(Received for publication, May 26, 1995; and in revised form, August 7, 1995)
From the
Atrial natriuretic peptide (ANP) treatment of rat aortic smooth
muscle cells suppressed both I-ANP binding and
ANP-dependent cGMP accumulation, suggesting reductions in the type C
(NPR-C) and type A (NPR-A) natriuretic peptide receptor populations,
respectively. NPR-A, but not NPR-C, mRNA levels were reduced in a
dose-dependent fashion by ANP. The latter effect appeared to be due, at
least in part, to suppression of NPR-A gene promoter activity. ANP
effected a dose- and time-dependent reduction in a transiently
transfected NPR-A luciferase reporter (-1575LUC). Analysis of 5`
deletion mutants of the NPR-A promoter demonstrated that the
ANP-dependent sequence lies between -1575 and -1290
relative to the transcription start site. Inhibition of the ANP
promoter was also effected by brain natriuretic peptide, type C
natriuretic peptide, and 8-bromo-cGMP, but not by the NPR-C-selective
ligand cANF. In the case of 8-bromo-cGMP, the responsive element(s) was
localized to the same 285-base pair region linked to the ANP effect
above. These findings indicate that ANP autoregulates its own receptors
in these cells and, at least in the case of NPR-A, it does so through
suppression of receptor gene expression and receptor synthesis. This
suppression may operate through a cGMP-dependent element located more
than a kilobase upstream from the transcription start site.
Atrial natriuretic peptide (ANP) ()is a cardiac
hormone which has profound effects in the kidney, vasculature, and
nervous system (1) . It has vasorelaxant activity in vascular
smooth muscle(2) , and it promotes urinary excretion of sodium
and water by the kidney(3) . It also has antimitogenic activity
in renal mesangial cells(4) , vascular smooth muscle (5) and endothelial cells(6) , glial cells of the
central nervous system(7) , and fibroblasts of the cardiac
interstitium(8) . Thus, it possesses hemodynamic and antigrowth
properties which oppose those of vasoconstrictors like the
-adrenergic agonists, endothelin, and angiotensin.
ANP is one
member of a family of natriuretic peptides which also includes brain
natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP and
BNP are both made in and secreted from the heart, circulate in plasma,
and share many of the same biological activities. CNP, on the other
hand, is made primarily in non-cardiac tissues, does not circulate at
appreciable levels in plasma, and has a different activity profile at
the level of target tissues(9) . At a molecular level these
ligands interact with one or more of three different receptor subtypes.
The type A natriuretic peptide receptor (NPR-A) has an extracellular
ligand-binding domain which recognizes both ANP and BNP. This domain is
linked through a trans-membrane region with a carboxyl-terminal
guanylyl cyclase domain which serves as the effector portion of the
receptor. The type B receptor (NPR-B) shares a similar structural
topology with NPR-A but binds selectively to CNP. The C-type receptor
(NPR-C) is structurally distinct from the guanylyl cyclase receptors.
It contains a large extracellular ligand-binding domain followed by a
short trans-membrane segment; however, its intracellular domain
contains only 38 amino acids and lacks guanylyl cyclase activity. It is
the predominant receptor on most vascular cells in
culture(10) . This, together with its limited intracellular
structure, led investigators early on to speculate that it functioned
predominantly in a clearance mode, responsible for removing natriuretic
peptides from the circulating plasma and processing them to inactive
forms. Direct experimental testing supports this
hypothesis(11, 12) . Additional evidence suggests that
NPR-C may also have a signaling function. cANF, an analogue of ANP
which associates relatively selectively with NPR-C, has been shown to
inhibit adenylyl cyclase activity in a number of different whole cell
and membrane preparations (13, 14) . It also at least
partially mimics the actions of ANP in suppressing
[H]thymidine incorporation (as an index of
mitogenic activity) in vascular smooth muscle cells(5) , glial
cells(7) , and cardiac fibroblasts(8) . Such studies
raise the possibility that NPR-C may possess important regulatory
activity operating either independently or in parallel with that of the
guanylyl cyclase-linked receptors(15) .
