(Received for publication, October 13, 1994; and in revised form, January 13, 1995)
From the
In bovine lung membranes, atrial natriuretic peptide (ANP)
showed temperature-dependent binding to guanylate cyclase-natriuretic
peptide receptor (NPR-GC). Photoaffinity labeling of the receptors with
4-azidobenzoyl (AZB)-I-ANP and competitive binding
studies with
I-ANP, ANP, and atriopeptin I (API) revealed
that NPR-GC was detected as the predominant ANP-binding protein at 0
°C, whereas at 37 °C natriuretic peptide clearance receptor
(NPR-C) was detected as the predominant protein. The ratio of NPR-GC
and NPR-C was 89:11 at 0 °C for 40 min, respectively, whereas 6:94
at 37 °C. AZB-
I-ANP bound to NPR-GC dissociated from
the binding site within 5 min at 37 °C but not at 0 °C, whereas
ANP bound to NPR-C did not dissociate from the binding site at 0 and 37
°C. The dissociated AZB-
I-ANP rapidly rebound to
NPR-GC at 37 °C but not to NPR-C, and the dissociated NPR-GC was
capable of binding. Some AZB-
I-ANP was hydrolyzed by a
membrane-bound proteinase(s). Phosphoramidon inhibited the hydrolysis
of AZB-
I-ANP. Thus, the dissociated
AZB-
I-ANP rebound to NPR-GC and NPR-C. These results
suggest that usually intact ANP repeatedly binds to NPR-GC until
hydrolysis. Furthermore, the majority of ANP bind to NPR-GC before
binding to NPR-C under physiological temperature.
The natriuretic peptide receptor family has been identified and
characterized from many
sources(1, 2, 3, 4, 5) .
Analysis of the primary structures deduced from the cloned cDNA have
identified three receptor proteins(6, 7, 8) .
At present, two subtypes of guanylate cyclase-natriuretic peptide
receptor (NPR-GC) ()termed NPR-A and NPR-B have been
described. These receptors are similar in their size and
structure(7, 8) . NPR-GC consists of a single
polypeptide with a molecular mass of approximately 130 kDa and contains
an extracellular ligand-binding domain, a single transmembrane domain,
and an intracellular signaling domain that is composed of a protein
kinase-like and a guanylate cyclase catalytic
domain(7, 8) . Although the ligand selectivity of
NPR-A is different from NPR-B(9, 10) , both NPR-A and
NPR-B have high affinity with intact natriuretic peptides but low
affinity with truncated natriuretic
peptides(2, 9, 11, 12) . Binding of
intact natriuretic peptides to NPR-GC stimulates guanylate cyclase to
produce cGMP(13, 14) . Therefore, it has been believed
that NPR-GC is a biological receptor for natriuretic peptides. Another
natriuretic peptide receptor is a non-guanylate cyclase-coupled
receptor termed clearance receptor or C-receptor
(NPR-C)(6, 15) . The extracellular binding domain of
NPR-C has approximately 30% homology to that of NPR-A and
NPR-B(7, 8) , but NPR-C has a much shorter
intracellular domain than NPR-A and
NPR-B(6, 7, 8) . In contrast to NPR-GC, NPR-C
has high affinity with intact natriuretic peptides and their truncated
analogs(2, 9, 11, 12) . These
observations imply that this receptor may not be coupled to any of the
known biological actions of ANP. Therefore, it has been proposed that
NPR-C functions as a clearance receptor to remove excess natriuretic
peptides and their truncated peptides from the
circulation(15) . The physiological and pharmacological
functions of NPR-C are still under investigation. However, recent
studies have demonstrated that NPR-C is negatively coupled to adenylate
cyclase via G
and decreases cAMP accumulation in several
tissues(16) .
