INTRODUCTION

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ANG II HAS A KEY ROLE IN the renal control of Na⁺, acid-base, and water balance not only by controlling aldosterone secretion and affecting renal hemodynamics but also by acting directly on tubular transports. In the proximal tubule, in which ANG II has basolateral and apical receptors, low ANG II concentrations (10¹⁰ M) stimulate but high ANG II concentrations (10⁷ M) inhibit NaCl, NaHCO₃, and water absorption (reviewed in Ref. 7). The latter effects occur through several transduction pathways acting on various carriers. For example, the proximal apical Na⁺/H⁺ antiport system, which is critical to NaHCO₃, NaCl, and water absorption, is stimulated by low ANG II concentrations through both decrease in cAMP production and activation of protein kinase C (PKC), and it is inhibited by high ANG II concentrations through activation of arachidonic acid metabolism via the cytochrome P-450/monooxygenase pathway (reviewed in Ref. 7). These ANG II experimental effects are physiologically or pathophysiologically relevant, since ANG II concentrations up to 2 × 10⁸ M have been measured in the proximal tubular fluid, concentrations that are much higher than the 10¹¹ to 5 × 10¹⁰ M plasma ANG II concentrations because of intrarenal generation of ANG II (6, 24, 25).

ANG II may also act at tubular sites distal to the proximal tubule because specific ANG II binding sites and mRNA for ANG II receptors are present in the distal segments of the nephron (22, 23, 27), but possible distal tubular effects of ANG II have been directly addressed in only a few studies. Recent work has established that ANG II stimulates Na⁺, HCO₃, and water absorption in the rat distal tubule accessible to micropuncture (18, 19, 29). In the rabbit cortical collecting duct microperfused in vitro, 10⁷ M ANG II stimulates luminal alkalinization (HCO₃ secretion by B-type intercalated cells) (30). No direct evidence is available, to our knowledge, regarding possible ANG II effects on thick ascending limb (TAL) transepithelial transports. However, specific ANG II binding sites and mRNA for ANG II receptor subtype 1 (AT₁) are present in the TAL, particularly in the medullary TAL (MTAL) (22, 23, 27). In that segment, the Na⁺-K⁺-2Cl cotransporter is a major apical carrier responsible for luminal uptake and thus transcellular absorption of NaCl and for much of the luminal step of transcellular MTAL NH⁺₄ absorption (10, 11) because it can function in a Na⁺-NH⁺₄-2Cl mode (2, 3, 14); the Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity thus contributes to the degree of medullary hyperosmolality and thus water excretion as well as to the process of NH₃-NH⁺₄ accumulation in the renal medulla, which is critical to NH⁺₄ and thus net acid urinary excretion.

These considerations prompted us to directly assess possible effects of ANG II on Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity of freshly harvested MTAL cells. To this purpose, we have monitored intracellular pH (pH_i) in suspensions of rat MTALs by use of the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF); rates of pH_i changes due specifically to NH⁺₄ transport were determined to quantify the bumetanide-sensitive cotransport activity, as previously reported (1). The results show for the first time that ANG II inhibits the MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity compared with untreated controls at the very low concentration of 10¹² M but that higher ANG II concentrations (5 × 10¹¹ M) progressively stimulate cotransport activity compared with the level seen with 10¹² M ANG II; the inhibitory basal effect is caused by cytochrome P-450/monooxygenase-derived products of arachidonic acid [20-hydroxyeicosatetranoic acid (20-HETE)], whereas the stimulatory effect is mediated by PKC.

