control (asterisks, p < 0.05). Control values are 130.3 ± 7.9 nM at t = 50 min in A, and 78.0 ± 6.6 nM at t = 30 min in B. Values represent the average of data acquired from individual cells in multiple dishes for experiments in A, and the average of data acquired from individual dishes in B. A, control, n = 160 cells from 8 dishes; bafilomycin, n = 202 cells from 11 dishes; B, control, n = 8 dishes; ryanodine + bafilomycin, n = 9 dishes. Replicates were performed using cells prepared from 4 individual animals.

Effect of GRGDSP on cADPR Levels—The pyridine nucleotide, cADPR, is thought to be the endogenous activator of ryanodine receptors (30). To test whether GRGDSP affected cADPR levels and thereby verify the pharmacological results of Figs. 6A and 7, we measured cADPR directly in cultured PASMCs upon exposure to GRGDSP. In unstimulated PASMCs, the concentrations of cADPR ranged from 0.5 to 2 pmol/mg of protein, with an average of 1.2 ± 0.3 pmol/mg of total protein. Treatment of cultured PASMCs indeed increased cADPR levels as early as 30 s after treatment with GRGDSP (Fig. 8B). Maximum levels were reached by 1 min, and were sustained through 5 min, but were decreased by 45 min post-application. GRGDSP treatment elevated cADPR levels by 70% compared with untreated controls.

CD38 is a transmembrane glycoprotein residing on the plasma membrane that possesses ADP ribosyl cyclase activity (30). Cultured PASMCs were shown to express CD38 via Western analysis (Fig. 8A). Lane 1 shows post-nuclear supernatant of PASMCs probed with the anti-CD38 antibody, whereas lane 2 shows the same protein probed with the antigen-pretreated anti-CD38 antibody. A broad band of 45 kDa (arrow) that is observed in lane 1 is absent in lane 2, indicating the specific band.

View larger version (24K): [in this window] [in a new window]   FIGURE 8. GRGDSP causes elevation of cADPR levels. A, representative immunoblot (IB) showing the presence of CD38 in cultured PASMCs (passage 4). Total protein (25 µg) separated by 10% SDS-PAGE were probed with the anti-CD38 antibody (lane 1) or the anti-CD38 antibody that had been preadsorbed with the antigenic peptide (lane 2). Arrow indicates the presence of a 45-kDa immunoreactive band to the anti-CD38 antibody (lane 1) that is absent upon preadsorption with the antigenic peptide (lane 2). B, cADPR levels in response to GRGDSP (1.25 mM) treatment. Values are normalized to untreated controls at time 0, and reflect the average of 4 separate experiments, except for the 5-min time point that was performed 3 times. Asterisks indicate p < 0.05 relative to normalized values before treatment, and were obtained using a two-tailed t test.

Most intriguingly, treatment of PASMCs with GRGDSP (Fig. 10) affected Lysotracker Red fluorescence intensities in a concentration-dependent manner. The decrease in Lysotracker Red fluorescence elicited by GRGDSP was transient in nature as it showed a partial recovery with time. Effects of photobleaching were evident from control experiments in which vehicle (Tyrode's solution, Fig. 10B) was applied, but significantly differed from the peptide-induced effects (Fig. 10C). The degree to which GRGDSP affected the Lysotracker Red fluorescence, as well as its transient nature differed from the effect elicited by bafilomycin A1 (Fig. 9), which dissipates the lysosomal pH gradient through inhibition of the V-ATPase. These data directly demonstrate the modulation of the properties of lysosomerelated acidic organelles by GRGDSP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Here we characterize intracellular Ca²⁺ signaling by integrins in rat PASMCs. We show the presence of multiple integrins in PASMCs, and the initiation of intracellular Ca²⁺ signaling by soluble ligands. The Ca²⁺ response is independent of Ca²⁺ entry and IP₃ receptorgated intracellular Ca²⁺ stores, but dependent on both RyR-gated Ca²⁺ stores and the lysosome-related acidic organelles. We further demonstrate the ability of soluble integrin ligands to directly affect RyR-gated Ca²⁺ stores by increasing levels of cADPR, as well as alter the properties of lysosome-related acidic compartments through Lysotracker Red imaging. These data illustrate a novel mode of Ca²⁺ regulation by integrins, and underscore both the RyR-gated Ca²⁺ store and lysosome-related acidic organelle as important players in influencing intracellular Ca²⁺ homeostasis in PASMCs.

