INTRODUCTION

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POTENT ACTIONS OF PURINE nucleotides and nucleosides were first reported over 60 years ago by Drury and Szent-Gyorgyi (7). The role of intracellular ATP as a source of energy, a phosphate group donor in phosphorylation reactions, and a substrate of ATPases and adenylate cyclase was established many years ago (9).

Cells possess complex mechanisms for the synthesis of ATP and for its control at the cytosolic level. ATP can be released during neurotransmission into the synaptic space or from "dense granules" during blood platelet activation (24), and stimulation with Ba²⁺ and high K⁺ concentration causes the release of adenine nucleotides from a cultured sympathetic neuron (20). ATP and norepinephrine are co-released from an isolated rat kidney upon renal nerve stimulation (2). In addition, cell damage during tissue injury (e.g., vascular rupture) may also lead to the release of ATP into the extracellular space. Such is the case in the thymus, where lymphocyte differentiation induces extended proliferation and cell death, which may lead to very high ATP concentrations, particularly in the cortex and the corticomedullary junction (13). Therefore, based on the above sources, it appears that extracellular ATP concentrations can attain active levels for extracellular ATP receptor stimulation. The local ATP content will depend on the amount released, the effect of dilution, and the efficiency of adjacent ectonucleotidases. Ectonucleotidases are responsible for extracellular ATP hydrolysis (29); three different enzymes (ecto-ATPase, ecto-ADPase, and ecto-5'-nucleotidase) sequentially dephosphorylate ATP to adenosine. These ectoenzymes might play a role in the modulation of extracellular ATP-induced functional changes. It is well known that extracellular ATP mediates various physiological actions via specific receptors on the surface of cells, termed purinergic receptors or purinoceptors. In 1990, Burnstock (4) proposed a basis for discriminating the two types of purinergic receptor, which was based largely on an analysis of information contained in many previous reports regarding the actions of purine nucleotides and nucleosides on a wide variety of tissues (4). Adenosine and AMP have a higher affinity for the P1 purinoceptor than do ATP and ADP. In contrast, ATP and ADP have a higher affinity for the P2 purinoceptor than do AMP and adenosine. The P1 purinoceptors were subdivided into A₁/R_i and A₂/R_a subtypes according to their relative affinity for a series of adenine analogs and to whether adenylate cyclase activity is increased (A₂/R_a) or decreased (A₁/R_i) in the presence of adenosine (4).

P2 purinoceptors have been subdivided into P2X and P2Y subtypes on the basis of their relative affinity for synthetic ATP and ADP derivatives (16). There are many receptor subtypes, which are P2X₁ through P2X₇ and P2Y₁ through P2Y₈ (25).

Regarding the kidney, much less is known concerning the pharmacological and physiological events involved in P2 purinoceptor action. However, it has recently been demonstrated that P2 purinoceptors stimulate the formation of inositol 1,4,5-trisphosphate (IP₃) in the rat renal cortex (23), release arachidonic acid and metabolites in MDCK cells (11), mediate an increase in intracellular free calcium concentration ([Ca²⁺]_i) in the LLC-PK₁, and cultured glomerular endothelial and mesangial cells (3, 15, 27, 28), and mediate the regulation of adenylate cyclase and protein kinase C activity (1). However, the localization of ATP receptors and the determination of their functional activity in specific nephron segments has not been carried out satisfactorily to date.

We investigated the effects of ATP on [Ca²⁺]_i in freshly isolated nephron segments from rat kidneys and attempted to evaluate the mechanisms by which ATP alters [Ca²⁺]_i in the renal tubule segments. Several types of purinoceptor agonists and antagonists were used for the identification of ATP-responsive purinoceptor subtypes.

	MATERIALS AND METHODS

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Animals. Male Sprague-Dawley rats weighing 200-240 g were purchased from Saitama Experimental Laboratories (Saitama, Japan) and housed in commercial equipment in a conventional environment for at least 3 days prior to use. The rats were provided with a pelleted standard diet (Oriental Yeast, Osaka, Japan) and tap water ad libitum.

