Vasopressin-dependent Inhibition of the C-type Natriuretic Peptide Receptor, NPR-B/GC-B, Requires Elevated Intracellular Calcium Concentrations*

Natriuretic peptides bind their cognate cell surface guanylyl cyclase receptors and elevate intracellular cGMP concentrations. In vascular smooth muscle cells, this results in the activation of the type I cGMP-depend-ent protein kinase and vasorelaxation. In contrast, pressor hormones like arginine-vasopressin, angiotensin II, and endothelin bind serpentine receptors that interact with G q and activate phospholipase C (cid:1) . The products of this enzyme, diacylglycerol and inositol trisphosphate, activate the conventional and novel forms of protein kinase C (PKC) and elevate intracellular calcium concentrations, respectively. The latter response results in vasoconstriction, which opposes the actions of natriuretic peptides. Previous reports have shown that pressor hormones inhibit natriuretic peptide receptors NPR-A or NPR-B in a variety of different cell types. the mechanism for this inhibition it that PKC component of because of PKC mimic inhibitory we in A10 smooth muscle cells, chronic PKC down-regulation nor specific PKC the AVP-dependent desensitization of both processes In contrast, the that

The natriuretic peptide family consists of atrial natriuretic peptide (ANP), 1 B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (1,2). ANP and BNP are stored primarily in the cardiac atria and ventricles, respectively, and are released into the circulation upon an increase in cardiac wall stretch that usually results from increased blood pressure. CNP is found in reasonably high quantities in cytokine-treated vascular endothelial cells (3), porcine seminal plasma (4), the brain (5), and bone tissue (6 -9). Unlike ANP, CNP is not stored in granules. Instead, it is regulated at the level of transcription by various signaling molecules, such as transforming growth factor-␤ and tumor necrosis factor-␣ (3, 10) as well as shear stress (11,12).
⌻he physiological responses elicited by natriuretic peptides are similar but not identical. In general, ANP and BNP counterbalance the renin-angiotensin-aldosterone system (1,2). Acutely, they decrease blood pressure by increasing renal sodium and water excretion, stimulating vascular vasorelaxation, and inhibiting aldosterone and renin secretion. Similarly, CNP has been implicated in a "vascular natriuretic peptide system," but in this scenario it signals in an autocrine/ paracrine manner (3). CNP binding to NPR-B relaxes phenylephrine-contracted rat aortic rings and, unlike ANP, is equally effective at relaxing veins and arteries (13). Furthermore, CNP inhibits the proliferation of vascular smooth muscle cells (14) and has been shown by many groups to inhibit balloon angioplasty-induced coronary artery restenosis (15)(16)(17)(18).
CNP also regulates the growth of long bones (19). In mice, transgenic overexpression of BNP results in skeletal overgrowth (8), and CNP, but not ANP, increases the height of the proliferative and hypertrophic chondrocyte zones in cultured tibia preparations (9). Consistent with these findings, mice lacking NPR-C display increased natriuretic peptide half-lives and skeletal overgrowth (20), whereas mice lacking either CNP (6) or type II cGMP-dependent protein kinase (21) exhibit dwarfism.
The signaling receptors for natriuretic peptides are cell surface guanylyl cyclases, which catalyze the synthesis of the intracellular messenger cGMP (22)(23)(24). Natriuretic peptide receptor A (NPR-A) is activated by both ANP and BNP, whereas the B-type natriuretic peptide receptor (NPR-B) is activated by CNP. Both NPR-A and -B are constitutively phosphorylated when expressed in tissue culture cells (25)(26)(27)(28), and receptor phosphorylation is absolutely essential for hormonal activation (29,30). The dephosphorylation of NPR-A and NPR-B in response to hormone binding has been shown to correlate with the declining activity of these receptors in whole cells (25,27,28), suggesting that receptor dephosphorylation mediates the homologous desensitization of these receptors. Consistent with this idea, a mutant version of NPR-A that cannot be dephosphorylated is resistant to ANP-dependent desensitization in whole cells and in membrane preparations (31,32).
