P2 receptor regulation of [Ca 2+ ] i in cultured mouse mesangial cells

【摘要】  Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca 2+ ] i ) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca 2+ ] i averaged 102 ± 2 nM ( n = 346). One hundred micromolar concentrations of ATP, ADP, 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), ATP- -S, and UTP in normal Ca 2+ medium evoked peak increases in [Ca 2+ ] i of 866 ± 111, 236 ± 18, 316 ± 26, 427 ± 37, and 808 ± 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca 2+ ] i, whereas identical concentrations of adenosine, AMP, and, -methylene ATP (, -MeATP) had no detectable effect on [Ca 2+ ] i. Removal of Ca 2+ from the extracellular medium had no significant effect on the peak increase in [Ca 2+ ] i induced by ATP, ADP, BzATP, ATP- -S, or UTP compared with normal Ca 2+; however, Ca 2+ -free conditions did accelerate the rate of decline in [Ca 2+ ] i in cells treated with ATP and UTP. [Ca 2+ ] i was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X 2, P2X 4, P2X 7, P2Y 2, and P2Y 4 receptors. No evidence of P2X 1 and P2X 3 receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X 1, P2X 2, P2X 3, P2X 4, P2X 7, P2Y 2, and P2Y 4 receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca 2+ ] i by stimulating calcium influx from the extracellular medium and through mobilization of Ca 2+ from intracellular stores in cultured mouse mesangial cells.

【关键词】  PX receptors PY receptors Ca + signaling

RECENT STUDIES SUGGEST THAT P2 receptors play important roles in regulating renal hemodynamics, autoregulation, and tubular function ( 3, 11, 13, 15, 16, 20, 30, 31, 40, 45, 49 - 51, 57 ). P2 receptors mediate autoregulatory adjustments in preglomerular resistance and, along with P1 receptors, have been implicated in tubuloglomerular feedback (TGF)-dependent responses ( 3, 13, 15, 16, 40, 45 ). Autoregulatory control of preglomerular vascular resistance is largely responsible for controlling glomerular capillary pressure and thus glomerular filtration ( 28 ). Mesangial cells purportedly play an important role in regulating glomerular filtration, possibly by modulating the glomerular ultrafiltration coefficient ( K f ) and perhaps by communicating TGF signals from the macula densa to afferent arteriolar smooth muscle ( 1, 28 ).

Mesangial cells are located in the intra- and extraglomerular regions of the kidney. The function and pharmacology of P2 receptors expressed by rat mesangial cells have been extensively characterized in previous studies ( 2, 10, 12, 24, 34 - 36, 48, 52, 54, 56, 60, 62 ). Regulation of many glomerular processes, including the synthesis of extracellular matrix and modulation of K f, is thought to arise from mesangial cells interspersed throughout the glomerular tuft ( 1, 52 ). ATP-mediated activation of P2 receptors in mesangial cells produces an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) via influx pathways involving activation of ligand-gated P2X receptors or mobilization of intracellular Ca 2+ through activation of G protein-coupled P2Y receptors ( 10, 24, 34 - 36, 54, 62 ). Most studies investigating the role of P2 receptors in mesangial cell function utilize cells obtained from rat kidneys, but P2 receptor expression and function in mouse mesangial cells have not been examined. With the advent of genetic manipulation in mice, mouse models are being employed more frequently to examine the role of P2 receptors in regulating intrarenal vascular, hemodynamic, and tubular function. Given that mesangial cells form an important interface between macula densa cells, vascular smooth muscle cells, and the glomerular capillaries, it is important to understand the molecular pharmacology of these cells. The purpose of these studies was to determine the P2 receptors expressed by mouse mesangial cells and to establish the calcium signaling capability associated with their activation. Accordingly, we examined the expression and calcium signaling capabilities of cultured mouse mesangial cells to determine whether their P2 receptor expression profile was similar to, or different from, the rat. This information will be applicable to other studies utilizing mouse models to investigate renal hemodynamic questions. The findings of these studies indicate that mouse mesangial cells express a unique compliment of P2 receptors. Activation of these receptors results in variable calcium signaling responses ranging from robust to undetectable.

METHODS

Preparation of mouse mesangial cells. Mouse mesangial cells (MES 13) were obtained from American Type Culture Collection (Manassas, VA) at passage 27 and grown to confluency (37°C; 95% air-5% CO 2 ) in medium containing three parts DMEM (Cellgro, Herndon, VA), 1 part Ham's F12 (Cellgro), 15% fetal bovine serum (Sigma, St. Louis, MO), 1% penicillin/streptomycin (Cellgro), and 14 mM HEPES. Cells were generated from isolated mouse glomeruli from SV40 transgenic mice and exhibit features similar to normal cultured mesangial cells ( 25, 41, 42 ). Cells exhibit a stellate appearance with a centrally located nucleus when grown in low confluence culture. They are described as having prominent actin cytoskeleton with abundant fibrils oriented in parallel. The cells are negative for Factor VIII-related antigen and cytokeratin. Cells were released from the culture surface using a solution containing of 0.25% trypsin/0.2% EDTA, washed, pelleted by low-speed centrifugation, and resuspended in the culture medium described above. Aliquots of cell suspension were transferred to 60 x 15-mm polystyrene dishes containing a sterile glass coverslip (no. 1, 22 x 40 mm). Coverslips of cells were maintained in culture for a minimum of 16 h before use.