Materials. The materials used were purchased from the following sources: fura 2-AM was from Dojin Laboratories (Kumamoto, Japan); ATP, ADP, and UTP were from Boehringer (Mannheim, Germany); 2-MeS-ATP and ,-Me-ATP were from Research Biochemicals (Natick, MA); AMP, adenosine, bovine serum albumin (BSA, fraction V), neomycin, [Arg⁸]vasopressin, bradykinin (BK), reactive blue 2 (RB-2), ionomycin, EGTA, and collagenase were from Sigma Chemical (St. Louis, MO); fetal calf serum (FCS) was from Life Technologies (Bethesda, MD); suramin was from Wako (Tokyo, Japan); [Asn¹,Val⁵]ANG II and endothelin-1 (ET-1) were from Protein Research Foundation (Osaka, Japan). All other chemicals were of the highest grade available.

Preparation of nephron segments and tubule suspensions. Techniques for the microdissection of various nephron segments have been previously described (32). Briefly, male Sprague-Dawley rats were decapitated, and the left kidneys were perfused with modified Hanks' solution consisting of (in mM) 127 NaCl, 0.8 MgSO₄, 0.33 Na₂HPO₄, 0.44 KH₂PO₄, 1 MgCl₂, 10 CH₃COONa, 10 Trizma base, 1 CaCl₂, (pH 7.4) containing 2 pyruvate, 2 DL-aspartic acid, 2 L-glutamic acid, 0.1% (wt/vol) BSA (fraction V) and 0.1% (wt/vol) collagenase (type I, 220 U/mg). Perfused kidneys were removed and cut into 1-mm-thick slices on the corticomedullary axis. The collagenase treatment was carried out at 37°C for 20 min in the modified Hanks' solution. The microdissection of the nephron segments was carried out at 4°C using a fine siliconized stainless steel needle. The nephron segments were identified as previously reported (32). As the predominant segment used, the early proximal tubule (S1) was identified by its attachment to a glomerulus. The cortical and outer medullary collecting tubule (CCT and OMCT, respectively) were dissected from the medullary ray of the cortex and the inner stripe of the outer medulla, respectively.

Tubule suspensions were prepared according to the following procedures (5). Perfused kidneys were divided into the cortex and medulla. The cortex or medulla was cut into small pieces and incubated for 20 min at 37°C aerobically in modified Hanks' solution containing BSA (1 mg/ml) and collagenase (1 mg/ml). The tissue suspension was filtered gently through three layers of gauze to remove the undigested pieces. Tubule suspensions were prepared after washing the sediment twice with the same solution containing neither collagenase nor BSA.

Measurement of [Ca²⁺]_i. The methods for the [Ca²⁺]_i measurements have been previously described (19). Briefly, isolated nephron segments placed on the center of a serological glass were incubated at 25°C for 20 min in modified Hanks' solution containing 10 µM fura 2-AM, 2 mM pyruvate, 2 mM aspartate, 2 mM glutamate, 10% FCS, and 0.5 mM CaCl₂. To remove the extracellular fura 2-AM, the nephron segments were rinsed three times with the same solution containing neither FCS nor fura 2-AM. The nephron segments were then transferred to coverslips (0.12-0.17 mm thickness) and placed in a two-wavelength microscopic fluorometer (model CAM 220, JASCO). The iris of the source of fluorescent light was adjusted to the diameter of each nephron segment. Excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm were used for the fura 2 fluorescence. In all experiments, autofluorescence at both 340- and 380-nm wavelengths was extracted from the total fluorescence intensity increased by agonists. The fluorescence ratio (R) was obtained by dividing the fluorescence excited at 340 nm by that at 380 nm, and the [Ca²⁺]_i was calculated based on the formula described by Grynkiewicz et al. (14). [Ca²⁺]_i = [(R  R_min)/(R_max  R)] × (F_max/F_min) × K_d, where R_min is the ratio at zero calcium in the medium, and R_max is the ratio at saturation calcium concentration, F_min is the fluorescence at 380 nm at zero calcium concentration in the medium, and F_max is the fluorescence at 380 nm at saturation calcium concentration. The effective dissociation constant (K_d) of 224 nM for the fura 2-Ca²⁺ complex was used as reported by Grynkiewicz et al. (14).

