Apical ANG II-stimulated PLA2 activity and Na+ flux: a potential role for Ca2+-independent PLA2

B. N. Becker, H.-F. Cheng, and R. C. Harris

Department of Medicine, Vanderbilt University School of Medicine and the Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232-2372

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Type 1 angiotensin II (ANG II) receptors (AT1R), which mediate proximal tubule (PT) salt and water reabsorption, undergo endocytosis and recycling. Prior studies in a PT-like model (LLC-PKCl4 cells expressing rabbit AT1R) (LLC-PK-AT1R cells) determined that quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor, and the haloenol lactone suicide substrate (HELSS), a Ca2+-independent PLA2 inhibitor, attenuated apical (AP) AT1R recycling. Further studies were undertaken to examine the association between AT1R endocytotic movement and PLA2 activity in this model. AP ANG II (100 nM) increased [3H]arachidonic acid ([3H]AA) release 4.4 ± 0.38-fold in LLC-PK-AT1R cells cultured on permeable supports. Basolateral (BL) ANG II had no significant effect. Reversed-phase high-performance liquid chromatography confirmed that AP ANG II stimulated free [3H]AA release. Quinacrine, HELSS, and palmitoyl trifluoromethyl ketone, another Ca2+-independent PLA2 inhibitor, inhibited AP ANG II-stimulated [3H]AA release, as did inhibiting AP AT1R internalization with phenylarsine oxide. The role of HELSS-inhibitable AA release in ANG II-mediated 22Na flux was examined, given the effects of AT1R-mediated PLA2 activity on salt and water reabsorption. AP ANG II (100 nM) stimulated 22Na flux (AP right-arrow BL), a response inhibited by HELSS. Thus, in this model, AP AT1R activated PLA2 with concomitant 22Na flux (AP right-arrow BL), suggesting a link between AP AT1R endocytotic movement, AT1R-stimulated PLA2 activity, and 22Na flux in this model. The effects of HELSS suggest that Ca2+-independent PLA2 activity may be involved in this AP ANG II response.

angiotensin II; type 1 angiotensin II receptor; phospholipase A2 activity; haloenol lactone suicide substrate; sodium

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

PHOSPHOLIPASE A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to liberate free fatty acids and lysophospholipids. PLA2 plays an important role in numerous cellular processes, including phospholipid digestion and metabolism and signal transduction. PLA2s are actually a diverse class of enzymes that can be categorized on the basis of tissue and cellular localization, regulation, sequence and structural components, and divalent cation requirements. In addition to cytosolic and secretory PLA2s (29), recent studies have also described Ca2+-independent PLA2 activity in canine myocardium (44), macrophage-like P388D1 cells (2), and in rabbit proximal tubule cells (31). Such Ca2+-independent PLA2 activity may play a significant role in endocytic vesicle fusion, as the mechanism-based inhibitor of Ca2+-independent PLA2 activity [haloenol lactone suicide substrate (HELSS)] inhibits this process (4, 27).

Proximal tubule epithelial cells are endowed with a high degree of endocytic capacity to cope with the glomerular filtrate. Proteins and other components of the filtrate are adsorbed to apical membranes and internalized via endocytic uptake for delivery to subcellular compartments. Efficient recycling of membrane back to the plasma membrane must occur to balance membrane loss from internalization. This endocytic pattern is duplicated by certain hormonal, nutrient, and growth factor receptors, including angiotensin II (ANG II) receptors (42).

Tubular ANG II receptors are relatively unique as they are expressed at apical and basolateral membranes (5, 6). By coupling to numerous signal-transduction pathways, including PLA2 (13), these receptors play a significant role in proximal tubule salt and water reabsorption (18). We have previously shown in a model of proximal tubule epithelia that apical (AP) type 1 ANG II receptor (AT1R) and basolateral (BL) AT1R displayed differential rates of endocytosis and recycling (3). Furthermore, HELSS, the calcium-independent PLA2 inhibitor, preferentially decreased AP AT1R recycling (3). We therefore examined the effects of AP or BL ANG II treatment on PLA2 activity, the effects of AT1R internalization on PLA2 activity, and whether ANG II-mediated PLA2 activity affected vectorial 22Na flux in this cell culture model.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Materials. [3H]arachidonic acid ([3H]AA, 100 Ci/mmol) and 22Na were obtained from NEN (Boston, MA). ANG II, penicillin (10,000 units)-streptomycin (10 mg/ml) solution, trypsin-EDTA, Dulbecco's modified Eagle's medium-Ham's F-12 (DME/F12) medium mixture, bovine serum albumin, quinacrine, A-23187, phorbol 12-myristate 13-acetate (PMA), and pertussis toxin (PTX) were obtained from Sigma (St. Louis, MO). HELSS and palmitoyl trifluoromethyl ketone (PACOCF3) were purchased from Calbiochem (Santa Cruz, CA). Geneticin was obtained from GIBCO (Grand Island, NY). DuP-753 (losartan) was provided by DuPont-Merck (Wilmington, DE), and CGP-42112A was obtained from Ciba-Geigy (Basel, Switzerland). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) was purchased from Biomol (Plymouth Meeting, PA). Fetal calf serum was obtained from Hyclone (Logan, UT).

Cell culture. Cell culture was performed as previously described (3). Briefly, LLC-PKCl4 cells, an LLC-PK1 clone that does not express endogenous AT1 receptors, were stably transfected with the rabbit AT1R. These cells, descendants of the CL4 clone (16), polarize Na+-dependent glucose transport and L-glutamate transport to the apical compartment and ouabain-sensitive 86Rb uptake to the basolateral compartment when grown on filter supports (16). Previous studies also determined that these cells formed domes when grown on plastic and, when grown on permeable filters, expressed Na+/H+ exchangers with differential sensitivities to ethylisopropylamiloride at AP and BL surfaces (8) and displayed differential signaling when exposed to AP or BL ANG II (7). Preliminary studies further characterizing LLC-PKCl4 cells demonstrated that 79% of ouabain-sensitive 86Rb uptake occurred from the BL surface (n = 2).