The pressor hormones arginine vasopressin, angiotensin II, and endothelin, which stimulate phospholipase C-␤, oppose the actions of natriuretic peptides (33). Therefore, from a teleological point of view, it is reasonable that all three pressor peptides decrease natriuretic peptide-dependent cGMP elevations in cultured cell lines (34 -39). Three primary observations have implicated PKC in this inhibitory response. First, all hormones that inhibit natriuretic peptide signaling activate PKC via phospholipase C. Second, direct pharmacologic activation of PKC with phorbol esters mimics the desensitizing effect of the hormones on natriuretic peptide receptors (36, 37, 39 -47). Third, in a few instances relatively specific inhibitors of PKC, like H7, block all or part of the hormone-dependent desensitization (35,36,42). Hence, for more than 15 years it has been generally assumed that PKC is obligatory component in the heterologous desensitization of natriuretic peptide signaling. In this report, we provide evidence for a calcium-dependent desensitization pathway that appears to be distinct from the previously characterized PKC-dependent desensitization pathway.
Cell Culture and Preparation of Crude Membranes-A10 rat aortic smooth muscle cells (CRL-1476) were purchased from American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Mediatech, Inc.) in an atmosphere of 95% air and 5% CO 2 at 37°C. 15-cm plates of ϳ95% confluent cells were washed and incubated for at least 4 h with serumreplete DMEM. To prepare crude membranes, cells were washed twice with phosphate-buffered saline, scraped into 0.5 ml of phosphatase inhibitor buffer (25 mM HEPES, 20% glycerol, 50 mM NaCl, 50 mM NaF, 2 mM EDTA, 1 M microcystin, 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 g/ml pepstatin), sonicated for 1-2 s with a Misonix Sonicator XL2020, and centrifuged at 20,000 ϫ g for 20 min at 2°C. Pellets were resuspended in phosphatase inhibitor buffer at a protein concentration of 1-2 mg/ml. Protein concentrations were estimated using the Coomassie Plus Protein Assay Kit (Pierce).
Whole Cell Stimulations-Cells plated in 12-well dishes were grown to 90% confluency and incubated at least 4 h in serum-free media. The dishes were then placed on a slide warmer maintained at 37°C for 1 h. The medium was aspirated and replaced with 0.5 ml of DMEM containing 20 mM HEPES to stabilize the pH at room atmosphere, 0.5 mM 1-methyl-3-isobutylxanthine to inhibit phosphodiesterase activity, and various hormones or drugs. After a 30-min incubation, the medium was aspirated and replaced with the same medium containing CNP. The cells were then stimulated for 3 min and stopped by aspirating the medium and adding 1 ml of ice-cold 80% ethanol. The ethanol extract was transferred to 1.5-ml tubes and centrifuged for 10 min at 20,000 ϫ g to remove any particulate matter. The supernatant was transferred to borosilicate 12 ϫ 75-mm tubes, and the ethanol was evaporated to dryness in a Speedvac apparatus. The amount of cGMP contained in each sample was estimated by radioimmunoassay according to the manufacturer's instructions (PerkinElmer Life Sciences).
Guanylyl Cyclase Assays-Guanylyl cyclase assays were performed in the presence of 25 mM HEPES, 50 mM NaCl, 0.1% bovine serum albumin, 0.5 M 1-methyl-3-isobutylxanthine, 1 mM EDTA, 5 mM creatine phosphate, and 0.1 mg/ml creatine kinase (as a nucleotide regen-erating system), 1 M microcystin, 0.1-0.2 Ci of [␣-32 P]GTP, and 0.1 or 1 mM GTP. Activator mixtures consisted of 1 mM ATP, 1 M CNP, and 5 mM MgCl 2 or 1% Triton X-100 and 5 mM MnCl 2 . Between 25 and 50 g of crude membranes were assayed for 3 min at 37°C by the addition of a mixture containing the above reagents to a total volume of 100 l. The reactions were initiated by the addition of a mixture containing the substrate and terminated with 500 l of 110 mM zinc acetate. To purify the cGMP, 0.5 ml of sodium carbonate was added to the mixture, and the sample was vortexed and centrifuged at 3000 ϫ g for 10 min at 2°C. The supernatant was added to chromatography columns (Bio-Rad model 731-1550) containing ϳ0.5 g of dry neutral alumina resin (Sigma, A9003) acidified with 5 ml of 1 N perchloric acid. The columns were then washed with 10 ml of 1 N perchloric acid followed by 10 ml of water. The purified [␣-32 P]GTP was then eluted with 5 ml of freshly prepared 200 mM ammonium formate and quantitated using the Cerenkov method in a Beckman 3801 scintillation counter.
