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J Am Soc Nephrol 13:313-321, 2002
© 2002 American Society of Nephrology

Nitric Oxide Induces Resensitization of P2Y Nucleotide Receptors in Cultured Rat Mesangial Cells

Ruisheng Liu*, Antonio M. Gutiérrez*, Avi Ring*{dagger} and A. Erik G. Persson*

*Department of Physiology, University of Uppsala, Uppsala, Sweden; and {dagger}Norwegian Defense Research Establishment, Oslo, Norway.

Correspondence to Professor A. Erik G. Persson, Department of Physiology, University of Uppsala BMC, Box 572, S-75123, Uppsala, Sweden. Phone: +46-18-4714180; Fax: +46-18-4714938; E-mail: erik.persson{at}fysiologi.uu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Receptor desensitization of G protein–coupled receptors (GPCRs), which occurs during short-term (seconds to minutes) exposure of cells to agonists, is mediated by phosphorylation and receptor endocytosis. Recycling of the receptors is a requisite for resensitization of the response. The mechanisms that attenuate signaling by GPCRs are of considerable importance to regulation of intercellular signaling and maintenance of their ability to respond to agonists over time. This study evaluates the effect of nitric oxide (NO) on P2Y nucleotide receptor resensitization in cultured rat glomerular mesangial cells. The NO production in cultured mesangial cells was measured by using confocal microscopy and the fluorescence NO indicator 4,5-diaminofluorescein diacetate (DAF-2 DA). L-arginine increased and N{omega}-nitro-L-arginine methyl ester (L-NAME) decreased NO production significantly (P < 0.05). Calcium responses to ATP were measured with fura-2 and imaging techniques. Repeated stimulation with ATP results in receptor desensitization that is characterized by lower calcium peak amplitude. Desensitization was induced by challenging mesangial cells with four consecutive 2-min pulses of ATP (0.1 mM) separated by 4.5-min control perfusions. Intracellular calcium concentration ([Ca2+]i) increase evoked by second, third, and fourth ATP challenges were about 40%, 26%, and 18% of the first one. The NO precursor, L-arginine (10 mM), and the NO donors, spermine-NONOate (500 µM) and sodium nitroprusside (SNP) (1 mM), were added before and during a fourth ATP challenge. Spermine-NONOate and L-arginine induced a recovery of the [Ca2+]i response to the fourth ATP challenge (P < 0.01 and 0.05, respectively). The NO synthase inhibitor, L-NAME (5 mM), applied along with ATP, was shown to enhance desensitization. 1H-(1,2,4)oxadiazolo(4,3-{alpha})quinoxalin-1-one (ODQ, 30 µM), an inhibitor of guanylate cyclase, was used along with L-arginine, SNP, or spermine-NONOate. There was no significant difference with or without ODQ. Neither ODQ nor 8-Br-cGMP, an analog of cGMP, at different concentrations showed effects on ATP-stimulated [Ca2+]i. There was no elevation of [Ca2+]i when the cells were challenged by different concentrations (1 µM, 100 µM, 1 mM, 20 mM, and 30 mM) of caffeine, caffeine plus ATP (0.1 mM), and 4-chloro-3-ethylphenol (100 µM, 500 µM, and 1 mM), a new agonist of ryanodine receptors. The results indicate that NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells by acting through a cGMP-independent pathway. No evidence was found for the existence of ryanodine-sensitive intracellular calcium stores in rat mesangial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATP is considered to be an important paracrine regulator of renal hemodynamics (1). ATP binds to cell surface receptors that are designated as P2 nucleotide receptors and are expressed in many cell types. Several P2 receptors have recently been cloned and can be divided into two groups: (1) P2X receptors, which are ATP-regulated ion channels and (2) P2Y receptors, which are G protein–coupled receptors (GPCRs) (2). It was shown that ATP can influence the nephron tubular function in all segments by its action on P2Y receptors subtypes (3). Rat mesangial cells express P2Y receptors (4,5) as well as low levels of P2X receptors; several studies have demonstrated an abundance of P2Y2, but P2Y4 receptors have also been found (5). The P2Y receptors respond to nucleotide stimuli with an IP3-dependent transient increase in intracellular calcium concentration ([Ca2+]i) and a slower phase that is related to calcium influx (6,7).

