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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 436-445, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0012OC

Regulator of G-Protein Signaling Protein 2 Modulates Purinergic Calcium and Ciliary Beat Frequency Responses in Airway Epithelia

Marie-Christine Nlend, Richard J. Bookman, Gregory E. Conner and Matthias Salathe

Division of Pulmonary and Critical Care Medicine, Department of Molecular and Cellular Pharmacology, and Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida

Address correspondence to: Dr. Matthias Salathe, Division of Pulmonary and Critical Care Medicine (R-47), University of Miami School of Medicine, 1600 NW 10th AVE, RMSB 7063, Miami, FL 33136. E-mail: msalathe{at}miami.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In ciliated airway epithelial cells, purinergic stimulation increases both intracellular calcium ([Ca2+]i) and ciliary beat frequency (CBF). Because regulator of G-protein signaling protein 2 (RGS2) terminates G{alpha}q-mediated phospholipase C activation, we examined its role in regulating purinergic signaling in human and ovine airway epithelial cells. RT-PCR of both human and ovine epithelial cell RNA yielded fragments of expected size (~ 491 bp) and sequence, confirming RGS2 message. Immunofluorescence demonstrated RGS2 protein expression in cultured airway epithelial cells of both species. Overexpression of an EGFP-RGS2 fusion protein (increasing RGS2 protein levels 1.8 times control, n = 28 cells) resulted in a reduced [Ca2+]i and CBF response to 10 µM ATP (human: 58 ± 9% and 49 ± 8% lower, respectively; n = 8 measurements, 4 cells; ovine: 56 ± 12% and 53 ± 16% lower, respectively; n = 5 measurements, 4 cells). Reducing RGS2 protein levels using antisense oligonucleotides increased the response of both [Ca2+]i and CBF to ATP in human cells by 57 ± 10% and 47 ± 11%, respectively (n = 10 measurements, 6 cells), and in ovine cells by 88 ± 13% and 48 ± 9%, respectively (n = 10 measurements, 5 cells). These data provide functional evidence that RGS2 modulates purinergic signaling in human and ovine ciliated airway epithelial cells.

Abbreviations: air–liquid interface, ALI • ciliary beat frequency, CBF • Dulbecco's modified Eagle's medium, DMEM • enhanced green fluorescent protein, EGFP • Hanks' balanced salt solution, HBSS • intracellular calcium concentration, [Ca2+]I • phospholipase C-ß, PLC-ß • regulators of G-protein signaling, RGS • region of interest, ROI • tetramethylrhodamine isothiocyanate, TRITC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Purinergic activation of apical P2Y receptors on airway epithelial cells is one of the strongest stimuli that transiently increase CBF in a variety of mammalian species (1–5). P2Y receptors signal largely through G{alpha}q, thereby stimulating the ß isoform of phospholipase C (PLC-ß) to produce inositol 1,4,5-trisphosphate (6), which in turn mobilizes calcium from internal stores to increase the intracellular calcium concentration ([Ca2+]i). We have shown that transient increases in [Ca2+]i are associated with kinetically similar CBF changes in ovine and, after PKA inhibition, human airway epithelial cells (7–9). Receptor-mediated transient [Ca2+]i increases are tightly controlled in airway epithelial cells, suggesting that such signaling has multiple points of regulation, not all of which are known.

Members of the family of regulators of G-protein signaling (RGS) proteins have been shown to terminate G-protein signaling, and more than 20 such RGS proteins have been identified thus far (10–13). These proteins differ widely in their overall size and amino acid sequence, and thus possess a remarkable variety of structural domains and motifs. But they also share a highly conserved 130 amino acid region that exhibits GTPase-activating activity, binds directly to activated G{alpha} subunits, and effectively ends G{alpha}-mediated signaling. In addition, RGS proteins can attenuate G-protein signaling by directly interfering with G protein/effector interactions (14, 15). Among the known RGS proteins, RGS2, RGS3, and RGS4 bind to and inhibit G{alpha}q-mediated PLC-ß activation (15). RGS2, a small 24 kD protein, has been shown to inhibit G{alpha}q-mediated phosphoinositide hydrolysis ten times more potently than RGS4 in vitro (16). We therefore wondered whether RGS2 represents one of the points of regulation of P2Y-receptor signaling in mammalian airway epithelial cells. We show here that ovine and human ciliated airway epithelial cells express RGS2 and, using direct physiologic measurements, that RGS2 plays a role in regulating purinergically mediated, transient increases in CBF and [Ca2+]i in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Lechner and La Veck medium, stock 4, and stock 11 were purchased from Biosource International (Rockville, MD); Dulbecco's phosphate-buffered saline, Opti-MEM, Ham's nutrient F-12, and Hanks' balanced salt solution (HBSS) from Gibco BRL Laboratories (Grand Island, NY); fura-2/AM from Molecular Probes (Eugene, OR); myristoylated protein kinase A inhibitory peptide 14–22 (PKI14–22) from Calbiochem (La Jolla, CA); rabbit serum from Chemicon (Temecula, CA); tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-goat IgG from Kirkegaard and Perry Laboratories (Gaithersburg, MD); affinity-purified goat polyclonal antibodies raised against a peptide near the carboxy terminus of human RGS2 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); GeneFECTOR from VennNova Inc. (Miami, FL); all other reagents were from Sigma Chemicals (St. Louis, MO).