Fluorescence measurements. Fluorescence experiments were performed using monochromator-based fluorescence spectrophotometry (Photon Technology International, London, Ontario, Canada), as previously described ( 17 ). Excitation wavelengths were set at 340 and 380 nm and emitted light was collected at 510 ± 20 nm. Fluorescence intensity was collected at 10 data points/s and analysis of these data was accomplished using FeliX software (Photon Technology International). Calibration of fluorescence data was accomplished in vitro using the method of Grynkiewicz et al. ( 9 ).

Measurements of [Ca 2+ ] i obtained from clusters of two to five mouse mesangial cells were acquired as previously described ( 17 ). Coverslips of cells were transferred to a fura 2 loading solution containing 5 µM fura 2 acetoxymethyl ester (fura 2-AM; Molecular Probes, Eugene, OR) in serum-free DMEM for 30-45 min at 37°C and 5% ambient CO 2. Coverslips of fura 2-loaded cells were mounted to a perfusion chamber (Warner Instrument, Hamden, CT) and affixed to the stage of an Olympus IX50 inverted light microscope. The cells were continuously superfused (1.4 ml/min; 25°C) with a normal Ca 2+ PSS (in mM): 140 NaCl, 3 KCl, 1 MgCl 2 ·6H 2 0, 1.8 CaCl 2, 10 glucose, and 20 HEPES. Nominally Ca 2+ -free solute ions were prepared, without EGTA, and by excluding Ca 2+ ( 7, 17, 61 ). High K + solutions were prepared by equimolar substitutions of KCl for NaCl. Fluorescence data were collected with background subtraction. A new coverslip of cells was used for each experiment.

Series 1: control responses to agonist stimulation. Cells were monitored continuously for 100 s during exposure to control buffer followed by a 200-s period of agonist exposure (100-300 s). Agonists examined included ATP, ADP, AMP, adenosine, UTP, UDP, 2-methylthio ATP (2MeSATP),, -methylene ATP (, -MeATP), ATP- -S, 2',3'(benzoyl-4-benzoyl)-ATP (BzATP), and KCl. Each period of agonist treatment was followed by a 100-s recovery period (300-400 s).

Series 2: agonist-induced responses in Ca 2+ -free conditions. In the second series of experiments, cells were exposed to control medium for a period of 50 s before the superfusate was changed to a similar, Ca 2+ -free medium (50 s) to establish baseline fluorescence under in the nominal absence of extracellular calcium. Previous studies using freshly isolated preglomerular vascular smooth muscle showed that extracellular calcium averages 38 nM and results in no elevation of cytosolic calcium during KCl depolarization ( 7, 17 ). Each experiment was concluded with a Ca 2+ -free (50 s) and normal calcium (100 s) recovery period.

Series 3: Western blot analysis of P2 receptor expression. Mouse mesangial cells were grown to confluence and harvested. The collected cells were mixed with RIPA lysis buffer (pH 7.4, 0°C) containing 50 mM Tris·HCl, 150 mM NaCl, 0.25% deoxycholate, 1.0% NP-40, 1 mM EDTA, and a cocktail of protease inhibitors phenylmethylsulfonyl fluoride (1.0 mM), aprotinin (2.0 µg/ml), leupeptin (1.0 µg/ml), and sodium orthovanadate (Na 3 VO 4, 1.0 mM). The cells were homogenized, sonicated, and centrifuged (20,800 g ) at 4°C for 15 min to remove cellular debris. The protein concentration in the supernatant was determined using the method of Lowry et al. ( 22 ). Protein samples were diluted with a Laemmli sample buffer containing 62.5 mM Triz·HCl (pH 6.8), 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.01% bromophenol blue and heated (5 min; 100°C) using a dry bath incubator with intermittent vortexing. Proteins were separated electrophoretically in polyacrylamide gels and transferred to nitrocellulose membranes. Transfer membranes were washed with PBS and incubated with a blocking solution containing 5% nonfat dry milk and PBS + 0.1% Tween 20. After being blocked, the transfer membranes were incubated overnight with primary antibody (P2X 1, P2X 2, P2X 3, P2X 4, P2X 7, P2Y 2, P2Y 4; rabbit anti-rat; Alomone Labs, Jerusalem, Israel). The characteristics of these antibodies are detailed in Table 1. Dilution factors were 1:300 for P2X 1 and 1:200 for P2X 2, P2X 3, P2X 4, P2X 7, P2Y 2, and P2Y 4. Transfer membranes were washed with PBS + 0.1% Tween 20 and incubated with secondary antibody (1:15,000 dilution; goat anti-rabbit IgG horseradish peroxidase conjugate, Sigma) for 1.5 h. Immunoreactivity was detected by enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). Membranes were reprobed for -actin as a loading control. After being washed with PBS + 0.3% Tween 20 and being blocked with 5% nonfat dry milk, membranes were incubated with mouse anti- -actin antibody (1:5,000 dilution; Sigma) for 1 h. Membranes were washed and incubated with secondary antibody (1:2,000 dilution; goat anti-mouse IgG horseradish peroxidase conjugate, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Antibody-specific control antigens (Alomone Labs) were used as negative controls to determine specific binding. The primary antibody was incubated with the control antigen for 1 h before incubation with the membrane.