Immunoblot Analysis-NPR-B present in crude membranes was fractionated on an 8% SDS-polyacrylamide gel and blotted to polyvinylidene difluoride (Immobilon P) membrane using a BioRad Trans-Blot semidry transfer cell. The membrane was then incubated for 1 h in TBST (20 mM tris(hydroxymethyl)aminomethane, 500 mM NaCl, and 0.05% polyoxyethylene sorbitan monolaurate, pH 7.5) containing 3% bovine serum albumin followed by two 5-min washes with TBST. The primary antiserum was diluted 1:2,500 in TBST and incubated with the membrane for 2 h followed by four washes for 5 min with TBST. The specific antisera were raised against synthetic peptides corresponding to the last 17 or 10 carboxyl-terminal amino acids of NPR-A (antiserum 6326) or NPR-B (antiserum 6328), respectively, which were conjugated to keyhole limpet hemocyanin. These antisera are specific for each receptor and do not cross-react (see Fig. 1, bottom panel). The membrane was then incubated with donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) diluted 1:10,000 in TBST for 45 min. After four washes for 5 min with TBST, the NPR-B antibody complex was visualized by chemiluminescence using the ECL Western blot detection system (Amersham Biosciences).
Calcium Imaging-A10 cells were plated on 15-mm glass coverslips and grown until they formed a monolayer. The cells were washed with Hanks' balanced salt solution (HBSS) and incubated at 37°C for 30 min with 5 M fura-2-acetoxymethyl ester (a ratiometric fluorescent Ca 2ϩ indicator). The coverslip was then washed with HBSS and placed on a 150-l open slide chamber (RC-25F, Warner Instruments) mounted on the stage of a Nikon Diaphot inverted microscope. The chamber was perfused at ϳ2.5 ml/min at room temperature with HBSS. In experiments using ionomycin or CNP, the reagents were added directly to the chamber without constant perfusion. fura-2-loaded cells were alternately excited at 340 and 380 nM with a digitally controlled filter wheel (DG-4, Sutter Instrument Co.). The fluorescence emissions at 510 nM were collected with a cooled CCD 12-bit digital camera (Princeton Scientific Instruments). The digital camera output was then analyzed by a digital computer (Universal Imaging). Fluorescent signals were determined from regions of interest and the images corrected for system background, shading errors, and the very low autofluorescence of the unloaded cells. In each experiment, fura-2-loaded cells were exposed to 1 M 4-bromo-A23187 and 10 mM EGTA to obtain maximum and minimum F340/F380 ratios, respectively. Calcium concentrations were calculated using an in vitro calibration method as previously described (48).
Data Analysis and Statistics-The data were graphed and IC 50 values estimated with GraphPad Prism for the MacIntosh. In Fig. 2, the dose-response curve was fit using the equation: Y ϭ Bottom ϩ (Top Ϫ Bottom)/(1 ϩ 10 (logEC50ϪX) ). The "Top" is the best-fit highest value, which the program determined to be 4.990. The "Bottom" is the best-fit lowest value, given by 0.5670. With these two values, the logEC 50 was estimated at Ϫ10.5.

RESULTS
One of the primary goals of this study was to examine the heterologous regulation of a natriuretic peptide receptor in a physiological setting. Therefore, we chose rat vascular smooth A10 cells because vascular smooth muscle is a known target for natriuretic peptides (13,49). However, because of discrepancies in the literature regarding whether A10 cells express NPR-A (34,41,50) or NPR-B (51-53), we first determined the expression profile of natriuretic peptide receptors in these cells. Initially, we examined the sensitivity of whole A10 cells to CNP or ANP by measuring cGMP elevations in response to increasing concentrations of each peptide. Cells treated with CNP responded with dose-dependent increases in intracellular cGMP ( Fig. 1, top panel, squares). Statistically significant elevations in cGMP concentrations were first detected at 1 nM CNP, and the maximum dose (1 M), resulted in a 230-fold increase in cGMP concentrations above basal levels. In contrast, 100 nM concentrations of ANP were required to detect an increase in cGMP levels, and 1 M ANP only stimulated cGMP concentrations 34-fold above basal levels ( Fig. 1, top panel, circles). The ANP-dependent dose response in the A10 cells is similar to that observed in cell lines only expressing NPR-B (54, 55). Therefore, it most likely results from ANP cross-activation of NPR-B. In complete agreement with these data, Western blots on membranes prepared from A10 cells or 293 cells stably expressing NPR-A (293-NPR-A) or NPR-B (293-NPR-B) indicated that A10 cells express NPR-B but no detectable NPR-A (Fig. 1, bottom panel). Together, these data indicate that NPR-B is the primary and probably the only natriuretic peptide receptor expressed in A10 cells.