LLC-PKCl4 cells transfected with rabbit AT1R (LLC-PK-AT1R cells) expressed the rabbit transcript as determined by reverse transcription-polymerase chain reaction. When grown on permeable supports (6- or 12-well Transwell plates; Corning Costar, Boston, MA), they displayed specific 125I-labeled ANG II binding at apical and basolateral membranes. Cells were cultured in DME/F12 medium containing 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and geneticin (0.5 µg/ml). SV40-immortalized rabbit proximal tubule epithelial cells were cultured in similar growth medium excluding geneticin. Before assays were performed, cells grown on permeable supports were assayed for [3H]inulin leak as previously described. Any well with >3% apical-to-basolateral leak (AP right-arrow BL) was discarded from further analysis.

AA release. Cells were incubated in serum-free medium 48 h prior to assaying for AA release. Eighteen hours before experimentation, cells were washed three times in medium supplemented with 1 mg/ml bovine serum albumin, then incubated in 1 ml of medium containing [3H]AA (4 µCi/ml). After overnight incubation, this medium was aspirated, and the cells were washed five times with nonradioactive media and then incubated in nonradioactive media for 30 min at 37°C. After this incubation, cells were exposed to AT1R or PLA2 antagonists in either AP or BL medium or in both for 10 min prior to treatment with ANG II for the indicated times at the noted concentrations. For PTX studies, cells were incubated overnight with PTX (500 ng/ml) in the radioactive medium. For studies with BAPTA-AM, cells were incubated with BAPTA-AM for 30 min following the nonradioactive medium incubation prior to study. After the assay period, an aliquot of AP or BL medium was removed and centrifuged at 12,000 g to pellet cellular debris. The supernatant was transferred to a scintillation vial with 3 ml Aquasol, and radioactivity released into the medium was determined by liquid scintillation spectrometry. The remaining media was aspirated, and cells were washed and then digested with the addition of 0.05 M NaOH. An aliquot of the digest was used for protein determination, and the remainder was assayed for total cellular incorporated radioactivity.

Reversed-phase high-performance liquid chromatography (HPLC). To verify that the radioactive species in the supernatant represented arachidonic acid, cells cultured on permeable supports, loaded for 16 h with [3H]AA, were treated with vehicle or apical ANG II (100 nM) for 10 or 20 min. Media and cells were centrifuged at 5,000 g for 5 min, and the supernatant was mixed with 50 µl of glacial acetic acid. AA and metabolites were obtained by repeated ethyl acetate extraction. After the third extraction, samples were evaporated under N2. Samples were resuspended in 1 ml of absolute ethanol, and 200 µl of this solution were added to 50 µl of 2 N KOH. This solution was incubated at 50°C for 30 min. After this incubation, 20 µl formic acid and 2 ml sterile H2O were added to this solution, which was vortexed and subjected to repeat ethyl acetate extraction as above. After the third extraction, the sample was evaporated under N2, and resuspended in 70 µl of ethanol-butylated hydroxytoluene solution. This sample was then analyzed as described by Capdevila et al. (9).

22Na flux. ANG II-stimulated sodium flux was studied in LLC-PK-AT1R cells as previously described (8). Growth medium was removed, and the apical or basolateral surface was exposed to a solution containing (in mM) 117 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 5.6 glucose, 0.9 NaH2PO4, 25 NaHCO3, and 1 phenylmethylsulfonyl fluoride (PMSF), pH 7.4, along with 22Na (2 µCi/ml) in the presence or absence of ANG II (100 nM) and/or PLA2 inhibitors. The contralateral surface was incubated in buffer lacking ANG II, 22Na, and PMSF. The incubation continued for up to 30 min on a rocking platform at 23°C. Buffer pH was maintained at 7.4 by continuous exposure to 5% CO2-95% O2. Fifty-microliter aliquots were removed from the contralateral well buffer at indicated times, and 22Na was quantified by scintillation spectrometry. At least one well per plate was used as a control (no ANG II), and values were normalized to protein determination of the studied wells.

For assays to determine whether apical ANG II induced nonspecific transport processes, cells were incubated with [3H]inulin with or without apical ANG II (100 nM). After 45 min, aliquots of basolateral medium were removed, and radioactivity in the medium was determined by liquid scintillation spectrometry.

Protein determination. Proteins were quantitated by the method of Smith et al. (41), using bincinchonic acid reaction reagents (Pierce, Rockford, IL).

Statistical analyses. Results are reported as means ± SE. Results of AA release experiments are reported as counts released per milligram protein or as percentage of control values. Statistical comparisons were made with analysis of variance or Student's t-test and the Bonferroni correction when indicated. P < 0.05 was considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Given previous data that AP AT1R recycling in this proximal tubule-like model was selectively attenuated by pharmacological inhibitors of PLA2 activity (3), LLC-PK-AT1R cells were grown on permeable supports and assayed for [3H]AA release. LLC-PKCl4 cells are known to polarize certain membrane proteins and phospholipids when grown on permeable supports (16). This characteristic appeared to be important for ANG II-mediated PLA2 activity as addition of ANG II did not significantly stimulate [3H]AA release in cells grown on plastic [at 20 min, in counts · min released-1 · mg protein-1: untreated, 4,013 ± 292; ANG II (100 nM), 3,780 ± 470; n = 4, not significant (NS)]. This response was similar to the lack of ANG II-stimulated [3H]AA release in nontransfected LLC-PKCl4 cells (3,612 ± 597; n = 2, NS vs. untreated transfected). In contrast, AP ANG II stimulated a significant increase in [3H]AA release in cells grown on permeable supports compared with untreated cells [20 min counts · min released-1 · mg protein-1: untreated, 4,453 ± 422; AP ANG II (100 nM), 16,788 ± 2,712; n = 20, P < 0.0005 vs. untreated]. BL ANG II stimulated only a minimal increase in [3H]AA release compared with controls [BL ANG II (100 nM), 4,114 ± 1,210; n = 3; NS vs. untreated]. The majority of the counts released in response to AP ANG II were present in the AP medium (AP, 82 ± 21%; BL, 18 ± 14%; n = 3). This distribution of [3H]AA release also was evident for unstimulated cells, and, therefore, [3H]AA release in the remaining studies is reported as total counts released.