	METHODS

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Isolation of rat MTAL tubules. The method used has been previously described in detail (17). In brief, eight kidneys of four anesthetized male Sprague-Dawley rats (200-300 g body wt) were bathed in situ for 1-2 min with ice-cold dissecting solution before rapidly removing them to avoid anoxic damage to medullary tissues and to improve cell viability. The kidneys were then cut into thin slices along the corticopapillary axis into ice-cold Hanks' solution supplemented with 24 mM HCO₃ and bubbled with 95% O₂-5% CO₂, pH 7.38. Small tissue pieces of inner stripes of outer medulla were then subjected at 37°C to successive 6-min periods of collagenase digestion (0.40 g/l); the MTAL tubules were harvested by sieving the supernatants through a nylon mesh (75-µm opening) to separate MTAL fragments from isolated cells and small fragments of other medullary tissues and were resuspended in an appropriate volume of the desired medium. The final suspension contained almost exclusively MTAL tubules 75-200 µm in length (>95%), occasional thin descending limb fragments, very few medullary collecting tubules, and virtually no isolated cells. Because the present study deals with possible tubular effects of ANG II, we have checked by electron microscopy that there were no proximal tubule fragments in this MTAL suspension obtained from carefully dissected inner stripes of outer medulla. (Electron microscopy was performed by Gerard Feldmann and Alain Moreau, Institut National de la Santé et de la Recherche Médicale Unité 327, Paris, France.) MTAL fragments were suspended at 37°C in a CO₂-free medium composed of (in mM) 125 NaCl, 15 choline chloride, 3 KCl, 0.8 K₂HPO₄, 0.2 KH₂PO₄, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 5 glucose, and 5 L-leucine; this solution was adjusted to pH 7.4 with Tris, contained 0.1 g/l BSA, and was bubbled with 100% O₂; in some solutions, 10 mM BaCl₂ isosmotically replaced choline chloride.

Measurements of pH_i. pH_i was estimated with use of BCECF as previously described in detail (17). In brief, aliquots of BCECF-loaded tubules were diluted into glass cuvettes containing 2 ml of the experimental medium, and BCECF fluorescence was monitored with a Jobin Yvon JY3D spectrofluorometer equipped with a water-jacketed, temperature-controlled cuvette holder and magnetic stirrer. Fluorescence intensity was recorded at one emission wavelength, 525 nm, whereas the excitation wavelength was alternated manually at regular intervals between two wavelengths, 504 and 450 nm. The fluorescence ratio values (F₅₀₄/F₄₅₀) were converted into pH_i values with calibration curves that were established daily by the high-K⁺ medium-nigericin and/or Triton X-100 methods, as described previously (17).

Measurements of intracellular Ca²⁺ concentration. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was estimated with use of the Ca²⁺-sensitive fluorescent probe fura 2 exactly as previously described (5). In brief, fura 2-loaded tubules were diluted into glass cuvettes placed in the spectrofluorometer described above, and fluorescence intensity was recorded at a 495-nm wavelength (10-nm bandwidth), whereas the excitation wavelength was manually changed at 3-s intervals between 340- and 380-nm wavelengths (4-nm bandwidth). At the end of each run (<90 s), 5 mM EGTA was added first, to estimate and correct for the fluorescence of fura 2 that leaked out of the cells, as previously described (5). Then 15 mM Tris and 6.5 µM digitonin were added to permeabilize the cell plasma membrane and determine the minimum fura 2 fluorescence ratio, which was followed by addition of 5.5 mM CaCl₂ to determine the maximum fura 2 fluorescence ratio. The signals generated by fura 2-loaded cells were corrected for autofluorescence determined at the two excitation wavelengths on a fura 2-free MTAL suspension aliquot. Values of [Ca²⁺]_i were calculated with the usual equation (5).

Materials. Collagenase CH grade II was obtained from Boehringer Mannheim (Maylan, France). BCECF-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and fura 2-AM were obtained from Molecular Probes (Eugene, OR). 20-HETE, 17-octadecynoic acid (17-ODYA), oleyloxyethyl phosphorylcholine, RHC-80267, U-73122, U-73343, W-7, and W-5 were from Biomol Research Laboratories (Plymouth Meeting, PA). N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) and SKF-525A were from Calbiochem (La Jolla, CA). ANG II, 8-bromo-cAMP, staurosporine, calphostin C, 4-bromophenacyl bromide, amiloride, furosemide, ouabain, bumetanide, nigericin, and all other chemicals were obtained from Sigma-Chimie (La Verpillière, France). Nonpeptide ANG II receptor antagonists losartan (DUP-753) and PD-123319 were provided by Dr. R. D. Smith from DuPont and Dr. J. A. Keiser from Parke-Davis, respectively.