Arginine-vasopressin (AVP) has previously been shown to decrease ANP-dependent cGMP elevations in A10 cells (34,50). However, based on the observation that micromolar concentrations of ANP were not able to saturate the cGMP response in these cells (34,50) combined with the expression data shown in Fig. 1, it is likely that previous investigators were actually studying the regulation of NPR-B and not NPR-A as they suggested. This was a reasonable oversight because CNP and NPR-B had not yet been identified at the time their studies were conducted. Consistent with this hypothesis, we found that AVP reduced CNP-dependent cGMP elevations in these cells. Incubation of A10 cells with 1 M AVP for 30 min reduced cGMP concentrations at every CNP dose tested ( Fig. 2A). Cells stimulated with the highest concentration of CNP tested (5 M) produced 12.5 pmol cGMP/well, whereas the same CNP concentration resulted in only 3.0 pmol cGMP/well after treatment with AVP, which equals 24% of the control values. AVP exposure had no effect on basal cGMP concentrations, which suggests that the reduced cGMP concentrations are not mediated through increased cGMP degradation. The ability of AVP to decrease cGMP elevations was also dose-dependent (Fig. 2B). Diminished CNP-dependent cGMP elevations were first apparent at picomolar concentrations of AVP, and the maximum desensitization was reached around 10 nM AVP. The IC 50 for the response was estimated to be 0.03 nM AVP.
To distinguish between the possibilities that the reduced cGMP concentrations could have resulted from increased phosphodiesterase activity or from decreased guanylyl cyclase activity, we performed guanylyl cyclase assays. Crude membranes prepared from A10 cells treated in the presence (circles) or absence (squares) of 100 nM AVP for 30 min were assayed for CNP-dependent (1 M CNP, 5 mM MgCl 2 and 1 mM ATP) guanylyl cyclase activity for 5 and 10 min (Fig. 3). Consistent with the whole cell stimulation data, AVP substantially reduced NPR-B activity, decreasing CNP-dependent cGMP for-

FIG. 2. AVP decreases CNP-dependent cGMP elevations in whole A10 cells.
A, AVP decreases CNP-dependent cGMP elevations at all CNP concentrations tested. Confluent, serum-starved A10 cells were incubated in the presence (squares) or absence (circles) of 1 M AVP for 30 min. The medium was aspirated, and the cells were stimulated with increasing concentrations of CNP for 3 min. The reaction was then terminated by aspirating the medium and adding 1 ml of 80% ethanol. Cellular cyclic GMP levels were determined by radioimmunoassay. Values are the mean of cells from two separate wells (Ϯ range) from one representative experiment. Where error bars are not visible, the error is contained within the data point. This experiment was repeated at least three times with similar results. B, AVP inhibits CNP-dependent cGMP elevations in a dose-dependent manner. Confluent A10 cells were serum-starved and treated with increasing concentrations of AVP for 30 min. The medium was then removed, and the cells were stimulated with 20 nM CNP for 3 min. The reaction was then terminated, and cellular cyclic GMP levels were determined by radioimmunoassay. The error bars indicate the S.E. of four separate well determinations. Where error bars are not visible, they are contained within the data point. This experiment is representative of at least three experiments. mation to ϳ35% of that observed in membranes isolated from cells not exposed to AVP. These results indicate that the decreased cGMP concentrations observed in whole cells were primarily due to reductions in guanylyl cyclase activity and not to increased degradation of cGMP by cyclic nucleotide phosphodiesterases.