Cellular responses to agonists of GPCRs are usually rapidly attenuated (8). The mechanisms that attenuate signaling by GPCRs are of considerable interest from several viewpoints. In the healthy organism, they govern the ability of cells to respond to hormones and neurotransmitters regulating intercellular signaling. Agonist removal from the extracellular fluid, receptor desensitization, and receptor endocytosis prevent uncontrolled stimulation of cells. On the other hand, receptor resensitization is also critical because it allows cells to maintain their ability to respond to agonists over time. Receptor desensitization, which occurs during short-term (seconds to minutes) exposure of cells to agonists, is mediated by (1) phosphorylation, which causes uncoupling of activated receptors from G proteins, a process that effectively terminates the signal and (2) receptor endocytosis, which depletes the plasma membrane of high-affinity receptors. This receptor internalization is the first step of receptor recycling, which is a requisite for resensitization of the response. Receptor downregulation is a loss of receptors from a cell that results from long-term (hours to days) continuous exposure of cells to agonists (9,10).

These regulatory mechanisms are also important from a therapeutic viewpoint. Over half of all medicines used today exert their effects through signaling pathways that involve G proteins. In particular, stimulation of the P2Y2 receptor has been proposed as a secretagogue therapy for cystic fibrosis disease (11). New findings indicate a complex signaling pattern of P2Y receptors (12). Desensitization of P2Y2 receptors has been demonstrated in different cell lines and preparations (11,13). Elucidation of the mechanisms involved in P2Y receptor desensitization and resensitization, as well as identification of the specific enzymes that take part, will be important for the understanding of the physiologic role of extracellular nucleotides and will be crucial for any possible use in therapy.

There are multiple interactions between the Ca2+ and the nitric oxide (NO) signaling system. These interactions have been reported in a variety of cell types and preparations and suggest that almost all regulatory mechanisms involved in the control of Ca2+ homeostasis are modulated by NO (14). It was recently shown that extracellular ATP interacts with NO regulation in mesangial cells by inhibiting inducible NO synthase (iNOS) (4). They found that ATP (10-3 M) inhibited 24-h nitrite production induced by lipopolysaccharide/interferon-{gamma} as well as induction of iNOS protein and mRNA. The suppression of iNOS is mediated via activation of protein kinase C through stimulated P2Y2 receptors. Recycling of phospholipase C–ß–coupled receptors has been studied in detail in a few cases (15), but the effect of NO on the recycling process, and thus the calcium response, has not been reported previously. The aim of this study was to characterize the effect of NO on the desensitization-resensitization cycle of the ATP-induced [Ca2+]i response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and Culture of Mesangial Cells
Rat glomerular mesangial cells were cultured as described previously in our lab (7,16). In short, both kidneys from male Sprague Dawley rats (120 to 170 g) were removed and decapsulated under sterile conditions. Cortical tissue was cut away from the medulla and minced in isolation buffer solution (IBS) containing 5 mM KCl, 2 mM CaCl2, 130 mM NaCl, 10 mM glucose, 20 mM sucrose, and 10 mM Tris (pH 7.4; osmolality, 290 mOsm). Glomeruli were isolated by sequential sieving and collected on a 50-µm sieve. After incubation with 0.1% collagenase in IBS for 30 min at 37 C° to remove epithelial cells and obtain glomerular cores consisting mostly of mesangium and capillary loops (17). The glomeruli suspension was centrifuged at 2200 rmp for 7 min at room temperature. The pellet was resuspended with 10 ml of RPMI 1640 medium supplemented with 18% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 0.66 U/ml insulin. The RPMI 1640 medium contained D-valine instead of L-valine. D-valine inhibits fibroblast growth (18). Aliquots of the glomeruli were placed in 25-cm2 tissue flasks (Nunclon; NUNC, Roskilde, Denmark) containing 6 ml of supplemented RPMI 1640 medium. The flasks were incubated at 37 C° and 5% CO2 in a humidified atmosphere in a CO2-controlled incubator for 3 to 6 wk. The medium was changed every third day. Epithelial- and endothelial-specific staining (cytokeratin and factor VIII, respectively) in the confluent cultures of mesangial cells were negative, excluding any contamination.