Measurement of IP₃. Because of the limited sensitivity of the IP₃ assay using isolated S1 and OMCT, we used renal cortical and medullary tubule suspensions (19). In brief, the cortical and medullary tubules suspended in Hanks' medium containing 10 mM LiCl were preincubated at 25°C for 10 min. To analyze the IP₃ production, the tubule suspensions were incubated at 25°C for 20 s following their addition to the agonists. The reaction was terminated by the addition of trichloroacetic acid (15% wt/vol), followed by titration to pH 7.50. The aqueous layer was separated by centrifugation (3,000 g) for 15 min at 4°C. An aliquot (100 µl) of supernatant was transferred to a scintillation vial (polypropylene, 12 × 75 mm) for quantitation of the IP₃. The IP₃ contents in the supernatant were determined using a radioimmunoassay kit (Amersham, UK).

	RESULTS

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P2 purinoceptor distribution in a single nephron. To clarify the P2 purinoceptor distribution in isolated nephron segments, the [Ca²⁺]_i was determined. As shown in Fig. 1, ATP at 10⁵ M caused a significant and immediate increase in [Ca²⁺]_i in isolated rat glomerulus, S1, CCT, and OMCT. The peak values of ATP-induced [Ca²⁺]_i increases were as follows (as % of their basal levels): glomerulus, 147 ± 12%; S1, 139 ± 9%; CCT, 150 ± 10%, OMCT, 275 ± 16%. In contrast, ATP had no appreciable effects on the [Ca²⁺]_i of the proximal straight tubules, the medullary thick ascending limb of Henle's loop, the cortical thick ascending limb of Henle's loop, and distal convoluted tubules (Table 1). UTP (10⁵ M)-induced [Ca²⁺]_i increases were observed in glomerulus, CCT, and OMCT (Table 1). Changes in fura 2 fluorescence showed a transient increase to a peak between 5-15 s after adding agonists and decreased until a steady level was reached between 90-120 s after the peak in the glomerulus and S1. In contrast, sustained increases in the [Ca²⁺]_i in the CCT and OMCT were maintained for longer than 20 min following the initial transient increase. The percent increases in [Ca²⁺]_i in the nephron segments were in the decreasing order of the OMCT > CCT > glomerulus = S1.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Representative tracings of ATP-induced increases in intracellular free calcium concentration ([Ca2+]i) in isolated rat glomerulus (Glm, A), early proximal convoluted tubule (S1, B), cortical collecting tubule (CCT, C), and outer medullary collecting tubule (OMCT, D). ATP was applied as a bolus (arrowheads) at a final concentration of 105 M.

ATP-induced [Ca²⁺]_i changes in S1 and OMCT. Figure 2, A and B, illustrates a typical series of tracings and the dose-response curve of ATP for the [Ca²⁺]_i increases in isolated rat S1 and OMCT, respectively. ATP concentrations required to mediate an increase in [Ca²⁺]_i differed between the S1 and OMCT. [Ca²⁺]_i increase in the S1 was observed only at relatively high concentrations (10⁵ M) of ATP (top of Fig. 2A). At 10³ M ATP, the peak [Ca²⁺]_i of ATP-induced [Ca²⁺]_i increases was calculated as 202 ± 17% of the basal [Ca²⁺]_i in the S1 (Fig. 2B). In contrast, the [Ca²⁺]_i in OMCT could be increased by ATP at lower concentrations (10⁶ M) as shown in the lower part of Fig. 2A. The peak [Ca²⁺]_i in the presence of 10³ M ATP in the OMCT was 356 ± 21% of basal [Ca²⁺]_i (Fig. 2B). The EC₅₀ value of ATP for [Ca²⁺]_i increases in OMCT was estimated at 4 × 10⁶ M. 

