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(Circulation Research. 2003;93:338.)
© 2003 American Heart Association, Inc.

Cellular Biology

ATP-Dependent Mechanism for Coordination of Intercellular Ca²⁺ Signaling and Renin Secretion in Rat Juxtaglomerular Cells

Jian Yao, Michihiro Suwa, Bing Li, Kazuko Kawamura, Tetsuo Morioka, Takashi Oite

From the Department of Cellular Physiology, Institute of Nephrology, Postgraduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan.

Correspondence to Dr Takashi Oite, Department of Nephrology, Institute of Nephrology, Postgraduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. E-mail oite{at}med.niigata-u.ac.jp

	Abstract

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A change in intracellular Ca²⁺ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca²⁺ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca²⁺ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca²⁺ wave to up to 11.9±4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7±0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8±0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca²⁺ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P<0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca²⁺ signals.

Key Words: juxtaglomerular cells • calcium • ATP • renin • mechanical strain

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The renin-angiotensin system is a major regulatory system controlling extracellular fluid volume and blood pressure. The rate-limiting enzyme in this hormonal cascade is renin, which is secreted into the circulation by renal juxtaglomerular (JG) cells of the afferent arteriole. Renin secretion is controlled by a number of factors, such as blood pressure, sodium chloride load, sympathetic nerves, hormones, cytokines, and vasoactive materials.^1–3 Although the signal pathways underlying the actions of these factors differ, it is generally accepted that a change in intracellular Ca²⁺ is the common final pathway through which renin secretion from JG cells is governed. In contrast to other secretory cells in which an increase of Ca²⁺ usually promotes secretion, renin secretion is inversely related to the Ca²⁺ concentration in JG cells. This view is based on the following evidence: Ca²⁺-mobilizing agents inhibit renin secretion¹; Ca²⁺ influx via store-operated Ca²⁺ channels suppresses renin secretion⁴; and lowering the extracellular Ca²⁺ stimulates renin secretion.⁵ Therefore, information relating to the generation, control, and processing of Ca²⁺ signals in JG cells is essential for understanding the behaviors of these cells under various pathophysiological conditions.

Recent studies have indicated that Ca²⁺, besides acting as an intracellular signal, is also able to act as an intercellular signal. Ca²⁺ can spread out in a population of cells as an intercellular Ca²⁺ wave, and groups of cells can coordinate their oscillations, thereby generating synchronous oscillatory Ca²⁺ signals.^6–8 Intercellular Ca²⁺ waves have been demonstrated in a variety of cell types and are presumably responsible for integrated multicellular behaviors. The mechanisms underlying the propagation of intercellular Ca²⁺ waves are thought to be either diffusion of an intracellular messenger through gap junctions or paracrine effects of extracellular nucleotides, such as ATP or UTP.^6–11 Synchronization of Ca²⁺ fluctuations is especially important in secretory units (such as the liver, pancreas, and salivary and lacrimal glands), where Ca²⁺ plays a central role in the initiation, facilitation, and maintenance of cellular secretory functions.¹² Indeed, participation of intercellular communication in the coordination and amplification of insulin secretion in the pancreas,^11,13 bile release in the liver,¹⁴ and catecholamine production in adrenal tissues,¹⁵ has been documented. Because JG cells share many of the properties of secretory cells, it is reasonable to speculate that intercellular communication between these cells may provide them with a pathway for signal transduction as well as for coordination of multicellular secretory functions. The purpose of this study was to address the above hypothesis. Specifically, we asked (1) whether JG cells could communicate with each other to harmonize their intracellular Ca²⁺ signals; and (2) whether the mediator implicated in the coordination of Ca²⁺ transients could alter renin secretion.

Using an in vitro model of mechanical stimulation, our study demonstrated that ATP, but not gap junction channels, was responsible for synchronizing intercellular Ca²⁺ signals in JG cells. Furthermore, ATP was identified as a potent inhibitor of renin secretion in isolated perfused kidneys. It is therefore proposed that ATP released by JG cells under mechanical stimulation might be an important mediator implicated in the control of renin secretion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of Rat JG Cells
Rat JG cells were cultured according to the method described by Carey et al.¹⁶ Briefly, the cortical tissues of male Wistar rats were taken, minced, and digested with enzymatic solution. Cells were separated on a Percoll density gradient, resuspended in culture medium, and seeded onto collagen-precoated plastic tissue culture dishes or special glass-bottom microwell dishes for primary culture. The JG cells within 96 hours of culture were used for experiments.