Measurement of Mesangial Cell NO Production
The primary culture cells were subcultured by incubating in Ca2+- and Mg2+-free phosphate buffered saline–Dullbecco’s modified Eagle medium containing 0.01% ethylenediaminetetraacetic acid (EDTA) and 0.125% trypsin at 37°C for 5 min. After centrifugation, the pellet was resuspended in 6 ml of medium, and aliquots (0.5 ml) were placed onto 24-well dishes, each well containing a 13-mm glass coverslip (Menzel-Gläser, Braunschweig, Germany). A concentration of 9% FCS was used during subculturing and further incubation.

A cell-permeable fluorescence NO indicator, 4,5-diaminofluorescein diacetate (DAF-2 DA) was used to detect NO production in mesangial cells. The cells were incubated with 10 µM DAF-2 DA (1% DMSO) in 9% FCS medium for 30 min in the incubator. The cells were then rinsed twice with the standard experimental solution (control solution) containing 135 mM NaCl, 1.3 mM CaCl2, 1 mM MgSO4, 1.6 mM KH2PO4, 5 mM glucose, and 20 mM Hepes (pH, adjusted to 7.4; osmolality adjusted with sucrose, 290 mOsm). The coverslip was transferred to an experimental chamber mounted on the stage of an inverted microscope (Eclipse; Nikon, Tokyo, Japan) that was connected to a laser confocal system (Noran Instruments Inc., Middleton, WI), which was equipped with an argon-ion laser. Confocal slit widths were 15 nm. Photobleaching was kept at a minimum by maintaining laser intensity below 30% of maximum and by using a software-controlled shutter during intervals of each image. The confocal system is controlled by a Silicon Graphics workstation (Mt. View, CA). The image acquisition was limited to 30 frames/s. When necessary, image noise was reduced by 16 to 32 images jump average. The sampling time for any pixel was 100 ns. DAF-2 DA esther is converted to the acid form DAF-2 by intracellular estherases (19). DAF-2 fluorescence was excited by using a 488-nm argon-ion laser while emitted fluorescence was recorded simultaneously at wavelengths of 510 nm through a 510-nm long pass filter. Relative changes in the NO concentration were expressed relative to the resting level: F - Frest/Frest). The acid form, DAF-2, was used to calibrate the system with the standard NO solution.

The cells were perfused with control solution for 5 min, and one of the following agents was then applied separately in separate experiments: (1) 10 mM L-arginine was added to the control solution for 5 min; (2) NO-synthase inhibitor, 5 mM L-NAME, was added to the control solution for 5 min; (3) 5 mM L-NAME was applied for 5 min and then 10 mM L-arginine plus 5 mM L-NAME were added for another 5 min.

Measurement of Mesangial Cell [Ca2+]i
The subculturing procedure was the same as for the NO measurement. [Ca2+]i was determined with a fluorescence digital imaging system using the intracellular fluorescence indicator Fura 2-AM. The cells were incubated with 10 µM Fura 2-AM (1% DMSO) in 9% FCS medium for 40 min in the incubator. The loaded cells on the coverslip were rinsed twice with the standard experimental solution. The coverslip was transferred to an experimental chamber mounted on the stage of an inverted microscope (Nikon Eclipse). The digital imaging system used was QC-700 (Applied Imaging, Sunderland, England). This system allows for independent evaluation of each of the cells in the field of view (8 to 15 cells). We performed one experiment per coverslip. The cells were excited alternately at 340 nm and 380 nm, and the emission was measured at 510 nm. The 340/380 emission ratio was used to determine the intracellular calcium concentration after calibration in vitro.

Experimental Protocols
The experiments were performed at 37°C with a continuous perfusion rate of 6 to 7 ml/min and consisted of four consecutive ATP challenges: three challenges to induce desensitization and a fourth challenge to check for resensitization after different treatments. ATP challenges consisted of a 2-min perfusion with the standard experimental solution containing 0.1 mM ATP. The challenges were separated by 4.5 min resting periods, i.e., perfused with standard experimental solution.