Table 1. Characteristics of the antibodies used to detect P2 receptor expression

Series 4: RT-PCR analysis for detection of P2 receptor mRNA. P2X and P2Y receptor mRNA expression was identified in mouse mesangial cells by RT-PCR. Cells were grown to confluence and harvested. Total RNA was isolated using an RNeasy Mini Kit (QIAGEN Sciences, Valencia, CA), according to the manufacturer's instructions. Extraneous DNA was degraded using RNase-Free DNase set (QIAGEN Sciences) and the extracted RNA was quantified by spectrophotometer (260 nm; Beckman DU Series 600).

P2X and P2Y receptor mRNA sequences were obtained from GenBank ( http://www.ncbi.nlm.nih.gov/Genbank ) and primer sequences were selected using Beacon Designer 5.0 software (Premier Biosoft International, Palo Alto, CA). The primer sequences were checked against the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) to establish their specificity to the target genes. The sense and antisense primer sequences (20 pM) used for RT-PCR are listed in Table 2. One microgram of total RNA was reverse transcribed at 42°C for 30 min in a 20-µl reaction volume, using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The cDNA template (5 µl) was added to a 50-µl reaction volume including Taq DNA Polymerase (cycle number and annealing temperatures are detailed in Table 2; Taq PCR Core Kit QIAGEN Sciences). Water controls were run for selected receptors and were found to be negative. The amplified PCR products were run on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide and visualized with a Bio-Rad Molecular Image Gel Doc XR system. Product size was estimated with 100-bp DNA ladder (Invitrogen, Carlsbad, CA). PCR products were purified (QIAquick PCR Purification kit, QIAGEN Sciences) and sequenced by the Medical College of Georgia genomic core laboratory. Sequence data were compared with BLAST sequences to verify the identity of the RT-PCR products.

Table 2. Primer sequences used for detection of P2 receptor mRNA

Statistical analysis. Data are presented as means ± SE. Within-group comparisons of basal [Ca 2+ ] i with peak [Ca 2+ ] i were assessed by a paired t -test. Differences between groups were analyzed either by unpaired t -test or one-way ANOVA, as appropriate. Post hoc tests were performed using Newman-Keuls multiple range test. Statistical probabilities <0.05 ( P < 0.05) were considered significantly different.

RESULTS

A total of 346 coverslips of cells were examined for this study. Basal [Ca 2+ ] i averaged 102 ± 2 nM. Agonists and concentrations used for this study are included in Table 3.

Table 3. Baseline and peak [Ca 2+ ] i in P2 receptor agonist treatment groups

Series 1. The first series of experiments determined the effect of selected purine and pyrimidine receptor agonists on [Ca 2+ ] i in cultured mouse mesangial cells. Representative traces for each agonist are provided in Figs. 1, 2, and 3. Table 3 presents the basal and peak values for [Ca 2+ ] i in mouse mesangial cells for all treatment groups. Baseline [Ca 2+ ] i remained stable during the control period and was similar across all treatment groups. No detectable increase in [Ca 2+ ] i was observed in cells treated with, -MeATP ( n = 14), adenosine ( n = 26), or AMP ( n = 14; 100 µM; Fig. 1, A - C; Table 3 ). In cases where no detectable fluorescence response was observed ( Fig. 1, A - C ), cell viability and responsiveness to P2 receptor stimulation were verified by application of ATP. In each case, ATP elicited a typical [Ca 2+ ] i response. Significant increases in [Ca 2+ ] i occurred upon exposure of mouse mesangial cells to ATP, UTP, ATP- -S, BzATP, and ADP (100 µM; Fig. 1, D - H, Table 3 ). Responsive cells typically exhibited a rapid initial increase in [Ca 2+ ] i followed by a gradual recovery toward baseline.

Fig. 1. Representative traces for mouse mesangial cells treated with selected P1 and P2 receptor agonists. Data represent the fura 2 fluorescence traces for cells treated with 100 µM, -MeATP ( A ), adenosine ( B ), AMP ( C ), ATP ( D ), UTP ( E ), ATP- -S ( F ), BzATP ( G ), and ADP ( H ). In cases where no detectable fluorescence response was observed ( A - C ), cell viability and responsiveness to P2 receptor stimulation were verified by application of ATP. Agonist exposure is depicted by the black bar above the x -axis.

Fig. 2. Representative traces for mouse mesangial cells treated with 100 µM UDP. A : typical trace from mouse mesangial cells exhibiting a modest response upon treatment with UDP. B : trace of mouse mesangial cells exhibiting no response upon treatment with UDP. In cases where no detectable fluorescence response was observed, cell viability and responsiveness to P2 receptor stimulation were verified by application of ATP. Agonist exposure is depicted by the black bar above the x -axis.