To determine the timing of ANG II-mediated AA release, cells loaded with radiolabeled AA were incubated with ANG II (100 nM) at the AP or BL surface for 0-30 min. BL ANG II did not significantly affect [3H]AA release for up to 30 min following treatment (5 min control, 5,113 ± 600; BL ANG II, 4,229 ± 700; 10 min control, 3,331 ± 405; BL ANG II, 4,005 ± 1,330; 20-min control, 4,956 ± 560; BL ANG II, 4,154 ± 1,220; 30 min control, 5,123 ± 820; BL ANG II, 4,335 ± 990; n = 3 for all BL values, n = 5 for 5 and 10 min control; n = 22 for control 20 min; n = 15 for control 30 min; NS) (Fig. 1). However, AP ANG II stimulated a significant increase in [3H]AA release at 10 min, and this increased further by 20 min, remaining significantly elevated up to 30 min following AP ANG II treatment (AP ANG II, 5 min, 5,842 ± 2,044; n = 4, NS; AP ANG II, 10 min, 7,338 ± 1,015; n = 3, P < 0.005; AP ANG II, 20 min, 16,788 ± 2,712; n = 20, P < 0.0005; AP ANG II, 30 min, 17,820 ± 3,223; n = 7, P < 0.0005) (Fig. 1).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of angiotensin II (ANG II)-mediated [3H]arachidonic acid ([3H]AA) release in LLC-PKCl4 cells stably transjected with AT1 receptors (LLC-PK-AT1R cells). Cells grown on transwells were loaded with [3H]AA in the growth medium overnight, then treated as described in METHODS. Cells incubated with ANG II (100 nM) at the apical (AP) or basolateral (BL) surface for indicated times at 23°C were assayed for [3H]AA release. Data are expressed as [3H]AA counts released/mg protein; and n = 3-10 for each BL concentration, n = 7-22 for each AP concentration. * P < 0.005 vs. control and BL ANG II. ** P < 0.0005 vs. control and BL ANG II.

To assess whether ANG II-stimulated [3H]AA release occurred at a threshold concentration or in a concentration-responsive manner, LLC-PK-AT1R cells cultured on permeable supports were incubated with increasing concentrations of AP or BL ANG II for 20 min. AP ANG II at low concentrations had minimal effects on [3H]AA release [untreated, 5,570 ± 1,573; ANG II (100 pM), 6,335 ± 1,660; ANG II (1 nM), 6,568 ± 810; n = 3, NS] (Fig. 2). However, higher concentrations stimulated a significant increase in [3H]AA release [ANG II (10 nM): 12,264 ± 1,894, n = 3, P < 0.05 vs. untreated; ANG II (100 nM): 17,102 ± 2,520, n = 3, P < 0.005]. The apparent Michaelis constant for AP ANG II-stimulated [3H]AA release in this model was 3.4 ± 2 nM, consistent with the apical dissociation constant for ANG II in this model (6.4 ± 1 nM) (3). BL ANG II did not stimulate a significant increase in [3H]AA release in the concentrations tested (100 pM to 100 nM) (n = 3 for each; Fig. 2).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Dose dependence of ANG II-mediated [3H]AA release in LLC-PK-AT1R cells. Cells grown on transwells were loaded with [3H]AA in the growth medium overnight then treated as described in METHODS. Cells were treated with the noted concentrations of ANG II at the AP or BL surface for 20 min at 23°C and assayed for [3H]AA release. Data are expressed as [3H]AA counts released/mg protein; n = 3-10 for each BL concentration, and n = 4-22 for each AP concentration, except n = 2 for AP (1 mM). * P < 0.05 vs. AP untreated. ** P < 0.0005 vs. AP untreated.

To verify that ANG II-stimulated [3H]AA release in this model occurred as a result of PLA2 activity, cells were incubated in radiolabeled medium overnight and treated with ANG II as described in METHODS. Subsequently, the radiolabeled material was extracted from medium and cells and analyzed by reversed-phase HPLC. As shown in Fig. 3, AP ANG II induced the specific release of AA. Moreover, reversed-phase HPLC failed to demonstrate any other AA metabolites in the medium and cells following ANG II treatment (Fig. 3).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Reversed-phase high-performance liquid chromatography (HPLC) of radiolabeled material extracted from medium and LLC-PK-AT1R cells following treatment with AP ANG II (100 nM). Radiolabeled lipids were extracted from medium and cells following treatment with AP ANG II as described in METHODS. Samples were analyzed by reversed-phase HPLC with an injection time of 45 min. Retention time for AA was 32 min. A representative assay is shown. A: untreated control. B: peak following 20 min AP ANG II (100 nM) treatment. CPM, counts/min.

To ascertain whether this response was unique to our cell model, SV40 immortalized rabbit proximal tubule epithelial cells cultured on permeable supports were incubated with [3H]AA overnight and treated with AP or BL ANG II (100 nM). AP ANG II stimulated [3H]AA release (untreated, 2,084 ± 1,073; AP ANG II, 4,279 ± 1,689; n = 4; P < 0.02 vs. untreated). This response could be inhibited by losartan (1 µM) (2,369 ± 954; n = 3, NS vs. untreated). BL ANG II, however, did not significantly stimulate [3H]AA release (2,512 ± 1,514; n = 4, NS vs untreated).

In cultured cardiac myocytes, type 2 ANG II receptors (AT2R) have been associated with ANG II-mediated AA release (26). However, LLC-PK-AT1R cells express exclusively the rabbit AT1R (3). To verify that ANG II-stimulated [3H]AA release in LLC-PK-AT1R cells occurred through AT1R, cells loaded with [3H]AA were incubated with losartan (100 nM), an AT1R antagonist, at the AP surface prior to ANG II treatment. Losartan inhibited AP ANG II-stimulated [3H]AA release at 20 min [losartan + AP ANG II (100 nM), 4,382 ± 455; n = 6; P < 0.005 vs. AP ANG II] (Fig. 4). CGP-42112A (100 nM), an AT2R antagonist, had no significant effect on AP ANG II-mediated [3H]AA release (CGP + AP ANG II, 13,697 ± 2,450; n = 6, NS vs. AP ANG II) (Fig. 4).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of ANG II receptor antagonists, pertussis toxin (PTX), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) on AP ANG II-stimulated [3H]AA release. LLC-PK-AT1R cells were pretreated as described in METHODS with losartan (100 nM), an AT1R antagonist, CGP-42112A (100 nM), an AT2R antagonist, BAPTA-AM (10 µM), or treated overnight with pertussis toxin (500 ng/ml) prior to assaying for ANG II-mediated [3H]AA release. AP ANG II (100 nM) was used for these assays. Data are expressed as counts [3H]AA released/mg protein and represent n = 6-22 for each. * P < 0.005 vs. AP ANG II alone.