ANP receptor levels
and ANP receptor activity (assessed predominantly through ANP-dependent
guanylyl cyclase activity) are regulated by a number of factors,
including
hormones(16, 17, 18, 19, 20) ,
growth
factors(21, 22, 23, 24, 25) ,
neurotransmitters(26) , physiological (27, 28) and pathophysiological
perturbations(29, 30) , and changes in extracellular
ion composition(31) . ANP, itself, is an important regulator of
NPR activity. Physiological changes in the intact animal, which are
known to raise circulating ANP levels, have been shown to decrease
levels of I-ANP binding in the
kidney(27, 28) . In vitro, ANP pretreatment
has been shown to diminish NPR-C density in vascular endothelial (32) and smooth muscle cells (33, 34, 35) and to abrogate ANP-dependent
cGMP synthesis in the latter(33, 36, 37) .
Studies carried out with cells stably transfected with the NPR-A
receptor suggest that homologous desensitization of guanylyl cyclase
activity by ANP is linked to dephosphorylation of key serine and
threonine residues in the receptor protein(22, 38) .
The present study demonstrates that the fall in cyclase activity is
also associated with a reduction in steady-state levels of NPR-A mRNA
and transcriptional activity of the NPR-A gene. This implies that ANP
exerts both transcriptional and post-transcriptional effects to
regulate the activity of its receptors in target cells.
To preclude the possibility that the ANP-mediated reduction in binding activity resulted from receptor occupancy with unlabeled ligand, we washed ANP-pretreated cells with acidified DMEM (pH 5.0) prior to running the binding assay as described above. These wash conditions remove >95% of prebound ligand from surface ANP receptors (data not shown).
Pretreatment with ANP led to a significant decrease in I-ANP binding in cultured RASM cells. As shown in Fig. 1, ANP pretreatment effected a dose- and time-dependent
reduction in binding which was maximal between 10 and 100 nM ANP after 24 h of exposure. Since the majority of ANP receptors in
RASM (85-95%) are of the NPR-C or clearance receptor class, by
definition this group of receptors is suppressed by ANP. This
inhibition was reproduced in cultures subjected to the acid wash
(without wash: 12.3 ± 1.5% of control binding; with wash: 33.9
± 1.5% of control binding after 24 h. treatment with 0.1
µM ANP), indicating that the decrease in binding could not
be accounted for exclusively by prior receptor occupancy with ligand.
Figure 1:
Effect of ANP on I-ANP
binding in cultured RASM cells. Confluent cells were incubated in the
presence or absence of increasing concentrations of ANP for the times
indicated. Cells were then washed and subjected to the binding assay.
I-ANP (0.5 nM) in the absence or presence of 0.2
µM unlabeled ANP was used to measure total and nonspecific
binding, respectively. All values are normalized to specific binding
measured in untreated cells (counts/min/microgram of protein). Data are
presented as mean ± S.D.; n = 4. *, p < 0.05; +, p < 0.01 versus untreated
groups.
To determine whether ANP had independent effects on levels of the guanylyl cyclase-linked receptors, we examined the effect of ANP pretreatment on the subsequent ability of ANP to trigger the synthesis of cGMP in these cultures. As shown in Fig. 2, there was a dose-dependent reduction in ANP-stimulated cGMP generation (largely reflective of NPR-A activity) following pretreatment with the peptide, suggesting that ANP suppresses or down-regulates activity of both NPR-A and NPR-C in these RASM cells.
Figure 2: Effect of ANP pretreatment on ANP-stimulated cyclic GMP production in cultured RASM cells. Confluent cells were preincubated with 10 or 100 nM ANP for 48 h. Cells were then washed extensively and re-challenged with increasing concentrations of ANP in the presence of IBMX (0.5 mM) for 10 min. All values are normalized to cGMP levels found in untreated cells. Data are presented as mean ± S.D.; n = 3. *, p < 0.05;**, p < 0.01 versus corresponding point(s) in untreated groups.
We next attempted to determine
whether either of these effects were operative at the level of receptor
synthesis. To approach this question we isolated RNA from RASM cells
pretreated with increasing concentrations of ANP and subjected them to
Northern blot hybridization. As seen in Fig. 3, ANP pretreatment
effected a dose-dependent decrease in NPR-A mRNA levels in these cells.
Maximal inhibition to 48% of control levels was seen at 10M ANP. Levels of the housekeeping transcript for
glyceraldehyde-3-phosphate dehydrogenase were unchanged by the
treatment. Interestingly, levels of the NPR-C transcript were also
unaffected by ANP treatment. This suggests that the reduction in NPR-A
activity results, at least in part, from a decrease in receptor
synthesis, whereas the coincident reduction in NPR-C levels is
dependent upon a post-synthetic mechanism.