To characterize the time course of AVP-dependent inhibition of NPR-B activity, A10 cells were serum-starved for 4 h and then treated with AVP for 0, 2, 5, 10, 20, or 40 min. Crude membranes were prepared from these cells and assayed for guanylyl cyclase activity in the presence of CNP (1 M CNP, 5 mM MgCl 2 and 1 mM ATP; squares) or detergent (10% Triton X-100, 5 mM MnCl 2 ; circles) (Fig. 4). The latter treatment maximally activates NPR-B independently of CNP and is an excellent indicator of the total amount of NPR-B present in any given membrane preparation. The effect of AVP was rapid, with a t1 ⁄2 of ϳ3 min, and the majority of the desensitization was achieved by 10 min. Importantly, since the detergent-dependent activity was unaffected by AVP, this indicates that the reduced CNP-dependent activity was not due to increased phosphodiesterase activity or degradation of the NPR-B catalytic domain.
Once we had clearly shown that cellular AVP treatment inhibits NPR-B activity, we assessed the requirement for PKC in this process. First, A10 cells were incubated for 1 h in the presence of vehicle (Me 2 SO) or 1 M GF-109203X, a cell-permeable PKC inhibitor that acts as a noncompetitive ATP inhibitor of the ␣1, ␤1, ␤2, ␦, ⑀, and ␥ PKC isoforms (56). The cells were then treated in the presence or absence of 1 M concentrations of the synthetic PKC activator phorbol 12-myristate 13-acetate (PMA) or 1 M AVP for 30 min. Crude membranes were prepared from these cells and assayed for NPR-B guanylyl cyclase activity in the presence of its physiologic activators. As shown in Fig. 5A, PMA (open triangles) or AVP (open circles) exposure potently desensitized NPR-B, resulting in membranes that contained only ϳ25% of the cyclase activity measured in membranes from cells treated with medium alone (Control, open squares). Consistent with the target of PMA being a member of the PKC family, cellular pretreatment with 1 M GF-109203X abolished the PMA-induced inhibition of NPR-B (filled triangles). The effect of GF-109203X is specific for PMAdependent responses because it had no effect on membranes isolated from cells treated in the absence of PMA (filled squares). In surprising contrast to PMA, GF-109203X was completely ineffective in blocking the ability of AVP to inhibit NPR-B (filled circles, dotted line). In fact, the graphical results corresponding to the 1 M AVP and 1 M AVP ϩ GF-109203X responses are superimposable.

FIG. 4. The AVP-dependent desensitization of NPR-B is rapid.
Confluent, serum-starved A10 cells were incubated with 100 nM AVP for the indicated period of time, and then crude membranes were prepared from the treated cells and assayed for guanylyl cyclase activity in the presence of 1 mM ATP, 1 M CNP, and 5 mM MgCl 2 (squares) or 1% Triton X-100 and 5 mM MnCl 2 (circles) for 3 min. Control values for CNP-dependent and detergent-dependent activities are 0.4 and 1.5 nmol cGMP/mg/3 min, respectively. Values are the mean of assays determined on membranes prepared from two separate tissue culture plates, which were assayed in duplicate (Ϯ range) from one representative experiment. Where error bars are not visible, they are contained within the data point. The data represent one of at least three experiments with similar results. To further investigate the potential role of PKC in the AVPdependent desensitization of NPR-B, we performed a second set of experiments in which A10 cells were incubated for 24 h in serum-deficient medium containing vehicle (Me 2 SO) or 1 M PMA. The latter treatment is widely used to down-regulate phorbol ester-sensitive PKC isoforms (57). Following the incubation, the cells were treated in the absence or presence of 100 nM PMA or 100 nM AVP for 30 min. Crude membranes were then isolated and assayed for CNP-dependent guanylyl cyclase activity. In membranes isolated from control cells exposed to AVP or PMA for 30 min, NPR-B activity was decreased to less than 50% of control values (black bars). As anticipated, chronic PMA exposure (24 h) completely abolished subsequent PMAinduced decreases in CNP-dependent cGMP production, confirming that PKC was down-regulated (Fig. 5B). In contrast, AVP-induced inhibition of NPR-B was preserved despite the inactivation of PKC. Together, these two experiments indicate that AVP-induced desensitization of NPR-B does not require GF-109203X-sensitive or phorbol ester down-regulatable protein kinase C isoforms.