We examined the largest number of integrin subtypes expressed in native rat PASM, and detected ₁, ₂, ₃, ₄, ₅, ₇, ₈, ₁, ₃, and ₄. To date, integrins ₁, ₂, ₃, ₄, ₅, ₆, ₇, ₈, ₉, _v, ₁, ₃, ₄, and ₅ have been identified in vascular smooth muscle cells (5). A few studies have probed for integrin expression in the pulmonary vasculature (31-39). Our data largely corroborate previous reports showing the presence of ₁, ₂, ₃, _v, and ₃ in human PSM, as well as in the rat SMC line PAC-1, which possesses ₁, ₃, ₄, ₅, ₇, _v, and ₁ (33, 34, 36, 38). The presence of ₈, _v, and ₃ is also consistent with that reported in rat lung tissues (32, 35, 37, 39). The lack of ₄ and ₅ in human PSM disagree with this study as well as those in mouse and PAC-1, presumably due to species differences (31, 33, 34). Another minor difference is the absence of ₂ in PAC-1, perhaps due to the effects of cell culture (34). Of all subtypes examined, only aortic ₄ was repeatedly negative. This is reminiscent of the aortic SMC line, suggesting subtle differences in integrin expression between pulmonary and systemic arteries (34).

Of the vascular smooth muscle integrins, ₄₁, ₅₁, _v3, and _v5 have thus far been implicated in the regulation of vascular tone, in which soluble and insoluble ligands elicit [Ca²⁺]_i changes to modulate contractile properties (5). For example, soluble cyclo-RGD causes an _v3-dependent decrease in [Ca²⁺]_i and subsequent vasodilation of rat cremaster arterioles. In rat renal afferent arterioles, soluble GRGDSP increases SMC [Ca²⁺]_i resulting in vasoconstriction (8-11). Ligation of ₅₁ by insoluble ligands also increases [Ca²⁺]_i in rat skeletal SMCs (40).

View larger version (59K): [in this window] [in a new window]   FIGURE 9. Bafilomycin A1 causes loss of organellar acidification in lysosome-related organelles of PASMCs. A, representative image of bafilomycin A1 (lower panel) disrupting the pH gradient of acidic organelles at 0, 2, and 30 min post-treatment, as compared with treatment with vehicle containing Me2SO (upper panel). Images were visualized by staining with Lysotracker Red. B, average Lysotracker Red fluorescence intensities over time. Time 0 indicates the point immediately prior to addition of vehicle or bafilomycin A1 (arrow). Dashed lines correspond to the time points shown in the images of A. C, bar graph summarizing the average fluorescence intensities at time 2 and 30 min post-treatment. Treatment of PASMCs with bafilomycin A1 caused an approximate 80% decrease in the average Lysotracker Red fluorescence intensity. Values are the average of data acquired from individual cells in 5 separate dishes (i.e. vehicle: n = 46 cells; bafilomycin n = 68 cells). Replicates were performed using cells prepared from 2 individual animals. Asterisks indicate significant difference (p < 0.05) between vehicle and bafilomycin Al-treated cells.

Integrin ligation causes [Ca²⁺]_i changes in many cell types, but the source of Ca²⁺ mobilized is both integrin and cell-type specific. For example, endothelial integrin ligation causes Ca²⁺ influx and Ca²⁺ release from IP₃-sensitive intracellular stores (42, 43). In Jurkat T cells, integrin ligation activates IP₃ and cADPR-sensitive stores as well as capacitative Ca²⁺ entry (44). Smooth muscle Ca²⁺ signaling by integrins has as yet only been studied in rat cremaster arterioles and renal SMCs. In rat cremaster arterioles, integrins differentially regulate the L-type Ca²⁺ channel, where ₄₁ and ₅₁ activate and _v3 inhibits the L-type Ca²⁺ channels, causing vasoconstriction and vasodilation, respectively (8, 12, 40). Stimulation of rat renal vascular smooth muscle cells by soluble GRGDSP causes Ca²⁺ release entirely from RyR-sensitive stores (10, 11).

Our investigations into the source of Ca²⁺ mobilized by GRGDSP offer unique information on signaling by integrins. Specifically, Ca²⁺ entry and IP₃-sensitive Ca²⁺ stores are uninvolved, whereas inhibition of the RyR-gated Ca²⁺ store and the lysosome-related acidic organelle each reduce the GRGDSP-mediated Ca²⁺ response by 50%. Furthermore, simultaneous inhibition of the RyR-gated Ca²⁺ store and the lysosome-related acidic organelle is essential to completely abolish the GRGDSP-induced response.