Statistics. Results are expressed as means ± SE. Statistical significance between experimental groups was assessed by Student's t-test or by one-way ANOVA completed by a t-test using the within-groups residual variance of one-way ANOVA, as appropriate.

	RESULTS

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Concentration-dependent effects of ANG II. In the absence of NH⁺₄ and in the presence of 0.1 mM furosemide to block Na⁺-K⁺-2Cl cotransport, 10¹² and 10⁶ M ANG II had no effect on resting pH_i or on the pH_i recovery following intracellular acidification caused by abrupt addition of 40 mM potassium acetate to MTAL suspensions (Fig. 1), a condition in which pH_i recovery occurs through Na⁺/H⁺ antiport and H⁺-ATPase activities; ANG II also had no effect on pH_i recovery in the presence of 200 nM bafilomycin A₁ added to block the MTAL H⁺-ATPase (not shown). As shown in Figs. 2-4, ANG II did not change the magnitude of the initial NH₃-induced cell alkalinization following abrupt exposure to NH₄Cl but affected the Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity. In the presence of 1 µM amiloride plus 10 mM Ba²⁺, 10¹⁶ to 10¹² M ANG II reduced the NH⁺₄-induced dpH_i/dt by 30-50% compared with untreated controls (P < 0.005; Figs. 2A and 3).¹ However, higher ANG II concentrations (5 × 10¹¹ M) progressively stimulated the NH⁺₄-induced dpH_i/dt compared with the level observed with 10¹² M ANG II (Fig. 3); eventually, 10⁶ M ANG II significantly stimulated the NH⁺₄-induced dpH_i/dt above the level of untreated controls (Fig. 3). Thus it appeared that ANG II activated an inhibitory transduction pathway at very low concentrations (10¹² M) and that another stimulatory pathway was activated by 5 × 10¹¹ M ANG II; 10¹² and 10⁶ M ANG II are used hereafter as representative of the inhibitory and stimulatory ANG II concentrations, respectively. In contrast, ANG II at 10¹² and 10⁶ M (Figs. 2B and 4) had no effect on NH⁺₄-induced cell acidification in the presence of 0.1 mM bumetanide, a condition in which NH⁺₄ enters the cell through transport pathways other than Na⁺-K⁺(NH⁺₄)-2Cl cotransport. Taken together, these results demonstrate that the ANG II effects described above resulted from alterations of the Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity specifically but not from effects on other NH⁺₄ or H⁺ carriers or on NH₃ permeability. To assess whether ANG II acts on Na⁺-K⁺(NH⁺₄)-2Cl cotransport directly or indirectly through effects on other Na⁺, K⁺, or Cl MTAL carriers and attendant changes in intracellular ion concentrations, ANG II was applied after MTAL cells were exposed to 1 mM ouabain for 3 min to block the Na⁺-K⁺-ATPase. Under this experimental condition, 10¹² M ANG II still inhibited ~26% (P < 0.007) and 10⁶ M ANG II still stimulated ~28% (P < 0.008) the NH⁺₄-induced dpH_i/dt in the presence of 1 µM amiloride plus 10 mM Ba²⁺ compared with untreated controls (Fig. 4). Note that control NH⁺₄-induced dpH_i/dt values were lower in the presence of ouabain than in its absence (1.01 ± 0.06 vs. 1.78 ± 0.05 pH units/min; P < 0.05) as a result of Na⁺-K⁺-ATPase blockade and subsequent changes in intracellular Na⁺, K⁺, and Cl concentrations. Thus effects observed in the presence of 1 µM amiloride plus 10 mM Ba²⁺ are hereafter referred to as effects on the Na⁺-K⁺(NH⁺₄)2Cl cotransport activity.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Cell intracellular pH (pHi) recovery from an acid load. Medullary thick ascending limb (MTAL) cells were incubated in a CO2-free medium, and pHi was monitored in presence of 0.1 mM furosemide; at time 0, cells were acutely acidified by entry of acetic acid after addition of potassium acetate. There was no difference in basal pHi or in initial rate of pHi recovery (in pH units/min) between control cells (1.03 ± 0.10, n = 7) and cells exposed for 5 min to 1 pM (1.08 ± 0.07, n = 8) or 1 µM ANG II (1.08 ± 0.08, n = 7).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Cell acidification caused by NH+4 entry. MTAL cells were incubated in a CO2-free medium in presence or absence of 1 pM ANG II for 5 min; addition at time 0 of NH4Cl caused an immediate cell alkalinization (NH3 entry) followed by cell acidification due to NH+4 entry (see METHODS for detailed explanations). A: in presence of amiloride + Ba2+, initial rate of acidification (dpHi/dt, in pH units/min) due to Na+-NH+4-2Cl cotransport activity was decreased by ANG II (1.29 ± 0.17, n = 14) vs. control (1.93 ± 0.23, n = 12) (P < 0.001). B: in presence of bumetanide to block Na+-NH+4-2Cl cotransport, dpHi/dt was not affected by ANG II [2.08 ± 0.23 vs. 2.20 ± 0.30; not significant (NS)].