To clarify whether ANG II-mediated AA release in this model occurred as a consequence of other AT1R-mediated signaling pathways, cells pretreated with PTX (500 ng/ml) for 16 h in the presence of radiolabeled AA were assayed for ANG II-mediated [3H]AA release. Previous studies had established that, although PTX ADP-ribosylated G proteins in LLC-PK-AT1R cells, it did not affect AT1R internalization (3). PTX treatment did not significantly affect AP AT1R-mediated [3H]AA release (AP ANG II + PTX, 18,611 ± 4,540; n = 8, NS vs. AP ANG II) (Fig. 4). In addition, to determine whether intracellular calcium movement influenced AP or BL ANG II-stimulated [3H]AA release, cells loaded with [3H]AA were incubated with the intracellular calcium chelator, BAPTA-AM (10 µM) for 30 min prior to ANG II treatment. BAPTA-AM did not significantly affect AP ANG II-stimulated [3H]AA release (17,663 ± 4,733; n = 5, NS vs. AP ANG II) (Fig. 4).

To investigate further a potential role for calcium in ANG II-stimulated AA release in this model, cells were incubated with PMA (300 nM) + A-23187 (0.25 µM) for varying time periods to activate potential cytosolic Ca2+-dependent PLA2 activity. PMA + A-23187 elicited a 1.8-fold increase in [3H]AA release compared with unstimulated cells following treatment for 20 min at 37°C [5 min untreated, 4,474 ± 612; PMA + A-23187, 4,743 ± 129 (n = 4, NS); 10 min untreated, 3,312 ± 446; PMA + A-23187, 5,962 ± 1,943 (n = 4, P < 0.05); 20 min untreated, 3,926 ± 510; PMA + A-23187, 6,674 ± 944 (n = 14, P < 0.025)] (Fig. 5A). Pretreatment with quinacrine (250 µM), a nonspecific inhibitor of PLA2 activity, attenuated this response (20 min quinacrine + PMA + A-23187, 2,290 ± 402; n = 3, P < 0.02 vs. PMA + A-23187 alone) (Fig. 5B), but HELSS (10 µM), an inhibitor of calcium-independent PLA2 activity that affected AT1R endocytic movement (4), had no effect on PMA + A-23187-mediated [3H]AA release (20 min HELSS + PMA + A-23187, 6,474 ± 676; n = 3, NS vs. PMA + A-23187 alone) (Fig. 5B).


View larger version (21K):
[in this window]
[in a new window]
 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   A: effect of PMA + A-23187 on [3H]AA release in LLC-PK-AT1R cells. Cells were treated with PMA (300 nM) + A-23187 (0.25 mM) for the indicated times as described in METHODS and assayed for [3H]AA release. Data are expressed as counts [3H]AA released/mg protein and represent n = 4-14. * P < 0.05 vs. untreated cells. ** P < 0.025 vs. untreated cells. B: effect of PLA2 inhibitors on PMA + A-23187-stimulated [3H]AA release in LLC-PK-AT1R cells. Cells were treated as described in METHODS with PMA (300 nM) + A-23187 (0.25 µM) with or without quinacrine (Quin, 250 µM) or haloenol lactone suicide substrate (HELSS, 10 µM), then assayed for [3H]AA release at 20 min. Data are expressed as counts [3H]AA released/mg protein; n = 3 for each. * P < 0.02 vs. PMA + A-23187 alone.

The selective effect of HELSS to inhibit AP AT1R recycling in this model strongly suggested that calcium-independent PLA2 activity played a potential role in AP ANG II-mediated [3H]AA release in LLC-PK-AT1R cells. To investigate this possibility, cells were pretreated with HELSS, then incubated with AP ANG II and assayed for [3H]AA release. HELSS had no effect on baseline release of [3H]AA from unstimulated cells. However, HELSS significantly inhibited AP ANG II-mediated [3H]AA release [AP ANG II (100 nM), 16,695 ± 1,250; ANG II + HELSS, 6,003 ± 1,170 (n = 8, P < 0.005)] (Fig. 6). Quinacrine (250 µM) also significantly decreased AP ANG II-mediated [3H]AA release [AP ANG II (100 nM), 15,063 ± 2,520; AP ANG II + quinacrine, 2,758 ± 704 (n = 3, P < 0.025) vs. AP ANG II], as did another inhibitor of calcium-independent PLA2 activity, PACOCF3 (50 µM) (2) [AP ANG II (100 nM), 16,810 ± 3,230; AP ANG II + PACOCF3, 7,046 ± 2,250 (n = 5, P < 0.05) vs. AP ANG II] (Fig. 6). Furthermore, inhibiting AT1R internalization with phenylarsine oxide (PAO) pretreatment (50 µM) also inhibited AP ANG II-stimulated [3H]AA release [AP ANG II (100 nM), 16,810 ± 3,230; AP ANG II + PAO (50 µM), 3,235 ± 1,145 (n = 4, P < 0.05) vs. AP ANG II] (Fig. 6), suggesting that a signaling step related to endocytosis was involved in AP AT1R-mediated [3H]AA release.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of PLA2 inhibitors and phenylarsine oxide (PAO) on AP ANG II-stimulated [3H]AA release. LLC-PK-AT1R cells were pretreated as described in METHODS with the nonspecific PLA2 inhibitor, quinacrine (250 µM), inhibitors of calcium-independent PLA2 activity, HELSS (10 µM) PACOCF3 (50 µM), or the endocytosis inhibitor, PAO (50 µM), as described in METHODS prior to assaying for ANG II-mediated [3H]AA release. AP ANG II (100 nM) was used for these assays. Data are expressed as counts [3H]AA released/mg protein and represent n = 3-8 for each. * P < 0.05 vs. AP ANG II. ** P < 0.005 vs. AP ANG II. **# P < 0.025 vs. AP ANG II.