Neutral endopeptidase 24.11 is also believed to reduce intact ANP levels by degradation(17) . However, it is puzzling that intact natriuretic peptides are hydrolyzed by cell surface endopeptidases in the target organs without binding to NPR-GC and stimulating guanylate cyclase. The present study was, therefore, designed to test the hypothesis that binding of natriuretic peptides to NPR-GC is a passing interaction, and natriuretic peptides repeatedly bind to NPR-GC before binding to NPR-C or degradation under physiological conditions.
Photo-reactive radioligand,
AZB-I-ANP-(4-28), can be covalently linked to the
binding proteins even at 0 °C using an ultraviolet lamp. However,
if it is cleaved in any peptide bond, the labeled proteins are not
detected under reducing conditions(18, 22) . When the
bovine lung membranes were incubated with
AZB-
I-ANP-(4-28) at 0 °C for 40 min, NPR-GC was
labeled as the predominant ANP-binding protein (Fig. 1). The
prolonged incubation at 0 °C for 80 and 160 min resulted in neither
increase nor decrease in both NPR-GC and NPR-C bands (Fig. 1).
In contrast, NPR-C was labeled as the predominate protein at 37 °C
for 40 min, and no significant increase in the NPR-GC and NPR-C bands
were observed over 80-160 min (Fig. 1). The quantitative
analysis revealed that the ratio of NPR-GC and NPR-C was 89:11 at 0
°C for 40 min, respectively, whereas at 37 °C, the ratio was
6:94. The incubation at 37 °C resulted in a marked decrease in the
bound radioactivity that was about one-fourth as compared with the
radioactivity at 0 °C. The results indicate that although the
incubation at 37 °C causes decrease in the bound radioactivity, the
interaction of ANP with its receptors in the bovine lung membranes is a
temperature-dependent event.
Figure 1:
Effect of temperature on ANP binding to
NPR-GC and NPR-C in bovine lung membranes. The lung membrane suspension
containing 0.1 mg in 0.4 ml of the assay buffer was incubated with
approximately 10M AZB-
I-ANP-(4-28) (300,000 cpm) in the presence
or absence of 5
10
M ANP-(4-28) (cold ANP) at 0 °C (ice water) or 37 °C
for varying periods of time (40, 80, and 160 min) in the dark. After
centrifugation, the pellet was suspended in 0.4 ml of the assay buffer
and subjected to photoaffinity labeling as described under
``Experimental Procedures.'' The membranes were collected by
centrifugation, and the membrane pellet was solubilized with the SDS
buffer under reducing conditions and boiled. The solubilized whole
membranes were analyzed by SDS-polyacrylamide gel electrophoresis using
a 7.5% gel and autoradiographed as described under ``Experimental
Procedures.''
Competitive binding studies with I-ANP-(4-28), unlabeled ANP-(4-28), and API
was used to determine the ANP receptors in the bovine lung membranes at
0 or 37 °C. Binding of
I-ANP-(4-28) to the
bovine lung membranes was rapid and reached an apparent equilibrium by
20 min at 0 °C. The binding studies showed that
I-ANP-(4-28) was competitively displaced by
increasing concentrations of unlabeled ANP-(4-28) (Fig. 2). In contrast to ANP-(4-28), API displaced the
radioligand from 21% of the total binding sites at 0 °C and 93% at
37 °C (Fig. 2). These results indicate that NPR-GC is
approximately 80% of the total binding sites in the bovine lung
membranes at 0 °C, whereas at 37 °C NPR-GC is less than 10% of
the total binding sites, and NPR-C is more than 90%.
AZB-
I-ANP-(4-28) was similar to
I-ANP-(4-28) in the affinity with NPR-GC and NPR-C
in the bovine lung membranes (data not shown). This suggests that AZB
is not likely to affect the affinity with the receptors. Therefore, the
competitive binding showed similar results to the autoradiography by
photoaffinity labeling.