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent effects of ANG II on Na+-NH+4-2Cl cotransport. Initial rate of cell acidification (dpHi/dt) caused by Na+-NH+4-2Cl cotransport activity in presence of 1 µM amiloride + 10 mM Ba2+ was maximally reduced by 1014 M ANG II; from this low level, higher ANG II concentrations (5 × 1011 M) were stimulatory. Absolute value of control dpHi/dt (100%) was 1.78 ± 0.05 pH units/min (n = 39); each point in presence of ANG II represents mean ± SE of 5-12 values expressed as % of mean control value of each of 14 MTAL suspensions. * P < 0.003, compared with untreated control;  P < 0.10 and § P < 0.0001, compared with 1012 M ANG II.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Direct effects of ANG II on Na+-NH+4-2Cl cotransport. Inhibition by 1 pM and stimulation by 1 µM M ANG II of Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of amiloride + Ba2+] were still observed in cells treated with ouabain 3 min before ANG II. No effect of ANG II was observed on dpHi/dt in presence of bumetanide to block Na+-K+(NH+4)-2Cl cotransport. Each bar in presence of ANG II represents mean ± SE of 6-12 values expressed as % of mean control value (0).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of ANG II receptor antagonists. The AT1 antagonist losartan (Los.), but not the AT2 antagonist PD-123319 (PD.), abolished inhibitory and stimulatory effects of 1 pM and 1 µM ANG II, respectively, on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+]. C, control; each bar represents mean ± SE of 8-12 values expressed as % of mean control value.

The ANG II inhibitory effect is mediated through the arachidonic acid pathway. We first established that the cAMP/cAMP-dependent protein kinase (cAMP/PKA) pathway does not contribute to the ANG II effects under our experimental conditions. To this purpose, MTAL cells were exposed to 0.5 mM 8-bromo-cAMP or 15 µM H-89 3 min before ANG II; we have previously shown that 8-bromo-cAMP stimulates the cotransport activity through a PKA-dependent mechanism and that 15 µM H-89 completely blocks PKA in MTAL cells (1). As shown in Fig. 6, 10¹² M ANG II still decreased and 10⁶ M ANG II still increased the cotransport activity despite the presence of either 8-bromo-cAMP or H-89.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   cAMP/protein kinase A (cAMP/PKA) pathway has no role in ANG II effects. Pretreatment of MTAL cells with 0.5 mM 8-bromo-cAMP or 15 µM H-89, an inhibitor of PKA, did not prevent 1 pM and 1 µM ANG II from inhibiting and stimulating, respectively, Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+]. Each bar represents mean ± SE of 9-11 values expressed as % of mean control value. Absolute control values were 1.97 ± 0.12 and 0.81 ± 0.07 pH units/min (P < 0.001) in presence of 8-bromo-cAMP and H-89, respectively.