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A: typical signalings of ATP-induced [Ca2+]i increases in the S1 and OMCT. ATP was applied as a bolus (arrowheads) at the stated final concentrations. B: dose-response curves of peak values in [Ca2+]i increases evoked transiently as a function of ATP concentrations in isolated rat S1 and OMCT. Fura 2-loaded tubule segments were placed in a 0.5 mM CaCl2-contained medium. ATP at each concentration was added to the medium from a stock solution to give the final agonist concentrations indicated. Each of the peak values was measured 5-15 s after the addition of ATP. Ordinate represents the relative changes in [Ca2+]i with respect to basal [Ca2+]i in the tubules. Basal [Ca2+]i values of S1 and OMCT were 101 ± 11 and 69 ± 5 nM, respectively. Values are means ± SE for 6 experiments.

4

5

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A: representative tracings of ATP-induced [Ca2+]i increases under conditions where the medium contained no calcium in isolated rat S1 and OMCT. To confirm the extracellular Ca2+-free conditions, 1 mM EGTA was added to the media not containing CaCl2 90 s before addition of ATP. B: effects of calcium removal from the medium on peak heights of ATP-induced [Ca2+]i transients in isolated rat S1 and OMCT. Each column represents the relative change in [Ca2+]i with respect to the basal [Ca2+]i in each sample. Values are means ± SE for 4-7 experiments. TMB-8, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride. * P < 0.05 (vs. ATP alone). ** P < 0.01.

Effects of extracellular ATP, ATP metabolites, ATP analogs, and UTP on [Ca²⁺]_i increases. In this series of experiments, we measured [Ca²⁺]_i in both the S1 and the OMCT using ATP, ADP, AMP, adenosine, UTP, 2-MeS-ATP (P2Y-selective agonist), and ,-Me-ATP (P2X-selective agonist) for the characterization of P2 purinoceptor subtype. ATP, ADP, UTP, and 2-MeS-ATP induced increases in these [Ca²⁺]_i values in a dose-dependent manner, but AMP and adenosine failed to induce an increase in [Ca²⁺]_i in both of these nephron segments (Fig. 4, A and B). In contrast, an ,-Me-ATP-induced [Ca²⁺]_i increase was detected only at 10³ M (170 ± 9% in S1 and 223 ± 19% in OMCT). Although in OMCT, the average of maximal response to UTP was larger than that to ATP, it was not significantly larger. Similarly, the average [Ca²⁺]_i increases induced by ATP, UTP, and ADP were larger than that induced by 2-MeS-ATP. Concentrations of ATP higher than 10³ M could not be used because of fluorescent disturbance. The EC₅₀ values in OMCT were estimated to be 6.2 × 10⁷ M for 2-MeS-ATP, 4 × 10⁶ M for ATP, 5 × 10⁶ M for ADP, and 1.5 × 10⁵ M for UTP.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Dose-response curves of [Ca2+]i increase induced by nucleotides, in isolated rat S1 (A) and OMCT (B). Various agonists were added to the medium after recording of basal values of [Ca2+]i. Ordinate represents the relative changes in calculated [Ca2+]i with respect to the basal [Ca2+]i before the addition of agonists to the medium. Basal [Ca2+]i values of S1 and OMCT were 98 ± 6 and 69 ± 7 nM, respectively. Values are means ± SE for 5-9 experiments.