Seven different groups of experimental series were performed as follows:

  1. Control experiment: standard experiment solution was used throughout the experiment (Figure 2).
  2. Before and during the three desensitizing challenges, cells were perfused with the standard experimental solution. Before and during challenge 4, L-arginine, D-arginine, spermine-NONOate or SNP were added (Figure 3).
  3. Before and during the three desensitizing challenges, cells were perfused with the standard experimental solution containing NO-synthase inhibitor (5 mM L-NAME). Before and during challenge 4, L-NAME was removed and either L-arginine, D-arginine, spermine-NONOate, or SNP was added (Figure 4).
  4. 30 µM ODQ was present throughout the experiment. Before and during challenge 4, L-arginine or spermine-NONOate was added (Figure 5).
  5. Before challenging the cells for the first time, they were perfused for 5 min with either of L-NAME, L-arginine, or spermine-NONOate. In this series, the cells were challenged only once.
  6. Dose response of 8-Br-cGMP and ODQ (Figure 6).
  7. Ryanodine receptor challenge: the cells were challenged by (1) caffeine from 100 nM to 50 mM, (2) ATP (0.1 mM) plus caffeine (0 to 50 mM) (Figure 7), and (3) 4-chloro-3-ethylphenol (100 µM to 1 mM).



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  		   Figure 2. ATP-induced changes of intracellular calcium concentration ([Ca2+]i) in mesangial cells. In control saline solution, four successively smaller ATP-stimulated [Ca2+]i peaks occur due to receptor desensitization. (A) A single-cell response curve and the experimental protocol. (B) The average of all control experiments (n = 246).
 


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  		   Figure 3. The effects of different NO donors and D-arginine on ATP-challenged calcium changes. After the third ATP challenge, different NO donors or D-arginine were added. Compared with the control, spermine-NONOate (n = 43) and L-arginine (n = 126) could significantly recover the fourth ATP-stimulated calcium release. SNP (n = 64) and D-arginine (n = 48) had no significant effect. (A) A representative cell response to L-arginine and the experimental protocol. (B) The average change graph.
 


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  		   Figure 4. The effects of L-NAME, D-NAME, and different NO donors on [Ca2+]i. In the presence of L-NAME, the [Ca2+]i responses to the second, third, and fourth ATP challenges was greatly decreased. After the third ATP challenge, different NO donors were used instead of L-NAME. Spermine-NONOate (n = 173) and L-arginine (n = 149) could significantly recover the fourth ATP-stimulated [Ca2+]i. Sodium nitroprusside (SNP) (n = 98) and D-NAME (n = 71) showed no significant effect. (a) A response of a representative cell to L-NAME and spermine-NONOate, which also shows the experimental protocol. (B) The average data graph.
 


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  		   Figure 5. The effects of 1H-(1,2,4)oxadiazolo(4,3-{alpha})quinoxalin-1-one (ODQ). Different NO donors were added after the third ATP challenge, but ODQ was used throughout the experiment. There were no significant difference in ATP-challenged [Ca2+]i with and without ODQ.
 


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  		   Figure 6. Dose response of 8-Br-cGMP and ODQ on calcium release of mesangial cells. Different concentrations of 8-Br-cGMP and ODQ were added after the third ATP challenge. There were no significant differences among them.
 


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  		   Figure 7. The effects of caffeine plus ATP. Different concentrations of caffeine plus 10-4 M ATP were used to challenge the mesangial cells. The results showed that caffeine could inhibit the calcium release effect of ATP in a dose-response way. The data were from second ATP/caffeine challenge.
 
Chemicals
RPMI 1640 media, FCS, trypsin-EDTA, and PBS were obtained from Biochrom KG, Berlin, Germany. Fura-2 a.m. was from Molecular Probes Inc., Eugene, OR. Spermine-NONOate and DAF-2 DA were from Calbiochem, San Diego, CA. All other chemicals were obtained from Sigma, St. Louis, MO. 4-chloro-3-ethylphenol was a gift from Dr. Shahidul Islam, Karolinska Institute, Sweden.

Statistical Analyses
Nonpaired t test (two-tail) and the Newman-Keuls method were used where appropriate. P < 0.05 was set as the significance level. Data are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NO Production in Mesangial Cells
Loaded mesangial cells were clearly visualized in the confocal microscope, compared with the unloaded cells (Figure 1). Three experiments verified the presence of NO-synthase in the cells: a) the intensity of the fluorescence increased 20% ± 2.0% after L-arginine was added (n = 42). b) When L-NAME was used, the intensity was decreased 8% ± 1.1% (P < 0.05) (n = 51). c) The intensity did not increase after first using L-NAME then adding L-arginine plus L-NAME (n = 47).