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View larger version (17K): [in this window] [in a new window]   Fig. 7.   Role of arachidonic acid pathway in inhibitory ANG II effect. Blockade of cytochrome P-450-dependent monooxygenase by 3 µM SKF-525A (SKF) abolished inhibitory effect of 1 pM ANG II on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+]; 1 pM ANG II had no additional inhibitory effect in presence of 20-hydroxyeicosatetranoic acid (20-HETE), one of the main monooxygenase-derived products of arachidonic acid in MTAL, which itself inhibited Na+-NH+4-2Cl cotransport activity. Each bar represents mean ± SE of 6-8 values expressed as % of mean control value. Absolute control values were 0.87 ± 0.09 (left) and 1.02 ± 0.12 (right) pH units/min.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Diacylglycerol (DAG) lipase, but not phospholipase A2 (PLA2), is involved in inhibitory ANG II effect. Inhibitory effect of 1 pM ANG II on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+] was abolished by the DAG lipase blocker RHC-80267 (RHC; 50 µM) but not by the PLA2 blockers 4-bromophenacyl bromide (BPB; 3.3 µM) and oleyloxyethyl phosphorylcholine (OPCH; 5 µM); RHC, BPB, and OPCH had no effect per se. Each experimental bar represents mean ± SE of 6-11 values expressed as % of mean control value for each experimental series. Absolute control values (n = 23) were 0.87 ± 0.05 (n = 6), 1.05 ± 0.08 (n = 6), and 1.34 ± 0.11 pH units/min (n = 11) for BPB, OPCH, and RHC experiments, respectively.

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PKC is responsible for the stimulatory effect of ANG II. We have previously shown that pharmacological activation of PKC by phorbol 12,13-dibutyrate stimulates the MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity (1). We thus raised the hypothesis that PKC could be responsible for the stimulation caused by ANG II. Indeed, results obtained in our laboratory have established that ANG II increases [Ca²⁺]_i in rat MTAL cells through AT₁ receptor occupancy (abolished by losartan but not by PD-123319; G. Lazar, unpublished results); for example, Fig. 9 shows that 10⁶ M ANG II caused [Ca²⁺]_i to rapidly increase, which was followed by a return toward the basal value. The ANG II-induced increase in [Ca²⁺]_i was abolished by 30 µM W-7, an inhibitor of Ca²⁺/calmodulin-dependent protein kinase (Fig. 9). This suggests that the ANG II-induced increase in [Ca²⁺]_i was due to liberation of Ca²⁺ from intracellular organelles because W-7 has been shown to deplete intracellular D-myo-inositol 1,4,5-trisphosphate-sensitive Ca²⁺ stores (28). In contrast, the ANG II-induced Ca²⁺ spike persisted when extracellular free Ca²⁺ was removed by 5 mM EGTA; the ANG II-induced Ca²⁺ spike was nevertheless reduced by ~34% under the latter experimental condition (P < 0.05; not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effect of ANG II on intracellular Ca2+ concentration ([Ca2+]i). At time 0, 1 µM ANG II was added to MTAL cells pretreated or not for 6 min with 30 µM W-7, a Ca2+/calmodulin-dependent protein kinase blocker; ANG II-induced rise in [Ca2+]i was abolished by W-7; n = 8 in absence and 4 in presence of W-7.

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View larger version (20K): [in this window] [in a new window]   Fig. 10.   Role of PKC in ANG II effects. Stimulatory effect of 1 µM ANG II on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+] was turned into an inhibitory effect by the PKC blocker staurosporine (10 nM); inhibitory effect of 1 pM ANG II was not affected by staurosporine. Each bar in presence of staurosporine represents mean ± SE of at least 8-9 values expressed as % of mean control value. Data in absence of staurosporine are those of Fig. 4. Absolute control value in presence of staurosporine was 1.28 ± 0.08 pH units/min.

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View larger version (25K): [in this window] [in a new window]   Fig. 11.   Role of cytosolic Ca2+. Stimulatory effect of 1 µM ANG II on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+] was prevented partially in BAPTA- and W-7-treated MTAL cells compared with complete abolition (significant inhibition being unmasked) caused by 10 nM staurosporine (PKC blockade); W-7 had no further effect in presence of staurosporine. W-5, an inactive structural analog of W-7, did not prevent stimulation of Na+-NH+4-2Cl cotransport activity by 1 µM ANG II. Each bar represents mean ± SE of 6-9 values expressed as % of mean control value. Absolute control values were 0.95 ± 0.24 (left), 1.22 ± 0.08 (middle), and 1.01 ± 0.03 pH units/min (right).

