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Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago Illinois 60637
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart.
cardiomyocytes; fibroblasts; smooth muscle; guanylate cyclase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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C-TYPE NATRIURETIC PEPTIDE (CNP) is a member of the family of NP hormones that also includes atrial NP (ANP) and brain NP (BNP) (26). ANP is synthesized in cardiac myocytes, where it is secreted constitutively in both the ventricle and atrium as well as in a regulated manner from granules in the atrium. First discovered in the brain, CNP has also been found in other tissues as well, e.g., in bone, reproductive tissue, and heart tissue (6, 22). CNP is secreted by endothelial cells in the heart (25), but its role in myocardial function is much less clear than that of ANP. There are presently three known NP receptors (NPR): NPR-A, -B, and -C. In an anomaly of nomenclature, NPR-A binds ANP and BNP in the nanomolar range and CNP in the micromolar range, whereas NPR-B binds CNP in the nanomolar range but binds ANP and BNP only in the micromolar range (17). Both of these transmembrane receptors express guanylyl cyclase (GC) activity in their cytoplasmic domains and are also known as GC-A and GC-B (27). NPR-C, found most abundantly in the kidney, has a truncated cytoplasmic tail, has no cyclase activity, and is believed to be involved predominantly in NP clearance (21), although a role in Gi-dependent signaling has recently been proposed (15).
mRNA for all three receptor types was detected by RT-PCR in myocytes isolated from the adult rat ventricle, but expression at the protein level was convincingly shown for GC-A, but not GC-B (9). In particular, whether GC-B and responses to CNP in cardiomyocytes play any role in adult myocardial function is unclear. In one study, CNP, but not ANP, significantly induced production of cGMP in adult rat ventricular myocytes in the absence of phosphodiesterase (PDE) inhibition (1). This effect was kinetically parallel to phosphorylation of the inhibitory subunit of troponin I and phospholamban (PLN) as well as a strong positive lusitropic (relaxing) effect and a weak negative inotropic effect in the heart papillary muscle. The effect on relaxation of contractility was associated with an increase in Ca2+ pump activity, presumably through attenuation of PLN inhibition of the pump due to the PLN phosphorylation. These effects resemble those recently found in neonatal rat cardiomyocytes in which a weak negative inotropic effect was associated with CNP-dependent cGMP accumulation (16). However, other studies (9) suggest that CNP has virtually no effect on cGMP in adult rat cardiomyocytes even in the presence of PDE inhibitors, whereas ANP and BNP lead to substantial increases in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). In the latter case, a substantial CNP response was only measured in cultured fibroblasts derived from isolated hearts, but whether such a response might lead to effects on proliferation of these cells is debatable (12). In addition to myocytes and fibroblasts, CNP produced in the vascular endothelia has been shown to have vasodilatory effects on smooth muscle cells of the coronary vasculature (25, 28).
