INTRODUCTION

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THE ATRIAL NATRIURETIC peptide (ANP) is a cardiac hormone that displays potent vasorelaxant and natriuretic activity in a variety of species. Its natriuretic action has been attributed to a number of hemodynamic and direct tubular effects in the kidney (2, 3). These include alterations in intraglomerular pressure, changes in glomerular permeability, shifts in inner medullary blood flow, and inhibition of sodium transport in both proximal and distal nephron segments. In the latter instance, it has been shown to inhibit sodium reabsorption in the inner medullary collecting duct (IMCD), a nephron segment that handles less than 5% of the filtered sodium load, yet plays a pivotal role in establishing the final sodium concentration of the urine (30).

The natriuretic peptides have been shown to exert their biological effects through binding to one or more of a small group of natriuretic peptide receptors (NPR). NPR-A and NPR-B are single transmembrane domain receptors that link an extracellular peptide binding domain to a carboxy terminal guanylyl cyclase catalytic domain located inside the cell (17). The latter represents the effector arm of the liganded receptor. NPR-A binds predominantly to ANP and brain natriuretic peptide (BNP), whereas NPR-B recognizes C-type natriuretic peptide (CNP) as its cognate ligand (18). A third receptor, NPR-C, is truncated just distal to its single transmembrane-spanning domain and lacks the guanylyl cyclase activity of the other receptors. It binds to all three natriuretic peptides and functions in a clearance mode, taking up and internalizing peptide from the extracellular space (12, 20). It may also possess signaling activity that is independent of the cGMP generation intrinsic to the other receptors (1, 16).

The guanylyl cyclase receptors are the predominant receptor subtypes found in the IMCD (15), and very little NPR-C is thought to be present in this segment. It is assumed that ANP, present in circulating plasma or in the tubular fluid itself, binds to these receptors and increases cellular cGMP levels. cGMP, either directly or through activation of the cGMP-dependent protein kinase (19), suppresses sodium transport through the amiloride-sensitive sodium channel on the luminal surface of the IMCD cell and, thereby, increases urinary excretion of sodium.

We have shown previously that NPR-A expression and activity in rat aortic smooth muscle cells (RASM) are suppressed by prior exposure to the natriuretic peptides (i.e., ANP, BNP, or CNP) (5). RASM cells are known to express all three NPRs (27). We sought to determine whether a similar downregulation of NPR-A activity would be conserved in IMCD cells, which display a more limited receptor repertoire and, if so, whether this downregulation is accompanied by a decrease in NPR gene expression.

	MATERIALS AND METHODS

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Materials. ANP [rat ANF-(128)], brain natriuretic peptide [BNP-(132), porcine], C-type natriuretic peptide [CNP-(122), porcine], cANF {des-[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹, Gly²²]ANP-(423)-NH₂, rat}, and urodilatin [cardiodilatin/ANP-(95126)] were purchased from Peninsula Laboratories (Belmont, CA). IBMX, reagents for cGMP radioimmunoassay (cGMP standard and antibody), and actinomycin D were purchased from Sigma Chemical (St. Louis, MO). ¹²⁵I-cGMP and [-³²P]dCTP were purchased from New England Nuclear (Wilmington, DE). Lipofectamine (GIBCO) was obtained from Life Technologies (Gaithersburg, MD). The luciferase assay kit was purchased from Promega (Madison, WI). Collagenase was purchased from Worthington Biochemical (Freehold, NJ). The rat NPR-A and NPR-B cDNAs were generously provided by David Garbers. The bovine NPR-C cDNA was a gift from Gordon Porter, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was from Seigo Izumo. All other chemicals and cell culture reagents were purchased from standard commercial suppliers.