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View larger version (24K): [in this window] [in a new window]   Fig. 12.   Role of PLC in ANG II effects. Inhibition of PLC by 1 µM U-73122 abolished both inhibitory and stimulatory effects of 1 pM and 1 µM ANG II, respectively, on Na+-NH+4-2Cl cotransport activity [NH+4-induced initial rate of cell acidification (dpHi/dt) in presence of 1 µM amiloride + 10 mM Ba2+]; U-73343, an inactive structural analog of U-73122, did not prevent ANG II effects. Each bar represents mean ± SE of 10-13 values expressed as % of mean control value. Absolute control values were 0.78 ± 0.04 (left) and 0.68 ± 0.05 pH units/min (right).

	DISCUSSION

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This study is the first, to our knowledge, in which possible acute (5 min) effects of ANG II on rat MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport were tested for. Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity was assessed in rat MTAL CO₂-free suspensions as the bumetanide-sensitive NH⁺₄-induced initial rate of cell acidification caused by abrupt exposure to NH₄Cl. We have previously documented the validity of this approach in which the acute NH⁺₄-induced initial pH_i changes are not affected by the activity of other pH_i regulatory mechanisms including H⁺ carriers (1, 2). In addition, ANG II specifically altered the Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity, since no effect of ANG II was observed in the presence of bumetanide (or furosemide) on basal pH_i, NH⁺₄-induced cell acidification, or pH_i recovery from an acid load. That ANG II did not affect basal pH_i and the global response to an acid load, which we wanted to ascertain, does not exclude the possibility that ANG II might affect one H⁺ carrier or another specifically; for example, it is known that Na⁺/H⁺ exchange (NHE) activity of MTAL cells in suspension is mediated by both NHE-1 and NHE-3 (4), carriers that may be affected by ANG II in opposite directions to maintain cell pH.

The Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity was inhibited by very low ANG II concentrations, i.e., maximal inhibition (~50%) compared with untreated controls was observed with 10¹⁴ M ANG II; higher ANG II concentrations (5 × 10¹¹ M) progressively stimulated cotransport activity from the low level seen with 10¹⁴ to 10¹² M ANG II. Because plasma or intrarenal ANG II concentrations are 10¹¹ M, an in vivo increase in the medullary ANG II concentration, even moderate, should stimulate MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport, a hypothesis that is consistent with the previous demonstration of a primary antidiuretic action of ANG II (16). The ANG II effects occurred through AT₁ receptor occupancy inasmuch as they were abolished by losartan but insensitive to PD-123319; this result is consistent with the previously established presence in the rat MTAL of specific ANG II binding sites (23) and mRNA for AT₁ receptors (22, 27). However, a biphasic effect of ANG II occurred over a very wide range of ANG II concentrations, which could suggest that different types of AT₁ receptor are present in the MTAL. Under this hypothesis, high- and low-affinity receptors would be activated by 10¹² M and 5 × 10¹¹ M ANG II, respectively, with the low-affinity receptor corresponding to classical AT₁ receptors. Further studies are needed to address this issue. ANG II appeared to act on the cotransport activity directly and not indirectly through changes in intracellular ion concentrations, since the ANG II effects were observed virtually unchanged in the presence of ouabain added 3 min before ANG II. Furthermore, the cAMP/PKA pathway was not involved in the ANG II effects, since these effects were still observed in the presence of exogenous cAMP or H-89 that blocks PKA. We have previously demonstrated that the rat MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity is controlled not only by cAMP/PKA but also by arachidonic acid products and PKC (1).