Figure 2:
Competition for I-ANP-(4-28) to bovine lung membranes with
ANP-(4-28) or API. The lung membrane suspension containing 0.1 mg
in 0.4 ml of the assay buffer was incubated with 130 pM
I-ANP-(4-28) (30,000 cpm) and varying
concentrations of unlabeled ANP-(4-28) (
) or API (
)
at 0 °C (A) or 37 °C (B). Binding assay was
described under ``Experimental Procedures.'' Binding data
were corrected for nonspecific binding measured in the presence of
10
M ANP-(4-28). Data from two
experiments are represented as mean ± S.D. of duplicate
determination.
To determine whether the detection of NPR-GC
at 0 °C was due to a lower degradation rate of intact ANP, the
AZB-I-ANP-bound membranes were incubated with several
proteinase inhibitors such as chymostatin, bestatin, antipain,
pepstatin, leupeptin, EDTA, and phosphoramidon at 0 or 37 °C for 40
min. As shown in Fig. 3, NPR-GC was labeled as the predominant
ANP-binding protein at 0 °C (Fig. 3A), whereas at
37 °C NPR-C was labeled as the predominant protein even in the
presence of the proteinase inhibitors (Fig. 3B). The
results suggest that these proteinase inhibitors do not prevent the
temperature-dependent conversion of the ANP receptors. Interestingly,
the membranes, incubated at 37 °C in the presence of chymostatin,
EDTA, or phosphoramidon, showed the considerable binding to both NPR-GC
and NPR-C (Fig. 3B, lanes3, 8, and 9). Among these proteinase inhibitors,
phosphoramidon was the most effective agent to detect the NPR-GC and
NPR-C bands (Fig. 3B).
Figure 3:
Detection of NPR-GC and NPR-C in bovine
lung membranes at 0 or 37 °C in the presence of proteinase
inhibitors. The lung membrane suspension containing 0.1 mg in 0.4 ml of
assay buffer was incubated with AZB-I-ANP in the presence (lane1) or absence (lanes2-9) of 5
10
M ANP at 0 °C for 40 min in the dark. The membranes were
collected by centrifugation and suspended in 0.4 ml of the assay
buffer. The AZB-
I-ANP-bound membranes were again
incubated with 5
10
M ANP-(4-28), control (none), 10 µg/ml chymostatin, 20
µg/ml bestatin, 10 µg/ml antipain, 10 µg/ml pepstatin, 10
µg/ml leupeptin, 4 mM EDTA, and 20 µg/ml
phosphoramidon (in lanes1-9, respectively) at
0 °C for 40 min (A) or at 37 °C for 40 min (B). After photoaffinity labeling and centrifugation, the
whole membranes were solubilized with 0.15 ml of the SDS buffer and
analyzed as described under ``Experimental Procedures.'' Gels
were exposed for 2 (panelA) or 3 days (panelB).
Phosphoramidon may function
as a potent proteinase inhibitor to prevent intact ANP from
hydrolyzing. To elucidate the effects of phosphoramidon on the
detection of NPR-GC and NPR-C at 37 °C, the
AZB-I-ANP-bound membranes were analyzed using a
reverse-phase C
column after their incubation at 0 °C (Fig. 4A), 37 °C (Fig. 4B), or 37
°C with phosphoramidon (Fig. 4C). As shown in Fig. 4, the majority of AZB-
I-ANP-(4-28)
remained intact at 0 °C (Fig. 4A), whereas at 37
°C approximately 70% of the radioactive ANP was hydrolyzed (Fig. 4, A and B), and the rest of the
radioactivity was the intact AZB-
I-ANP-(4-28). At
37 °C in the presence of phosphoramidon, the hydrolysis of the
radioactive ANP was significantly less than that at 37 °C in the
absence of phosphoramidon, and approximately 80% of the radioactive ANP
remained intact (Fig. 4, B and C). These
results indicate that phosphoramidon prevents ANP from hydrolyzing as a
potent proteinase inhibitor for a membrane-bound proteinase(s) in
bovine lung membranes.
