ANG II controls Na+-K+(NH+4)-2Clminus cotransport via 20-HETE and PKC in medullary thick ascending limb

Hassane Amlal1,2, Christian LeGoff1, Catherine Vernimmen1, Manoocher Soleimani2, Michel Paillard1, and Maurice Bichara1

1 Physiologie et Endocrinologie Cellulaire Rénale, Institut National de la Santé et de la Recherche Médicale Unité 356, Université Pierre et Marie Curie and Hôpital Broussais, 75270 Paris Cedex 06, France; and 2 Department of Internal Medicine and Molecular Genetics, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na+-K+(NH+4)-2Cl- cotransport. ANG II at 10-16 to 10-12 M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10-12 M ANG II level cotransport activity; eventually, 10-6 M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10-12 M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10-12 M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10-6 M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus <= 10-12 M ANG II inhibits via 20-HETE, whereas >= 5 × 10-11 M ANG II stimulates via PKC Na+-K+(NH+4)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.

intracellular pH; intracellular calcium; cytochrome P-450-dependent monooxygenase; phospholipase C; diacylglycerol lipase; protein kinase C; angiotensin II receptors; DUP-753; PD-123319

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 (<= 10-10 M) stimulate but high ANG II concentrations (>= 10-7 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 × 10-8 M have been measured in the proximal tubular fluid, concentrations that are much higher than the 10-11 to 5 × 10-10 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+, HCO-3, and water absorption in the rat distal tubule accessible to micropuncture (18, 19, 29). In the rabbit cortical collecting duct microperfused in vitro, 10-7 M ANG II stimulates luminal alkalinization (HCO-3 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 (pHi) in suspensions of rat MTALs by use of the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF); rates of pHi 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 <= 10-12 M but that higher ANG II concentrations (>= 5 × 10-11 M) progressively stimulate cotransport activity compared with the level seen with 10-12 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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Introduction
Methods
Results
Discussion
References

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 HCO-3 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.

Because the purpose of the present study was to assess possible acute effects of ANG II, this hormone (or its vehicle for control studies) was applied to the cells at 37°C 5 min before the measurements. To assess the mechanism of the observed effect, inhibitors (or their vehicles) were in some cases applied 3 min before ANG II.

Measurements of pHi. pHi 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 pHi values with calibration curves that were established daily by the high-K+ medium-nigericin and/or Triton X-100 methods, as described previously (17).

The Na+-K+(NH+4)-2Cl- cotransport activity was assessed by estimating the bumetanide-sensitive rate of intracellular acidification caused by entry into the cells of NH+4 after abrupt application of 4 mM NH4Cl to the cells (1-3). After NH4Cl addition, a very rapid cellular alkalinization first occurred (see Fig. 2) due to immediate predominant NH3 entry, which ended when intracellular and extracellular NH3 concentrations were equal (NH3 equilibrium); this was followed by a relatively rapid and profound fall in pHi, the initial rate of which is determined almost exclusively by the rate of NH+4 entry that causes H+ accumulation within the cells because the intracellular NH3 amount that tends to rise above the NH3 equilibrium value leaves the cell as fast as NH+4 enters (the extracellular NH3 concentration, which determines the NH3 equilibrium value, is constant in our experiments). Indeed, it is generally accepted that NH3 readily permeates plasma membranes by lipid phase diffusion (at least the basolateral membrane in the MTAL), which thus may not be rate limiting during the secondary acidification phase. In the presence of 1 µM amiloride plus 10 mM Ba2+ to block MTAL NH+4 carriers other than Na+-K+ (NH+4)-2Cl- cotransport [NH+4 conductance, K+/NH+4(H+) antiport, and NH+4(K+)-Cl- cotransport; Refs. 1-3], a noticeable cell acidification followed the initial cell alkalinization after NH4Cl addition (see Fig. 2); cell acidification was due to Na+-K+(NH+4)-2Cl- activity because, in the presence of 1 µM amiloride plus 10 mM Ba2+ + 0.1 mM furosemide (or bumetanide), 4 mM NH+4-induced cell acidification was abolished (Fig. 1D in Ref. 2). We have previously demonstrated that the NH+4-induced initial rate of acidification (dpHi/dt) is not affected by changes in the activity of the other MTAL pHi regulatory mechanisms such as Na+/H+ antiport (1, 2). Therefore, the Na+-K+(NH+4)-2Cl- cotransport activity was defined as the bumetanide-sensitive 4 mM NH+4-induced dpHi/dt observed in the presence of 1 µM amiloride plus 10 mM Ba2+. In the experiments to be described, the NH+4-induced dpHi/dt was taken to directly reflect changes in the transmembrane H+-equivalent flux (= dpHi/dt × beta i × cell volume, where beta i is the intrinsic cell buffering power) because the beta i × cell volume product is constant in these acute experiments that lasted only a few minutes, as previously demonstrated (1). The results concerning the NH+4-induced dpHi/dt are presented both as absolute values in the text and the legends of Figs. 1-3, 6-8, and 10-12 and as percent of the mean control value of each experiment in Figs. 3-8 and 10-12 to facilitate comparisons.