Preparation of Submerged Human and Ovine Tracheal Epithelial Cultures
Ovine tracheas were purchased from Pel-Freez Biologicals (Rogers, AR) and five different tracheas were used for the experiments. Human tracheas and bronchi were obtained from organ donors whose lungs were deemed not suitable for transplant through the organ procurement team of the University of Miami School of Medicine with appropriate consent procedures approved by the local institutional review board. Cells from four donors were used in this study.

Primary cultures of human and ovine tracheobronchial epithelial cells were prepared as previously described (7, 9). Briefly, the mucosa was dissected from the underlying cartilage under sterile conditions and incubated in 0.05% protease (type XIV; Sigma Chemicals) in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL Laboratories, Grand Island, NY) overnight at 4°C. After protease treatment, epithelial cells were released by vigorous shaking and cells were harvested by centrifugation. The cells were plated on collagen-coated glass coverslips (human placental collagen, type VI) (Sigma Chemicals) at a density of 0.5 x 106 cells/cm2 in a minimal volume of 100 µl/cm2 (1 ml per 35-mm dish). The medium was made of 50% DMEM, 50% Ham's nutrient F-12 supplemented with 10 µg/ml of insulin, 5 µg/ml of transferrin, 0.36 µg/ml of hydrocortisone, 20 ng/ml of triiodothyronine, 7.5 µg/ml of endothelial cell growth supplement, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The media were changed every other day. Cells from these cultures were used for [Ca2+]i and CBF measurements between 3 and 14 d after plating.

Preparation of Air–Liquid Interface Cultures of Human Tracheal Epithelial Cells
Human tracheobronchial epithelial cells, harvested as described above, were plated on collagen-coated plastic dishes, grown to confluence in bronchial epithelial growth medium yielding undifferentiated airway epithelial cells, and passaged after enzyme dissociation with trypsin (17). Cells from passage 1 or 2 were plated onto 24-mm Transwell-clear culture inserts (Corning Costar Corporation, Cambridge, MA), coated with human placental collagen (Sigma). The culture medium was adapted from published methods (17–21) and contained 50% DMEM and 50% LHC basal medium supplemented with insulin (5 µg/ml), hydrocortisone (0.072 ng/ml), epidermal growth factor (0.5 ng/ml), triiodothyronine (T3, 6.5 ng/ml), transferrin (10 µg/ml), epinephrine (0.6 µg/ml), phosphorylethanolamine (0.5 µM), ethanolamine (0.5 µM), bovine pituitary extract (1% vol/vol), bovine serum albumin (0.5 mg/ml), CaCl2 (0.08 mM), trace elements (1x), stock 4 (1x), stock 11 (1x), penicillin/streptomycin (100 µg/ml), and retinoic acid (1 µM). Cells were grown in an incubator at 37°C in ambient air supplemented with 5% CO2. Their apical surface was exposed to air as soon as they reached confluence until they reached full redifferentiation (~ 4 wk); they were then used for experiments.

RT-PCR Analysis of RGS2 mRNA
To investigate expression of RGS2 mRNA in airway epithelial cells, total RNA was extracted either from human and ovine tracheobronchial epithelial cells freshly isolated or from expanded human cells redifferentiated for 28 d at the air–liquid interface (ALI) according to the methods of Chomczynski and Sacchi (22) with Ultraspec (Biotecx Laboratories Inc., Houston, TX). Total RNA (1–3 µg) was used for first-strand synthesis using SuperScript RT (Gibco BRL, Grand Island, NY) and oligo-dT–coupled primers. PCR was performed with specific oligonucleotide primers designed according to the published human RGS2 cDNA sequence (23). These oligonucleotides were: 5'-GAC CCG TTT GAG CTA CTT CTT G (forward) and 5'-CCG TGG TGA TTT GTG GCT TTT TAC (reverse). PCR reactions (35 cycles; 45 s at 95°C; 45 s at 55°C; 1 min at 72°C; followed by an extended elongation of 8 min at 72°C) were performed using Taq DNA polymerase (Gibco BRL). The predicted size of the RT-PCR product was 491 bp. Control reactions were performed in the absence of RT. cDNA libraries, prepared in our laboratory from human and sheep trachea, were also screened by PCR for RGS2 with the same primers.

PCR products were separated on agarose gels containing ethidium bromide and products of expected size (~ 491 bp) were sequenced by the University of Miami DNA Core Laboratory. Obtained sequences were compared with the published RGS2 cDNA sequences (human: GenBank accession #L13463.1; rat: GenBank accession #AY043246.1; mouse: GenBank accession #NM_009061.1) using the Wisconsin Package (GCG, Madison, WI).