Fig. 3. Representative traces for mouse mesangial cells treated with 100 µM 2MeSATP. A : typical trace from mouse mesangial cells exhibiting a modest response upon treatment with 2MeSATP. B : trace of mouse mesangial cells exhibiting no response upon treatment with 2MeSATP. In cases where no detectable fluorescence response was observed, cell viability and responsiveness to P2 receptor stimulation were verified by application of ATP. Agonist exposure is depicted by the black bar above the x -axis.

Exposure of mouse mesangial cells to either 100 µM UDP or 100 µM 2MeSATP yielded either modest transient increases in [Ca 2+ ] i or no change in [Ca 2+ ] i. Of the 18 UDP-treated coverslips, 10 exhibited a slight increase in [Ca 2+ ] i, as illustrated in Fig. 2 A, whereas the remaining coverslips exhibited no change in [Ca 2+ ] i ( Fig. 2 B ). Similarly, of the 25 coverslips of mouse mesangial cells treated with 2MeSATP, eight coverslips exhibited small increases in [Ca 2+ ] i, ( Fig. 3 A ), whereas the remaining 17 coverslips exhibited no change in [Ca 2+ ] i ( Fig. 3 B ). A positive control challenge with 100 µM ATP was included in cases where no response was observed to establish the viability, and responsiveness, of these cells when an appropriate agonist is used ( Figs. 2 B and 3 B ). Clearly, P2 receptor-mediated elevations in [Ca 2+ ] i could be evoked when a receptor-appropriate agonist was applied.

Series 2. The next series of experiments was designed to determine the basic calcium access pathways involved in P2 receptor-mediated elevation in [Ca 2+ ] i. Initially, we determined the potential role for voltage-dependent calcium influx as a means of elevating [Ca 2+ ] i in mouse mesangial cells. Cells were challenged with a high-K + solution containing 143 mM KCl and were subsequently treated with 100 µM ATP after a recovery period. As shown in Fig. 4, elevation of extracellular K + had no effect on [Ca 2+ ] i, whereas the same cells yielded a rapid and robust response when subsequently challenged with ATP. From a sample of 29 coverslips, [Ca 2+ ] i averaged 96 ± 5 and 100 ± 7 nM during the control and KCl periods, respectively. Subsequent exposure of these cells to 100 µM ATP increased [Ca 2+ ] i from 98 ± 6 nM to a peak of 806 ± 202 nM. These data indicate that voltage-dependent Ca 2+ influx is not a mechanism by which P2 receptor activation increases [Ca 2+ ] i in these cells.

Fig. 4. Representative trace of mouse mesangial cells treated with 143 mM KCl. ATP was applied as a second agonist treatment to verify cell viability. KCl and ATP exposures are depicted by the black bars above the x -axis.

The contribution of extracellular calcium to the calcium response evoked by selected P2 receptor agonists was also examined using a nominally Ca 2+ -free medium. The results of these studies are provided in Figs. 5 and 6. Removal of Ca 2+ from the extracellular medium had no significant effect on either basal calcium levels or on the peak increase in [Ca 2+ ] i induced by ATP, UTP, ADP, ATP- -S, or BzATP. Interestingly, the rate of return [Ca 2+ ] i toward baseline immediately following the peak response was accelerated under Ca 2+ -free conditions in cells treated with ATP or UTP ( Figs. 5, A and B, and 6 ), whereas no significant change was noted for BzATP, ATP- -S, or ADP. The reduction in the area under the curve for ATP and UTP reflects an accelerated return to the baseline, as the peak responses were similar in the presence or absence of extracellular calcium ( Figs. 5 and 6 ).

Fig. 5. Comparison of P2 receptor-mediated increases in intracellular calcium concentration in mouse mesangial cells in the presence of 1.8 mM Ca 2+ (black traces) and in nominally Ca 2+ -free conditions (gray labels and traces). Responses are shown for cells treated with ATP, UTP, ATP- -S, and BzATP (100 µM; A - D, respectively). Agonist exposure is depicted by the black labels and black bars. Nominally calcium-free conditions are depicted by the gray bars and gray 0 Ca 2+ labels above the x -axis labeled 0 Ca 2+.

Fig. 6. Area under the curves mouse mesangial cells exposed to selected P2 receptor agonists. Responses were analyzed for cells bathed in calcium-containing solutions (1.8 mM Ca 2+, black bars) and in nominally Ca 2+ -free media (gray bars). Bars depict the average total area under the traces elicited by exposure to 100 µM ATP, UTP, BzATP, ATP- -S, and ADP. The numbers shown in the bars reflect the number of coverslips studied.

Series 3: experiments. Western blot analysis was performed to determine P2 receptor expression in cultured mouse mesangial cells ( Figs. 7 and 8 ). No banding was detected for mouse mesangial cells probed with anti-P2X 1 and anti-P2X 3 antibody despite strong banding in positive control samples ( Fig. 7 ). Clear evidence of specific banding was evident in membranes probed with antibodies against P2X 2 ( 95 kDa), P2X 4 ( 52 kDa), P2X 7 ( 78 kDa), P2Y 2 ( 51 kDa), and P2Y 4 ( 80 kDa) receptors. In each case, the molecular weights estimated from the Western blot analysis tend to be larger than those predicted ( Table 1 ) by sequence data. In the case of P2X 2 and P2Y 4, the estimated molecular weights are nearly double the predicted molecular weights. The explanation for these discrepancies is not clear. Certainly, posttranslational modifications such as glycosylation and/or receptor dimerization could be involved, but the exact explanation remains to be determined. The specificity of the antibody banding was verified by preincubating the primary antibody with the respective control antigen. In each case, preincubation with the control antigen eliminated antibody staining consistent with specific banding under normal conditions ( Figs. 7 and 8, right; Ab + CA). Preincubation with the control antigen also eliminated antibody staining for the positive control samples. Examples of this are shown for P2X 1 and P2X 3 receptor proteins, where no bands were observed with mesangial cell extracts. Reprobe for -actin protein was positive for all membranes.