Because PKC was not required for the AVP response, we investigated the role of the inositol trisphosphate/calcium arm of the phospholipase C pathway in this process. As a first step, we determined the ability of AVP to elevate intracellular calcium concentrations in our A10 cells. We studied the response of a population of A10 cells that had been grown on glass coverslips and loaded with 5 M fura-2-AM for 30 min. In this representative experiment, basal intracellular calcium concentrations in these cells ranged from 150 to 350 nM, and exposure to 1 M AVP resulted in elevated calcium concentrations that ranged between 1.5 M and 5 M (Fig. 6A), which is similar to previously reported AVP-dependent calcium elevations in A10 cells (58). To further explore the involvement of intracellular calcium elevations in NPR-B regulation, we validated that the common calcium ionophore ionomycin could elevate intracellular calcium concentrations and that the cell-permeable calcium chelator, BAPTA-AM, could inhibit free calcium elevations. Treatment of A10 cells with 1 M ionomycin mimicked the effect of AVP on calcium elevations, increasing the average net (maximum peak to average basal) intracellular Ca 2ϩ concentration to above 3000 nM (Fig. 6B). In contrast, preincubation with 50 M BAPTA-AM completely blocked the AVP-dependent intracellular calcium elevations. These experiments authenticated the use of these compounds in modulating intracellular calcium concentrations in A10 cells and suggested that they could be effective in assessing the requirement of calcium in AVP-dependent inhibition of NPR-B.
To determine whether increases in intracellular calcium were sufficient for NPR-B desensitization, A10 cells were treated with 1 M ionomycin for increasing periods of time. Crude membranes prepared from the treated cells were then assayed for guanylyl cyclase activity in the presence of CNP (Fig. 7, black bars) or Triton X-100 (gray bars). Membranes isolated from cells treated with ionomycin for 5 or 10 min contained 68 or 48%, respectively, of the CNP-dependent guanylyl cyclase activity measured in membranes from untreated cells (Fig. 7A). Similar to AVP-treated cells, ionomycin did not reduce the amount of NPR-B protein as evidenced by guanylyl cyclase measurements obtained in the presence of detergent. To verify that the ionomycin-induced inhibition of NPR-B was due to elevated intracellular calcium concentrations, cells were preincubated with 50 M BAPTA-AM, a cell-permeable calcium chelator (Fig. 7B). In the presence of APTA-AM, ionomycin was a completely ineffective desensitizing agent, which suggest that elevated intracellular calcium concentrations are required and sufficient for the heterologous desensitization of NPR-B.
Next, we used BAPTA-AM pretreatment to test the requirement for calcium elevations in AVP-induced inhibition of NPR-B. A10 cells were treated with or without 7.5 or 75 M BAPTA-AM for 30 min before exposing them to 100 nM AVP for an additional 30 min (Fig. 7C). Crude membranes isolated from the treated cells were then assayed for CNP (black bars) or detergent (gray bars) dependent NPR-B guanylyl cyclase activity as before. AVP treatment reduced hormone-dependent activity to less than 50% of the control activity. Pretreatment with 7.5 M BAPTA-AM had no effect on the control activity but slightly decreased AVP-dependent inhibition of NPR-B. Similarly, 75 M BAPTA-AM did not alter NPR-B activity in membranes isolated from cells not exposed to AVP. However, it completely blocked the AVP-dependent decreases in hormonedependent guanylyl cyclase activity without affecting the detergent-dependent activity. These data clearly indicate that intracellular calcium elevations are required for AVP-dependent desensitization of NPR-B in A10 cells.