Transient Transfections of Airway Epithelial Cells
The plasmid encoding an EGFP-RGS2 fusion protein (based on pEGFP-N3 from Clontech) was a generous gift of Dr. Rory A. Fisher (24). Ovine and human tracheobronchial airway epithelial cells attached to collagen-coated glass coverslips were grown in submerged culture for 2–5 d before transfection. Airway epithelial cells were first incubated in Opti-MEM containing 1 mM EGTA for 20 min at 37°C (25–27). Plasmid DNA (pEGFP-RGS2 or pEGFP-N3 as control) was complexed with liposomes (GeneFECTOR) at room temperature in serum-free OPTI-MEM for 20 min. After washing out EGTA with OPTI-MEM, the cells were transfected with plasmid–liposome complexes for 6 h. Transfection efficiency after 48 h was ~ 30% of ciliated cells (50 cells counted per coverslips) with an average viability > 80% as evidenced by continued ciliary beating.

FITC-labeled morpholino oligonucleotides (RGS2-antisense and 4-base mismatched control oligonucleotides) were designed and ordered from GeneTools (Corvallis, OR). The 25-mer antisense oligonucleotide was targeted to the region of human RGS2 (GenBank accession #L13463.1) from 19 bp before to 6 bp after the start codon (5'-CTTTGCATTATCGTTCTCCCGCTGG). The control oligonucleotide contained four base mismatches (5'-CTTaGCAaTATCGTTCTCgCGCaGG). Cellular delivery of these oligonucleotides was accomplished with ethoxylated polyethylenimine (EPEI), a weakly basic delivery reagent, according to the manufacturer's instructions (GeneTools). After incubation for 3 h, the delivery solution was removed from the cells and replaced with fresh culture medium. Because morpholino oligonucleotides are stable and nuclease resistant, there was no need for redelivery within a 48-h period (28, 29). This was confirmed in our cultures by continued FITC fluorescence in transfected cells over this period.

Immunofluorescence
For immunocytochemical detection of RGS2, transfected and nontransfected airway epithelial cells in submerged culture were fixed for 30 min with 4% paraformaldehyde in phosphate-buffered saline, pH 7.5, permeabilized with 100% methanol twice for 5 min each at –20°C and incubated twice for 5 min each with 50 mM NH4Cl to quench free aldehydes. Rabbit serum (100%) was used as a blocking agent. An affinity-purified, goat polyclonal antibody raised against an internal peptide near the carboxy-terminus of human RGS2 was used (2 µg/ml) as the primary antibody overnight at 4°C. The secondary antibody was a TRITC-conjugated rabbit anti-goat IgG (25 µg/ml). Whole goat serum, undiluted, served as the nonimmune control and competition with the antigenic peptide as the specificity control. Cells were imaged with a Nikon Eclipse E600FN upright microscope equipped with epifluorescence. The excitation filter used for FITC and EGFP fluorescence (assessment of transfection) had a 10-nm passband centered on 480 nm, whereas the one for TRITC was centered on 540 nm (Chroma Technology Corp., Brattleboro, VT). The emitted light was appropriately filtered and recorded for 600 ms through a 60x objective lens with a cooled black and white CCD camera (Quantix; Photometrics, Tucson, AZ), set to the same, fixed gain for all experiments. All immunofluorescence pictures were pseudo-colored using Photoshop (Adobe, San Jose, CA).

CHO-M3 Cell Culture and Transfection
CHO cells stably expressing M3 receptors were a kind gift of Dr. Mark Brann (University of Vermont, Burlington, VT) through the laboratories of Drs. D. Flynn and L. Potter (University of Miami School of Medicine, Miami, FL). CHO cells were plated onto protamine-coated coverslips. The medium consisted of DMEM, supplemented with 10% fetal bovine serum, nonessential amino-acid solution, glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and geneticin (0.5%). The medium was exchanged every other day. The day after plating, cells were transiently transfected with EGFP-RGS2, pEGFP-N3 alone, or cotransfected with pEGFP-N3 and RGS2 (using two different plasmids) using GeneFECTOR. The transfection took place overnight (16–18 h) at 37°C.

CBF Measurements
Human and ovine airway epithelial cells cultured on coverslips in submerged cultures were mounted at room temperature onto the stage of a Nikon Eclipse E600FN upright microscope with a water-immersion lens in a Warner Instrument RC-25F perfusion chamber (Warner Instrument Corporation, Hamden, CT) with 150 µl of working volume. Ciliated cells were imaged with infrared differential interference contrast optics using a total gain of 600x. For online CBF measurements, the light path was directed to a CCD video camera (CCD 100; Dage MIT, Michigan City, IN) and a box of 3 x 3 pixels from the live, digitized, contrast-enhanced video image was selected (where one pixel samples an area of 180 x 180 nm). The magnitude spectra from a fast Fourier transform (FFT) of each of the pixels' intensity signals were computed online and displayed on the monitor for immediate adjustments. The intensity signals, representing the movement of one or a small group of cilia on a single cell, were recorded and later used for analysis according to published methods (8) using a sliding FFT window approach (128 frames per FFT, sliding the FFT window through the data set by 100 frames at a time), providing a frequency resolution of at least 0.23 Hz and a time resolution of ~ 3 s. The individual FFT magnitude spectra were peak extracted for graphing (8).