To address whether GC-B is more highly expressed in myocytes or nonmyocytes in adult rat hearts, we compared the degree of expression of GC-B in acutely isolated cardiac myocytes, acutely isolated cardiac nonmyocytes, primary cultures of isolated cardiac fibroblasts, and the A7r5 rat smooth muscle cell line. We employed confocal immunofluorescence microscopy of frozen heart sections, as well as biochemical extraction and immunoblotting, and also the responsiveness of GC-B to CNP by measurement of cGMP accumulation in isolated cells. We further investigated whether there is an interactive relationship between GC-B responsiveness to CNP and proliferation-related mitogen-activated signal transduction pathways in fibroblasts isolated from the heart.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials
A rabbit polyclonal antibody, Z658, made to a peptide consisting of the 10 most COOH-terminal residues of the rat CNP receptor (NPR-B) was the gift of Dr. David L. Garbers, University of Texas Southwestern Medical Center. This antibody does not recognize the closely homologous ANP receptor GC-A. An antibody to NH2-terminal residues 2-18 of the rat caveolin-3 sequence was made commercially (QCB, Hopkinton, MA) (5). Polyclonal and monoclonal antibodies to caveolin-1 and -3 were obtained from Transduction Laboratories (Louisville, KY). Anti-phospho extracellular signal-related kinase (ERK) and anti-pan ERK antibodies were from Cell Signaling Technologies (Beverly, MA). Monoclonal antibodies to desmin and vimentin were from Sigma (St. Louis, MO). Fluorescently tagged secondary antibodies were from Molecular Probes (Eugene, OR). Kits for the measurement of cGMP and cAMP were purchased from Amersham (Piscataway, NJ). The rat smooth muscle cell line (A7r5 at passage 30) was a gift from Dr. Ken Byron, Loyola University Medical Center (Maywood, IL).Preparation of Heart Cells
Myocytes. Rat ventricular cells were dispersed by collagenase treatment of whole heart via perfusion on the Langendorff cannula utilizing DMEM medium (GIBCO-BRL, Gaithersburg, MD). Myocytes were initially separated from nonmyocytes by differential centrifugation. A myocyte-enriched fraction was obtained in a pellet by centrifugation at 120 g for 1 min in IEC tabletop clinical centrifuge. A nonmyocyte-enriched fraction was obtained by centrifuging the supernatant 570 g for 13 min. Viable myocytes were further purified by Percoll density gradient centrifugation (11).
Fibroblasts. To help assess the origin of nonmyocyte GC-B, a primary culture of fibroblasts was obtained by plating the nonmyocyte fraction (above) suspended in DMEM, 10% fetal bovine serum (FBS), pen/strep, and fungizone for 2 h. After cell attachment, the plates were washed and cultured until fibroblasts reached confluency. Cells were subcultured by trypsinization and used at passage 4 for experiments. At this stage, the population was considered to be free of nonfibroblast contaminants, as determined below.
Whole cell lysates of acutely isolated cells of the two populations, prepared by Percoll gradient and differential centrifugation, were fractionated by SDS-PAGE and immunoblotted.Detergent Extraction and Gradient Centrifugation of Cell Proteins
Fibroblasts were extracted with cold Triton X-100 and centrifuged in a sucrose density gradient according to established procedures (10) to determine whether GC-B protein resided in detergent-soluble or -insoluble membranes. The entire Triton X-100-treated cell sample was loaded in the bottom 6 ml of the gradient of 42% sucrose. Three milliliters each of 5 and 30% sucrose were layered above the sample.Confocal Immunofluorescence Microscopy
Pieces of the rat ventricle were frozen in liquid nitrogen, sectioned, and prepared for confocal microscopy as previously described (19). After quenching of aldehyde groups with 150 mM ammonium chloride in PBS, the sections were permeabilized with 0.2% Triton X-100 and 0.02% Tween detergents and blocked with 10% normal goat serum (13). Sections were incubated with anti-GC-B polyclonal antibody (1:50). Isolated ventricular fibroblasts, prepared and passaged as described above, were grown on coverslips as previously described (19) during the final passage. They were rinsed with PBS and fixed with methanol for 5 min at
20°C. Primary
antivimentin monoclonal antibody (1:100) as a marker for fibroblasts
and antidesmin monoclonal antibody (1:20) as a marker for smooth muscle
cells were followed by fluorescently labeled secondary antibodies. In
all microscopic applications, rabbit and mouse nonimmune IgGs at
similar concentrations were used as controls in place of primary antibodies.