Figure 4:
Analysis of hydrolyzed peptides from
AZB-I-ANP-(4-28) by HPLC. The
AZB-
I-ANP-bound bovine lung membranes in 0.4 ml of the
assay buffer were incubated at 0 °C (A), 37 °C (B), or 37 °C with 20 µg/ml phosphoramidon (C). The incubated radioactive membranes were terminated by
adding 0.3 ml of acetic acid. After centrifugation using a 0.2-µm
centrifuged membrane filter, 0.1 ml of the filtered solution was
analyzed using HPLC as described under ``Experimental
Procedures.'' The separation was achieved in linear 15 to 60%
acetonitrile gradient over 5-35 min (dottedline). *ANP represents intact
AZB-
I-ANP-(4-28).
Although phosphoramidon inhibited the
hydrolysis of ANP at 37 °C, the ratio of NPR-GC and NPR-C in the
presence of phosphoramidon was 32:68, respectively (Fig. 3B, lane9). The finding may be
due to either a cleavage of the ANP-bound NPR-GC or a dissociation of
the radioactive ANP from the binding sites of NPR-GC. To examine in
detail, the AZB-I-ANP-bound membranes were incubated for
varying periods of time at 37 °C. As shown in Fig. 5, A and C, the NPR-GC band decreased with increasing time at
37 °C, whereas the NPR-C band did not increase at any time point.
The results suggest that ANP bound to NPR-GC may dissociate from the
binding site at 37 °C, since the decrease in the NPR-GC band was
not associated with a corresponding increase in the NPR-C band. To
confirm this, the
I-ANP covalently-linked membranes were
incubated at 37 °C for varying periods of time. The autoradiograph
showed neither rapid decrease in the NPR-GC band nor increase in the
NPR-C band (Fig. 5, B and D). These results
indicate that the 65-kDa band in the bovine lung membrane does not
arise from a proteolytic cleavage of the ANP-bound NPR-GC. Therefore,
the decrease of NPR-GC is caused by the dissociation of ANP from the
binding site of NPR-GC.
Figure 5:
Time-dependent dissociation of
AZB-I-ANP-(4-28) from NPR-GC in bovine lung
membranes. A, the AZB-
I-ANP-bound membranes in
0.4 ml of the assay buffer were incubated at 37 °C for varying
periods of time (0, 5, 10, 20, and 40 min in lanes2-6, respectively) and then covalently linked by
photolysis. B, AZB-
I-ANP-bound membranes were
covalently linked by photolysis before incubation at 37 °C. The
I-ANP covalently-linked membranes were centrifuged, and
the membrane pellet was suspended in 0.4 ml of the assay buffer. The
I-linked membranes were incubated at 37 °C for
varying periods of time as described above. After solubilization with
0.15 ml of the SDS buffer, the whole membranes were analyzed as
described under ``Experimental Procedures.'' Lane1 was performed with AZB-
I-ANP-(4-28)
and 5
10
M ANP-(4-28) to
detect nonspecific bands. The relative intensity of the NPR-GC and
NPR-C bands in the autoradiographs A and B was
analyzed using a densitometer (C and D,
respectively).
To elucidate this further, the
AZB-I-ANP-bound membranes were incubated at 37 °C in
the presence of phosphoramidon. As shown in Fig. 6A,
the NPR-GC band gradually decreased with increasing time, whereas the
NPR-C band concomitantly increased. The increase in the NPR-C band with
time is caused by the decrease in degradation of ANP and by its binding
to NPR-C. However, when the incubation was carried out in the presence
of phosphoramidon and excess unmodified ANP, the NPR-GC band rapidly
disappeared, and the NPR-C band did not show any significant change (Fig. 6, B and D, lanes3-6). These results clearly showed that the
radioactive ANP dissociated from NPR-GC within 5 min at 37 °C,
whereas it did not dissociate from NPR-C at 37 °C over 40 min,
since the dissociated radioactive ANP was competitively displaced by
excess ANP or hydrolyzed peptides. These and the results shown in Fig. 5A suggest that usually the dissociated intact ANP
rebind to NPR-GC but not to NPR-C. Therefore, the NPR-GC band detected
at 37 °C over 10-40 min (Fig. 5A and
6A) can be derived from the ANP-rebound NPR-GC, since the
majority of the bound ANP dissociate from NPR-GC within 5 min (Fig. 6B).