[Ca2+]I MEASUREMENTS
Human and ovine airway epithelial cells from submerged cultures or CHO-M3 cells were loaded at room temperature with 4 µM fura-2/AM in HBSS, 10 mM HEPES, pH 7.4, containing 2.5% fetal calf serum (Hyclone, Logan, UT) for 60 min on a rocking table. The dishes were washed three times with HBSS/HEPES and used for measurements after a minimum of 30 min.

For [Ca2+]i measurements with fura-2, a lambda DG4 excitation system (Sutter, Novato, CA) was used, which was controlled by Isee software from Inovision, Inc. (Durham, NC). Ratiometric calcium estimates were made using 10-nm-wide filters centered on 340 nm and 380 nm, capturing the emitted light (510–600 nm) at each excitation wavelength for 600 ms with a cooled CCD camera (Quantix; Photometrics). Using Inovision's Isee software, individual cells were identified as regions of interest (ROIs). The fura-2 ratio within each ROI was computed on a pixel-by-pixel basis (after background fluorescence correction). Ratios were computed every 10 s. Average fura-2 ratio values for each ROI were written to disk for later analysis and graphing.

Because we were not able to perform reliable calibrations of the calcium indicator dye fura-2 intracellularly in ciliated cells (cells exposed to 10 µM ionomycin did not change their intracellular calcium concentration when exposed to different extracellular calcium concentrations), conversions of the fura-2 ratio data into [Ca2+]i using in vitro calibration procedures would have given rough, but not accurate estimates of the true calcium values. We therefore decided against conversions and report here the original ratiometric fura-2 data. Rough estimates indicated the baseline [Ca2+]i in ciliated cells from both human and ovine tracheas to be between 70 and 100 nM and peak responses to 10 µM ATP to be between 200 and 350 nM.

Simultaneous Measurement of CBF and [Ca2+]I
Both the CBF analysis software and the Isee ratio software ran on an SGI O2 workstation (Silicon Graphics, Mountain View, CA). By using a dual-image module and guiding the infrared signal for CBF measurements to the CCD camera while sending all fluorescence signals (< 600 nm) to the cooled CCD camera, we were able to measure recordings of CBF and calcium of the same single cell simultaneously (8).

Incubation Protocols/Experimental Procedures
We have found that CBF in human airway epithelial cells stays elevated for a prolonged period of time after short-term ATP stimulation due to PKA activation (9). Thus, all physiologic experiments described herein using human airway epithelial cells were performed after inhibition of PKA with 1 µM of the cell membrane–permeable, myristoylated protein kinase A inhibitory peptide 14–22 (PKI14–22) (9, 30, 31). Cells mounted on the stage were perfused continuously with HBSS/HEPES, pH 7.4, using perfusion pumps (Harvard Apparatus Model 22; Harvard Apparatus, South Natick, MA) that allowed a constant flow rate of 250 µl/min. After recording baseline signals for 5–10 min, 10 µM ATP was added for 1 min at a flow rate of 1,000 µl/min and then washed away with buffer at 250 µl/min. Cells were left to return to the baseline in HBSS/HEPES, pH 7.4, for 10 min. In most cells, the stimulation with ATP was repeated once. Flow rates were changed to accelerate the full exchange of the bathing solution during short-term agonist application (complete after 1 minute). These changes in flow rates did not influence CBF or [Ca2+]i.

Statistics
All data were always compared with date- and culture-matched controls. Responses of both CBF and [Ca2+]i to 10 µM ATP showed a statistically insignificant rundown when comparing the second response to the first one. Therefore, all measurements were combined for final analysis. Because only two groups were compared, the statistical analysis was done with an unpaired t test using JMP software from SAS Institute Inc. (Cary, NC). A P < 0.05 was considered to be significant. Data were expressed as mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RGS2 mRNA Expression in Human and Ovine Airway Epithelial Cells
To demonstrate RGS2 mRNA expression in human and ovine airway epithelial cells, RT-PCR was performed as described in MATERIALS AND METHODS. RT-PCR products of expected size (~ 491 bp) were amplified using RNA either from both human and sheep freshly isolated airway epithelial cells or from expanded human cells redifferentiated at the ALI for 28 d. After gel purification, all RT-PCR products were sequenced in both directions. The sequence of the ovine product was similar to the published human, rat, and mouse RGS2 sequences, with a nucleotide and amino acid identity score of at least 94% and 97%, respectively (Figure 1). Sequence analysis of all products from human airway epithelial cells confirmed that they were a fragment of the published human RGS2, perfectly matching positions 158–649 of the full-length cDNA (or positions 43–205 of the protein sequence). The fragment contained only sequences from exons 2, 3, 4, and 5 of the human RGS2 gene, confirming the amplification of human mRNA rather than genomic DNA (Figure 1). Thus, these results demonstrate that ovine and human airway epithelial cells, either freshly isolated or redifferentiated in culture at the ALI, express RGS2 mRNA.



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  		Figure 1. Sequence alignment of RT-PCR products using native human and ovine airway epithelial cell total RNA and RGS2-specific primers. Shown is the sequence alignment using NCBI BLAST of the RT-PCR products (~ 491 bp) amplified from sheep and human airway epithelial cell mRNA. The RGS2 fragment amplified from human cells perfectly matched the full human published sequence (L13463.1: GOS8). The RGS2 fragment obtained from ovine airway epithelial cells showed a 94% identity to the published human RGS2 sequence and a high similarity to mouse and rat RGS2. Both PCR products contained fragments from exons 2, 3, 4, and 5 of RGS2.