To determine whether ANG II-stimulated [3H]AA release was a nonspecific response following an internalization event or a selective effect of AP ANG II stimulation, LLC-PK-AT1R cells loaded with [3H]AA were treated with AP ANG II (100 nM) or transferrin (5 µM) and assayed for [3H]AA release. Transferrin receptors are constitutively maintained in an endocytic compartment, internalizing from the cell membrane and cycling back to reinsert into the cell surface (33). [3H]AA release following transferrin treatment was not significantly different from control cells (Fig. 7) (5 min control, 4,625 ± 1,110; 5 min transferrin, 4,887 ± 930; NS; 10 min control: 3,533 ± 1,100; 10 min transferrin, 2,969 ± 1,200, NS; 20 min control, 4,968 ± 700; 20 min transferrin, 5,562 ± 1,304, NS; n = 4-12 for each). AP ANG II, however, stimulated a significant increase in [3H]AA release at 10 and 20 min following treatment (Fig. 7).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Time-dependent determination of [3H]AA release following exposure to transferrin (5 µM) or AP ANG II (100 nM). LLC-PK-AT1R cells were exposed to transferrin or AP ANG II for the indicated times and assayed as described in METHODS for [3H]AA release. Data shown are expressed as counts [3H]AA released/mg protein; n = 4-12 for each time point; * P < 0.02 vs. control and transferrin. ** P < 0.001 vs. control and transferrin.

These data suggested that AP AT1R-mediated [3H]AA release in LLC-PK-AT1R cells was related to endocytosis. AP AT1R endocytosis has been previously linked to ANG II-mediated Na+ flux by Schelling and Linas (39) in studies using rat proximal tubule cells. They noted that PAO pretreatment to inhibit AP AT1R internalization significantly attenuated Na+ flux (AP right-arrow BL). However, PAO treatment had no effect on BL ANG II-mediated Na+ flux. Because PAO treatment inhibited AP AT1R-stimulated [3H]AA release in our model, studies were undertaken to determine whether AP AT1R-mediated [3H]AA release was important in mediating Na+ flux (AP right-arrow BL). ANG II (100 nM) was utilized for these studies, because this concentration yielded the maximal release of [3H]AA in the previous studies. AP ANG II stimulated a time-dependent increase in Na+ flux (AP right-arrow BL) in LLC-PK-AT1R cells (Fig. 8) (22Na counts appearing in BL media/mg protein: 15 min control, 20,104 ± 2,220; 15 min AP ANG II, 28,650 ± 3,210, P < 0.025 vs. control; 20 min control, 25,200 ± 2,625; 20 min AP ANG II, 39,400 ± 2,875, P < 0.025; 30 min control, 28,740 ± 4,450; 30 min AP ANG II, 34,300 ± 2,105, P < 0.05; n = 4-7 for each). Moreover, HELSS (10 µM) pretreatment inhibited the ANG II-mediated increase in 22Na flux (AP right-arrow BL) (15 min, 20,620 ± 3,529; 20 min, 25,866 ± 3,433; 30 min, 25,244 ± 2,755; n = 4 for each; each NS vs. control), suggesting that HELSS-inhibitable AA release and calcium-independent PLA2 activity were involved in ANG II-stimulated 22Na flux (AP right-arrow BL).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8.   AP ANG II-stimulated 22Na flux (AP right-arrow BL) in LLC-PK-AT1R cells. Cells were assayed for 22Na flux (AP right-arrow BL) as described in METHODS. At least 1 well/plate was left untreated but incubated with radioactive buffer and assayed for control 22Na flux. Assays were carried out for 30 min, then radioactive buffer was removed, and cells were lysed with 0.05 M NaOH. For experiments using HELSS, wells were pretreated with vehicle or HELSS for 10 min at 37°C prior to initiating assay. Data shown are expressed as 22Na counts appearing in BL medium · time point-1 · mg protein-1; n = 4-7 for each time point. * P < 0.05 AP ANG II vs. other conditions. ** P < 0.025 AP ANG II vs. other conditions.

To verify that this was not related to a nonspecific increase in ANG II-mediated transport (AP right-arrow BL), cells were incubated with [3H]inulin and AP ANG II (100 nM). In control wells not exposed to ANG II, average nonspecific [3H]inulin leak was 0.082% at 45 min (n = 4). AP ANG II did significantly alter [3H]inulin leak (ANG II, 0.085%; n = 6).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

PLA2 activation appears to be an important signal-transduction pathway for proximal tubule AT1R (13, 37). Experimental data from Yanagawa and others (24, 37) suggest that vectorial ion and water flux is directly related to ANG II-mediated PLA2 activity. The studies presented here demonstrate that ANG II-mediated [3H]AA release is associated with AT1R endocytosis/recycling, at least in part, through calcium-insensitive PLA2 activity. Moreover in our model, this event appears linked to a physiological response to ANG II as ANG II-mediated 22Na flux (AP right-arrow BL) occurred concomitant with ANG II-stimulated [3H]AA release in LLC-PK-AT1R cells.

ANG II lipid-related signal transduction has been described in other cell models expressing ANG II receptors (26, 40, 43). Lokuta et al. (26) reported that ANG II-dependent [3H]AA release in neonatal rat cardiac myocytes was partially inhibited by the AT1R antagonist, losartan (100 nM), as well as the type 2 ANG II receptor (AT2R) antagonist, CGP-42112A. In those studies, CGP-42112A was a more potent inhibitor of ANG II-stimulated [3H]AA release, suggesting that more than one ANG II receptor subtype participated in ANG II-mediated [3H]AA release.