Effects of suramin or RB-2 on ATP-induced [Ca²⁺]_i change. To further characterize the receptor subtype mediating ATP responses in isolated rat S1 and OMCT, we used a selective P2Y agonist, 2-MeS-ATP, and the putative P2Y receptor antagonists, suramin and RB-2. As shown in Fig. 5, A and B, the pretreatment of both these nephron segments with suramin or RB-2 for 2 min dose-dependently inhibited ATP- or 2-MeS-ATP-induced (10⁴ M in S1 and 5 × 10⁶ M in OMCT) [Ca²⁺]_i increases significantly.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of suramin (SU) or reactive blue 2 (RB-2) on ATP- and 2-methylthio-ATP (2-MeS-ATP)-induced [Ca2+]i increases in isolated rat S1 (A) and OMCT (B). Each nephron segment was pretreated with suramin or RB-2 for 2 min before the addition of ATP to the medium.

Effects of extracellular ATP, ATP metabolites, ATP analogs, and UTP on IP₃ production. ATP, ATP metabolites, ATP analogs, and UTP were tested for their effects on IP₃ production in cortical (concentration of agonist = 10³ M) and medullary (concentration of agonist = 10⁴ M) tubule suspensions. ATP stimulated IP₃ formation at 252 ± 38% and 327 ± 62% of control levels within 20 s in cortical and medullary tubule suspension, respectively. The IP₃ concentrations in the controls were 1.6 ± 0.3 and 0.8 ± 0.2 pmol/mg protein for cortical and medullary tubule suspensions, respectively. Figure 6 shows the IP₃ production induced by ATP, ATP metabolites, ATP analogs, and UTP. The ratio of IP₃ production was significantly increased by the treatment of these tubule suspensions with ATP, ADP, UTP, or 2-MeS-ATP. Neither AMP, adenosine, nor ,-Me-ATP induced IP₃ production. Their efficacies with respect to IP₃ formation were similar to their efficacies with respect to inducing transient [Ca²⁺]_i increases.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of ATP, ATP metabolite, ATP analogs, and UTP on inositol 1,4,5-trisphosphate (IP3) production in renal tubule suspensions. Control IP3 contents of cortical (A, agonist = 103 M) and medullary (B, agonist = 104 M) tubule suspension were 1.6 ± 0.3 and 0.8 ± 0.2 pmol/mg protein, respectively. Values are means ± SE for 4 experiments. * P < 0.05, ** P < 0.01 (vs. respective control).

Inhibitory effect of neomycin on ATP-induced [Ca²⁺]_i increase and IP₃ production. To evaluate the possible involvement of phospholipase C (PLC) in ATP-induced transient [Ca²⁺]_i increases and IP₃ production, the effect of neomycin was investigated. The isolated rat S1 and OMCT and cortical and medullary tubule suspensions were pretreated for 15 min with neomycin (3 × 10⁴ M), a PLC inhibitor, before the addition of ATP. As shown in Figs. 7 and 8, ATP-induced transient [Ca²⁺]_i increases and IP₃ production were significantly inhibited by neomycin.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Representative tracings showing effects of neomycin on ATP-induced [Ca2+]i increase in isolated rat S1 (A) and OMCT (B). After the pretreatment of tubule segments with neomycin for 15 min, ATP was applied to each sample (n = 4).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effects of neomycin on ATP-induced IP3 production in renal tubule suspensions. Neomycin was pretreated for 15 min before ATP addition. Control IP3 contents of cortical and medullary tubule suspension were 1.4 ± 0.4 and 0.9 ± 0.3 pmol/mg protein, respectively. Values are means ± SE for 4 experiments. * P < 0.05 (vs. respective control).