Ryanodine receptor-gated intracellular Ca²⁺ stores have been implicated pharmacologically as contributing to integrinmediated [Ca²⁺]_i increases, as in the case of rat renal vascular smooth muscle cells, cardiomyocytes, and T cells (10, 11, 44, 45). However, our study is the first to directly demonstrate that integrin binding peptides increase the levels of cADPR, which is the endogenous activator of RyRs. It has been well established that cADPR contributes to the regulation of arterial smooth muscle tone by altering [Ca²⁺]_i homeostasis. In the pulmonary vasculature, cADPR plays important roles in the development of hypoxic pulmonary vasoconstriction (46). The resting cADPR levels obtained in this study from cultured PASMCs (1.2 pmol/mg of protein) is in agreement with that reported in the PASM tissue (1.5 pmol/mg of protein) (47). These concentrations are also similar to those described in airway smooth muscle (0.8 pmol/mg of protein) as well as uterine smooth muscle (1 pmol/mg of protein) (48-50). GRGDSP causes increases in cADPR levels as early as 30 s post-application. This time course is consistent with that observed for [Ca²⁺]_i measurements, where [Ca²⁺]_i levels were on the rise by 30 s, and increased further at 1 and 5 min. The [Ca²⁺]_i at 5 min post-GRGDSP treatment is generally double of that observed at 1-min post-application. This differs from that seen for [cADPR], whose normalized values vary only by 5% between 1 and 5 min post-treatment, and can be due to a contribution of the cADPR-independent, bafilomycin-sensitive intracellular Ca²⁺ store.

View larger version (32K): [in this window] [in a new window]   FIGURE 10. GRGDSP affects lysosome-related acidic organelles in PASMCs in a concentration-dependent manner. A, representative images of the effect of control (Tyrode's solution), 1.25 mM GRGDSP, and 5 mM GRGDSP on the Lysotracker Red staining of PASMCs before and 6 min after treatment. B, average Lysotracker Red fluorescence intensities over time. Time 0 indicates the point immediately prior to treatment (arrow). Dashed lines correspond to the time points shown in the images of A. An effect of dye photobleaching was seen in control experiments. C, bar graph summarizing the average fluorescence intensities at 6 min post-treatment. Values are the average of data acquired from individual cells in multiple dishes. Control, n = 51 cells from 7 dishes; 1.25 mM GRGDSP, n = 63 cells from 7 dishes; 2.5 mM GRGDSP, n = 58 cells, 7 dishes; 5 mM GRGDSP, n = 73 cells from 6 dishes. Replicates were performed using cells prepared from at least 3 individual animals. Asterisks, significant difference from control, p < 0.05.

The sensitivity of the GRGDSP-elicited [Ca²⁺]_i response to ryanodine and bafilomycin A1 is consistent with that observed by Evans and colleagues (25). They have proposed that NAADP evokes spatially localized "calcium bursts" that trigger RyR-mediated Ca²⁺-induced Ca²⁺-release through "lysosome-SR" junctions, because RyR inhibition eliminates the global Ca²⁺ transients without affecting Ca²⁺ bursts (24, 25). However, our results cannot be explained entirely by this model, because thapsigargin treatment or inhibition of RyRs alone did not eliminate the global [Ca²⁺]_i increase. Rather, in our preparation the RyR-gated Ca²⁺ store and lysosome-related acidic compartments each contributed to half of the GRGDSP-induced Ca²⁺ response, and had to be inhibited together to abrogate the response. Whereas it is conceivable that these two sources of intracellular Ca²⁺ may be functionally coupled, GRGDSP does not seem to evoke Ca²⁺-induced Ca²⁺ release from either store. Instead our results suggest that it activates Ca²⁺ release from these two stores via independent pathways.

Based on the fact that: 1) bafilomycin A1 reduces the intracellular Ca²⁺ response to GRGDSP, and 2) both bafilomycin A1 and GRGDSP directly affect the properties of acidic organelles as visualized by Lysotracker Red imaging, we can conclude that GRGDSP stimulates lysosome-related organelles. The seemingly transient nature of the GRGDSP-induced decrease in Lysotracker Red fluorescence (Fig. 10B) suggests that it causes a loss of organelle acidification, rather than disrupting the integrity of the lysosome as in the case of compounds such as glycyl-L-phenylalanine 2-napthylamide (58, 59). A correlation between lysosomal pH and lysosomal [Ca²⁺] ([Ca²⁺]_lys) has been shown in macrophages, where alkalinization of lysosomes resulted in proportional decreases in [Ca²⁺]_lys (28). The mechanism of [Ca²⁺]_lys regulation in macrophages was likened to that in yeast vacuoles, in which the V-ATPase maintains a proton gradient that in turn drives [Ca²⁺]_lys uptake through a Ca²⁺/H⁺ exchanger in the lysosomal membrane (28). The fact that GRGDSP reduces the Lysotracker Red fluorescence therefore suggests a proportional decrease in [Ca²⁺]_lys, which is accompanied by a transient alkalinization of the lysosome. Because bafilomycin A1 also promotes lysosomal alkalinization, it is possible that pretreatment of PASMCs with bafilomycin A1 (Figs. 6C and 7B) causes concomitant [Ca²⁺]_lys depletion, such that this Ca²⁺ source is no longer available for stimulation by GRGDSP. Given that NAADP evokes Ca²⁺ release from lysosome-related acidic organelles in a bafilomycin A1-sensitive manner, we hypothesize that GRGDSP mobilizes lysosomal Ca²⁺ in part via NAADP (24, 25, 29, 52). However, we have yet to establish a direct link between GRGDSP treatment and induction of the NAADP pathway. Future studies on the change in cellular NAADP concentrations upon GRGDSP treatment should address this pressing issue.