Fig. 7. Western blot analysis of P2X 1, P2X 2, P2X 3, P2X 4, and P2X 7 receptor expression in cultured mouse mesangial cells. Left : MW represents the molecular weight marker; lane 1 contains the positive controls for each receptor protein and include rat brain (P2X 1 ); mouse mesangial cells (P2X 2 ); rat dorsal root ganglion for P2X 3 and P2X 4, rat brain for P2X 7 (50 µg protein/lane); lanes 2 - 5 represent sample mouse mesangial cells (50 µg protein/lane). Right : effect of preincubation of the primary antibody (Ab) with the respective control antigen (Ab + CA) to verify specificity. Lane 1 depicts the antibody binding results using rat dorsal root ganglion (P2X 1 and P2X 3 ) or normal mouse mesangial cell extracts (P2X 2, P2X 4, P2X 7 ) and lane 2 depicts the antibody binding results following preincubation with the control antigen. Antibody dilutions of 1:300 for anti P2X 1 and 1:200 for anti P2X 2, P2X 3, P2X 4, and P2X 7 were used.

Fig. 8. Western blot analysis of P2Y 2, and P2Y 4 receptor expression in cultured mouse mesangial cells. Left : MW represents the molecular weight marker; lane 1 contains the positive controls for each receptor protein and include rat spinal cord (P2Y 2 ); rat dorsal root ganglion for P2Y 4 (50 µg protein/lane); lanes 2-5 represent samples mouse mesangial cells (50 µg protein/lane). Right : effect of preincubation of the primary antibody (Ab) with the respective control antigen (Ab + CA) to verify specificity. Lane 1 depicts the antibody binding results using normal mouse mesangial cell extracts and lane 2 depicts the antibody binding results following preincubation with the control antigen. Antibodies were diluted 1:200.

Series 4: experiments. RT-PCR analysis was performed to determine mRNA expression for selected P2X and P2Y receptors in cultured mouse mesangial cells ( Fig. 9 ). Mouse mesangial cells express mRNA for P2X 1, P2X 2, P2X 3, P2X 4, P2X 7, P2Y 2, and P2Y 4 receptors. Single bands were detected for each receptor using the primer sequences listed in Table 2 and product identity was confirmed by sequencing of the RT-PCR products. In each case, sequence homology was found to be greater than 98% compared with the predicted receptor sequence. Interestingly, despite the fact that mRNA was detected for P2X 1 and P2X 3 receptors, calcium signaling responses and Western blot analysis do not support detectable expression of these receptor proteins. In contrast, mRNA data parallel calcium signaling and Western blot analysis for P2X 2, P2X 4, P2X 7, P2Y 2, and P2Y 4 receptors.

Fig. 9. RT-PCR analysis of mRNA for P2 receptor expression by mouse mesangial cells. Each gel depicts typical RT-PCR records for 2 independent mesangial cell mRNA extracts. Left lanes provide the molecular weight marker. Primer sequences used for each mRNA are detailed in Table 2.

DISCUSSION

Experiments were performed to establish the pharmacological profile of P2 receptors expressed by cultured mouse mesangial cells and to determine the calcium signaling pathways invoked by purinoceptor activation. Results of these experiments demonstrate that mouse mesangial cells express multiple P2 receptors and that activation of P2 receptors with selected agonists stimulates a rapid increase in [Ca 2+ ] i in cultured mouse mesangial cells in a receptor-specific manner.

P2 receptors are a structurally distinct group of purine- and pyrimidine-sensitive receptors classified as either P2X or P2Y receptors. P2X receptors have two membrane-spanning domains that function as ligand-gated ion channels. Approximately seven P2X receptor subtypes (P2X 1-7 ) have been described ( 33 ). P2Y receptors are G protein-coupled receptors with seven membrane-spanning domains ( 59 ). Activation of P2Y receptors generally mobilizes calcium from intracellular stores, as well as stimulates capacitative calcium influx. Across all species, the P2Y receptor family includes the P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, P2Y 12, and P2Y 13 receptor subtypes ( 38, 44, 59 ).

The studies described here demonstrate that cultured mouse mesangial cells express a unique compliment of P2X and P2Y receptors that signal through calcium. Activation of these receptors results in variable calcium signaling responses ranging from robust to undetectable, and when present, occurs through mechanisms involving influx of extracellular calcium and mobilization of calcium from intracellular stores. We compared responses to selected P2 receptor agonists to functionally identify the likely P2 receptor subtypes expressed by mouse mesangial cells and then confirmed those findings by Western blot and PCR analysis, where possible.