 
Expression of RGS2 Protein in Tracheal Epithelial Cells in Cultures
We evaluated RGS2 protein expression using immunofluorescence with an antibody raised against the carboxy-terminus of human RGS2 as described in MATERIALS AND METHODS. Immunocytochemistry of both human and ovine tracheal epithelial cells in submerged culture showed low-level but specific staining for RGS2 (Figure 2). Interestingly, the staining had a nuclear predominance (see deconvoluted image in Figure 2). Although RGS2 expression was seen in all cells, careful evaluation of the coverslips revealed that ciliated cells showed a higher expression of RGS2 than other cells. Nonimmune serum did not reveal nonspecific staining. Furthermore, the staining could be eliminated by preabsorption with the antigenic peptide, again confirming specificity (Figure 2). Thus, both human and sheep airway epithelial cells express RGS2 protein.



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  		Figure 2. Expression of RGS2 in airway epithelial cells. Shown are the phase contrast images (A, F) and their corresponding TRITC fluorescent images (B, G) for human (A, B) and ovine (F, G) tracheal epithelial cells probed with anti-RGS2. RGS2 is preferentially expressed in ciliated cells with a nuclear predominance (deconvoluted image B'). C and E show control slides from human (C) and ovine (E) cells where the primary antibody has been replaced with nonimmune serum. D shows an additional control using human cells where the primary antibody has been preabsorbed with the antigenic peptide. Bars, 10 µm.

 
Effect of RGS2 Overexpression on CBF and [Ca2+]I Responses to 10 µM ATP in Ciliated Airway Epithelial Cells
To assess the effect of RGS2 overexpression on purinergic signaling in ciliated cells, we transfected human airway epithelial cells with an enhanced green flourescent protein (EGFP)-RGS2 fusion protein–encoding plasmid as described in MATERIALS AND METHODS. To evaluate only the calcium-dependent changes in CBF upon purinergic stimulation, human cells were preincubated with the PKA inhibitor PKI14–22 as indicated in MATERIALS AND METHODS (9). CBF and [Ca2+]i were measured simultaneously from single cells showing EGFP fluorescence, indicative of RGS-EGFP fusion protein expression, 48 h after transfection (Figure 3). ATP (10 µM) caused a transient increase in both [Ca2+]i and CBF with kinetically identical properties at the chosen time resolution of the measurement (Figure 4). Baseline CBF in cells expressing the EGFP-RGS2 fusion protein (as assessed by EGFP fluorescence) was 6.5 ± 0.8 Hz (n = 4 ciliated cells, 8 measurements) and was statistically indistinguishable from the baseline CBF of cells transfected only with EGFP (6.1 ± 1.2 Hz, n = 3 ciliated cells, 6 measurements; P > 0.05). In response to 10 µM ATP, CBF increased maximally by 1.4 ± 0.2 Hz in EGFP-RGS2–expressing cells. In contrast, cells expressing EGFP only increased CBF by 2.9 ± 0.2 Hz, a value significantly higher than cells expressing the EGFP-RGS2 fusion protein (P = 0.0008; Figure 4).



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  		Figure 3. Overexpression of RGS2 in human airway epithelial cells. Shown are images of fixed human airway epithelial cells 48 h after transfection with an EGFP-RGS2 fusion protein–encoding plasmid. (A) Phase contrast. (B) EGFP fluorescence. (C) immunofluorescence after probing with an RGS2 antibody. (D) Overlay image of B and C. Bar, 10 µm.

 


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  		Figure 4. Role of RGS2 protein overexpression in purinergic signaling in human airway epithelial cells. Simultaneously measured [Ca2+]i (A) and CBF (B) responses to 10 µM ATP are shown from human airway epithelial cells transiently transfected with an EGFP-RGS2 fusion protein–encoding plasmid (filled circles) or a plasmid solely encoding EGFP (open circles). Expression of the EGFP-RGS2 fusion protein resulted functionally in a reduced [Ca2+]i (filled circles in A) and CBF (filled circles in B) response to 10 µM ATP compared with transfection with EGFP alone (open circles). (C) Other ciliated cells on the same coverslips that did not express EGFP-RGS2 (filled squares) or EGFP (open squares) revealed a similar transient [Ca2+]i increase in response to 10 µM ATP. This transient was indistinguishable from the transient increase seen with EGFP expression (open circles in A).

 
The [Ca2+]i responses, as represented by the peak fura-2 ratio, behaved similarly to the CBF responses. It must be noted here that EGFP expression can influence the signal obtained from fura-2 because EGFP excitation can be elicited at 380 nm as well (32). Thus, we always compared cells with similar EGFP expression to avoid any problems in our analysis. ATP increased the fura-2 ratio by 0.65 ± 0.05 in cells transfected with EGFP only versus 0.27 ± 0.04 in cells expressing the EGFP-RGS2 fusion protein (P = 0.0003; Figure 4). These data were further confirmed by measuring additional [Ca2+]i responses from ciliated cells without simultaneous CBF assessment. In cells expressing only EGFP, ATP increased the fura-2 ratio by 0.6 ± 0.02 (n = 10 ciliated cells, 16 measurements), whereas the fura-2 ratio increased only by 0.23 ± 0.03 in cells expressing the EGFP-RGS2–fusing protein (n = 11 ciliated cells, 21 measurements; P < 0.0001).