Isolation and culture of cells. IMCD cells were isolated from freshly excised kidneys of adult Sprague-Dawley rats using the hypotonic lysis technique of Grenier et al. (14) with some modifications. The inner medullary tissue was dissected away from the outer medulla and minced into fine pieces with a scalpel blade. The minced tissue from six rats was incubated in 4 ml phosphate-buffered saline (PBS) containing 1 mg/ml collagenase, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37°C for 30 min with gentle agitation. The cell suspension was collected, and 1 ml FBS was added to each 4 ml suspension. Harvested cells were kept at 4°C. The process was repeated until the tissue was completely digested. A quantity of 7.5 ml of distilled water was then added to every 4 ml of cell suspension to give a final osmolality of 120 mosmol/kg, and the mixture was centrifuged immediately at 500 g for 4 min. This hypotonic shock lyses all but the medullary collecting duct cells in the preparation (14). The cell pellet was resuspended in PBS containing 10% BSA and centrifuged again at 500 g for 4 min. Cells were resuspended in medium-1 (1:1 mixture of DMEM and Ham's F-12 medium supplemented with 10% FBS, 42 mM sodium bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin). Cells were seeded into either 24-well (16-mm well diameter), 6-well (35-mm diameter), or 10-cm (diameter) Falcon culture plates. On the second day, the cells were transferred to K-1 medium [1:1 mixture of DMEM and Ham's F-12 medium supplemented with 10 mM HEPES (pH 7.4), 42 mM sodium bicarbonate, 5 µg/ml insulin, 50 nM hydrocortisone, 5 µg/ml transferrin, 5 pM triiodothyronine, 100 IU/ml penicillin, and 100 µg/ml streptomycin] and cultured for 3-4 days until the cells attained confluence.

cGMP measurement. Confluent IMCD cells, cultured in 24-well plates, were pretreated with the agents described in the legends to Figs. 1-7. For measurement of ANP-stimulated cGMP accumulation, the cells were washed (3 times) with prewarmed PBS or acidic medium (pH of the medium was reduced to 5.0 by the addition of 4% acetic acid) (21), as indicated, and incubated with 0.5 ml of DMEM containing 0.5 mM IBMX and 10 mM HEPES, pH 7.4, for 10 min at 37°C. ANP (100 nM except where indicated) was added to the medium, and the incubation continued for another 10 min. The reaction was terminated by aspiration of media and addition of 0.3 ml of 12% trichloroacetic acid (TCA). The extraction was continued for 30 min at 4°C. The precipitated proteins were removed by centrifugation, and the supernatant was extracted four times with 0.5 ml of water-saturated ether. Aliquots were lyophilized and resuspended in 0.1 ml of 50 mM sodium acetate buffer. Soluble protein was measured using the Coomassie protein assay reagent (Pierce Chemical). cGMP levels were determined by radioimmunoassay after acetylation of the samples and standards, using a commercially available antibody and ¹²⁵I-labeled cGMP. For measurements of basal cGMP levels, the pretreatment was terminated by aspiration of culture media and the addition of 0.3 ml of 12% TCA. Subsequent steps were identical to those described above.

RNA isolation and Northern hybridization. Confluent IMCD cells (10-cm dishes) were washed with cold PBS, and total cellular RNA was extracted using the guanidinium thiocyanate-CsCl technique (8). A quantity of 30 µg RNA was electrophoresed through a 1% agarose/2.2 M formaldehyde gel, transferred to a nitrocellulose membrane (GeneScreen Plus, New England Nuclear) and fixed to the membrane by ultraviolet irradiation (DNA transfer lamp, Fotodyne). A 1.2-kb EcoR I fragment from the 5' end of the rat NPR-A cDNA (7), 1.3-kb EcoR I-BamH I fragment from the rat NPR-B cDNA (25), or a full-length bovine NPR-C cDNA (2.1-kb Hind III-EcoR I fragment) (12) was labeled with [-³²P]dCTP using the random primer technique. Hybridizations were performed overnight at 42°C using conventional techniques. To standardize for variations in RNA loading and transfer, the filters were stripped of label with 2× SSC (0.3 M sodium chloride and 0.03 M sodium citrate)/0.5% SDS followed by 0.1× SSC (0.015 M sodium chloride and 0.0015 M sodium citrate)/0.5% SDS at 100°C. They were then reprobed with a 1.3-kb Pst I fragment from the human GAPDH cDNA. Hybridization signal was detected by autoradiography and quantified by densitometry.