cGMP and cAMP Assays
Fibroblasts and A7r5 cells were grown to confluence in 12- well plates. Cells were washed two times with PBS and incubated for 15 min with HEPES-DMEM at 37°C. Of the 11 PDEs presently identified (23), four that cleave cGMP have been localized to the heart (PDE1, -2, -3, and -9). PDEs -1, -2, and -3 are all inhibited by IBMX and selectively by various other xanthine analogs. PDE inhibitors IBMX (100 µM, nonselective inhibitor) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 20 µM; inhibits type 2), cilostamide (0.7 µM; inhibits type 3), or 8M-IBMX (40 µM; inhibits type 1) were added for 15 min, and then agonist (CNP or ANP) was added at concentrations designated in the text for 10 min. For these experiments, we routinely used 100 nM CNP, which should not activate NPA receptors (i.e., GC-A) (17). For control samples, neither inhibitor, peptide, nor inhibitor but no peptide was added. At the concentrations used, PDE inhibitors in the absence of peptide did not significantly raise cyclic nucleotide levels over control values. After incubation and washing, 0.5 ml of ice-cold 65% EtOH was added to each well. Lysate was transferred to microfuge tubes and each well was washed with an additional aliquot of 65% EtOH. Extracts were dried in a Savant SpeedVac and then resuspended in EIA buffer before assay. cGMP and cAMP EIA assay kits (Amersham) were used to quantitate cyclic nucleotide levels. NaOH (0.1 N) was added to each well to dissolve cellular debris before protein assay (Pierce Coomasie Plus protein assay). Experimental results were analyzed by one-tailed unpaired t-test or one-way ANOVA analysis (version 3.0; Prism, San Diego, CA).Ventricular myocytes were suspended in HEPES-DMEM after isolation and aliquoted into microfuge tubes. PDE inhibitors were added for 15 min followed by agonists for 10 min. Samples were cooled on ice and cells were pelleted for 2.5 min at 1,000 g in the cold. Supernatant was aspirated and 1 ml of 65% ice-cold EtOH was added to these cells. Samples were mixed, and the cell debris was pelleted by centrifugation for 10 min at 16,000 g. The supernatant extract was transferred to clean microfuge tubes and dried down as above. NaOH (0.1 N) was added to the remaining cellular debris before protein assay.
ERK Phosphorylation in Cardiac Fibroblasts
The procedure was based on Chrisman and Garbers (3), with modifications. Briefly, confluent cells in 12-well plates were incubated in DMEM with 0.5% FBS (GIBCO-BRL) for 24 h and then in DMEM without FBS for 18 h. After 18 h, fresh HEPES-DMEM was added to the wells and the cells were allowed to equilibrate for an additional hour. CNP (100 nM), with or without IBMX (100 µM), or vehicle was added. After an hour, the appropriate amount of FBS was added, and the plates were incubated at 37°C for 15 min to stimulate ERK phosphorylation. Cells were washed twice with Tris-buffered saline (TBS) on ice, and then 200 µl of ice-cold lysis buffer containing (in mM) 2 NaVO4, 10 Na4P2O7, 1 NaF, 0.5 benzamidine, 1 phenylmethylsulfonyl fluoride, and 1 O-phenathroline plus 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.8 µg/ml pepstatin were added to each well. Lysate was transferred to microfuge tubes and allowed to sit on ice for 20 min. Samples were cleared by centrifugation for 10 min at 16,000 g and the resulting supernatant was transferred to new microfuge tubes. Sample protein was fractionated by 8% SDS-PAGE gel and transferred to nitrocellulose filters for immunoblotting as previously described (5). BSA (5%) was used to block the filters probed with anti-phospho-ERK (1:5,000) and anti-pan-ERK (1:1,000) antibodies.Measuring the Effects of Preincubation with FBS on CNP- Stimulated cGMP
The procedure was based on Chrisman and Garbers (3). Briefly, fibroblasts were grown to confluency in 12-well plates. Cells were incubated for 24 h in DMEM with either 10 or 0.5% FBS. Fresh HEPES-DMEM-FBS was added to the wells for 5 min before CNP (100 nM) and IBMX (100 µM) was added. Incubation continued for 10 min after which the cells were washed with TBS, and cGMP was extracted and assayed as described above.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Differential Localization and Concentration of GC-B in Rat Heart Ventrical Myocytes and Fibroblasts
We determined the distribution of the CNP receptor by confocal immunofluorescence microscopy in thin sections of frozen rat left ventricle wall (Fig. 1) and papillary muscle (data not shown) with the use of anti-GC-B antibody. As shown in Fig. 1, strong staining for GC-B was found in regions interstitial to cardiomyocytes, presumably due to fibroblasts, and in ring-like structures corresponding to vascular cross section, presumably due to smooth muscle cells and/or fibroblasts in an arteriole. Staining of the myocyte surface, visualized in these experiments by double labeling with the bona fide sarcolemma marker caveolin-3, was not apparent. Staining patterns in sections obtained from the ventricle left wall were similar to those from papillary muscle.