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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Abstract
Introduction
Methods
Results
Discussion
References

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, 10-12 and 10-6 M ANG II had no effect on resting pHi or on the pHi recovery following intracellular acidification caused by abrupt addition of 40 mM potassium acetate to MTAL suspensions (Fig. 1), a condition in which pHi recovery occurs through Na+/H+ antiport and H+-ATPase activities; ANG II also had no effect on pHi 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+, 10-16 to 10-12 M ANG II reduced the NH+4-induced dpHi/dt by 30-50% compared with untreated controls (P < 0.005; Figs. 2A and 3).1 However, higher ANG II concentrations (>= 5 × 10-11 M) progressively stimulated the NH+4-induced dpHi/dt compared with the level observed with 10-12 M ANG II (Fig. 3); eventually, 10-6 M ANG II significantly stimulated the NH+4-induced dpHi/dt above the level of untreated controls (Fig. 3). Thus it appeared that ANG II activated an inhibitory transduction pathway at very low concentrations (<= 10-12 M) and that another stimulatory pathway was activated by >= 5 × 10-11 M ANG II; 10-12 and 10-6 M ANG II are used hereafter as representative of the inhibitory and stimulatory ANG II concentrations, respectively. In contrast, ANG II at 10-12 and 10-6 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, 10-12 M ANG II still inhibited ~26% (P < 0.007) and 10-6 M ANG II still stimulated ~28% (P < 0.008) the NH+4-induced dpHi/dt in the presence of 1 µM amiloride plus 10 mM Ba2+ compared with untreated controls (Fig. 4). Note that control NH+4-induced dpHi/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.


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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).


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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)].


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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 10-14 M ANG II; from this low level, higher ANG II concentrations (>= 5 × 10-11 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; ddager  P < 0.10 and § P < 0.0001, compared with 10-12 M ANG II.


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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).

To establish by which ANG II receptor these effects occurred, we used the nonpeptide ANG II receptor antagonists losartan and PD-123319 to block AT1 and AT2 receptors, respectively. As shown in Fig. 5, 10 µM losartan abolished both the inhibitory and stimulatory ANG II effects, whereas 10 µM PD-123319 did not prevent them from occurring. These results are consistent with all ANG II effects being mediated through AT1 receptor occupancy.


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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, 10-12 M ANG II still decreased and 10-6 M ANG II still increased the cotransport activity despite the presence of either 8-bromo-cAMP or H-89.


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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.