Transfection of IMCD cells. Cells were plated in 6-well plates and grown to 60-80% confluence over 2-3 days. For each individual transfection, the following solutions were prepared: solution A contained 2 µg plasmid DNA diluted in 0.1 ml OPTI-MEM I reduced serum medium (GIBCO-BRL); solution B contained 0.01 ml of Lipofectamine reagent diluted in 0.1 ml of OPTI-MEM I reduced serum medium. Solutions A and B were combined, mixed gently, and incubated at room temperature for 30 min to allow DNA-liposome complexes to form. Cells were washed once with 2 ml of OPTI-MEM I medium. A quantity of 0.8 ml of the same medium was added to 0.2 ml of DNA-liposome complex. After gentle agitation, the mixture was overlaid onto the washed cells. Cultures were incubated at 37°C in 5% CO₂ for 5 h, at which point 1 ml of K-1 medium was added. The incubations were continued for another 20 h. The medium was then replaced with complete K-1 culture medium supplemented with the additives indicated. Seventy-two hours after the start of transfection, cells were harvested and luciferase activity was measured using a commercial kit (Promega).

Statistics. Data were analyzed by one-way analysis of variance with the Newman-Keuls test for significance. Pooled data were drawn from 2-5 experiments with three independent samplings per experiment.

	RESULTS

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Previous studies have shown that IMCD cells harbor almost exclusively the biologically active (i.e., particulate guanylyl cyclase-associated) NPRs with little or no clearance receptor activity (15). Identification of the precise NPR subtypes present in the IMCD has been controversial, with some investigations supporting the presence of both NPR-A and NPR-B in this segment (9, 28) and others demonstrating only NPR-A (10). To determine which of the biologically active receptors (NPR-A vs. NPR-B) predominate in our in vitro preparations of IMCD cells, we challenged them with either ANP or CNP (selective ligands for the A and B receptor, respectively) and measured cellular cGMP levels. As shown in Fig. 1A, these cells displayed a robust response to ANP but not to CNP, implying a predominance of NPR-A in the cultures. This was confirmed by Northern analysis of RNA isolated from these cells. When total RNA was blot-hybridized with cDNAs for the different NPRs, only that probed for NPR-A displayed a detectable signal (Fig. 1B) under conditions which have readily detected NPR transcripts in RNA prepared from other cell types (4, 6).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Natriuretic peptide receptor (NPR) subtypes in primary cultures of inner medullary collecting duct (IMCD) cells. A: confluent cells were washed and preincubated in medium containing 0.5 mM IBMX and 10 mM HEPES, pH 7.4, for 10 min, then challenged with 100 nM atrial natriuretic peptide (ANP) () or C-type natriuretic peptide (CNP; ) for an additional 10 min. Reactions were terminated with 12% TCA, and cGMP levels were measured by radioimmunoassay. Data are means ± SD (n = 3). B: total RNA was collected from confluent cells as described in MATERIALS AND METHODS. RNA, 30 µg, was size-fractionated, transferred to a nitrocellulose filter by capillary action, hybridized with [-32P]dCTP-labeled NPR-A (lane 1), NPR-B (lane 2), or NPR-C (lane 3) cDNA, and autoradiographed at 70°C for 5 days. Results in each panel are representative of 2 independent experiments.

Next, we asked whether pretreatment of IMCD cells for varying periods of time with the receptor's cognate ligand (i.e., ANP) would alter the subsequent response to ligand. As shown in Fig 2A, there was a rapid reduction in ANP-stimulated cGMP levels in these cultures after as little as 2-h exposure to the ligand. NPR-A activity remained suppressed throughout the remainder of the experiment (48 h). This desensitization did not result from continued occupancy of the receptor by ligand. If ligand was removed from the cells with a mild acid wash (21) prior to challenge with ANP, then the response to the latter was still suppressed (Fig. 2B) to a level equivalent to or less than that seen in the conventional cultures.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   ANP inhibits NPR-A activity in IMCD cells. A: IMCD cells were pretreated with 100 nM ANP for varying periods of time, washed with PBS (3 times), and ANP-stimulated cGMP (ANP-s-cGMP) was measured as described in MATERIALS AND METHODS. Pooled data represent means ± SD (n = 6). Zero time point represents 263 ± 34 pmol cGMP/mg soluble protein. B: confluent cultures were pretreated with varying concentrations of ANP for 48 h. Cells were then washed 3 times with either acidic DMEM-Ham's F-12 (pH 5.0) () or PBS () and incubated in fresh medium containing 0.5 mM IBMX and 10 mM HEPES (pH 7.4) for 10 min. ANP-stimulated cGMP levels were determined as described in MATERIALS AND METHODS. Pooled data are presented as means ± SD (n = 6). * P < 0.05 and ** P < 0.01 vs. untreated group. Control cGMP levels in absence of ANP were 147 ± 45 pmol/mg soluble protein in the PBS-washed cultures and 213 ± 17 pmol/mg soluble protein in the acid-washed cultures.