Immunocytochemistry
Immunocytochemical staining for Cx43 and renin in cultured JG cells was performed as described previously.^17–19 For double-label staining, cells were incubated simultaneously with both goat anti-renin and rabbit anti-Cx43 antibodies, followed by sequential incubation with secondary antibodies, CY2-conjugated donkey anti-goat IgG, and tetramethyl rhodamine B isothiocyanate-conjugated goat anti-rabbit IgG, respectively.

Measurement of Gap Junctional Intercellular Communication (GJIC)
GJIC was assessed by transfer of the membrane-impermeable fluorescent dye, Lucifer yellow (LY), after single-cell microinjection with an automated microinjection system (Zeiss), using a method described previously.^17–19

Single-Cell Mechanical Stimulation
Mechanical stimulation of a single JG cell was achieved by briefly distorting the apical surface of the cells with a blunted prepulled Eppendorf micropipette.¹⁸

Measurement of Ca²⁺
Cultured JG cells were loaded with fura-2 by incubation with 5 µmol/L fura-2 acetoxymethyl ester (Fura-2 AM) in Hanks’ balanced salt solution (HBSS) containing 2.0 mmol/L CaCl₂ and 1.0 mmol/L MgCl₂ at room temperature. Ca²⁺ was determined by the ratio method as reported previously.¹⁸

Mass Mechanical Stimulation and Collection of Extracellular Samples
Mass mechanical stimulation and collection of extracellular samples were performed as previously published with minor modifications.^20,21 Briefly, primary cultures of JG cells in 35-mm culture plates were washed three times with HBSS, and cultured in 0.5 mL of HBSS with or without 10 mg/dish glass microbeads (30 to 50 µm; Polysciences). The dishes were gently tilted to 45 degrees and then returned to a normal position to initiate mechanical stimulation. Twenty seconds after mechanical stimulation, the HBSS supernatants were collected and immediately used for ATP determination.

ATP Measurement
ATP was measured using a luciferin/luciferase bioluminescence assay kit (Molecular Probes) and a luminometer (Luminosenser-PSN.AB-2200).

Isolated Kidney Perfusion
Male Wistar rats weighing 350 to 400 g were purchased from Charles River Japan (Yokohama, Kanagawa, Japan), and the study was approved by the Committee on the Guidelines for Animal Experiments at Niigata University. The kidney perfusion system was established as previously described.^4,22

Renin Activity Assays
To determine the renin activity, perfusate samples were incubated with an excess of rat renin substrate (plasma from 36-hour bilaterally nephrectomized rats). The amount of Ang I generated was determined by a radioimmunoassay using a Renin Riabead kit (Dainabot Co Ltd).

Statistics
Values are expressed as either the mean±SEM or mean±SD. Statistical analyses were performed by unpaired, two-tailed Student’s t test. A value of P<0.05 was considered statistically significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of the Cultured Rat JG Cells
All the experiments in this study were performed on JG cells within 72 to 96 hours of primary culture. During this period, the adherent layer was nearly confluent and composed of cells with prominent cytoplasmic granules (Figure 1A). Immunofluorescent staining of the cultures using a goat anti-renin antibody demonstrated that >90% of the adherent cells exhibited a granular cytoplasmic pattern characteristic of renin particles (Figure 1B).

View larger version (35K): [in this window] [in a new window]   Figure 1. Characterization of cultured JG cells. JG cells at 96-hour culture were used for immunohistological characterization. A, Micrograph of cultured JG cells. Note the prominent cytoplasmic granules. Magnification x600. B, Cells reacted with a goat anti-renin antibody (1:400) followed by Cy2-labeled anti-goat secondary antibodies. Note the positive punctate cytoplasmic staining. C, No labeled cells were observed when the cells were reacted with an irrelevant goat IgG followed by Cy2-labeled secondary antibodies. Magnification x400.