Figure 3:
Northern blots show effect of ANP
pretreatment on expression of mRNA for natriuretic peptide receptor
subtypes A (NPR-A) and C (NPR-C) in cultured RASM
cells. A, cells were pretreated with increasing concentrations
of ANP for 48 h in serum-free medium and total RNA was collected. 30
µg of RNA from each group was size-fractionated, transferred to a
nitrocellulose filter, blot-hybridized simultaneously with
[-
P]dCTP-labeled cDNAs for NPR-C and
glyceraldehyde-3-phosphate dehydrogenase and then autoradiographed at
-70 °C for 3 days. Lane 1, control; lanes
2-4, 10
, 10
, and
10
M ANP, respectively. The same membrane
was stripped of bound probe, rehybridized with
[
-
P]dCTP-labeled NPR-A cDNA probe, and
autoradiographed at -70 °C for 7 days. B,
autoradiographs from two independent experiments were scanned by laser
densitometry and the average density plotted arbitrarily as a function
of the relevant control signal.
To explore this further
we constructed a series of 5` deletion mutants of the rat NPR-A gene
promoter, linked them to a luciferase reporter, introduced them into
RASM cells by transient transfection, and examined the effects of ANP
treatment on reporter expression. As seen in Fig. 4, treatment
of cells transfected with -1575LUC, a construct harboring 1575
base pairs of NPR-A gene 5` FS linked to luciferase coding sequence,
with ANP led to a dose- and time-dependent reduction in reporter
activity. Maximal inhibition was seen at 10M ANP after 48 h of treatment. To localize the cis-acting element
responsible for conferring this inhibitory activity, we introduced the
5`-deletion mutants described above into RASM cells and examined their
expression in the presence or absence of ANP treatment (Fig. 5).
Based on the deletion profile presented in this figure, it is clear
that NPR-A gene transcription is positively regulated by at least three
regions in the promoter positioned between -1575 and -1290,
-716 and -387, and -273 and -77 relative to the
transcription start site. ANP exerted a significant negative effect on
the -1575LUC reporter, as described above; however, truncation of
the reporter to -1290 and smaller derivatives resulted in a loss
of the ANP-dependent inhibition. By inference this suggests that the
element responsible for suppressing transcriptional activity from the
NPR-A gene promoter is located between -1290 and -1575
relative to the transcription start site.
Figure 4: Effect of ANP pretreatment on -1575LUC activity. Cultured RASM cells were co-transfected with 20 µg of -1575LUC and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and treated with serum-free DMEM containing indicated concentrations of ANP for different periods of time. All values are expressed as a percentage of luciferase activity in control (untreated) group. Values represent mean ± S.D. from six different experiments. *, p < 0.01 versus control; +, p < 0.05; ++, p < 0.01 versus corresponding point in 6-h group.
Figure 5: Effect of ANP pretreatment on 5` deletion mutants. Cells were transfected with 20 µg of each of the receptor constructs indicated and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and exposed to serum-free DMEM containing 10 nM ANP for 48 h. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from six different experiments. *, p < 0.01 versus control (untreated) group.
The suppression of NPR-A
promoter activity was not limited to ANP. Both BNP and CNP, at
comparable concentrations of ligand, effected similar reductions in
-1575LUC activity (Fig. 6). Each of these ligands is
capable of increasing cGMP levels in these cells (Table 1). cANF,
an ANP homolog which lacks the guanylyl cyclase-activating function and
binds predominantly to the type C or clearance receptor, failed to
suppress NPR-A promoter activity. This suggests that the reduction in
transcription requires occupancy of a receptor system capable of
triggering the activation of guanylyl cyclase and production of cGMP (i.e. activation of NPR-A by ANP or BNP or activation of NPR-B
by CNP). To explore this further we pretreated
-1575LUC-transfected cells with one of two different
phosphodiesterase inhibitors (PDEI) in the presence or absence of ANP
and assessed the effect on reporter activity (Fig. 7). Both the
nonselective PDEI, IBMX, and the type V (cGMP-selective) PDEI, M&
22948, would be predicted to slow the degradation rate of cGMP in these
cells (confirmed in Table 1) and, therefore, amplify the effects
of agonists (e.g. ANP) operating through a guanylyl
cyclase-dependent mechanism. Each of these agents independently reduced
NPR-A promoter activity and, furthermore, each amplified the effect of
simultaneous ANP treatment. These findings are compatible with a
requirement for ANP-dependent cGMP generation in initiating and/or
maintaining the inhibitory effect. Next, we examined the effect of
8-bromo-cGMP, a membrane-permeable analogue of the cGMP second
messenger, on NPR-A promoter activity (Fig. 8). 8-Bromo-cGMP
effected a dose- and time-dependent reduction in -1575LUC
reporter activity similar to that seen with ANP. The inhibition was
maximal at 10M and required between 6 and
48 h to appear. To determine whether this effect operated through the
same region of the NPR-A gene promoter targeted by ANP, we examined a
number of the same promoter deletion mutants described in Fig. 5for suppressibility by 8-bromo-cGMP. As shown in Fig. 9, the dose-dependent inhibition of NPR-A promoter activity
was observed only with the -1575LUC construct and was not seen
with any construct containing 1290 or fewer base pairs of NPR-A gene 5`
FS. This finding further supports the hypothesis that the inhibitory
activity of the natriuretic peptides is cGMP-dependent and that the
genomic target for this activity is contained within 285 base pairs of
promoter sequence lying between -1575 and -1290 relative to
the transcription start site.