This study raises interesting questions regarding the mechanism of integrin-mediated Ca²⁺ signaling. In particular, how does integrin activation couple to Ca²⁺ mobilization from both RyR-gated stores and acidic organelles? One plausible explanation is that GRGDSP activates ADP-ribosyl cyclase. ADP-ribosyl cyclase synthesizes both cADPR and NAADP from -NAD and -NADP, respectively, and its activation by GRGDSP may thus explain the near equivalent contribution of these two Ca²⁺ stores to the peptide-induced response (60). High levels of ADP-ribosyl cyclase activity have in fact been reported in the PASM tissue (47). We have also evidenced robust levels of ADP-ribosyl cyclase activity in cultured PASMCs, using NGD⁺ as an alternative substrate to measure cyclic GDP ribose formation (data not shown), and have, furthermore, shown the expression of CD38 in these cells (this study). The lung is among a group of tissues containing a novel enzyme with ADP-ribosyl cyclase activity in addition to CD38 (61-63). This notion is supported by data from CD38^-/- mice containing an appreciable amount of cADPR in the lung albeit at lower levels than in the wild type, as well as the fact that cADPR synthesis has been measured in microsomal fraction of PASM homogenates (47, 63, 64).

Stimulation by GRGDSP may result in Ca²⁺ mobilization from the RyR-gated stores and acidic organelles, alternatively, by causing integrin clustering and subsequent protein recruitment to form focal adhesion complexes, which tether integrins to the cytoskeleton. Many components of the focal adhesion complex are either substrates of tyrosine kinases or tyrosine kinase themselves, and are activated upon integrin ligation in various cell types, including endothelial and vascular smooth muscle cells (65, 66). Moreover, in rat cremaster arterial cells, [Ca²⁺]_i modulation through the activation of tyrosine kinase pathways has been shown for ₄₁ and ₅₁ stimulation (12, 67).

In summary, we have demonstrated the modulation of [Ca²⁺]_i in PASMCs by integrin agonists. The information obtained adds a level of complexity in PASMC regulation, uncovering a unique mode of Ca²⁺ signaling by integrins in which RyR-gated Ca²⁺ stores and acidic organelles are recruited. Modulation of PASMCs by integrin ligands has important implications in pulmonary diseases including chronic obstructive pulmonary disease, pulmonary fibrosis, and pulmonary hypertension, where ECM remodeling as well as induction of matrix metalloproteinases that degrade ECM molecules is observed (68, 69). ECM remodeling and degradation expose cryptic integrin binding sites including RGD motifs (69). Therefore our work on [Ca²⁺]_i regulation by soluble RGD peptides highlights the importance of the ECM in influencing pulmonary function and may provide valuable insights in the progression of pulmonary disease.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants HL075134 and HL071835 (to J. S. K. S.), a Pulmonary Hypertension Association fellowship (to A. U.), and funds from the American Heart Association (to E. N. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 5501 Hopkins Bayview Circle, JHAAC, Baltimore, MD 21224. Tel.: 410-550-7751; Fax: 410-550-2612; E-mail: jsks{at}welchlink.welch.jhu.edu.

² The abbreviations used are: ECM, extracellular matrix; PASMC, pulmonary arterial smooth muscle cell; RGD, arginine-glycine-aspartic acid; LDV, leucine-aspartic acid-valine; cADPR, cyclic ADP-ribose; HBSS, HEPES-buffered salt solution; PBS, phosphate-buffered saline; PBST, PBS containing Tween 20; IP₃, inositol trisphosphate; RyR, ryanodine receptor; NAADP, nicotinic acid adenine dinucleotide phosphate; V-ATPase, vacuolar H⁺ ATPase; SMC, smooth muscle cell; RT, reverse transcriptase.

	ACKNOWLEDGMENTS

 
We thank Drs. M. J. Lin and X. R. Yang for assistance throughout the course of this work, and Lavanya Balasubramanian for comments on the manuscript.

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