Influx of extracellular calcium is a prominent aspect of P2X receptor activation. The rank order for activation of recombinant P2X 1 receptors cloned from rat vas deferens and human and mouse urinary bladder is 2MeSATP, ADP ( 38 ). In the current report, ATP potently stimulated increases in intracellular calcium, but the P2X 1 /P2X 3 agonist,, -MeATP, did not. Furthermore, ADP also potently increased the [Ca 2+ ] i. 2MeSATP had only a modest effect, as evident by slight elevations in [Ca 2+ ] i being detected in only 8 of 25 coverslips of cells. ATP evoked robust calcium responses in each case where no response was detected by the receptor selective agonist, establishing the viability and responsiveness of the cells when resident receptors were stimulated. Although PCR revealed message for P2X 1 receptors, Western blot analysis did not reveal evidence of P2X 1 receptor protein despite strong banding for dorsal root ganglion used as a positive control. Thus our data indicate that cultured mouse mesangial cells do not express P2X 1 receptors.

Similarly, P2X 3 receptors cloned from rat dorsal root ganglion, -MeATP ( 38 ). Our data reveal a significant calcium response to ATP and a diminished response to ADP, while there is no response to, -MeATP or 2MeSATP. Additionally, exposure to 100 µM ATP, following stimulation with either 2MeSATP or, -MeATP, revealed robust calcium responses. Western blot analysis did not reveal any detectable staining for P2X 3 receptor protein despite the fact that staining was evident in samples of rat dorsal root ganglion, and mRNA for the P2X 3 receptor was detected in mouse mesangial cell extracts. These data suggest that cultured mouse mesangial cells do not express P2X 3 receptors.

P2X 2 receptors exhibit strong permeability to calcium ( 33 ), but there are no agonists that are selective for P2X 2 receptors. Consequently, rank order potency profiles and functional responses are used to characterize P2X 2 receptors. The rank order potency profile for the P2X 2 receptors cloned from rat pheochromocytoma shows that ATP, ATP- -S, and 2MeSATP are equipotent and stimulate a nonselective inward cation current ( 33, 38 ). Although an increase in [Ca 2+ ] i to 2MeSATP was not observed in this study, both ATP and ATP- -S elicited significant responses as shown in Table 3 and Figs. 1, 5, and 6. PCR analysis revealed clear evidence for P2X 2 receptor mRNA. Furthermore, Western blot analysis revealed clearly detectable banding of membranes treated with anti-P2X 2 antibody. Accordingly, the functional and biochemical analysis supports the conclusion that cultured mouse mesangial cells express P2X 2 receptors.

Previous studies describe extensive P2X 4 receptor expression throughout the central and peripheral nervous system ( 18, 33, 38 ). A more recent report from Bo and co-workers ( 5 ), using a monoclonal antibody directed at an extracellular sequence of the P2X 4 receptor, suggests a ubiquitous distribution involving ductal epithelium, airways, uterine endothelium, fat cells and expression by smooth muscle of the bladder, gastrointestinal tract, uterus, and arteries. Homomeric P2X 4 receptors are activated by ATP, but not by, -MeATP ( 33 ). In the current report, we demonstrate marked increases in intracellular calcium concentration in response to ATP, but no detectable responses to, -MeATP. Furthermore, Western blot analysis revealed significant banding of membranes treated with anti-P2X 4 antibody, and mRNA for the receptor was detected by RT-PCR. Consequently, our data support the expression of functional P2X 4 receptors by cultured mouse mesangial cells.

Rapid inward cation currents are exhibited upon activation of P2X 5 receptors in rat coelic ganglia and by P2X 6 receptors in rat superior cervical ganglion ( 38 ). In these tissues, both P2X receptor subtypes exhibit similar potency profiles with ADP with, -MeATP being inactive ( 38 )., -MeATP was inactive in stimulating an increase in cytosolic calcium, whereas significant calcium responses were evoked by ATP and ADP while 2MeSATP elicited only small responses in 8 of 25 attempts (32%). Furthermore, the increase in calcium elicited by ADP occurs mainly though mobilization of calcium from intracellular stores, rather than through influx of extracellular calcium ( Fig. 6 ). Accordingly, based on the observations that the rank order profiles P2X 5 and P2X 6 receptors are not recapitulated in cultured mouse mesangial cells and that the responses to ADP do not primarily arise from the influx of extracellular calcium, it is not likely that P2X 5 and P2X 6 receptors play a major role on the response of these cells to ATP.