The suppression of NPR-A activity was shared solely by those natriuretic peptides capable of interacting with these receptors in IMCD cells. As shown in Fig. 3A, ANP, BNP, and urodilatin, all ligands with high affinity for NPR-A, promoted a dose-dependent reduction in the NPR-A response to a subsequent challenge with ANP. CNP and cANF, ligands which selectively bind to NPR-B and NPR-C, respectively, were ineffective. The activities of the individual ligands were related to their ability to activate cGMP production in these cells, presumably through occupancy of the NPR-A receptor. As shown in Fig. 3B, ANP and BNP pretreatment was accompanied by a significant elevation in basal cGMP levels. This was not seen with CNP nor with the clearance receptor ligand.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of individual natriuretic peptides on NPR-A activity. Confluent IMCD cells were treated with varying concentrations of the natriuretic peptide indicated for 48 h. A: cells were washed, and ANP-stimulated cGMP (ANP-s-cGMP) was measured as described in MATERIALS AND METHODS. Control cGMP levels in absence of natriuretic peptide were 162 ± 25 pmol/mg soluble protein. B: in parallel cultures, steady-state cGMP levels were measured immediately following the 48-h incubation. Control cGMP levels in absence of natriuretic peptide were 3.5 ± 0.6 pmol/mg soluble protein. Pooled data are presented as means ± SD. * P < 0.05 and ** P < 0.01 vs. untreated group (n = 6).

The rapid kinetics of ANP-dependent desensitization implied that at least the early reduction of NPR-A activity was independent of changes in NPR-A gene expression. This is supported by the fact that pretreatment of the cultures with 5 µg/ml actinomycin D for 1 h, a condition which reduced [³H]uridine incorporation in these cells to very low levels (data not shown), failed to block the response to ANP (Fig. 4). Thus suppression of NPR-A gene transcription did not prevent the ligand-dependent reduction of NPR-A activity. This implies that the ligand acts, at least early on, through a posttranscriptional mechanism in reducing NPR-A activity.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Actinomycin D (Act D) fails to prevent early ligand-dependent downregulation of NPR-A activity. IMCD cells were preincubated in presence or absence of 5 µg/ml actinomycin D for 1 h. ANP (100 nM) was then added, and incubation was continued for another 2 h. ANP-stimulated cGMP production was measured as described in MATERIALS AND METHODS. Pooled data are presented as the means ± SD (n = 14). Experimental data have been normalized to that obtained in untreated control cells. Control cGMP levels in absence of natriuretic peptide were 121 ± 16 pmol/mg soluble protein. * P < 0.05 and ** P < 0.01 vs. untreated control (Ctrl) cultures.

At later time points, however, definite effects on NPR-A gene expression were observed. An ANP-dependent reduction in NPR-A mRNA levels was seen after 48 h, but not after 2 h of treatment with the ligand (Fig. 5, A and B). Of interest, this reduction could also be effected by treatment of the cultures with 8-bromo-cGMP with similar kinetics of response, suggesting that the second messenger rather than ANP itself provides the ultimate signal for decreased NPR-A gene expression. The reduction in NPR-A mRNA levels was dose-dependent with maximal inhibition (~50%) seen at 100 nM ANP (Fig. 6, A and B).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Time course of ANP-dependent reduction of NPR-A mRNA levels. A: IMCD cells were pretreated with 100 nM ANP or 100 µM 8-bromo-cGMP (8-br-cGMP) for 2 or 48 h, and total cellular RNA was collected. RNA, 30 µg, from each group was analyzed for NPR-A transcripts by Northern blot hybridization followed by autoradiography. B: NPR-A transcript levels were quantitated by densitometric scanning, and normalized values are plotted as a bar graph. Data are pooled from 3 independent experiments. * P < 0.01 vs. untreated control group.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Dose-dependent suppression of NPR-A gene expression by ANP. A: cells were treated with increasing concentrations of ANP for 48 h, and total cellular RNA was collected. RNA, 30 µg, from each group was analyzed for NPR-A and GAPDH transcript levels by Northern blot hybridization followed by autoradiography. B: NPR-A transcript levels were quantitated by densitometric scanning, and normalized values were plotted as a bar graph. Data are pooled from 3 independent experiments. ** P < 0.01 vs. control.