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  		Figure 1. NO concentration measured with 4,5-diaminofluorescein diacetate (DAF-2 DA) using confocal microscopy in mesangial cells. (A) Unloaded mesangial cells were investigated by using the same laser intensity as in B, C, and D. (B) The mesangial cells loaded with DAF-2 were in resting condition. (C) The loaded cells were applied with 10 mM L-arginine. The intensity increased. (D) The loaded cells were applied with 5 mM N{omega}-nitro-L-arginine methyl ester (L-NAME), the intensity decreased.

 
[Ca2+]i Measurement
Rat glomerular mesangial cells responded to extracellular ATP with a sharp increase in [Ca2+]i. This response desensitized upon consecutive administration of the nucleotide. Figure 2A shows the result for a representative control experiment for a cell challenged four consecutive times with 0.1 mM ATP. It can be observed that the response to the fourth challenge is practically absent in this particular cell. Control experiments were performed for each of the experimental series. There was no difference among the groups of control experiments. Figure 2B shows the average of all control experiments (n = 246). [Ca2+]i reached 1025 ± 48 nM in response to the first ATP challenge. The response to subsequent challenges diminished to 407 ± 15, 270 ± 12, and 186 ± 10 nM, respectively. These values represent 40%, 26%, and 18% of the first response.

Effects of Endogenous and Exogenous Nitric Oxide in Desensitized Cells
The cells for this series of experiments were desensitized with three ATP challenges. At the end of the third challenge the cells were perfused with L-arginine (10 mM), D-arginine (10 mM), or one of the NO donors, spermine-NONOate (500 µM) or SNP (1 mM). Figure 3A shows a representative experiment where L-arginine was added. The figure also illustrates the experimental protocol. It can be observed that after a marked desensitization, L-arginine (endogenously increased NO) induced a recovery of the response. A summary of the results of this experimental series is shown in Figure 3B. L-arginine (n = 126) and spermine-NONOate (n = 43) were able to induce resensitization. The response for both agents to the fourth ATP challenge was significantly higher than that of control. No effects were observed when SNP (n = 64) or D-arginine (n = 48) were used.

Effects of Nitric Oxide Synthase Inhibition
The cells for this series of experiments were also desensitized with three ATP challenges, and iNOS was inhibited with 5 mM L-NAME. In the presence of L-NAME, the response to the second, third, and fourth ATP challenges were significantly lower. At the end of the third challenge, L-NAME was removed and either L-arginine, D-arginine, or one of the NO donors, spermine-NONOate or SNP, was added. Figure 4A shows a representative cell from an experiment in which spermine-NONOate was added. This figure also illustrates the experimental protocol. In the presence of L-NAME, desensitization was enhanced (Figure 4B) and spermine-NONOate (exogenously increasing NO) induced a substantial resensitization of the response to ATP. Figure 4B shows a summary of the results from this experimental series. D-NAME (n = 71) had no effect on the desensitization process. After removal of the inhibitor, L-arginine (n = 149) and spermine-NONOate (n = 173) were able to induce resensitization, but SNP (n = 98) had no effect.

Effects of Guanylate Cyclase Inhibition and a cGMP Analog
Experiments were performed in the presence of ODQ to inhibit guanylate cyclase during the whole experiment. Figure 5 shows the summary of the results. It can be seen that inhibition of guanylate cyclase had no effect on either the desensitization or the NO-induced resensitization. Figure 6 shows the effects of different concentrations of ODQ and the cGMP analog, 8-Br-cGMP, on the fourth ATP challenges. Neither ODQ nor 8-Br-cGMP at different concentrations showed any significant effect on ATP-stimulated [Ca2+]I, indicating that the effects of NO are not mediated through activation of protein kinase G (PKG). It is possible that the effects are due to the interaction with superoxide and formation of the highly reactive peroxynitrite species.