Siragy and Carey (40) described renal AT1R-mediated prostaglandin E2 (PGE2) production in sodium-depleted rats, a physiological state characterized by upregulation of the renin-angiotensin system. AT1R-mediated PGE2 production in this model was potentiated by AT2R pharmacological blockade and inhibited by AT1R blockade. In our model, the apical ANG II receptor appeared to be the primary mediator of ANG II [3H]AA release, similar to the recent report from Jacobs and Douglas (21). However, in contrast to their data and in keeping with the observations of Siragy and Carey (40), this AP ANG II response was mediated by AT1R as losartan (100 nM) significantly inhibited ANG II-mediated [3H]AA release. CGP-42112A (100 nM) treatment had no significant effect on this ANG II response, consistent with the absence of AT2R in LLC-PK-AT1R cells. Moreover, losartan inhibited this AP ANG II response in SV40-immortalized rabbit proximal tubule cells, further suggesting that AP ANG II [3H]AA release occurred through an AT1R in proximal tubule epithelia.

Although the exact mechanisms underlying ANG II-mediated AA release in LLC-PK-AT1R cells remain to be defined, the delayed release of AA in ANG II-treated LLC-PK-AT1R cells suggests that a downstream signaling event or coordinated concurrent signal transduction results in AA release. Such a scenario has been demonstrated for ANG II and platelet-derived growth factor receptors with late convergence of signaling pathways leading to tyrosine kinase activation (25). It is also possible, although as yet undetermined, that ANG II and/or AT1R maintain potential "signaling activity" following internalization similar to other G protein-coupled receptors, e.g., endothelin receptors (10) and cholecystokinin receptors (36). Finally, internalized ANG II may initiate intracellular signaling by binding to intracellular receptors, as recently suggested in experiments from Luft and associates (17). The timing of ANG II-mediated [3H]AA release and Na+ flux circumstantially argues for the possibility that this is the time period following internalization that a significant degree of AT1R recycling is likely occurring in cells (3).

Clearly, the combination of phorbol ester and calcium ionophore, although stimulating [3H]AA release, did not stimulate AA release to the extent that ANG II did in this proximal tubule-like model. The lack of an inhibitory effect of BAPTA-AM in our studies also suggested that intracellular calcium flux was not an integral component of ANG II-mediated AA release in LLC-PK-AT1R cells. Furthermore, HELSS did not affect PMA + A-23187-stimulated [3H]AA release but significantly inhibited ANG II-mediated [3H]AA release. Therefore, it is reasonable to postulate that calcium-independent PLA2 activity plays a role in ANG II-mediated AA release in LLC-PK-AT1R cells.

Calcium-independent PLA2 activity has been described in a number of different tissues, including epithelia and myocardium (14, 19, 31, 35). Lehman et al. (23) demonstrated HELSS-inhibitable [Arg8]vasopressin stimulated AA release in cultured vascular smooth muscle cells, demonstrating this form of PLA2 activity in a different cell type and characterizing an interrelationship between a G protein-coupled receptor and calcium-independent PLA2 activity. Although this form of PLA2 activity was described originally in the setting of myocardial ischemia (19), subsequent studies determined that calcium-independent PLA2 activity plays a role in endocytic vesicle fusion (4, 27, 30). HELSS inhibits this process in cell-free systems as well as in intact cells (4, 27). Hence, it is not surprising that this form of PLA2 activity is evident in transport, absorptive or secretory epithelia, e.g., proximal tubule cells (31), pancreatic acinar cells (35), and gastrointestinal epithelia (14).

Recent data suggest that AP AT1R may play an important role in proximal tubule transport in vivo, binding endogenously produced ANG II at the luminal surface (34). The polarity of the signaling response in our model was consistent with these data, as were previous studies (24, 38), suggesting that AP AT1R may be preferentially associated with PLA2 activity. The selective AP effect of PAO and HELSS also was similar to observations by Schelling et al. (38), that PAO treatment decreased AP but not BL ANG II-stimulated inositol trisphosphate generation and ANG II-induced Na+ flux (39) in proximal tubule cell primary cultures. Our model thus duplicates some features of AT1R in primary culture, including functionally distinct AT1R receptors in different intracellular compartments with differential rates of endocytosis, recycling, differential pharmacological responsiveness (3), and selective stimulation of AA release.

Some of these responses may be attributable to using a cultured cell line for our investigations. LLC-PK-AT1R cells lack endogenous cytochrome P-450 activity, consistent with the lack of cytochrome P-450 and cyclooxygenase activity in LLC-PK1 cells in general (12). This is evidenced in Fig. 3 by the absence of any AA metabolites. In studies in proximal tubule epithelium, ANG II-stimulated AA release occurred concomitant with the generation of a cytochrome P-450 metabolite that comigrated with 5,6-epoxyeicosotrienoic acid (13). This response appeared to inhibit ANG II-stimulated Na+ entry. Thus in vivo cytochrome P-450 lipid metabolism could potentially result in different cellular responses, i.e., inhibition of Na+ flux, from those demonstrated in the present experiments. Alternatively, the absence of cytochrome P-450 in LLC-PKC14 cells could "unmask" measurable ANG II-stimulated [3H]AA release, since cytochrome P-450 metabolites would not be present to provide feedback inhibition to AP ANG II-stimulated PLA2 activity. Furthermore, although reversed-phase HPLC verified ANG II PLA2 activity in our model, contributions of diacylglycerol lipase or potential PLA2 activating proteins (15) to ANG II lipid-related signal transduction and 22Na flux cannot be completely excluded based on the present studies. Finally, it should be recognized that AT1R expression in LLC-PK-AT1R cells is only 5-10% of that demonstrated for tubular cell AT1R in vivo. It is possible that a higher level of receptor expression might alter LLC-PK-AT1R lipid-related signaling. Further studies will be necessary to clarify how the unique characterstics of LLC-PKC14 cells may affect ANG II-generated signal transduction and cellular responses to ANG II.

Our studies indicate an important role for receptor-mediated endocytosis and/or receptor recycling in the ANG II signaling process related to 22Na flux in LLC-PK-AT1R cells. Endocytosis and recycling appear to be a key properties for AT1R in general. ANG II receptor-mediated endocytosis differentiates AP and BL AT1R in LLC-PK-AT1R cells (3). AP AT1R readily internalize ANG II bound at that surface and rapidly recover ANG II binding following endocytosis, a measure of AT1R recycling. BL AT1R internalize and recycle at a much slower rate. Furthermore, AP and BL AT1R are differentially affected by inhibitors of internalization and recycling. PAO significantly inhibited AP AT1R but not BL AT1R internalization (3) in LLC-PK-AT1R cells. The PLA2 inhibitors quinacrine and HELSS did not signficantly alter AT1R endocytosis, yet, each of these compounds significantly affected AP but not BL AT1R recycling (3). In the studies reported here, PAO and HELSS inhibited AP AT1R-mediated AA release and 22Na flux, suggesting that the endocytotic and recycling movement of AP AT1R may be important for physiological responsiveness to ANG II. It is therefore notable that ANG II receptor-mediated endocytosis is a requirement in various cell models for angiotensin degradation (11), diacyglycerol generation (22), and sustained intracellular calcium signaling (20).