ATP, but not Gap Junctions, Mediates the Mechanically Elicited Ca²⁺ Wave Propagation in JG Cells
To assess the synchronizing mechanisms of Ca²⁺ signaling in JG cells, we studied the mechanically induced propagation of intercellular Ca²⁺ waves in cell monolayers. Mechanical stimulation of a single JG cell using a micropipette induced an immediate elevation of Ca²⁺ in the targeted cell, followed by propagation of the Ca²⁺ signal to the surrounding cells. This wave was able to spread between clusters of cells that did not appear to have physical contact (Figure 2C, top). By analyzing the data from 22 separate Ca²⁺ wave experiments, it was found that the mechanical stress caused a 7-fold increase in the Ca²⁺ concentration in the targeted JG cells, from 30 nmol/L at the basal level to 205 nmol/L after stimulation (Figure 2A). To quantify the number of cells involved in the Ca²⁺ wave propagation, cells with an increase in intracellular Ca²⁺ to >100 nmol/L were arbitrarily designated as positive. According to this criterion, 11.9±4.1 JG cells (mean±SEM; n=22) participated in the Ca²⁺ wave propagation in the control (Figure 2B).

View larger version (105K): [in this window] [in a new window]   Figure 2. Intercellular Ca2+ wave transmission in cultured JG cells. A, Ca2+ concentrations in mechanically stimulated JG cells with or without treatment to remove the extracellular ATP-induced Ca2+ response. Data are the mean±SEM from 10 to 22 mechanically stimulated JG cells. B, Number of cells responding to mechanical stimulation in differently treated groups. Cells where the Ca2+ level increased to >100 nmol/L were designated as responding. Results are the mean±SEM from 10 to 22 separate experiments. C, Typical ratio images of Ca2+ obtained before and after stimulation of a single JG cell. Monolayers of JG cells were loaded with fura-2 AM, and the morphology and distribution of JG cells could be judged by their fluorescent images at 340 nm (I, II, and III). A single JG cell (arrow) was mechanically stimulated during fluorescence ratiometric imaging. Time in seconds before or after stimulation is indicated on each panel. Pseudocolor map represents the estimated Ca2+ concentrations. Top, Control JG cells without any treatment. Middle, JG cells pretreated with 100 µmol/L ATP for 15 minutes before mechanical stimulation. Bottom, JG cells in the presence of 50 U/mL apyrase.

Previous studies in various cell types have demonstrated that propagation of Ca²⁺ waves between cells can be mediated by either intercellular diffusion of messengers via GJIC, extracellular diffusion of cell-released ATP via activation of purinergic receptors, or simultaneously by both mechanisms.^{6–8,10,11,18} Because stimulation of JG cells could result in a Ca²⁺ wave that was able to spread between clusters of cells that did not appear to have physical contact, a mechanism involving diffusion of extracellular mediators is likely to be operating. We therefore focused on the potential role of ATP in the transmission of intercellular Ca²⁺ signaling. For this purpose, we manipulated the JG cell Ca²⁺ responses to extracellular ATP by either pretreatment of cells with high doses of ATP (100 µmol/L) 15 minutes before mechanical stimulation (to desensitize purinergic receptors) or addition of the ATP-degrading enzyme apyrase (50 U/mL) directly into the assay system. Both procedures were shown to be effective in abolishing the JG cell Ca²⁺ responses to the subsequent challenge with extracellular ATP, but did not interfere with the Ca²⁺ release from internal stores, as reflected by an increase in cytosolic Ca²⁺ after subsequent addition of thapsigargin (Figure 3). When mechanical stimulation was used to evoke intercellular Ca²⁺ wave propagation under the above conditions, the increases in Ca²⁺ in the targeted cells were not altered (Figure 2A); however, propagation of the Ca²⁺ wave from the stimulated cell to the surrounding cells was completely eliminated (control, 11.9±4.1, n=22; ATP-pretreated, 1.8±0.9, n=10; Apyrase addition, 1.7±0.7, mean±SEM, n=10) (Figure 2B). Typical sequences of Ca²⁺ ratio images obtained before and after stimulation of a single cell in the differently treated JG cells are depicted in Figure 2C.