Activation of the MTAL arachidonic acid pathway was responsible for the Na⁺-K⁺(NH⁺₄)-2Cl cotransport inhibition caused by low ANG II concentrations. We (1) and others (8) have shown that arachidonic acid inhibits Na⁺-K⁺(NH⁺₄)-2Cl cotransport in the MTAL via cytochrome P-450/monooxygenase-derived 20-HETE; in the MTAL, cyclooxygenase activity is low, lipoxygenase activity is absent, and there is no epoxyeicosatrienoic acid production (8, 20). In the present study, the inhibitory ANG II effect was quantitatively comparable to those of arachidonic acid and 20-HETE (1), was abolished by the monooxygenase inhibitors SKF-525A and 17-ODYA but not by PKA or PKC blockers, and was not additive to that of 20-HETE; furthermore, the stimulatory effect of high ANG II concentration was enhanced by cytochrome P-450/monooxygenase blockade. Because the ANG II inhibitory effect was abolished by PLC or DAG lipase but not PLA₂ blockade, it appears that very low ANG II concentrations stimulate PLC after AT₁ receptor occupancy sufficiently to produce DAG, which is in turn deacylated by DAG lipase to yield arachidonic acid; then the main cytochrome P-450/monooxygenase-derived products, 20-HETE and/or 20-carboxy-arachidonic acid in the MTAL, inhibit the Na⁺-K⁺(NH⁺₄)-2Cl cotransporter. Note that when PKC was blocked by staurosporine (Fig. 10) or calphostin C, 10⁶ M ANG II inhibited the cotransport activity to the same extent as did 10¹² M ANG II; this confirms that the cotransport is maximally inhibited through the arachidonic acid pathway by an ANG II concentration as low as 10¹⁴ to 10¹² M. Indeed, in a recent study (20), ANG II increased 20-HETE production in the rat MTAL to similar extents at 5 × 10¹¹ and 5 × 10⁸ M (to ~260 and ~210% of the control value, respectively). Thus arachidonic acid products would exert a basal fixed inhibitory tonus on Na⁺-K⁺(NH⁺₄)-2Cl cotransport, the activity of which would then be regulated by stimulatory influences such as those of the PKA and PKC pathways. The cellular mechanisms by which 20-HETE and/or 20-carboxy-arachidonic acid inhibits MTAL cotransport are unknown, but they do not, however, require Na⁺-K⁺-ATPase, PKA, or PKC activities, since they are not affected by the presence of ouabain, H-89, or staurosporine.

Activation of PKC was responsible for the stimulation of Na⁺-K⁺(NH⁺₄)-2Cl cotransport by ANG II. PKC activation by a phorbol ester stimulates the MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransport activity (1). In this study, the stimulatory ANG II effect was insensitive to the PKA blocker H-89 but was abolished by the PKC inhibitors staurosporine and calphostin C as well as by PLC blockade. Furthermore, stimulation of Na⁺K⁺(NH⁺₄)-2Cl cotransport by high ANG II concentrations was suppressed, although incompletely, in cells loaded with BAPTA or treated with W-7 in which the ANG II-induced rise in [Ca²⁺]_i was abolished; W-7 had no inhibitory effect additive to that of staurosporine. These results thus strongly suggest that, after AT₁ receptor occupancy and PLC activation, concentrations of ANG II 5 × 10¹¹ M stimulate MTAL Na⁺K⁺(NH⁺₄)-2Cl cotransport through DAG- and Ca²⁺-activated PKCs. Both Ca²⁺-responsive and Ca²⁺-unresponsive PKC isoforms appeared to be involved in the ANG II stimulatory effect, since the ANG II-induced rise in [Ca²⁺]_i is necessary for complete but not for partial stimulation. Results obtained on MTAL suspensions in our laboratory, with use of immunoblots on particulate and cytosolic fractions with antibodies specific to various PKC isoforms (13), have established that the Ca²⁺-responsive PKC and the Ca²⁺-unresponsive PKC, PKC, and PKC are present in rat MTAL cells (Z. Karim and J. Poggioli, unpublished results); in the rat proximal tubule in which the same isoforms of PKC are present, ANG II induces the translocation of PKC and PKC (13). The hypothesis of direct stimulation of the MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransporter by several PKC isoforms is consistent with the presence in renal apical cotransporter sequences of several consensus PKC phosphorylation sites within both the NH₂- and COOH-terminal domains (9).