Figure 6:
Time-dependent binding of dissociated
AZB-I-ANP-(4-28) from NPR-GC to NPR-C in the
presence of phosphoramidon. The AZB-
I-ANP-bound bovine
lung membranes in 0.4 ml of the assay buffer were incubated with 20
µg/ml phosphoramidon for varying periods of time at 37 °C (0,
5, 10, 20, and 40 min in lanes2-6,
respectively) (A). The AZB-
I-ANP-bound bovine
lung membranes in 0.4 ml of the assay buffer were incubated with both
20 µg/ml phosphoramidon and 5
10
M ANP-(4-28) for varying periods of time at 37 °C as
described above (B). After photoaffinity labeling, the whole
membranes were boiled and analyzed as described under
``Experimental Procedures.'' Lane1 was
performed with 5
10
M ANP-(4-28) to detect nonspecific bands as described in Fig. 5. The relative intensity of the NPR-GC and NPR-C bands in
the autoradiographs A and B was analyzed using a
densitometer (C and D,
respectively).
In the above study, the dissociated ANP
rebound to NPR-GC; however, it was unknown that the dissociated NPR-GC
was able to bind intact ANP. To test this, the membranes saturated with
excess unmodified ANP-(4-28) were incubated at 37 °C for
varying periods of time and then challenged with photoaffinity labeling
with AZB--ANP-(4-28). As shown in Fig. 7,
the NPR-GC band reappeared with increasing time (lanes4-10), whereas the NPR-C band was not detectable at
any time point (lanes 4-10), since the dissociated
ANP-(4-28) is readily hydrolyzed by a membrane-bound
proteinase(s) at 37 °C. The quantitative analysis also revealed
that only the NPR-GC band reappeared with increasing time (Fig. 7B). The results suggest that the dissociated
NPR-GC is capable to binding. Therefore, the binding of ANP to NPR-GC
is reversible, whereas the binding of ANP to NPR-C may be irreversible.
However, it is still unknown if the dissociated ANP rapidly rebinds to
the dissociated NPR-GC.
Figure 7:
Detection of ANP binding to NPR-GC after
saturation with excess ANP. 100 µl of the bovine lung membranes was
incubated with 10M ANP-(4-28) at 0
°C for 40 min as described in Fig. 1. The membranes were
collected by centrifugation and suspended in 1.0 ml of the assay
buffer. The membranes were washed twice with 1.0 ml of the assay
buffer. Finally, the membranes were centrifuged and resuspended in 0.4
ml of the assay buffer. The ANP-saturated membranes were incubated at
37 °C for varying periods of time (0, 5, 10, 20, 40, 60, and 80 min
in lanes4-10, respectively). After incubation,
the membranes were collected by centrifugation and suspended in 0.4 ml
of the assay buffer. These membranes were again incubated with
AZB-
I-ANP-(4-28) at 0 °C for 40 min in the
dark. After photoaffinity labeling and centrifugation, the whole
membranes were solubilized with 0.15 ml of the SDS buffer, boiled, and
analyzed as described under ``Experimental Procedures.'' Lanes1 and 3 were performed in the presence
of 5
10
M ANP-(4-28) to
detect nonspecific bands as described in Fig. 5. Lanes1 and 2 were performed as the same manner as lanes1 and 2 in Fig. 5,
respectively, but 25 µl of the volume were loaded onto gel. The
intensity of the NPR-GC and NPR-C bands in the autoradiograph was
analyzed using a densitometer, and the relative intensity of each band
was calculated as the percentage of the total intensity of the NPR-GC
and NPR-C bands in lane2.