To explore the locus of this reduction in steady-state NPR-A mRNA levels, we transfected the IMCD cells with a reporter construct linking 1,575 bp of 5'-flanking sequence from the rat NPR-A gene to bacterial luciferase (5), treated the cells with the agents indicated, and assayed reporter activity 72 h later. As shown in Fig. 7, pretreatment with either ANP or 8-bromo-cGMP resulted in a dose-dependent reduction in NPR-A promoter activity. This suggests that the reduction in NPR-A mRNA levels results, at least in part, from a decrease in NPR-A gene promoter activity and that the cis-acting element(s) which sense the ANP-dependent signal are contained within the 1,575 bp of 5'-flanking sequence present in this construct.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   ANP and 8-bromo-cGMP suppress the activity of the NPR-A gene promoter. Cells were transfected with 2 µg 1575 NPR-A LUC as described in MATERIALS AND METHODS. Cells were then exposed to different concentrations of ANP or 8-bromo-cGMP for 48 h. Cellular extracts were prepared and assayed for luciferase activity using a commercial kit. Pooled data from 3 independent experiments are presented as the means ± SD. * P < 0.05, ** P < 0.01 vs. untreated group.

	DISCUSSION

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We have shown previously that ligand-dependent downregulation of NPR-A is accompanied by a reduction in steady-state NPR-A mRNA levels and NPR-A gene promoter activity in cultured RASM cells (5). The findings presented above indicate that similar regulatory mechanisms are operative in IMCD cells of the rat kidney. Although RASM cells harbor all three NPR subtypes (i.e., NPR-A, -B, and -C), cultured IMCD cells appear to contain almost exclusively NPR-A (Fig. 1). As a result, treatment of RASM cells with either ANP or CNP, either of which is capable of increasing cGMP levels in these cells, results in a reduction of NPR-A activity, whereas only ANP is effective in IMCD cells. By inference, the cellular controls governing ligand-dependent downregulation of NPR-A activity in IMCD cells are more selective than those in RASM. This could have important physiological implications in that NPR-A activity in IMCD cells would be predicted to be largely independent of locally produced CNP from renal parenchyma (28) or vascular endothelial cells (26). Several recent reports have documented low level expression of the ANP gene in the distal convoluted tubule of the kidney (24). Local production of ANP, or its amino-terminal-extended homolog urodilatin (13, 29), would have the capacity not only to activate NPR-A but to control its expression as well. This would, in effect, create a local system for governing sodium excretion in distal tubular segments, operating in parallel with, but separate from, the systemic regulatory mechanisms that control sodium homeostasis.

A number of studies have shown that ANP regulates NPR-A activity at a posttranscriptional level (22, 23). Desensitization of NPR-A in a stably transfected COS cell system appears to result from selective dephosphorylation of the receptor protein. Other studies, however, have suggested that desensitization of the endogenous NPR-A is accompanied by an increase in receptor phosphorylation (11), so the precise mechanism underlying this reduction in receptor activity remains controversial. Based on the rapid kinetics of the ANP response and the failure of actinomycin D to eliminate the difference in NPR-A activity in control vs. ANP-treated cells, it is likely that a similar nontranscriptional desensitization of receptor activity is operative in the IMCD cell, at least early following administration of ligand. In addition, there appears to be a late downregulation of receptor activity that relies on a reduction in NPR-A mRNA levels and, hence, decreased template for de novo receptor synthesis. As in RASM cells, this latter effect is directly tied to a reduction in NPR-A promoter activity, implying a transcriptional locus for the inhibitory effect. This combination of transcriptional and posttranscriptional regulatory loci provides the IMCD cell with greater flexibility in both initiating and maintaining the suppression of NPR-A activity.

Collectively, these data imply that ANP and/or urodilatin control their own biological activity in the distal nephron. Experimental dissection of this system should provide us with a better understanding of the regulatory controls governing sodium handling in this nephron segment and, perhaps, provide clues to the underlying pathogenesis of disorders where regulation of sodium handling in this segment is altered.