Because we (1) and others (8) had previously demonstrated that arachidonic acid and its metabolites derived from the cytochrome P-450-dependent monooxygenase pathway inhibit Na+-K+(NH+4)-2Cl- cotransport activity, we tested for a possible involvement of that pathway in the inhibitory effect of low concentrations of ANG II in the following ways. First, as shown in Fig. 7, 3 µM SKF-525A, a compound that blocks the cytochrome P-450-dependent monooxygenase and abolishes the arachidonic acid effect in MTAL cells like econazole (1), suppressed the inhibitory effect of 10-12 M ANG II; we have previously shown that SKF-525A had no effect per se (1). Similarly, 17-ODYA, another inhibitor of the cytochrome P-450-dependent monooxygenase, had no effect per se but abolished the inhibitory effect of 10-12 M ANG II on the Na+-K+ (NH+4)-2Cl- cotransport activity [the NH+4-induced dpHi/dt (in pH units/min) was -1.94 ± 0.06 in control (n = 9), -1.94 ± 0.06 in the presence of 5 µM 17-ODYA (n = 5), and -1.97 ± 0.06 in the presence of 10-12 M ANG II + 5 µM 17-ODYA (n = 6)]. Second, 10-12 M ANG II caused no further inhibition in the presence of 1 µM 20-HETE, one of the main cytochrome P-450/monooxygenase-derived products of arachidonic acid in the MTAL (8), which itself inhibited (P < 0.004) the cotransport activity (Fig. 7). Thus the effects of 20-HETE and 10-12 M ANG II were not additive. Third, to determine whether ANG II causes the release of arachidonic acid from membrane lipids through activation of phospholipase A2 (PLA2) or diacylglycerol (DAG) lipase, we used known inhibitors of these enzymes. As shown in Fig. 8, 3.3 µM 4-bromophenacyl bromide and 5 µM oleyloxyethyl phosphorylcholine, which both inhibit PLA2 in MTAL cells (1), did not prevent 10-12 M ANG II from decreasing the Na+-K+(NH+4)-2Cl- cotransport activity; these two compounds had no effect per se. In contrast, 50 µM RHC-80267, an inhibitor of DAG lipase (26) that had no effect per se on Na+-K+(NH+4)-2Cl- cotransport activity, abolished the inhibitory effect of 10-12 M ANG II (Fig. 8). Finally, that 10-12 M ANG II activates phospholipase C (PLC) to produce DAG is shown below (see Fig. 12).


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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.


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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.

These results thus strongly suggest that 10-12 M ANG II enhances the production of DAG, which is in turn deacylated by DAG lipase to yield arachidonic acid that inhibits Na+-K+(NH+4)-2Cl- cotransport activity through 20-HETE.

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 10-6 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).


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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.

Figure 10 shows that 10 nM staurosporine, which blocks PKC but does not by itself affect PKA or Na+K+(NH+4)-2Cl- cotransport in MTAL cells at this concentration (1), not only abolished the stimulation by 10-6 M ANG II but also unmasked the underlying inhibitory influence of low ANG II concentrations, compared with untreated controls; in contrast, staurosporine did not affect the inhibitory effect of 10-12 M ANG II (Fig. 10). Similarly, calphostin C, another inhibitor of PKC, had no effect per se but turned the stimulatory effect of 10-6 M ANG II on the Na+K+(NH+4)-2Cl- cotransport activity into an inhibitory effect [the NH+4-induced dpHi/dt (in pH units/min) was -1.82 ± 0.04 in control (n = 9), -1.75 ± 0.09 in the presence of 2 µM calphostin C (n = 5; not significantly different from control value), and -1.38 ± 0.09 in the presence of 10-6 M ANG II + 2 µM calphostin C (n = 6; P < 0.005 compared with control or calphostin C)]. Conversely, blockade of cytochrome P-450-dependent monooxygenase by 3 µM SKF-525A slightly but significantly enhanced the stimulatory effect of 10-6 M ANG II [the NH+4-induced dpHi/dt increased from -1.84 ± 0.04 (10-6 M ANG II; n = 5) to -1.97 ± 0.03 pH units/min (10-6 M ANG II + 3 µM SKF-525A; n = 5); P < 0.03; not shown], as expected from the suppression of an inhibitory influence. Note that, in the presence of staurosporine, 10-12 and 10-6 M ANG II inhibited cotransport activity to the same extent (Fig. 10); these results confirm that the ANG II inhibitory effect via the arachidonic pathway described above was maximal at 10-12 M ANG II.


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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.

Usually, PKC is acutely activated by a hormonal agent after PLC activation and attendant simultaneous DAG accumulation within the plasma membrane and increase in [Ca2+]i triggered by D-myo-inositol 1,4,5-trisphosphate; both DAG accumulation and Ca2+ increase are able to activate PKC. To determine the contribution of Ca2+ to the activation of PKC by ANG II, we used BAPTA to clamp [Ca2+]i and W-7 to suppress the ANG II-induced increase in [Ca2+]i, as described above; cells were loaded with BAPTA by 15-20 min of preincubation in the presence of 50 µM BAPTA-AM and then washed before ANG II addition. As shown in Fig. 11, in cells pretreated with BAPTA as well as with 30 µM W-7, the stimulatory effect of 10-6 M ANG II was reduced significantly but incompletely, since the underlying inhibitory ANG II influence was not seen; W-5, an inactive structural analog of W-7, did not prevent significant stimulation by 10-6 M ANG II. Furthermore, W-7 had no additional effect in the presence of 10 nM staurosporine that totally suppressed the stimulatory effect of high ANG II concentrations (Fig. 11). It is thus clear that, under our experimental conditions, the stimulatory effect of 10-6 M ANG II is entirely due to PKC activation and that the PKC activation is due to the combined actions of DAG and Ca2+.