Desensitization of ATP- and vasoactive peptidyl hormone-induced [Ca²⁺]_i increases in isolated rat OMCT. Vasoactive peptidyl hormones such as vasopressin (AVP), ANG II, ET-1, and BK also caused [Ca²⁺]_i increases in isolated rat OMCT (Fig. 9A). There was no heterologous cross desensitization between the peptidyl hormones used. Thus we compared the effects of ATP on [Ca²⁺]_i with those of vasoactive peptidyl hormones in isolated rat OMCT. Peptidyl hormones at a maximal dose (10⁶ M) evoked [Ca²⁺]_i transiently (Table 2). The subsequent addition of the same peptidyl hormones caused no rises in [Ca²⁺]_i, suggesting the presence of homologous desensitization for each transient [Ca²⁺]_i increase by the same agonist (data not shown). The successive addition of ATP to the medium after pretreatment of the OMCT with the above hormones induced a [Ca²⁺]_i rise of almost the same magnitude as that of the [Ca²⁺]_i rise induced by a single addition of ATP (Fig. 9B; Table 2). Next, to determine whether ATP induced a depletion of the Ca²⁺ pool, heterologous cross desensitization and the effects of ionomycin on [Ca²⁺]_i were investigated under the calcium-free condition. Even under the calcium-free conditions, the peptidyl hormone-induced [Ca²⁺]_i increase was inhibited by the pretreatment of the OMCT with ATP. After the cross desensitization, ionomycin (10⁵ M) increased [Ca²⁺]_i transiently, and this ionomycin-induced [Ca²⁺]_i increase was much larger than that induced by ATP in extracellular calcium-free conditions (Fig. 9C).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   A: representative signalings of [Ca2+]i induced by peptidyl hormones, in isolated rat OMCT. B: representative signalings of cross desensitization between ATP and AVP in isolated rat OMCT. C: representative signalings of cross desensitization between ATP and AVP and the effect of ionomycin on changes in [Ca2+]i in isolated rat OMCT under the Ca2+-free conditions.

	DISCUSSION

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Previous studies have suggested the existence of both P1 and P2 purinoceptors in the kidney (23). The presence of ATP-responsive (P2) receptor subtypes in cortical slices (23), tubule suspension (5), cultured cells of renal tubular origin (1, 3, 15, 27) and isolated inner medullary collecting ducts (8) has been demonstrated. However, localization of ATP receptors and the identification of their functional significance in specific tubule segments have not been carried out to date.

Because it is well known that ATP induces [Ca²⁺]_i mobilization (3, 8, 15), our present study provides several lines of evidence for the presence of ATP-responsive receptor subtypes, primarily in the S1 and OMCT, using the fluorescent probe fura 2. At first, we demonstrated that ATP receptors are present predominantly in the OMCT and to a lesser extent in the CCT, glomerulus, and S1 of isolated nephron segments in the rat. And UTP-induced [Ca²⁺]_i increases were observed in the glomerulus, CCT, and OMCT. Similar to the response of OMCT to ATP, the response of isolated nephron segments to UTP was the largest in the OMCT. The minimally [Ca²⁺]_i-increasing effective concentrations of ATP were between 10⁶ and 10⁵ M in the S1 and 10⁸ and 10⁷ M in the OMCT. Although plasma ATP concentrations may vary, these concentrations of ATP ranging from 10⁸ to 10⁵ M are likely to exist under physiological and/or pathological conditions. Recently, Ecelbarger et al. (8) have reported the existence of nucleotide receptors in the rat terminal collecting duct. They demonstrated that P2U subtypes are not stimulated by ,-Me-ATP, ADP, or 2-MeS-ATP and that ATP-induced [Ca²⁺]_i increases are inhibited by pretreatment with indomethacin, which implies a coupling of the ATP-mediated signal pathway with cyclooxygenase (6). In addition to the report by Ecelbarger et al. (8), our results provide further information related to the intrarenal P2 purinoceptor distribution in rats.

The source of mobilized calcium was investigated under conditions whereby the extracellular medium contained no calcium. These results suggest that a large portion of the calcium in the initial rise in [Ca²⁺]_i induced by ATP in the S1 and OMCT originates from intracellular stores (Fig. 2A).