Effects of Nitric Oxide on Unactivated Receptors
The cells were superfused with L-NAME, L-arginine, or spermine-NONOate for a period of 5 min before and during the ATP challenge. In this way, we evaluated the effects of NO on receptors that had not been activated by ATP. The results are presented in Table 1. The responses observed in the three conditions were not different from the response in the control experiment.


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  		Table 1. Effects of nitric oxide on unactivated receptorsa
 
Ryanodine Receptors Challenges
Calcium response may be modulated by calcium-mediated calcium release; therefore, it was relevant to investigate the contribution of ryanodine-sensitive pools. There was no elevated [Ca2+]i when the cells were challenged by caffeine at concentrations of 1 µM, 100 µM, 1 mM, 20 mM, and 30 mM separately for 2 min. This was followed by a perfusion with the standard experimental solution for 5 min. Figure 7 shows the results of the cells challenged by caffeine (0, 100 nM, 1 mM, 20 mM) plus ATP (0.1 mM). Caffeine can inhibit the ATP effect at low concentrations (100 nM). At 20 mM caffeine almost totally blocks the calcium release effect of ATP. 4-chloro-3-ethylphenol (100 µM, 500 µM, and 1 mM), a new agonist of ryanodine receptors (20), showed no effect on [Ca2+]i increase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NO, a simple molecule synthesized from L-arginine by NOS, has been identified as playing an important role in cell communication, cell defense, and cell injury (21–23). NO is also important in the physiologic regulation of glomerular capillary pressure, glomerular plasma flow, and the glomerular ultrafiltration coefficient. Through its actions on glomerular pressures and flows, NO may also regulate the macromolecular and micromolecular traffic through the mesangium (24,25). Mesangial cells may be expected to be exposed to NO through the high production of NO from macula densa cells adjacent to the glomerular mesangial cells. Also, iNOS can be expressed in mesangial cells. When iNOS is induced, it produces large amounts of NO that can influence cell and tissue function and cause damage. DAF-2 DA is a newly developed indicator of NO (26). DAF-2 selectively traps NO between two amino groups in its molecule and yields triazolofluorescein, which emits green fluorescence when excited at about 490 nm. The fluorescence intensity is dependent on the amount of NO trapped by DAF-2. DAF-2 has been used as a specific NO indicator in different cells and tissues (27–30). We found that L-arginine can increase to a large extent and that L-NAME can significantly decrease the NO concentration, and the increase caused by L-arginine can be inhibited with L-NAME (Figure 1). This indicates that NO is produced by unstimulated mesangial cells and that NO production can be stimulated by application of L-arginine. This finding is also in agreement with other reports (31–35) regarding measurement of nitrite and/or nitrate, which are end-products of NO interaction with superoxide species. They found that there is a basal level of NO production in unstimulated mesangial cells. It has also been reported that NG-monomethyl-L-arginine or L-NG-monomethylarginine could reduce the nitrite concentration in mesangial cells in basal condition (36,37).

Care was taken in this study to accurately follow the designed time schedule. The duration of each ATP stimulation was exactly 2 min, and the recovery interval without ATP was exactly 4.5 min, because the receptor desensitization is dependent on the time during which the cells are exposed to the agonist (38). With the control solution, the [Ca2+]i stimulated by the first ATP challenge was about 1000 nM, but the [Ca2+]i stimulated by the second, third, and fourth ATP challenges were only about 40%, 26%, and 18% of the first peak (Figure 2). After three ATP challenges were performed, the receptors were desensitized to a large extent. When three timed ATP challenges were finished, different NO donors were added to the control solution. The result showed that spermine-NONOate (a class of sulfur-free compounds capable of releasing NO) and L-arginine can significantly increase the [Ca2+]i during the fourth challenges with ATP (Figure 3).