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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).

It thus appeared that both the inhibitory and stimulatory effects of the various concentrations of ANG II were possibly secondary to PLC activation. To confirm this point, we used the PLC inhibitor U-73122 (21). As shown in Fig. 12, 1 µM U-73122, but not its inactive structural analog U-73343, abolished both the inhibitory and stimulatory effects of 10-12 and 10-6 M ANG II, respectively.


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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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 pHi changes are not affected by the activity of other pHi 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 pHi, NH+4-induced cell acidification, or pHi recovery from an acid load. That ANG II did not affect basal pHi 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 10-14 M ANG II; higher ANG II concentrations (>= 5 × 10-11 M) progressively stimulated cotransport activity from the low level seen with 10-14 to 10-12 M ANG II. Because plasma or intrarenal ANG II concentrations are >= 10-11 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 <= 10-12 M and >= 5 × 10-11 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, 10-6 M ANG II inhibited the cotransport activity to the same extent as did 10-12 M ANG II; this confirms that the cotransport is maximally inhibited through the arachidonic acid pathway by an ANG II concentration as low as 10-14 to 10-12 M. Indeed, in a recent study (20), ANG II increased 20-HETE production in the rat MTAL to similar extents at 5 × 10-11 and 5 × 10-8 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 × 10-11 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 PKCalpha and the Ca2+-unresponsive PKCdelta , PKCepsilon , and PKCzeta 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 PKCalpha and PKCepsilon (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 × 10-11 M) and stimulated at a high concentration (5 × 10-8 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 <= 10-10 M but to enhance intracellular Na+ concentration at 10-9 to 10-7 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 10-14 to 10-12 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 (>10-12 M). These ANG II effects may be physiologically relevant, since the circulating and proximal tubular fluid ANG II concentrations range from 10-11 to 2 × 10-8 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.


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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 <= 10-12 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 × 10-11 M; low-affinity receptor), DAG accumulation and increase in [Ca2+]i progressively activate PKC, which counterbalances arachidonic acid inhibitory (-) pathway to stimulate (+) cotransport.

    ACKNOWLEDGEMENTS

We thank Dr. R. D. Smith (DuPont) and Dr. J. A. Keiser (Parke-Davis) for having kindly provided losartan and PD-123319, respectively.

    FOOTNOTES

This study was supported by grants from Institut National de la Santé et de la Recherche Médicale, Université Paris 6, Fondation pour la Recherche Médicale Française, and Fondation de France.

Parts of this work were presented at the 26th Annual Meeting of the American Society of Nephrology (Boston, MA, November 1993) and published in abstract form (H. Amlal, C. Vernimmen, C. LeGoff, M. Paillard, and M. Bichara. J. Am. Soc. Nephrol. 4: 862, 1993).

1 The time course of NH+4-induced cell acidification was curvilinear but could be well fitted from the 3rd to the 12th or 15th second to the linear equation form pHi = C - k · ln(t), in which C is pHi at time (t) = 1, k is a constant, and the rate of change in pHi at any time ti is dpHi/dt = -k/ti; r values from these linear fits were >= 0.90. Initial rate of pHi acidification at the third second was thus defined from the latter equation as dpHi/dt = -k/3 in pH units per second and expressed as pH units per minute, which thus represents the slope of a line tangent to the curve at the third second. Fitting the first 12 or 15 s of the pHi time course to a linear function relating pHi to ln(t) requires no assumption regarding the mechanisms of the NH+4-induced pHi response but merely provides a straightforward means of quantitatively comparing experimental groups.

Address for reprint requests: M. Bichara, INSERM U.356, Centre de Recherches Biomédicales des Cordeliers, 15 Rue de l'Ecole de Médecine, 75270 Paris Cédex 06, France.

Received 21 March 1997; accepted in final form 5 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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