In summary, our proximal tubule-like model suggests that AT1R cell biology is integrated with AT1R function. ANG II appears to maintain its cellular effects subsequent to surface binding, stimulating AA release. Indeed, the association between AT1R endocytosis/recycling and delayed AA release is somewhat similar in timing to the effects of ANG II on sustained intracellular calcium flux in adrenal glomerulosa cells (20). Yet, this association in LLC-PK-AT1R cells also appears to affect ANG II-mediated 22Na flux, providing an interesting link between receptor cell biology, signaling, and a cellular physiological response.

    ACKNOWLEDGEMENTS

We kindly acknowledge Jorge Capdevila for aid in completing the HPLC studies.

    FOOTNOTES

This work was supported in part by funds from the Department of Veterans Affairs (to R. C. Harris) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02420 (to B. N. Becker) and DK-39261 and DK-38226 (to R. C. Harris). R. C. Harris is a Clinical Investigator in the Career Development Program of the Veteran's Administration.

Address for reprint requests: B. N. Becker, Div. of Nephrology, Dept. of Medicine, Univ. of Wisconsin-Madison Medical School, H4/510 Clinical Science Center, 600 Highland Ave., Madison, WI 53792.

Received 1 November 1996; accepted in final form 12 June 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ackermann, E. J., K. Conde-Frieboes, and E. A. Dennis. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J. Biol. Chem. 270: 445-450, 1995[Abstract/Free Full Text].

2.   Ackermann, E. J., E. S. Kempner, and E. A. Dennis. Ca2+-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. J. Biol. Chem. 269: 9227-9233, 1994[Abstract/Free Full Text].

3.   Becker, B. N., H.-F. Cheng, K. D. Burns, and R. C. Harris. Polarized rabbit type 1 angiotensin receptors expressed in LLC-PK cells manifest differential rates of endocytosis and recycling. Am. J. Physiol. 269 (Cell Physiol. 38): C1048-C1055, 1995[Abstract/Free Full Text].

4.   Bobillo-Bette, P., and M. Vidal. Characterization of phospholipase A2 activity in reticulocyte endocytic vesicles. Eur. J. Biochem. 228: 199-205, 1995[Abstract].

5.   Brown, G. P., and J. G. Douglas. Angiotensin II binding sites on isolated rat renal brush border membranes. Endocrinology 111: 1830-1836, 1982[Abstract].

6.   Brown, G. P., and J. G. Douglas. Angiotensin II-binding sites in rat and primate isolated renal tubular basolateral membranes. Endocrinology 112: 2007-2014, 1983[Abstract].

7.   Burns, K. D., and R. C. Harris. Signaling and growth responses of LLC-PK1/C14 cells transfected with the rabbit AT1 ANG II receptor. Am. J. Physiol. 268 (Cell Physiol. 37): C925-C935, 1995[Abstract/Free Full Text].

8.   Burns, K. D., T. Homma, and R. C. Harris. Regulation of Na+-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F607-F616, 1991[Abstract/Free Full Text].

9.   Capdevila, J. H., J. R. Falck, E. Dishman, and A. Karara. Cytochrome P-450 arachidonate oxygenase. Methods Enzymol. 187: 385-394, 1990[Medline].

10.   Chun, M., H. Y. Lin, Y. I. Henis, and H. F. Lodish. Endothelin-induced endocytosis of cell surface ETA receptors. J. Biol. Chem. 270: 10855-10860, 1995[Abstract/Free Full Text].

11.   Crozat, A., A. Penhoat, and J. M. Saez. Processing of angiotensin II (AII) and (Sar1,Ala8)A-II by cultured bovine adrenocortical cells. Endocrinology 118: 2312-2318, 1986[Abstract].

12.   Dellipizzi, A. M., J. C. McGiff, and B. Escalante. Cytochrome P450 inhibitors attenuate the hypotonic shock-induced increases in K+ efflux in LLC-PK1 cells. Pharmacology 50: 348-356, 1995[Medline].

13.   Douglas, J. G., M. Romero, and U. Hopfer. Signaling mechanisms coupled to the angiotensin receptor of proximal tubule epithelium. Kidney Int. 38: S43-S47, 1990.

14.   Gassama-Diagne, A., J. Fauvel J, and H. Chap. Purification of a new calcium-independent, high-molecular-weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane. J. Biol. Chem. 264: 9470-9475, 1989[Abstract/Free Full Text].

15.   Goldberg, H., P. Maxwell, N. Hack, and K. Skorecki. Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release. Biochem. Biophys. Res. Commun. 198: 220-227, 1994[Medline].

16.   Haggerty, J. G., N. Agarwal, R. F. Reilly, E. A. Adelberg, and C. W. Slayman. Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1). Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988[Abstract].

17.   Haller, H., C. Lindschau, B. Erdmann, P. Quass, and F. C. Luft. Effects of intracellular angiotensin II in vascular smooth muscle cells. Circ. Res. 79: 765-772, 1996[Abstract/Free Full Text].

18.   Harris, P. J., and D. A. Young. Dose-dependent stimulation and inhibition of proximal tubular sodium reabsorption by angiotensin II in the rat kidney. Pflügers Arch. 367: 295-297, 1977[Medline].

19.   Hazen, S. L., L. A. Zupan, R. H. Weiss, D. P. Getman, and R. W. Gross. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. J. Biol. Chem. 266: 7227-7232, 1991[Abstract/Free Full Text].

20.   Hunyady, L., F. Merelli, A. J. Baukal, T. Balla, and K. J. Catt. Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells: a potential mechanism for receptor-operated calcium entry. J. Biol. Chem. 266: 2783-2788, 1991[Abstract/Free Full Text].