Figure 6: Effect of different natriuretic peptides on NPR-A promoter activity. Cells were transfected with 20 µg of -1575LUC and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and treated with serum-free DMEM containing the indicated concentrations of natriuretic peptide for 48 h. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from six different experiments. *, p < 0.01 versus control group.
Figure 7: Effect of PDE inhibition on NPR-A promoter activity. Cells were transfected with 20 µg of -1575LUC and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and exposed to serum-free DMEM containing 0.1 mM IBMX or 0.1 mM M& 22948 for 48 h. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from four different experiments. *, p < 0.01 versus control without PDE inhibition;**, p < 0.01 versus groups with ANP treatment alone; +, p < 0.05 versus corresponding groups without ANP treatment.
Figure 8: Effect of 8-bromo-cGMP on NPR-A promoter activity. Cells were transfected with 20 µg of -1575LUC and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and treated with serum-free DMEM containing increasing concentrations of 8-bromo-cGMP for the indicated periods of time. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from four different experiments. *, p < 0.01 versus control group.
Figure 9: Effect of 8-bromo-cGMP on NPR-A 5`-deletion mutants. Cultured RASM cells were transfected with 20 µg of each of the 5` deletion mutants and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and exposed to serum-free DMEM containing increasing concentrations of 8-bromo-cGMP for 48 h. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from six different experiments. *, p < 0.01 versus control group.
Recent studies (47) have suggested considerable crossover in the signaling mechanisms employed by cAMP and cGMP in VSMC. In fact, at least some VSMC cultures seem to harbor little, if any, cGMP-dependent protein kinase. Most cGMP-dependent activity, in this latter case, appears to operate through the cAMP-dependent protein kinase. To explore the potential crossover of these pathways at the level of the NPR-A gene promoter, we transfected cells with the same chimeric reporters described above, then treated them with 8-bromo-cAMP or forskolin for 48 h. As shown in Fig. 10, neither of these agents had an effect on NPR-A promoter activity, arguing that the inhibition is selective for cGMP.
Figure 10: Effect of 8-bromo-cAMP and forskolin on NPR-A 5`-deletion mutants. Cultured RASM cells were transfected with 20 µg of each of the 5` deletion mutants and 5 µg of pRSVCAT. After 24-h incubation in 10% FBS/DMEM, cells were washed and changed to serum-free DMEM containing 0.1 mM 8-bromo-cAMP (A) or 10 µM forskolin (B) for 48 h. Cells were harvested at that point and extracts assayed for luciferase and CAT activity. All values are expressed as a percentage of luciferase activity in control group. Values represent mean ± S.D. from three different experiments.
The results presented above convincingly demonstrate that
prior exposure to ANP results in down-regulation of I-ANP
binding on the surface of rat VSMC and desensitization of the
particulate guanylyl cyclase to subsequent challenge with the ligand.
ANP-dependent suppression of binding in these cells has been
controversial. While the reduction in binding activity is not disputed,
several investigators have argued that it results from prior receptor
occupancy with the ligand (i.e. persistent occupancy of
receptor sites by unlabeled ligand employed in the pretreatment phase
of the experiment) rather than a true decrease in receptor density on
the cell surface (19, 48) . However, other
investigators, by employing an acid wash of the pretreated cells to
remove associated ligand, have shown that the decrease in surface
I-ANP binding represents a true reduction in receptor
density(26, 33) . Our own findings support this
conclusion in that conditions predicted to remove more than 95% of
surface-associated ligand (26) failed to eliminate the
reduction in ligand binding. The reduction in ANP-coupled guanylyl
cyclase activity following prolonged exposure to the ligand, such as we
have noted here, is less
controversial(22, 30, 33, 37, 38) ,
although a number of investigators have failed to observe the
effect(32, 35, 49) . This discrepancy remains
unexplained, although it may reflect differences in passage number, ANP
pretreatment, or the conditions of cell culture.