There are inconsistent reports regarding the population of
natriuretic peptide receptors in cells and tissues. Generally, NPR-C is
the predominant ANP-binding protein in tissues and cultured
cells(23) . Although NPR-C was only identified by affinity
labeling at 22 °C and purified from bovine lung
membranes(3) , NPR-GC was also purified from the same source by
GTP affinity chromatography(1) . In a recent report, both
NPR-GC and NPR-C have been identified in bovine lung membranes by
photoaffinity labeling at 4 °C(20) . Moreover, the
competitive binding study has been suggested that two classes of
ANP-binding proteins are present in lung
membranes(10, 24) . These observations suggest that
NPR-GC and NPR-C are present in bovine lung membranes, but binding of
natriuretic peptides to their receptors could be a
temperature-dependent event. We investigated this proposition to
elucidate the binding mechanism of ANP to NPR-GC and NPR-C. We found
that when the photoaffinity labeling was carried out at 0 °C with
AZB-I-ANP-(4-28), NPR-GC was labeled as the
predominant ANP-binding protein in the bovine lung membranes, whereas
at 37 °C NPR-C was labeled as the predominant protein.
The
temperature-dependent detection of the ANP receptors is caused by the
dissociation of ANP from NPR-GC but not from NPR-C at a higher
temperature and by some hydrolysis of ANP. Therefore, the ratio of
NPR-GC and NPR-C depends upon labeling conditions. The competitive
binding study also shows that NPR-GC is determined as the predominant
ANP-binding protein at 0 °C, whereas at 37 °C NPR-C is
determined as the predominant protein. In a previous
paper(24) , NPR-C might be 73% of the total binding sites in
rabbit lung membranes, whereas NPR-GC might be 27% when the incubation
was carried out at 25 °C for 30 min. However, the K value for ANP obtained by the saturation binding studies of the
ANP receptors cannot be calculated at 37 °C in lung membranes,
since the bound ANP repeatedly dissociates and rebinds at a higher
temperature. Furthermore, ANP does not readily bind to NPR-C in bovine
lung membranes at 0 °C. This suggests that the binding sites of ANP
receptors in the tissue membranes vary according to the temperature.
Further studies are necessary to elucidate the mechanism.
The receptor selectivity for natriuretic peptides has been described (9, 10) . These observations demonstrate that NPR-GC in bovine lung membranes should be NPR-A. NPR-A has a high affinity with intact ANP but not to truncated analogs(2, 11, 12) . The observations are consistent with the results that ANP bound to NPR-A dissociates from the binding site, and the dissociated ANP rebinds to NPR-A. If the truncated natriuretic peptides bind to NPR-A as intact peptides and stimulate guanylate cyclase, the hormonal effects are prolonged by their rebinding to NPR-A. Therefore, the low affinity of NPR-A with the truncated analogs may be essential. The rapid dissociation of ANP from NPR-A at 37 °C has been described in cultured cells (25, 26) and may play a role in recycling of the receptors(26) . However, the ligand-mediated internalization of NPR-A is still under investigation, since there are inconsistent observations in cultured cells(25, 27) .
We showed that the rapid dissociation of ANP from NPR-A occurs at 37 °C and pH 7.4 in membrane preparations (Fig. 6, B and D). The dissociation is not caused by the ligand-receptor internalization. Therefore, usually the ligand-mediated internalization of NPR-A may not be essential to reuse of NPR-A. These findings are consistent with the results that usually NPR-A may not mediate ligand-receptor internalization(25) . It has been suggested that some cell surface receptors contain recognized sequences of amino acids for internalization in intracellular domain(28, 29) . However, NPR-A does not contain such recognized amino acid sequences and does not contain tyrosine or phenylalanine near the transmembrane domain(7, 30) . These observations suggest that NPR-A may not mediate ligand-receptor internalization. Therefore, the internalization of the ANP and NPR-A complexes in Leydig cells may be due to tumor cells(18, 26) . On the other hand, ANP does not dissociate from the binding site of NPR-C at 37 °C. This indicates that the ANP and NPR-C complexes may lead to their internalization. Indeed, it has been shown that the ligand and NPR-C complexes mediate internalization in cultured cells(25, 27, 31) .