The next series of experiments was designed to determine whether the calcium-dependent desensitization was being mediated through the same or a different pathway than the PMAdependent desensitization. In these studies, we treated cells individually or in combination with PMA, ionomycin, or AVP. We reasoned that if the effects of saturating concentrations of PMA and ionomycin were not greater than the effect of either agent alone, this would suggest that they are working through the same pathway. In contrast, if their combined effect was additive, then this would be most consistent with separate pathways. Membranes prepared from cells treated with 1 M PMA or ionomycin contained only 23 or 42% of the CNP- dependent guanylyl cyclase activity found in membranes isolated from untreated cells (Fig. 8). However, we observed maximum desensitization (13% of the control values) when cells were treated simultaneously with both agents, which suggests that intracellular calcium elevations and PKC activation are modulating different pathways. Interestingly, when cells were treated with AVP and PMA together, the desensitization was similar to that observed with AVP alone. We do not have a definitive explanation for these results, but because PMA inhibits AVP-dependent intracellular calcium elevations (data not shown), one possibility is that PMA is partially blocking the AVP-dependent activation of phospholipase C.
Finally, because AVP inhibited NPR-B in a calcium-dependent manner, we asked whether CNP inhibited AVP-dependent calcium concentrations in A10 cells. To this end, we plated A10 cells on glass coverslips, loaded them with fura-2-AM, and incubated the cells with 1 M CNP for 5 min before calcium imaging. CNP treatment had a minimal effect on basal calcium concentrations, decreasing levels from 345 nM Ϯ8 to 300 Ϯ 7 nM. However, when the CNP-treated cells were stimulated with 1 M AVP, their calcium elevations were markedly blunted compared with elevations observed in cells not exposed to CNP (Fig. 9). CNP decreased average AVP-stimulated calcium elevations from 2129 to 449 nM, a reduction of 79%. These data provide direct evidence for reciprocal antagonism between the CNP and AVP signaling pathways in vascular smooth muscle cells.

DISCUSSION
In this study we have shown that: 1) A10 cells express NPR-B and not NPR-A; 2) AVP decreases CNP-dependent but not basal cGMP levels in a time-and concentration-dependent manner; 3) the reduced cGMP concentrations are a result of decreased NPR-B guanylyl cyclase activity; 4) the NPR-B inhibition requires elevated intracellular calcium concentrations but not GF-109203X-sensitive forms of PKC or NPR-B degradation; and 5) CNP inhibits AVP-dependent intracellular calcium elevations. Together, these results reveal a dominant role for modulations of intracellular calcium in the reciprocal regulation of these two important vasoactive signaling pathways.
One surprising finding of this study was that of the two arms of the phospholipase C pathway, diacylglycerol/PKC or inositol trisphosphate/calcium, only the latter appears to be required for the AVP-dependent inhibition of NPR-B in A10 cells. This finding was unexpected because at least 15 published reports have suggested that PKC mediates the heterologous desensitization of natriuretic peptide receptors, whereas the role of calcium in this process has remained relatively unexplored (35-37, 40 -47, 59 -62). Nonetheless, because our experiments utilized two independent approaches, we believe that the notion of PKC being required for the heterologous desensitization of natriuretic peptide signaling has been severely weakened. On the other hand, we cannot rule out the possibility that a form of PKC that is not inhibited by GF-109203X or that is down-regulated by chronic PMA exposure participates in this process. Nonetheless, our results indicate for the first time that elevated intracellular calcium concentrations are sufficient to inhibit NPR-B activity, revealing an alternative pathway by which NPR-B can be regulated. Hence, with this report both products of the phospholipase C catalyzed reaction have now been shown to inhibit NPR-B. It is of interest that the effects of AVP on NPR-B activity are greater than that of ionomycin, even though ionomycin exposure results in greater calcium elevations. This suggests that maximum desensitization results from something in addition to increased calcium concentrations. One obvious possibility is that diacylglycerol-dependent activation of PKC is required. Using PKC inhibitors and PMA-dependent down-regulation of phorbol ester-sensitive PKC isozymes, we have been unable to document a measurable contribution of PKC to this process. Therefore, it is currently unclear whether PKC activation or another unknown signaling event is required for the maximum desensitization of NPR-B.
With respect to previous studies, our data are not consistent with a report showing that the protein kinase C inhibitor H7 could block angiotensin II-dependent reductions in ANPdependent guanylyl cyclase activity in primary glomerular mesangial cells (35). Similarly, Jaiswal used H7 to block the ability of endothelin to reduce ANP-dependent cGMP elevations in primary vascular smooth muscle cells (36). We do not know why our results differ from these previous studies, although one obvious reason is that NPR-A is regulated differently than NPR-B. This is clearly a possibility, but despite numerous attempts by our group as well as others, significant regulatory differences between these two receptors have not been identified. This is not completely unexpected given that the intracellular portions of these two receptors are 78% identical at the amino acid level. An alternative explanation may be related to the different cell systems employed in each study.