View larger version (27K): [in this window] [in a new window]   Figure 3. Modulation of JG cell Ca2+ responses to externally added ATP by pretreatment of JG cells with ATP or addition of apyrase into the assay system. A, JG cells were preincubated with 100 µmol/L ATP for approximately 15 minutes before being sequentially challenged with 100 µmol/L ATP and 200 nmol/L thapsigargin. B, JG cells were exposed to 100 µmol/L ATP and 200 nmol/L thapsigargin in the presence of 50 U/mL apyrase. Results are presented as both the ratiometric imaging (top) and dynamic traces of Ca2+ over time (bottom), representing the average level of Ca2+ in 25 cells in a single study. Similar results were obtained in 4 additional separate experiments.

These results suggest that the changes in Ca²⁺ in JG cells occurring during mechanically stimulated Ca²⁺ wave propagation are mediated by an ATP-dependent pathway. Consistent with this conclusion, the gap junction inhibitor heptanol (1 mmol/L) had no influence on the propagation of Ca²⁺ waves (data not shown). In addition, the major gap junction protein, connexin43, was not detected in the primary culture of JG cells by immunochemical staining (Figure 4). Furthermore, the dye transfer assay using a single-cell microinjection of Lucifer yellow also did not support the existence of functional gap junctional communication between these cells. In 10 separate experiments, no dye diffusion from the injected cell was observed (dye-coupled cell number=1±0; mean±SEM; n=10) (Figure 5). As a positive control for these experiments, cultured mesangial cells, which anatomically adjoin JG cells in vivo, possessed abundant Cx43 in the regions of cell-to-cell contacts and were coupled with each other via gap junctions (Figures 4 and 5).^17,18

View larger version (19K): [in this window] [in a new window]   Figure 4. Negative staining of Cx43 in cultured JG cells. Double-label immunofluorescence studies of JG cells with anti-renin and anti-Cx43 antibodies. Note the positive staining of renin particles (green, A) in these cells but no staining for Cx43 (red, B). As a positive control for Cx43, the characteristic punctate staining of Cx43 was found in both the cell-cell contact and perinuclear regions in cultured rat mesangial cells (red, C) under the same staining conditions. Magnification x400. Similar results were obtained in 4 additional separate experiments. No labeled cells were found after reactions with irrelevant primary or secondary antibodies (data not shown).

View larger version (111K): [in this window] [in a new window]   Figure 5. No diffusion of Lucifer yellow (LY) dye from microinjected JG. LY was pressure-injected into a single JG (top) or mesangial (bottom) cell. LY diffusion into adjacent cells was monitored within a 3-minute time period. A and C, Phase-contrast micrographs of cultured cells. B and D, LY diffusion. Magnification x320.

Release of ATP by Mass Mechanical Stimulation of JG Cells
To confirm that the mechanical strain induced the release of ATP from JG cells, a method involving massive mechanical stimulation of JG cells with microbeads was used,^20,21 and the amount of ATP in the supernatants after stimulation was measured using a sensitive luciferase bioluminescence assay. As expected, the mechanical strain caused a 3-fold increase in ATP (Figure 6) in samples collected from JG cultures (control=42±14 nmol/L; mechanically stimulated=125±17 nmol/L; mean±SEM; n=10; P<0.01). It should be noted that the actual concentration of ATP in the proximity of ATP-releasing JG cells was likely to have been much higher, because the released ATP would not have had time to equilibrate fully with the overlying medium. In addition, extracellular ATP is rapidly hydrolyzed by membrane-bound ectoenzymes. These results support the idea that ATP is the extracellular messenger mediating the propagation of intercellular Ca²⁺ signaling in mechanically stimulated JG cells.