The hydrolysis of ANP has been believed to regulate ANP concentrations. It has been suggested that the hydrolysis of ANP is attributable to neutral endopeptidase 24.11, and phosphoramidon is a potent proteinase inhibitor for the endopeptidase(17) . Indeed, in vivo studies demonstrated that proteinase inhibitor such as phosphoramidon elevated plasma ANP concentration and urine cGMP excretion(35, 36) . Besides, an amino peptidase and a kallikrein-like proteinase also hydrolyze intact ANP(32, 33) . Although the identity of the membrane-bound proteinase(s) in the lung membranes responsible is not clear, it may be the neutral endopeptidase 24.11, since our findings are similar to the previous studies (17, 34) that ANP was hydrolyzed by the neutral endopeptidase 24.11 in porcine membrane preparations and was inhibited 84% by 1 µM phosphoramidon. However, it may suggest that approximately 20% of intact ANP is hydrolyzed by a non-phosphoramidon-sensitive proteinase(s), and prolonged incubation with phosphoramidon causes more hydrolysis of ANP(34) . In the presence of phosphoramidon, the dissociated intact ANP gradually rebinds to NPR-C at 37 °C. This further exemplifies the finding that NPR-C may function as a clearance receptor to remove excess natriuretic peptides (15) .
Previous studies have shown that the cleavage of NPR-A was observed in bovine adrenal cortex plasma membranes(37, 38) . The cleavage of NPR-A might be attributed to a acid-metalloendopeptidase(s), which is associated with the plasma membranes. One of the cleaved fragments has an ANP binding activity with the molecular mass of 65 kDa and is similar to NPR-C in size and properties(37, 38) . Although it is not known if this 65-kDa ANP-binding protein is identical to NPR-C, it is tempting to speculate that NPR-A bound to ANP is internalized together with the membrane-bound metalloendopeptidase into endosomes and is cleaved by this endopeptidase upon exposure to acidic environment of the endosomal lumen. The internalization and proteolytic processes may play a role in ligand-induced down-regulation of NPR-A or desensitization of ANP-stimulated guanylate cyclase. However, our results suggest that the 65-kDa ANP-binding protein in bovine lung membranes does not arise by a proteolytic cleavage of NPR-A (Fig. 5B) since it originally exists as NPR-C. Therefore, further studies are necessary to elucidate the 65-kDa ANP-binding protein in bovine adrenal cortex plasma membranes.
It is also possible that the desensitization of ANP-stimulated guanylate cyclase is caused by phosphorylation (39, 40) in the same way as some cell surface receptors, which appear to be desensitized by phosphorylation(41) . Moreover, the agents that stimulate protein kinase C or phospholipase C cause a decrease in ANP-induced elevations of intracellular cGMP(42, 43) . However, a recent study (44) has shown that ANP causes a dephosphorylation of the NPR-A and desensitizes ANP-stimulated guanylate cyclase in NPR-A. These observations are opposite to the conclusion. Our results show that the dissociated NPR-A from ANP is capable of binding, and the binding of ANP to the dissociated NPR-A may be due to the empty binding site. Therefore, the rebinding of the dissociated NPR-A may not be caused by phosphorylation or dephosphorylation. Further studies are needed to elucidate the phosphorylation and dephosphorylation for the desensitization of ANP-stimulated guanylate cyclase and the rapid dissociation of ANP from NPR-A.
In conclusion, although the dissociated NPR-A is capable of rebinding, and a membrane-bound proteinase(s) hydrolyzes ANP, the majority of the dissociated ANP from NPR-A rebind to NPR-A at 37 °C. Hence, the intact ANP may selectively bind to NPR-A at 37 °C before hydrolyzing or binding to NPR-C. Phosphoramidon inhibits the hydrolysis of ANP, and the increase of ANP levels leads to its binding to NPR-C. Therefore, the proteinase and NPR-C may play an important role not only in regulation of intact ANP levels but also in reuse of ANP and NPR-A in target organs.