In terms of NPR-B regulation, the protein kinase C inhibitor Ro 31-8220 blocked only 63% of the ability of endothelin-3 to inhibit CNP-dependent cGMP elevations in C6 glioma cells, which is consistent with the existence of both PKC-dependent and independent inhibitory pathways (39). Unfortunately, the role of intracellular calcium elevation in the endothelindependent desensitization of NPR-B was not described in this report. In contrast, gonadotropin-releasing hormone was shown to inhibit CNP-dependent cGMP elevations in pituitary T3-1 cells in a manner that is mimicked by phorbol esters but not by the Ca 2ϩ ionophore A23187, which is completely opposite of our results (62).
Elevated intracellular calcium concentrations have been shown to decrease intracellular cGMP concentrations in several cell types (35,(63)(64)(65). Regardless of whether the calcium concentrations were elevated by hormones or ionophores, in most cases the decreased nucleotide concentrations resulted from increased phosphodiesterase activity, not reduced guanylyl cyclase activity. However, in mouse Leydig cells, Mukhopadhyay and colleagues (63) demonstrated that ionomycin decreased ANP-dependent, but not basal, cGMP concentrations in the presence of high concentrations of the general phosphodiesterase inhibitor isobutylmethyxanthine, suggesting that increased nucleotide degradation was not required for the diminished cGMP concentrations in these cells. On the other hand, these investigators were unable to detect any direct effect of calcium on ANP-dependent guanylyl cyclase activity in membranes from these cells, and the results of cyclase assays conducted on membranes isolated from cells treated in the presence or absence of ionomycin were not reported (63).
Although this study (63) suggests that PKC is not required for the desensitization of NPR-B in A10 cells, others and we have shown that PKC activation desensitizes NPR-B (39,42,44,59,62). Hence, it appears that both arms of the phospholipase pathway can lead to the inhibition of CNP-dependent guanylyl cyclase activity. The mechanism for this PKC-dependent loss of activity appears to involve the dephosphorylation of NPR-B at Ser-523, because the mutation of this residue to glutamate abrogates the effect (44). However, it is important to point out that this mechanism has not been shown to be required for any hormonal desensitization of NPR-B; it has been observed only upon pharmacologic activation of PKC by phorbol esters. In contrast, the mechanism for the calcium-dependent process is completely unexplored. We are currently investigating the role of NPR-B dephosphorylation in this process. However, the low concentration of NPR-B endogenously expressed in A10 cells combined with the low transfection efficiency of these cells has made this endeavor extremely difficult.
Finally, it is worth noting that the inhibitory effect of calcium on NPR-B is similar to the effect calcium has on the retinal guanylyl cyclases (RetGC-1 and RetGC-2/GC-E and GC-F) (66). These receptor cyclases have a predicted structural topology similar to NPR-A and NPR-B, but no specific extracellular activator of these receptors has been identified. It is known, however, that retinal cyclases are regulated by small intracellular calcium-binding molecules called guanylyl cyclase-activating proteins (GCAPs). Under low intracellular calcium conditions, GCAPs stimulate these receptors, whereas under elevated calcium concentrations the GCAPs inhibit them, presumably by causing a conformational change in the GCAP that is unfavorable to cyclase activation. Recently, visinlike protein-1 (VILIP-1), a member of the intracellular neuronal calcium sensor family that also includes GCAPs, was found to colocalize with NPR-B in cerebellar cell cultures. Unfortunately, the effect of altering intracellular calcium concentrations on this process was not investigated (67). It is tantalizing to speculate that VILIP-1 or perhaps a natriuretic peptide receptor-specific calcium-binding protein mediates the calciumdependent desensitization of NPR-B; however, this remains to be determined.
Acknowledgments-We are grateful to Dr. Mathur Kannan, Department of Veterinary Pathobiology, University of Minnesota, for generously helping with the calcium imaging experiments and to Alyssa Cody for conducting many of the whole cell stimulation and guanylyl cyclase assays.