In the isolated tubule preparations used in the present study, it is uncertain whether ATP acts on the basolateral or the apical side of the cells. Experiments on the in vitro perfusion of isolated S1 or OMCT are expected to yield an answer to this question. Therefore, further investigations are required.

In rat renal cortical slices (24) and LLC-PK₁ cells (14), the activation of P2 purinoceptors resulted in an increase in [Ca²⁺]_i that was attributable to the release of calcium from intracellular stores by stimulation of PLC activity (6, 23) and by entry from the extracellular medium (6). In rat renal cortical slices (18) and in cultured LLC-PK₁ cells (1), the activation of PLC may be insensitive to pertussis toxin. In other tissues, ATP has been demonstrated to mediate the opening of receptor-operated Ca²⁺ channels (10) and to stimulate the phospholipase A₂ pathway (12), although this may be a secondary effect of the increased [Ca²⁺]_i. Only PLC/Ca²⁺ as a possible second messenger system has been demonstrated to be activated by binding to P2 purinoceptors in the kidney; these receptors, however, have not been well studied. The results using neomycin, a PLC inhibitor (21), clearly showed that the P2Y₁ and P2Y₂ receptors were coupled with PLC (Fig. 7).

In this study, we could not determine whether PLC in both nephron segments was pertussis toxin sensitive because of the difficulty of a long-term incubation, i.e., 6 h for freshly prepared nephron segments. The viability of microdissected nephron segments was relatively stable for up to 2 h following microdissection. To resolve this problem, investigations with cultured cells are required.

To identify the receptor subtype in the isolated rat S1 and OMCT, the effects of ATP and its metabolites on [Ca²⁺]_i increase were compared. The order of potency obtained in this study, i.e., ATP  ADP > AMP = adenosine, clearly indicates that the receptors are P2 purinoceptors (16). At least five P2 purinoceptor subtypes have been proposed as a result of physiological and pharmacological analysis: P2X, P2Y, P2Z, P2T, and P2U (16, 25). Recently, however, there have been some change in terminology; P2Y, P2U, and P2Z are now referred to as P2Y₁, P2Y₂, and P2X₇, respectively (26). The P2U purinoceptor, which is activated by ATP and other nucleotides, such as UTP, has also been described. Studies using other tissues have shown that ,-Me-ATP is 10 times more potent than ATP in stimulating the P2X purinoceptor and is equally or less potent in stimulating the P2Y purinoceptor. Based on this information, our findings clearly exclude P2T as a possible receptor in the rat S1 and OMCT. In the present experiments, ATP, UTP, ,-Me-ATP (P2X-specific agonist), and 2-MeS-ATP (P2Y₁-specific agonist) were compared with respect to their ability to induce [Ca²⁺]_i increases to clarify the receptor subtype(s) in the S1 and OMCT. The order of potency with respect to [Ca²⁺]_i increases was 2-MeS-ATP > ATP  ADP > UTP in the S1 and OMCT. However, 2-MeS-ATP at a maximal dose (above 10⁴ M) was much less effective than ATP, ADP, and UTP in the OMCT (Fig. 4B). A similarly low apparent efficacy of 2-MeS-ATP has been reported for IP₃ formation in endothelial cells (27). According to the data, the ATP-mediating receptor to induce [Ca²⁺]_i increases in the S1 should be the P2Y₁ purinoceptor, and that in the OMCT may be the P2Y₁ and P2Y₂ purinoceptors. This conclusion is supported by the results of another experiment in which the antagonistic effects of suramin and RB-2, an anthraquinone-sulfonic acid derivative (17, 31), were investigated, and these compounds were found to inhibit ATP-induced transient [Ca²⁺]_i increases dose dependently. Therefore, the possibility that the P2Y₂ purinoceptor exists in the OMCT can be considered, because the dose-response curves of ATP and UTP were different, and 2-MeS-ATP could not fully inhibit the response to ATP and UTP (data not shown).