To evaluate RGS2 expression semiquantitatively, these coverslips were processed for RGS2 immunocytochemistry after the physiologic measurements and TRITC fluorescence intensity was measured by defining single ciliated cells as ROIs with NIH image V1.61 using the raw, nonmodified images (Figure 3). The single wavelength measurement was deemed adequate for this purpose because these cells grew as a single layer having approximately identical thickness. The average RGS2-immunofluorescence intensity was 93.8 ± 15.6 arbitrary units in ciliated cells expressing the EGFP-RGS2 fusion protein (as shown by the presence of EGFP fluorescence; n = 15 ciliated cells) compared with 51 ± 8.8 arbitrary units in cells transfected with EGFP only (n = 13 ciliated cells). Assuming a linear relationship between fluorescence and RGS2 protein presence, the expression of RGS2 was 1.8 times higher in cells expressing the fusion protein compared with EGFP controls (P = 0.02). Furthermore, the expression of RGS2 in cells with the fusion protein was ~ 2.5 times higher than in nontransfected cells on the same coverslips (n = 15 cells). In cultures transfected only with the EGFP-encoding plasmid on the other hand, all cells (whether or not expressing EGFP) had statistically indistinguishable RGS2 immunofluorescence intensity (n = 13 ciliated cells).

Similar results were obtained with ovine airway epithelial cells. Purinergic stimulation with 10 µM ATP caused kinetically similar increases in CBF and [Ca2+]i when measured simultaneously. CBF increased from a baseline of 7.1 ± 0.9 Hz by 1.4 ± 0.2 Hz in response to 10 µM ATP in cells expressing the EGFP-RGS2 fusion protein (n = 3 cells, 4 measurements). In contrast, CBF increased from a baseline of 7.0 ± 0.8 Hz by 3.0 ± 0.5 Hz in cells expressing EGFP only (n = 4 cells, 5 measurements; P > 0.05 for baseline, P = 0.04 for increase). ATP increased the fura-2 ratio by 0.36 ± 0.04 in cells transfected with EGFP only versus 0.15 ± 0.04 in cells expressing the EGFP-RGS2 fusion protein (P = 0.01).

These data are consistent with the hypothesis that increased RGS2 activity resulting from overexpression inhibits purinergic signaling in human and ovine airway epithelial cells as evidenced by both reduced CBF and [Ca2+]i responses to 10 µM ATP.

CHO Cell Confirmation of EGFP-RGS2 Construct Validity
To confirm that EGFP did not interfere with RGS2 function in the fusion protein construct, we either transfected CHO-M3 cells with the EGFP-RGS2 fusion protein, or cotransfected them with both EGFP and RGS2 using two separate plasmids. CHO cells express native P2Y receptors. The [Ca2+]i response to 10 µM ATP was recorded from multiple cells and comparisons were made only with cells expressing similar amounts of EGFP as assessed by EGFP fluorescence. Transfections with EGFP-RGS2 or cotransfections with EGFP and RGS2 using two plasmids resulted in similar reduction of the transient [Ca2+]i increase compared with transfection with EGFP alone: fusion protein transfected cells showed a 44 ± 8% reduction in peak height of the response (n = 8 cells, 24 responses) compared with cells transfected with EGFP alone and cells cotransfected with EGFP and RGS2 showed a 54 ± 6% reduction (n = 18 cells, 36 responses; P > 0.05; Figure 5). These results confirm that the EGFP-RGS2 fusion protein construct worked and did not alter the function of RGS2 in transfected cells.



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  		Figure 5. EGFP-RGS2 fusion protein transfection versus EGFP and RGS2 cotransfections in CHO cells. To show that the fusion protein construct did not interfere with the ability of RGS2 to terminate G{alpha}q signaling, CHO-M3 cells were either transfected with the EGFP-RGS2 fusion protein–encoding plasmid (A) or cotransfected with two separate plasmid encoding RGS2 and EGFP, respectively (B). Cells transfected with the fusion protein–encoding plasmid (filled circles in A) revealed a similar decrease in the transient [Ca2+]i to 10 µM ATP like cells cotransfected with two separate plasmids encoding RGS2 or EGFP, respectively (filled circles in B). Traces with open circles show control cells transfected only with the EGFP-encoding plasmid.

 
Inhibition of RGS2 Expression in Airway Epithelial Cells Using Antisense Oligonucleotides
Next we attempted to inhibit RGS2 protein expression in airway epithelial cells using antisense oligonucleotides as described in MATERIALS AND METHODS. The antisense and 4-base mismatch control oligonucleotides were labeled with fluorescein, enabling their detection inside single cells (Figure 6). Initial experiments revealed that fluorescein label was present in at least 37% of ciliated cells at 24, 48, 72, and 96 h after transfection in both ovine and human cultures. However, the most reproducible time point was 48 h after transfection, and therefore this time point was used for all simultaneous measurements of CBF and [Ca2+]i (Figure 7). Only cells containing fluorescein fluorescence, indicative of successful oligonucleotide transfection, were used for measurements.