View larger version (9K): [in this window] [in a new window]   Figure 6. Induction of ATP release from JG cells by massive mechanical stimulation. Primary cultures of JG cells in 35-mm culture plates were washed 3 times with HBSS and cultured in 0.5 mL of HBSS with or without glass microbeads. Dishes were gently tilted to 45 degrees and then returned to a normal position to initiate mechanical stimulation. Twenty seconds after mechanical stimulation, the HBSS supernatants were collected and immediately used for ATP determination. Values shown represent the mean±SEM from 10 separate experiments. *P<0.01 vs the control.

Inhibition of Renin Secretion by ATP in Isolated Kidneys
ATP can both induce intracellular Ca²⁺ and mediate synchronization of intercellular Ca²⁺ signaling in JG cells, and this led us to address the role of ATP in the control of renin secretion. To this end, we examined the effect of ATP on renin secretion in the isolated perfused kidney. We chose this experimental model instead of cell culture, because it is difficult to observe acute changes in renin in culture systems.

Isolated rat kidneys were perfused at a stable basal flow rate of 18 mL/min, and the perfusion pressure was around 70 to 90 mm Hg (80±15 mm Hg; mean±SEM; n=11). The renin secretion rates from the isolated kidneys reached stable plateaus 10 to 20 minutes after the onset of perfusion. Because the low renin secretion rate seen under basal conditions may hinder observation of small decreases in renin secretion after administration of ATP, the action of ATP on the renin secretion rate was observed under conditions of prestimulated renin secretion. Lowering the perfusion flow rate from the basal 18 mL/min to 5 mL/min led to a decreased perfusion pressure (from 80±15 mm Hg to 36±7 mm Hg; mean±SEM; n=11) and a more than two-fold increase in the renin secretion rate (from basal 23±8 to 56±9 ng Ang I per h · min^-1 · g^-1 after lowering the perfusion flow rate) (Figure 7B). When 1 mmol/L ATP was added to the perfusate 20 minutes after lowering the flow rate, there was an immediate increase in the renal vascular resistance, as reflected by the increased perfusion pressure (Figure 7A), which was transient and lasted for about 5 minutes (5.1±2.1 minutes; mean±SEM; n=6). The maximal pressure change was 46±25 mm Hg. In addition to the elevated renal vascular resistance, there were significant reductions in the renin secretion rates, which even decreased to levels below the basal values. Thirty minutes after ATP administration, the renin secretion rate was 21% of the untreated control (ATP-treated, 8.2±4.7; untreated, 38.6±7.3 ng Ang I per h · min^-1 · g^-1; P<0.01) and 35% of the basal value (P<0.01). In contrast to the short-term increase in renal vascular resistance seen, the inhibition of renin secretion by ATP was persistent and was sustained over the whole observation period of 30 minutes (Figure 7B).

View larger version (22K): [in this window] [in a new window]   Figure 7. Effects of ATP administration on perfusion pressure (A) and renin secretion rate (B). ATP (1 mmol/L) was administered into the perfusate 20 minutes after lowering the perfusion flow from the basal 18 mL/min to 5 mL/min. Perfusion pressure was monitored (A) and the renin secretion rate (B) was calculated as described in Materials and Methods. A, Representative examples of the change in renal perfusion pressure in the control (top) and ATP-treated (bottom) groups. B, Dynamic change in renin secretion rate. Results presented are the mean±SEM (n=5 for the control and n=6 for the ATP-treated group). #P<0.05, *P<0.01 vs the control; **P<0.01 vs both the control and the basal values.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increases in intracellular Ca²⁺ concentration that spread from cell to cell provide a mechanism for cells to coordinate many cellular activities. In this study, we investigated the mechanisms implicated in the transmission of intercellular Ca²⁺ signals in JG cells, using an in vitro model of single-cell mechanical stimulation. Our study demonstrated that ATP, rather than gap junction channels, mediates the transmission of mechanically elicited intercellular Ca²⁺ signaling in JG cells. The evidence supporting this conclusion is as follows: (1) the mechanically elicited Ca²⁺ wave was able to spread between clusters of cells that did not appear to be in physical contact; (2) desensitization of purinergic receptors with ATP or addition of an ATP-degrading enzyme directly into the assay system could completely eliminate the mechanically elicited propagation of the intercellular Ca²⁺ wave; (3) a mechanical stretch-dependent release of ATP from JG cells was detected; and (4) the gap junction inhibitor heptanol, at a concentration (1 mmol/L) that proved to be effective in blocking Ca²⁺ wave propagation in mesangial cells,¹⁸ did not exhibit any effects in JG cells (data not shown). Furthermore, our data support neither the presence of the major gap junction protein Cx43, nor functional gap junction communication in cultured JG cells. Thus, it seems unlikely that the Ca²⁺ waves in these cells were propagated by a gap junction-dependent mechanism.