P2X 7 receptors were formerly classified as P 2Z receptors, but based on their structural homology with other P2X receptors, it has been included as a member of the P2X receptor family ( 18, 38 ). P2X 7 receptors are implicated in apoptosis and necrosis of cultured rat mesangial cells ( 47 ). Brief applications (1-2 s) of extracellular ATP transiently open cation channels, whereas prolonged exposure to ATP causes the ligand-gated cation channel to convert to a pore, thus allowing passage of electrolytes like Na + and Ca 2+ in addition to small macromolecules ( 39 ). The currents passed by P2X 7 receptors are modulated by divalent cations, but concentrations approaching 5 mM Ca 2+ are required before significant P2X 7 receptor inhibition is observed ( 33, 58 ). P2X 7 receptors are not as sensitive to ATP as other P2X receptor subtypes. Usually ATP concentrations exceeding 100 µM are required for P2X 7 receptor activation. BzATP is frequently used as a selective agonist as it is up to 30 times more potent than ATP at P2X 7 receptors; however, it must be noted that BzATP is also an effective agonist for other P2X receptors at similar or lower concentrations ( 33, 38 ). In the current report, RT-PCR and Western blot analysis revealed prominent banding consistent with expression of P2X 7 receptor mRNA and protein by cultured mouse mesangial cells. Furthermore, BzATP elicited a rapid increase in [Ca 2+ ] i that was retained in nominally Ca 2+ -free medium, suggesting that the calcium response largely arose from the release of intracellular calcium ( Figs. 1, 5, and 6 ). Interestingly, there was a consistent tendency for [Ca 2+ ] i to rise when the bath solution was returned to a normal Ca 2+ concentration, as shown in Fig. 5. This increase in [Ca 2+ ] i suggests development of a slow-forming pore as is attributed to P2X 7 receptors or activation of capacitative-calcium influx mechanisms ( 6, 8, 26, 32, 37, 39, 46, 55 ). Taken together, these data support the contention that P2X 7 receptors are expressed by mouse mesangial cells.

We examined the possibility that cultured mouse mesangial cells express P2Y receptors. Activation of P2Y receptors generally mobilizes calcium from intracellular stores, as well as stimulates capacitative calcium influx ( 6, 8, 26, 37, 39, 46, 55 ). The human and rodent P2Y receptor families include P2Y 1, P2Y 2, P2Y 4, and P2Y 6 receptors. Humans also express P2Y 11 receptors ( 19, 53, 59 ).

Activation of P2Y 1 receptors in human brain, prostate, and ovary, bovine endothelium, rat insulinoma cells, rat ileal myocytes, turkey brain and chick brain all demonstrate that the potency of 2MeSATP ATP or ADP ( 38 ). Furthermore, UTP is typically an inactive, or extremely weak agonist for P2Y 1 receptors ( 38 ). In the current report, [Ca 2+ ] i was largely unaffected by exposure to 2MeSATP compared with responses evoked by ATP, ADP, or UTP. These data support the conclusion that P2Y 1 receptors are not expressed by mouse mesangial cells.

The renal vascular response to ATP, and related agonists, suggests vascular expression of P2X 1 and P2Y 2 receptors ( 13 - 17, 61 ). Based on agonist specificity, expression of P2Y 4 receptors is also possible ( 38, 59 ). Hence, we examined the expression of the P2Y 2 and P2Y 4 receptor subtypes by mouse mesangial cells. RT-PCR analysis indicated mRNA expression for P2Y 2 and P2Y 4 receptors in these cells. PCR data were substantiated by Western blot analysis, which demonstrated the presence of P2Y 2 and P2Y 4 receptor protein in extracts of cultured mouse mesangial cells. Functional evidence also supports expression of these receptors. P2Y 2 receptors are equipotently activated by ATP and UTP, but not ADP or UDP ( 23, 29 ). 2MeSATP and, -MeATP are considered weak or inactive agonists for P2Y 2 receptors. ATP- -S is an effective agonist for P2Y 2 receptors but tends to be less potent than ATP or UTP in mouse neuroblastoma cells ( 23, 38 ). Our data are consistent with this pharmacological profile. Both ATP and UTP were equipotent in stimulating an increase in [Ca 2+ ] i, while 2MeSATP and, -MeATP were weak or inactive. Furthermore, ATP- -S significantly increased [Ca 2+ ] i but the magnitude of the increase was less than half of the response evoked by ATP or UTP. Thus, based on the agonist response characteristics and the positive staining by Western blot, these data demonstrate clear expression of P2Y 2 receptors by cultured mouse mesangial cells.

P2Y 4 receptor profiles are more ambiguous. Distribution studies suggest that P2Y 4 receptors are expressed by the placenta with lower expression levels being detected in lung and vascular smooth muscle ( 38 ). Recombinant P2Y 4 receptors are activated equally by UTP and ATP ( 19, 53, 59 ) but no response is evoked by UDP or ADP ( 19 ). While P2Y 2 receptor-mediated responses are difficult to resolve from P2Y 4 receptor-mediated responses, data from the current study reflect the agonist responses profiles described for P2Y 4 receptors. UTP and ATP potently stimulate increases in cytosolic calcium concentration, whereas the dinucleotides UDP and ADP are either inactive or weakly active. RT-PCR and Western blot analysis indicate strong expression of P2Y 4 receptor mRNA and protein. Thus the data support expression of functional P2Y 4 receptors by cultured mouse mesangial cells.

Compared to other agonists, ATP is the least potent in the rank order potency profile for P2Y 6 receptors in human placenta and spleen and rat aortic smooth muscle ( 38 ). The rank order potency ATP ( 38 ). The rank order potency profile for rat aortic smooth muscle is similar to human ADP = 2MeSATP ATP ( 38 ). The data derived from this study show a significant increase in [Ca 2+ ] i when ATP is present in the extracellular environment and no increase in [Ca 2+ ] i when 2MeSATP is used. Therefore, it is reasonable to conclude that mouse mesangial cells do not express the P2Y 6 receptor.