21.   Jacobs, L. S., and J. G. Douglas. Angiotensin II type 2 receptor subtype mediates phsopholipase A2-dependent signaling in rabbit proximal tubular epithelial cells. Hypertension 28: 663-668, 1996[Abstract/Free Full Text].

22.   Kapas, S., J. P. Hinson, J. R. Puddefoot, M. M. Ho, and G. P. Vinson. Internalization of the type 1 angiotensin II receptor (AT1) is required for protein kinase C activation but not for inositol trisphosphate release in the angiotensin II stimulated rat adrenal zona glomerulosa cell. Biochem. Biophys. Res. Commun. 204: 1292-1298, 1994[Medline].

23.   Lehman, J. J., K. A. Brown, S. Ramanadham, J. Turk, and R. W. Gross. Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent phospholipase A2. J. Biol. Chem. 268: 20713-20716, 1993[Abstract/Free Full Text].

24.   Li, L., Y. P. Wang, A. W. Capparelli, O. D. Jo, and N. Yanagawa. Effect of luminal angiotensin II on proximal tubule fluid transport: role of apical phospholipase A2. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F202-F209, 1994[Abstract/Free Full Text].

25.   Linseman, D. A., C. W. Benjamin, and D. A. Jones. Convergence of angiotensin II and platelet-derived growth factor receptor signaling cascades in vascular smooth muscle cells. J. Biol. Chem. 270: 12563-12568, 1995[Abstract/Free Full Text].

26.   Lokuta, A. J., C. Cooper, S. T. Gaa, H. E. Wang, and T. B. Rogers. Angiotensin II stimualtes the release of phospholipid-derived second messengers through multiple receptor subtypes in heart cells. J. Biol. Chem. 269: 4832-4838, 1994[Abstract/Free Full Text].

27.   Mayorga, L. S., M. I. Colombo, M. Lennartz, E. J. Brown, K. H. Rahman, R. Weiss, P. J. Lennon, and P. D. Stahl. Inhibition of endosome fusion by phospholipase A2 (PLA2) inhibitors points to a role for PLA2 in endocytosis. Proc. Natl. Acad. Sci. USA 90: 10255-10259, 1993[Abstract].

28.   Morduchowicz, G. A., D. Sheikh-Hamad, B. E. Dwyer, N. Stern, O. D. Jo, and N. Yanagawa. Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: presence of local signal transduction system. J. Membr. Biol. 122: 43-53, 1991[Medline].

29.   Mukherjee, A. B., L. Miele, and N. Pattabiraman. Phospholipase A2 enzymes: regulation and physiological role. Biochem. Pharmacol. 48: 1-10, 1994[Medline].

30.   Nagao, T., T. Kubo, R. Fujimoto, H. Nishio, T. Takeuchi, and F. Hata. Ca2+-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro. Biochem. J. 307: 563-569, 1995[Medline].

31.   Portilla, D., and G. Dai. Purification of a novel calcium-independent phospholipase A2 from rabbit kidney. J. Biol. Chem. 271: 15451-15457, 1996[Abstract/Free Full Text].

32.   Portilla, D, S. V. Shah, P. A. Lehman, and M. H. Creer. Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules. J. Clin. Invest. 93: 1609-1615, 1994[Medline].

33.   Pytowski, B., T. W. Judge, and T. E. McGraw. An internalization motif is created in the cytoplasmic domain of the transferrin receptor by substitution of a tyrosine at the first position of a predicted tight turn. J. Biol. Chem. 270: 9067-9073, 1995[Abstract/Free Full Text].

34.   Quan, A., and M. Baum. Endogenous production of angiotensin II modulates rat proximal tubule transport. J. Clin. Invest. 97: 2878-2882, 1996[Abstract/Free Full Text].

35.   Ramanadham, S., M. J. Wolf, P. A. Jett, R. W. Gross, and J. Turk. Characterization of an ATP-stimulatable Ca2+-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the b-cell fuel sensor. Biochemistry 33: 7442-7452, 1994[Medline].

36.   Roettger, B. F., R. U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J. M. Larkin, and L. J. Miller. Dual pathways of internalization of the cholecystokinin receptor. J. Cell Biol. 128: 1029-1041, 1995[Abstract].

37.   Romero, M. F., U. Hopfer, Z. T. Madhun, W. Zhou, and J. G. Douglas. Angiotensin II actions in the rabbit proximal tubule. Angiotensin II-mediated signaling mechanisms and electrolyte transport in the rabbit proximal tubule. Renal Physiol. Biochem. 14: 199-207, 1991.[Medline]

38.   Schelling, J. R., A. S. Hanson, R. Marzec, and S. L. Linas. Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells. J. Clin. Invest. 90: 2472-2480, 1992[Medline].

39.   Schelling, J. R., and S. L. Linas. Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis. Am. J. Physiol. 266 (Cell Physiol. 35): C669-C675, 1994[Abstract/Free Full Text].

40.   Siragy, H. M., and R. M. Carey. The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3',5'-monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats. J. Clin. Invest. 97: 1978-1982, 1996[Abstract/Free Full Text].

41.   Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olsen, and D. C. Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85, 1985[Medline].

42.   Trowbridge, I. S., J. F. Collawn, and C. R. Hopkins. Signal-dependent membrane-protein trafficking in the endocytic pathway. Annu. Rev. Cell. Biol. 9: 129-161, 1993.

43.   Wen, Y., M. C. Cabot, E. Clauser, S. L. Bursten, and J. I. Nadler. Lipid signal transduction pathways in angiotensin II type 1 receptor-transfected fibroblasts. Am. J. Physiol. 269 (Cell Physiol. 38): C435-C442, 1995[Abstract/Free Full Text].

44.   Wolf, R. A., and R. W. Gross. Identification of neutral active phospholipase C which hydrolyzes choline glycerophospolipids and plasmalogen-selective phospholipase A2 in canine myocardium. J. Biol. Chem. 260: 7295-7303, 1985[Abstract/Free Full Text].


AJP Renal Physiol 273(4):F554-F562
0363-6127/97 $5.00 Copyright © 1997 the American Physiological Society