Our present study may support the 1995 report of Zegarra-Moran et al. (33) that two different purinoceptors (P2Y and P2U) are present in the apical membrane of canine kidney cells.

Recently, Valera et al. (30) have reported that molecular cloning data on a new class of ligand-gated ion channel could define the P2X receptor for extracellular ATP, and several molecular cloning investigations have revealed that P2U, P2Y, and P2Y₁ purinoceptors (from mouse neuroblastoma cells, mouse and rat insulinoma, and the embryonic chick whole brain cDNA library by hybridization screening, respectively) are coupled with G proteins that have seven transmembrane-spanning domains (22, 28, 31). In these reports, the orders of agonist potencies for P2U and P2Y₁ purinoceptors are given as ATP = UTP > ATPS  2-MeS-ATP = ADP > adenosine and 2-MeS-ATP  ATP > ADP >> ,-Me-ATP = UTP, respectively. These results indicate that molecular cloning studies are essential for the new classification of P2 purinoceptor subtypes.

The kidney is an important organ for controlling both the volume of body fluids and electrolytic balance. The distribution of renal gluconeogenic activities along the nephron has been determined (32). Yamada et al. (32) have reported that only the proximal tubules, mainly the S1 segments, had gluconeogenic ability. Glucose formed in the cortical tubule suspensions, therefore, should originate from the S1 segments. To elucidate the possible physiological significance of P2 purinoceptors, we demonstrated that gluconeogenesis in cortical tubule suspensions was stimulated by extracellular ATP and that this ATP-stimulated gluconeogenesis was inhibited by the treatment of the tubule suspensions with suramin (5). Because renal gluconeogenesis is localized exclusively in S1 (32), the regulation of gluconeogenesis is one of the functions of P2Y purinoceptors in the kidney.

In the signal transduction pathway in the kidney, vasoactive peptidyl hormones are very important. Therefore, the relationship between [Ca²⁺]_i induced by ATP and that induced by vasoactive peptidyl hormones was investigated. The ATP (10⁴ M)-induced [Ca²⁺]_i increase was not attenuated by the pretreatment of the OMCT with a maximal concentration (10⁶ M) of vasoactive peptidyl hormones, but the [Ca²⁺]_i increase induced by vasoactive peptidyl hormones was almost completely diminished by the pretreatment of the OMCT with a maximal dose of ATP (10⁴ M) (Table 2). Similar results were obtained under the conditions whereby the extracellular medium contained no calcium. As shown in Fig. 9, these results using ionomycin suggest that the ATP-activated calcium pool was overlapped with intracellular calcium pools activated by vasoactive peptidyl hormones and that ATP prominently caused the depletion of these overlapped intracellular calcium pools. This phenomenon suggests that the complete depletion of the calcium store activated ATP may play a role in any physiological response to agonist stimulation in OMCT.

The cytoplasmic ATP concentration in most cells is assumed to be over 5 mM, and a significant proportion of the cytoplasmic ATP can be released into the extracellular space due to sudden cell death; thus the concentrations of pericellular ATP could easily reach a high enough concentration to stimulate the P2 receptors. The local concentrations of ATP may depend on the amount of ATP released, the distributed fluid volume in the extracellular space, and the levels of activity of the catabolic enzymes, especially the ectonucleotidases present on adjacent cells. Teleological reasoning suggests that this possibility for increases in the extracellular ATP concentrations may be why the range of distribution of ectonucleotidases is wide (29).

In summary, the present study demonstrates that ATP mediates an increase in [Ca²⁺]_i in renal epithelial cells of the definite nephron segments like the glomerulus, S1, CCT, and OMCT. Rank-order efficacy studies of ATP analogs suggest that ATP exerts its action by activation of a P2Y₁ purinoceptor in isolated rat S1 and that there are two kinds of P2 purinoceptor (P2Y₁ and P2Y₂) in the OMCT. ATP might affect calcium mobilizations activated by the vasoactive peptidyl agonists.