Studies were performed to begin evaluating the mechanisms by which P2 receptor activation stimulates increases in [Ca 2+ ] i. Of the agonists tested, ATP, UTP, ATP- -S stimulated rapid and robust increases in [Ca 2+ ] i. ADP and BzATP produced more modest responses, and UDP, 2MeSATP, AMP, adenosine and, -MeATP yielded essentially no detectable change in [Ca 2+ ] i ( Fig. 1 and Table 3 ). Based on Figs. 5 and 6, the data argue that the majority of the [Ca 2+ ] i responses arise from the release of calcium from intracellular stores. This conclusion is based on the observation that stimulation of cells in nominally Ca 2+ -free medium had little effect on the peak change in [Ca 2+ ] i but did accelerate the return to baseline in cells treated with ATP and UTP. Nominally Ca 2+ -free bathing conditions had little effect on the temporal response profile for BzATP, ATP- -S, or ADP. These data suggest that ATP and UTP stimulate calcium release from intracellular stores as well as stimulate calcium influx from the extracellular medium. Calcium influx can occur directly through activation of P2X 2, P2X 4, or P2X 7 receptors, activation of voltage-dependent calcium influx, or activation of capacitive calcium influx pathways. Activated P2X receptors function as ligand-gated channels that allow passage of a nonselective cation current. Based on the agonist potency data, activation of P2X 2 and/or P2X 4 receptors seems the most likely consideration as P2X 7 receptor activation usually requires ATP concentrations exceeding 100 µM ( 33 ). P2X receptor-mediated cation currents typically involve influx of Na + and Ca 2+ ions from the extracellular medium and thus could contribute directly to elevation of [Ca 2+ ] i and membrane depolarization with subsequent activation of voltage-dependent calcium channels. Surprisingly, voltage-dependent calcium influx can be ruled out based on the determination that 90 mM (data not shown) and 143 mM KCl had no effect on [Ca 2+ ] i in these cells. Accordingly, it appears that voltage-dependent calcium influx mechanisms are not present in these cells.

Calcium mobilization plays a major role in the calcium response to many of the P2 receptor agonists tested here. Calcium mobilization occurs through IP3-dependent mechanisms or through calcium-induced calcium release ( 4 ). ATP, ADP, BzATP, ATP- -S, and UTP all stimulated significant elevations in [Ca 2+ ] i involving release of intracellular calcium. While the calcium release mechanisms involved are beyond the scope of this study, previous publications using rat mesangial cells demonstrate an important contribution of IP3-dependent calcium mobilization ( 34, 36 ). Liu and co-workers ( 21 ) reported that mouse mesangial cells do not appear to possess ryanodine-sensitive calcium stores based on a lack of sensitivity to challenges with caffeine. We observed a similar lack of calcium responses to caffeine by mouse mesangial cells (Inscho EW and Rivera I, unpublished observations).

Previous studies performed with rat mesangial cells reveal a different functional and molecular P2 receptor expression profile. PCR studies demonstrate message expression for P2X 2, P2X 3, P2X 4, P2X 5, P2X 7, P2Y 1, P2Y 2, P2Y 4, and P2Y 6 receptors ( 2, 43, 52 ). Functional and protein expression studies support the presence of P2X 7, P2Y 1, P2Y 2, and P2Y 4 receptors by cultured rat mesangial cells with little or no published evidence for other receptor subtypes ( 12, 27, 47, 52, 54, 60 ). In the current report, there is functional and molecular evidence supporting mouse mesangial cell expression of P2X 7, P2Y 2, and P2Y 4 receptors as is observed in the rat, plus the unique expression of P2X 2 and P2X 4 receptors. The presence of these receptors may influence the outcomes of studies designed to assess the physiological role of renal P2 receptors mouse models.

In conclusion, the results of this study demonstrate that cultured mouse mesangial cells express P2X 2, P2X 4, P2X 7, P2Y 2, and P2Y 4 receptor subtypes and do not express the P2X 1 or P2X 3 receptor subtypes, although mRNA for P2X 1 and P2X 3 receptors is detected. Furthermore, the data demonstrate that ATP, ADP, BzATP, ATP- -S, and UTP stimulate an increase in intracellular calcium in mouse mesangial cells that involves activation of calcium influx (ATP and UTP) and calcium mobilization (ATP, ADP, BzATP, ATP- -S, and UTP) pathways. Additional studies are needed to dissect the exact mechanisms that are linked to activation of individual receptors in these cells.

GRANTS

This study was supported by National Institutes of Health Grants HL-074167 and DK-44628 to E. W. Inscho, HL-082733 to M. H. Wang, and HL-058139 to M. B. Marrero, and American Heart Association Grant SDG-0435214N to T. Seki.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. R. Griner, Associate Professor at Augusta State University, for guidance of I. Rivera during the early stages of this work. We also thank Dr. S. Shaw for expert technical assistance. The authors also thank Dr. J. Imig, Dr. Z. Guan, and D. Osmond for thoughtful critique of the manuscript.

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作者单位：1 Department of Physiology and 2 Vascular Biology Center, Medical College of Georgia, Augusta, Georgia