The lack of gap junction-mediated cell-to-cell communication in JG cells is unexpected. Ultrastructural observations revealed the presence of gap junctions among renin-secreting cells.²³ Furthermore, histochemical data from our own studies,^18,19 as well as those from other investigators,^24–26 have shown the presence of abundant gap junction proteins, Cx43 and Cx40, in the juxtaglomerular region, although characterization of the proteins on a cellular basis has not been performed. The reason for the lack of functional gap junction communication in cultured JG cells is unclear. Loss of the differentiated cell phenotype in culture or the presence of gap junction proteins other than Cx43, which are less permeable to Lucifer yellow or Ca²⁺, might be possible explanations. The selective permeabilities of different connexin proteins to Ca²⁺ and dyes have recently been reported.^27,28 In this context, participation of gap junction channels in intercellular communication in vivo is still likely. A detailed structural and functional analysis of gap junctions in JG cells in vivo is needed for conclusive evidence.

Ca²⁺ waves are considered to coordinate multicellular processes, such as ciliary beating in tracheal epithelial cells,²⁹ bile expulsion in the liver,¹⁴ hormone secretion in the pancreas,³⁰ information processing in neural cells,³¹ and mesangial cell contraction in the kidney,¹⁸ and we therefore tried to elucidate the physiological relevance of the intercellular Ca²⁺ waves in JG cells. Because ATP was identified as mediating the propagation of Ca²⁺ waves in JG cells, the question equates to addressing the role of ATP in renin secretion. For this purpose, we examined renin release in isolated perfused kidneys after administration of exogenous ATP. We chose the perfusion system instead of a cell culture model for this study because it is impossible to detect acute changes in renin in culture with the assays that are presently available. As a signaling mediator implicated in the regulation of renin secretion, ATP should be able to affect renin secretion quickly. As expected, ATP induced a rapid and potent inhibition of renin release. This result is consistent with the generally accepted notion that Ca²⁺-mobilizing agents suppress renin secretion.^1,3 Because all the cells in the JGA are considered to possess purinergic receptors,^32–35 it can be envisaged that the activation of these receptors by ATP would elicit a rise in intracellular Ca²⁺ in these cells, thus leading to contraction of smooth muscle cells and reduced secretion of renin in JG cells. However, it is noteworthy that the factors and mechanisms actually contributing to the ATP-induced inhibition of renin secretion in this system might be multiple and complicated. For example, ATP can be rapidly and extensively degraded in vivo by membrane-bound ectoenzymes, and the metabolized product, adenosine, was reported to be capable of influencing renin secretion³⁶; furthermore, ATP-inducible endothelial-derived autocoids, such as NO and PGE2, were proven to be potent regulators of renin secretion.^3,37 In addition, an increase in perfusion pressure after ATP administration is also able to cause a subsequent reduction in renin secretion via the baroreceptor mechanism.³⁸ Therefore, the suppression of renin secretion by ATP observed in our study may reflect the overall actions of many known or unknown factors. Nevertheless, it does not alter our conclusion that ATP is able to modulate renin secretion.

It must be stressed that previous studies regarding the effect of ATP on renin secretion have produced inconsistent results. Using cultured mouse juxtaglomerular cells, Kurtz et al³⁹ reported that ATP had no effect on the basal renin secretory rate; however, it antagonized forskolin- and isoproterenol-stimulated renin release. Churchill and Ellis⁴⁰ reported that ATP stimulated renin secretion from rat renal cortical slices via a NO-dependent mechanism. These discrepancies could be due to the different experimental systems used. The exposure time to ATP, the presence or absence of heterocellular interactions, and the extent of ATP degradation are all potential factors influencing the results.