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  		Figure 6. Inhibition of RGS2 expression in human airway epithelial cells. Shown are images of human airway epithelial cells 48 h after transfection with antisense oligonucleotides (A, B, and C), and control, 4-base mismatch oligonucleotides (D, E, and F). Shown are the following images: phase contrast (A, D), FITC fluorescence of antisense (B) and 4-base mismatch oligonucleotides (E), and RGS2 immunofluorescence (C, F). Although quantification showed a statistically significant decrease after antisense oligonucleotide transfection, visual assessment of reduced expression in C versus F was not convincing. Bars, 10 µm.

 


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  		Figure 7. Effect of RGS2 antisense transfection on purinergic signaling in human airway epithelial cells. Simultaneously measured [Ca2+]i (A) and CBF (B) responses to 10 µM ATP are shown from human airway epithelial cells either transfected with RGS2 antisense (filled circles) or 4-base mismatch control (open circles) oligonucleotides. Antisense transfection resulted functionally in an enhanced [Ca2+]i (filled circles in A) and CBF (filled circles in B) response to 10 µM ATP compared with transfection with the control oligonucleotide (open circles). (C) Other ciliated cells on the same coverslips that did not reveal fluorescein fluorescence, indicative of failed transfection with antisense (filled squares) or control (open squares) oligonucleotide showed a similar transient [Ca2+]i increase in response to 10 µM ATP.

 
In all cells, ATP caused a rapid and transient increase in both [Ca2+]i and CBF with kinetically identical properties at the chosen CBF and calcium measurement resolution over time. In control cells transfected with the 4-base mismatch control oligonucleotide, baseline CBF was 7.4 ± 0.6 Hz (n = 6 ciliated cells, 10 measurements) and was statistically indistinguishable from the baseline CBF of cells transfected with the RGS2 antisense oligonucleotide (7.6 ± 0.8 Hz, n = 4 ciliated cells, 8 measurements; P > 0.05; Figure 7). In response to 10 µM ATP, CBF increased maximally by 2.5 ± 0.4 Hz in 4-mismatch transfected cells. In contrast, cells transfected with the antisense oligonucleotide increased CBF by 3.7 ± 0.4 Hz (P = 0.04; Figure 7). The [Ca2+]i responses, as represented by the fura-2 ratio, behaved similarly to the CBF responses. ATP increased the fura-2 ratio by 0.4 ± 0.06 in cells transfected with the control oligonucleotide versus 0.6 ± 0.06 in cells transfected with the antisense oligonucleotide (P = 0.02; Figure 7). These data were further confirmed by measuring additional [Ca2+]i responses from ciliated cells without simultaneous CBF assessment. In cells transfected with the control oligonucleotide, ATP increased the fura-2 ratio by 0.4 ± 0.04 (n = 10 ciliated cells, 19 measurements), whereas the fura-2 ratio increased by 0.6 ± 0.04 in cells transfected with the antisense oligonucleotides (n = 13 ciliated cells, 18 measurements; P = 0.0004). It is interesting to note that the fura-2 ratio increases were not higher in antisense oligonucleotide transfected cells in response to ATP compared with cells transfected with EGFP. However, because EGFP can interfere with fura-2 measurements as mentioned above (32) and different transfection procedures were used, direct comparisons of these values are not valid. Comparisons with 4-base mismatch controls, however, are valid and show significant effects of antisense transfection on [Ca2+]i responses to ATP.

Evaluation of RGS2 protein level changes in these cells by immunocytochemistry was complicated by intrinsically low RGS2 levels at baseline. Although quantification showed a statistically significant decrease after antisense oligonucleotide transfection compared with control cells (25.7 ± 2.1 arbitrary units in 34 cells from 6 experiments for antisense transfection versus 48.0 ± 2.5 arbitrary units in 56 cells from 6 experiments for 4-base mismatch control transfection; P < 0.001), visual confirmation of reduced expression was not entirely convincing. In summary, presumptive inhibition of RGS2 protein expression in ciliated airway epithelial cells (although difficult to validate due to the low signal levels) increased both the CBF and [Ca2+]i response to ATP 1.5-fold compared with control.

Similar results were obtained with ovine airway epithelial cells. CBF increased upon purinergic stimulation from a baseline of 7.9 ± 0.6 Hz by 3.4 ± 0.3 Hz in cells transfected with the RGS2 antisense oligonucleotide (n = 5 ciliated cells, 9 measurements) whereas CBF increased from a baseline of 7.9 ± 0.7 Hz by only 2.3 ± 0.3 Hz in cells transfected with the 4-base mismatch control oligonucleotide (n = 5 cells, 10 measurements; P > 0.05 for baseline; P = 0.02). In addition, ATP increased the fura-2 ratio by 0.3 ± 0.02 in antisense transfected cells versus 0.16 ± 0.03 in control cells (P = 0.02).