The NO synthase inhibitor, L-NAME, was shown to increase purine receptor desensitization. This effect is consistent with the assumption that cellular NO production is sufficient for a partial recovery of the response. It is also consistent with the observation that after iNOS inhibition by L-NAME, the effect of exogenous NO was more evident (enhanced). Unstimulated mesangial cells do not express constitutive NOS. It is therefore not possible to exclude that iNOS activitation seen in this report is due to the effect of cytokines or endotoxin during cell culture. Furthermore, it has been reported that in FCS-free medium, the increased nitrite and iNOS expression were detected in cultured mesangial cells (39,40). In this study, 18% FCS was used during culture, 9% FCS was used in subculture for 24 to 48 h, and the FCS-free solution was used during the experiment, which also might be another possibility for enhancing the activity of iNOS. Nevertheless, it is a condition of great relevance to conditions of inflammatory disease (4). Also, the effects of exogenous NO are independent of constitutive NOS and indicate a possible effect of NO production in macula densa cells close to mesangial cells. Among the NO donors, the spermine-NONOate had the greatest effect, although it was only tested at a single concentration. This is consistent with other reports (41,42). Though our data do not contradict the hypothesis that SNP induces resensitization, there is no significant difference to control (Figure 4). This finding could probably be attributed to the amount of NO released from different NO donors. The release rate of 1 mM SNP is only 4 nM/min, whereas 500 µM spermine-NONOate releases 116.5 nM/min (43,44).

The results of using D-arginine and D-NAME showed that there were no significant differences with D-arginine, D-NAME, and control (Figure 4). This finding supports the view that ATP-stimulated calcium release seems to be solely dependent on NO and not due to a charge effect.

From our data, it appears as if NO affects ATP-stimulated calcium release either through the intracellular calcium release cascade or/and through the receptor resensitization pathway. So far, NO has been identified as one of the key messengers that governs the overall control of Ca2+ homeostasis (14,45). NO can inhibit Ca2+ release from IP3-sensitive stores (the GPCR-PLC-IP3 cascade) through a cGMP-dependent pathway. This has been demonstrated in various cell systems (46–49), a mechanism operating through the activation of PKG. The results are contradictory in mesangial cells (50,51). In general, NO-stimulated activation of PKG is associated with a decrease in intracellular calcium, a mechanism consistent with decreased contractility of smooth muscle cells. To investigate the possible effects of PKG on the calcium response to ATP, the guanylate cyclase inhibitor, ODQ, and the cGMP analog, 8-Br-cGMP, were used to determine the contribution of the cGMP pathway. Our results indicate that there are no significant differences with or without ODQ and 8-Br-cGMP (Figures 5 and 6). NO may also play a role in controlling Ca2+ release from ryanodine-sensitive intracellular calcium stores (52). A recent study has indicated that NO can directly activate ryanodine receptors (53). Therefore, we attempted to identify the ryanodine-sensitive intracellular calcium stores in the cells. However, we have failed to see calcium release response when challenging with caffeine, caffeine plus ATP, and 4-chloro-3-ethylphenol, a new ryanodine receptor agonist (54,55). We found that a low dose of caffeine (100 nM) can inhibit calcium release effect by ATP (Figure 7), similar effects have been reported in the other kinds of cells and tissues through IP3-sensitive intracellular calcium stores (56,57). The mechanism is not clear, probably because caffeine inhibits the binding site of IP3 receptors and deactivates the receptors (58). Our results indicate that there are no ryanodine-sensitive intracellular calcium stores in rat mesangial cells.

Thus, we hypothesize that NO has its effect on changes in ATP-stimulated [Ca2+]i through effects on receptor recycling. To further test this hypothesis, we designed another experimental protocol. Different NO donors or L-NAME were perfused for 5 min separately before ATP was used. It has been shown that the receptor desensitization cascade is not activated until the receptors are stimulated by agonists (59). Thus we can exclude the contribution of receptor desensitization cascade in only one ATP challenge. We found that there was no significant difference in ATP-stimulated [Ca2+]i after a 5-min perfusion of different NO donors and L-NAME (Table 1). The lack of effects of NO before the first stimulus indicates that NO increases P2Y receptor resensitization in rat mesangial cells.

In summary, we have found that L-arginine and spermine-NONOate were able to induce P2Y receptor resensitization in rat mesangial cells, that the presence of L-NAME enhanced receptor desensitization, that these effects are mediated through a cGMP-independent pathway, and that it seems that there are no ryanodine-sensitive intracellular calcium stores in rat mesangial cells.


   Acknowledgments
 
This study was financially supported by the Swedish Medical Research Council (project number K99–14X-03522–28D), the Wallenberg Foundation, and the Ingabritt and Arne Lundberg Foundation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 27, 2001. Accepted for publication August 11, 2001.




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