Our findings may have significant physiological implications. (1) The results provide a coordinating mechanism for renin secretion. In contrast to other secretory cells, which are usually tightly packed in a well-organized structure, JG cells exhibit an irregular and variable arrangement in the wall of afferent arterioles. This structure has been suggested to be not ideal for sensing changes in the hydrostatic pressure.⁴¹ It is therefore unlikely that all the granular cells would be affected by a pressure increase to the same extent. The question arises as to how the coordinated response of JG cells is achieved under this condition, and our study appears to provide an acceptable explanation for this situation. Hydrostatic pressure on afferent arterioles would activate the most sensitive JG cells (just like the mechanically stimulated cells in our system), causing a rise in intracellular Ca²⁺ and a simultaneous release of ATP into the paracellular milieu. ATP-dependent Ca²⁺ propagation mechanisms would allow Ca²⁺ to spread from the most responsive cells to the other cells in the JGA and thereby coordinate their secretory function. Even if the cells are homogenously affected by the stimulus, the autocrine and/or paracrine action of the released ATP would further amplify and integrate individual Ca²⁺ response. (2) The results provide a potential mechanism for the mechanical strain–elicited inhibition of renin secretion. In this study, the Ca²⁺ wave was elicited by mechanical strain via deformation of a JG cell by an external device. This is somewhat similar to the mechanical deformation anticipated in vivo where increased renal perfusion pressure would lead to cellular elongation or stretching. Therefore, demonstration of the release of ATP induced by mechanical stress, as well as the inhibition of renin secretion by ATP, in this study may have significant relevance for the mechanically elicited inhibition of renin secretion. Indeed, the implication of ATP as a signaling mediator in the regulation of vasoconstriction of the afferent arteriole during myogenic responses is now being recognized.^32,33,42 Direct evidence regarding the effects of ATP on renin secretion during renal autoregulatory responses is still lacking. As two major reactions after elevated transmural pressure in the afferent arteriole, vascular constriction, and renin secretion may share the same controlling mechanisms, or at least, be somehow related to each other. Besides that produced by JG cells, ATP from other cell types may also participate in the control of renin secretion. Shear stress–dependent release of ATP has been demonstrated in a variety of cell types, including cells situated adjacent to JG cells, such as endothelial cells and smooth muscle cells.^43–45 Recently, a positive relationship between renal hydrostatic pressure and interstitial ATP concentrations has been established.^42,46 Taken together, these data support the critical role of ATP in mediating the inhibition of renin secretion under mechanical stimulation. Of note, a recent study by Bell et al⁴⁷ has demonstrated that ATP was released from macula densa cells in response to increased luminal NaCl concentration via a maxi anion channel at the basolateral membrane. The released ATP mediated the transmission of intercellular Ca²⁺ signal between macula densa cells and mesangial cells. It is therefore conceivable that a similar paradigm of interaction may exist between macula densa cells and renin-secreting cells, which might be involved in the inhibition of renin secretion seen in tubuloglomerular feedback (TGF).

In summary, our study implicated ATP as a paracellular mediator participating in the transmission of intercellular Ca²⁺ signaling and inhibition of renin secretion in JG cells. The findings may help us to understand the coordinating mechanisms of renin secretion, and may also have close relevance for the mechanically elicited inhibition of renin secretion observed under physiopathological conditions.

	Acknowledgments

 
This study was supported by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Naito Foundation, the Ichiro Kanehara Foundation, and the Promotion of Niigata University Research Project, as well as Grants-in-Aid for the encouragement of young scientists (A: No. 13770598) and for scientific research (C: No. 50303128; C: No. 12671032; and B: No. 15390266) from the Ministry of Education, Science, Sports and Culture, Japan. The authors wish to thank Dr S. Batsford, Institute of Medical Microbiology and Hygiene, Freiburg University, Germany, for a critical review and advice on the manuscript.

	Footnotes

 
Original received February 5, 2003; revision received July 2, 2003; accepted July 8, 2003.

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