The reduction in ANP-dependent cGMP synthesis is not likely to be a consequence of the reduction in NPR-C binding activity. In instances where it has been examined closely, reduction in NPR-C levels on the cell surface have more commonly been associated with an amplification of ANP-dependent cGMP generation(26, 50) , perhaps reflecting the increased availability of ligand as clearance of ANP decreases in parallel with NPR-C density.
VSMC are known to harbor both NPR-A and NPR-B receptors. The relative proportion of these two populations is tied to the growth status of the cells with NPR-B becoming predominant as the cells are passaged in culture(51) . Since ANP is the primary ligand for NPR-A, it is likely that the reduction in ANP-linked guanylyl cyclase activity is, in large part, related to a decrease in this receptor population. The associated fall in NPR-A mRNA levels implies that the fall in NPR-A activity results, at least in part, from a reduction in new receptor synthesis. However, this is unlikely to be the sole mechanism for desensitization of this receptor. Potter and Garbers (22, 38) recently showed that ANP effects a dephosphorylation of NPR-A in parallel with desensitization of guanylyl cyclase activity. Taken together with the findings presented here, this indicates that ANP operates through both transcriptional and post-transcriptional loci in regulating NPR-A activity. The decrease in NPR-C levels, on the other hand, was not accompanied by a decrease in NPR-C mRNA levels, a finding which has been reported by others(47) , implying that the ligand effect in this instance is confined to a post-synthetic locus (i.e. at the level of preformed receptor).
The decrease in NPR-A mRNA levels can be explained, at least partially, by a reduction in the transcriptional activity of the NPR-A gene promoter (see Fig. 4and Fig. 5). This inhibition is shared by all three natriuretic peptide ligands as well as 8-bromo-cGMP, and is amplified by PDE inhibition, but it is not shared by the NPR-C-selective ligand cANF. These findings argue that the reduction in promoter activity is mediated by elevations in cellular cGMP which are signaled by either NPR-A or NPR-B in these cells and that NPR-C is not directly involved. Participation of other non-guanylyl cyclase-signaled events from the NPR-A/B receptors cannot be formally excluded; however, co-localization of the ANP-sensitive and cGMP-sensitive region of the NPR-A promoter to the same 285 base pairs of DNA suggests that the inhibition is predominantly cGMP-signaled. Precise localization of the ANP/cGMP-sensitive element remains undefined; however, there are few regions with homology to known transcriptional regulatory elements in this segment of DNA and nothing bearing even remote homology to the cAMP-regulatory element (CRE), an important consideration given the potential for cross-activation of protein kinase A by cGMP (47) in these cells of interest, treatment of cells transfected with -1575LUC with either ANP or 8-bromo-cGMP reduced reporter activity to that of -1290LUC. This implies that the positive regulatory element(s) located in this region can be completely neutralized by elevations in cellular cGMP and may provide a clue as to the mechanism underlying the inhibitory effect. In any event this regulatory element is likely to be of interest since, in general, we know very little of the mechanism(s) underlying cGMP effects on eukaryotic gene promoters.
Given the reduction in both NPR-C and NPR-A activity in these cells, one is left with the question as to the predicted outcome at the level of the intact cell following prolonged exposure to agonist. A priori, one would anticipate that there would be a reduction in ANP-dependent activity given the impairment in the guanylyl cyclase effector mechanism; however, the reduction in NPR-C with presumed decrease in NP clearance would be expected to increase the availability of ligand in the microenvironment surrounding the target cell. Thus, it is possible that under some circumstances (e.g. greater reduction in NPR-C versus NPR-A activity) the combined effect would result in little effect, or perhaps a modest increase, in guanylyl cyclase-linked activity. Identification of those conditions (e.g. based on the concentration or kinetics of exposure to the natriuretic peptides) favoring one or the other of these phenomena could provide interesting insights into the physiology of the natriuretic peptides in the vascular wall and, by inference, identify situations where malfunction could lead to pathological consequences.