These data are consistent with the hypothesis that decreased RGS2 activity enhances purinergic signaling in human and ovine airway epithelial cells as evidenced by both increased CBF and [Ca2+]i responses to 10 µM ATP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study examined the role of RGS2 in purinergic signaling in ciliated ovine and human airway epithelial cells. We showed expression of RGS2 mRNA in freshly isolated human and ovine airway epithelial cells and in human cells redifferentiated at the ALI. By PCR, the RGS2 cDNA fragment obtained from human cells was identical to the published sequence and the fragment obtained from ovine cells was highly homologous (at least 94% identity by oligonucleotide and 97% by amino acid sequence). The PCR sequences included a large part of the C-terminus and the RGS core domain, the latter responsible for its GTPase-activating activity and the former for its previously reported direct interference with effectors (33, 34). The immunofluorescence experiments clearly showed RGS protein expression, albeit at a low level in human and ovine airway epithelial cells and, as published by other groups (24), a surprising "nuclear predominance." This finding suggests that RGS2 has functions beyond its regulation of membrane receptor/G-protein signaling. However, the goal of this study was to examine the role of RGS2 in purinergic signaling, and we did not investigate its nuclear role further.

We used two methods to influence RGS2 expression in ciliated airway epithelial cells: (i) transfection with a plasmid encoding an EGFP-RGS2 fusion protein to increase RGS2 protein expression, and (ii) transfection with RGS2 antisense oligonucleotides to decrease RGS2 protein expression. Although it was possible to validate the increase in RGS2 protein expression after transfection with the EGFP-RGS2 fusion protein-encoding plasmid using immunofluorescence (see RESULTS), we were not able to show decreases in protein expression levels convincingly after antisense oligonucleotide transfection. Despite the fact that quantitative analysis of RGS2 immunofluorescence suggested that its protein levels were decreased in cells transfected with the antisense oligonucleotide compared with 4-base mismatch oligonucleotide transfections and control, visual reproducibility of these numbers was not persuasive. Although the human eye is not a good linear detector of small intensity differences and thus the quantitative analysis is likely valid, we decided not to put too much emphasis on these data. However, the chosen time point of 48 h should have allowed the RGS2 protein expression levels to fall, even assuming a longer half-life than the one measured for RGS4, reported to be only 40–50 min (35). Other methods of validating the antisense approach, such as RT-PCR, were not attempted because the low transfection efficiency predicted that such an approach would not provide a conclusive answer either.

The physiologic measurements, however, provide clear evidence that both the antisense oligonucleotide and the plasmid transfections were successful. As predicted, purinergic signaling was suppressed by overexpression of RGS2 and enhanced by inhibiting RGS2 expression. Importantly, this was true for two signaling events downstream of ATP stimulation, namely the transient increases in intracellular calcium and CBF. In addition, all experiments provided consistent results whether examined in human or ovine ciliated airway epithelial cells.

It is important to point out that comparisons were only made with date-matched controls and transfection-appropriate controls. The importance of this issue is revealed by the fact that EGFP expression alone can alter ratio data obtained with fura-2, as indicated in RESULTS. Thus, direct comparisons of the fura-2 signals are only valid between appropriate experimental and control conditions.

Most of the published work on RGS proteins analyzed its biochemical behavior or examined its influence on signal transduction in vitro or in permeabilized cells, including patch clamp techniques. For instance, purified RGS4 has been shown to attenuate GTP{gamma}S-stimulated inositol lipid signaling in reconstitution studies with both purified G{alpha}q and NG-108 cell membranes (36). Among some of the physiologic data, RGS2 overexpression has been shown to result in a rightward shift of the morphine dose–response curve measured in a cell line system (37). The reported physiologic data in our paper on RGS2 and purinergic signaling directly demonstrate changes in physiologic function mediated by RGS. We only examined one RGS protein with specificity for G{alpha}q. Other RGS proteins with specificity for G{alpha}q are likely coexpressed in these cells as reported for other tissues (14, 16), and these may include RGS4. In fact, the role of RGS4 on G{alpha}q-mediated [Ca2+]i signaling has been examined in pancreatic acinar cells by measuring either agonist-dependent [Ca2+]i mobilization in streptolysin O-permeabilized cells or calcium-activated chloride current in patched cells (38). This latter study suggested that RGS4 and RGS2 inhibit signaling of several G{alpha}q-coupled receptors with different potency, suggesting that in vivo specificity of RGS proteins may be conferred by interaction with receptor complexes and not necessarily with specific G-proteins alone.

In conclusion, the data presented here provide an internally consistent set of results showing that RGS2 is expressed in human and ovine ciliated airway epithelial cells and that either up or down modulation of this protein changes [Ca2+]i and CBF responses to purinergic stimulation in a direction that supports the role of RGS2 as an inhibitor of G{alpha}q signaling.


   Acknowledgments
 
The elegant programming of Mr. Nenad Amodaj as well as the guidance of Dr. Adam Wanner is gratefully acknowledged. Supported in part by grants from NIH (HL-55341 to R.J.B.; HL-60644 and HL-67206 to M.S.).

Received in original form January 30, 2002

Received in final form May 24, 2002


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 Introduction
 Materials and Methods
 Results
 Discussion
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