In a recent study (20), ANG II inhibited at a low concentration (5 × 10¹¹ M) and stimulated at a high concentration (5 × 10⁸ M) the rat MTAL apical K⁺ channel; the inhibitory effect was mediated via the 20-HETE pathway, whereas the stimulatory effect appeared to involve nitric oxide but not PKC (20). In that work, ANG II was also shown, through undefined cellular pathways, to lower the intracellular Na⁺ concentration at 10¹⁰ M but to enhance intracellular Na⁺ concentration at 10⁹ to 10⁷ M. The latter effects could not be secondary to the ANG II actions on the apical K⁺ channel through modulation of luminal K⁺ recycling and subsequent secondary effects on Na⁺-K⁺-2Cl cotransport, since the tubules were not perfused, but were consistent with a direct regulation of Na⁺-K⁺-2Cl cotransport by ANG II as described in the present study. However, possible effects of ANG II on other Na⁺ carriers are not excluded.

In conclusion, the present results lead us to propose a working model for the AT₁- and PLC-mediated effects of ANG II on the MTAL Na⁺-K⁺(NH⁺₄)-2Cl cotransporter (Fig. 13). Very low ANG II concentrations through high-affinity receptor occupancy impose a basal inhibitory tonus via DAG lipase- and cytochrome P-450/monooxygenase-derived arachidonic acid products (20-HETE and/or 20-carboxy-arachidonic acid). This basal inhibitory tonus, which is maximal at 10¹⁴ to 10¹² M ANG II, is counterbalanced by the stimulatory influence of PKC, which is progressively activated through the combined actions of augmented DAG accumulation and increase in [Ca²⁺]_i caused by low-affinity receptor occupancy by higher ANG II concentrations (>10¹² M). These ANG II effects may be physiologically relevant, since the circulating and proximal tubular fluid ANG II concentrations range from 10¹¹ to 2 × 10⁸ M (6, 24, 25). ANG II could thus have important effects on MTAL transports because of the major role of the Na⁺-K⁺ (NH⁺₄)-2Cl cotransporter on the latter. As stated above, a rise in the local ANG II concentration should stimulate the Na⁺-K⁺(NH⁺₄)-2Cl cotransporter and enhance NaCl and NH⁺₄ absorption by the MTAL and consequently medullary osmolarity and medullary NH⁺₄ accumulation; this could explain the primary antidiuretic action of ANG II additive to that of antidiuretic hormone (16). By these effects on MTAL transports, ANG II could thus contribute to the renal control of water and acid-base balance, particularly in states of extracellular fluid volume contraction in which the renin-ANG system is activated. It must be emphasized that effects of ANG II on NaCl absorption by the MTAL could not contribute to the renal control of Na⁺ balance if they were not accompanied by similar effects of ANG II in the cortical segment of the TAL (CTAL), which is unknown at present. Indeed, changes in the NaCl load delivered to the CTAL may induce changes in CTAL transports that may balance those in the MTAL so that the total NaCl amount reabsorbed may remain constant (15); this is observed, for example, with vasopressin, which affects MTAL but not CTAL transports (15). Notably, it is unknown at present whether ANG II receptors are present in the basolateral or apical membrane of the MTAL (or both); further studies are needed to address this issue as well as that of the possible presence of different subtypes of AT₁ receptors in the MTAL.

View larger version (19K): [in this window] [in a new window]   Fig. 13.   Working model for ANG II effects in rat MTAL. ANG II effects on Na+-NH+4-2Cl cotransport are mediated by AT1 receptor occupancy and PLC activation that produces DAG and increases [Ca2+]i. At 1012 M (high-affinity receptor), ANG II maximally inhibits cotransport through arachidonic acid (A.A.; via cytochrome P-450/monooxygenase-derived products) produced from DAG by DAG lipase. At higher ANG II concentrations (5 × 1011 M; low-affinity receptor), DAG accumulation and increase in [Ca2+]i progressively activate PKC, which counterbalances arachidonic acid inhibitory () pathway to stimulate (+) cotransport.