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GC-B Protein in Heart Cells
To corroborate the immunofluorescence data, we assessed the relative quantity of GC-B protein in ventricular myocytes and nonmyocytes using a biochemical approach. As seen in Fig. 2A, the nonmyocyte-enriched population contained discernible GC-B protein, although at levels well below that seen in the rat brain preparation, which served as a positive control. The signal from the myocyte-enriched population was barely detectable. These results thus confirm the immunofluorescence data and suggest that GC-B is found at much higher levels in nonmyocytes than in cardiomyocytes in the rat ventricle.
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To confirm the presence of the protein in these cells and to assess
whether GC-B is associated with detergent-soluble or -resistant domains
of the plasma membrane, we treated whole cell lysates of fibroblasts
and A7r5 cells with cold Triton X-100 and fractionated the samples on
sucrose density gradients. Samples were then immunoblotted for GC-B.
GC-B was detected in the detergent-soluble fractions of fibroblast
samples (Fig. 2B) but was not detected in A7r5 cells or
myocytes (data not shown). No GC-B protein migrated to the middle of
the gradient in detergent-resistant membranes from the fibroblasts
(marked in these experiments with the caveolar protein caveolin-1),
suggesting that GC-B does not reside in caveolae or in Triton X-100
insoluble lipid rafts in these cells. We confirmed that our fibroblast
culture was not contaminated with smooth muscle cells by staining
samples with antibodies against vimentin (for fibroblasts) or desmin
(for smooth muscle cells) (Fig.
3A).
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Differential CNP-Induced Accumulation of cGMP in Rat Heart Ventrical Fibroblasts, Myocytes, and A7r5 Cells
To assess the relative functional response of GC-B receptors in these cell types, accumulation of cGMP due to incubation with 100 nM CNP in the presence or absence of PDE inhibitors was measured in primary cultures of rat heart fibroblasts of four passages or less (Fig. 3A), of A7r5 cells (Fig. 3B) and in acutely isolated ventricular myocytes (Fig. 4A). The highest IBMX-sensitive cGMP accumulation was found in the primary cardiac fibroblasts by an order of magnitude over A7r5 cells and two orders of magnitude over myocytes when expressed as per milligram of protein (Figs. 3 and 4A). When expressed as cGMP per cell, the values for myocytes increased relative to other cell types due to their relatively large size (Table 1). Because the values, although significant, were so low in myocytes, we tested whether these cells were indeed hormone responsive after acute isolation by stimulating them with isoproterenol (with or without accompanying IBMX) and measuring cAMP levels (Fig. 4B). The resultant values were comparable to previously reported results (8, 14) indicating that surface receptors were functional under our isolation and culture conditions. The paucity of GC-B response in acutely isolated myocytes is thus consistent with our microscopic and biochemical observations.
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Variable PDE effects on cGMP accumulation in response to CNP were observed among the three cell types. PDE1 inhibitor 8M-IBMX accounts for most although not all the IBMX-sensitive accumulation in fibroblasts. 8M-IBMX significantly enhances CNP induced-accumulation (n = 4, P < 0.001) but significantly less so than IBMX (P > 0.05; Fig. 3A). PDE2 inhibitor EHNA accounts for all the IBMX-sensitive accumulation in A7r5 cells (Fig. 3B). The low but significant IBMX-sensitive CNP-induced cGMP accumulation measured in myocytes is attributable to the presence of cilostamide-inhibited PDE3. In our myocyte-enriched preparation, we saw no statistically significant effect of 8M-IBMX or EHNA on cGMP accumulation, suggesting that the cGMP found in these preparations is unlikely to come from contaminating fibroblasts or smooth muscle cells.
To further test whether CNP cross-reacted with the GC-A receptor, we measured the cGMP accumulation in myocytes in response to 100 nM ANP, a concentration at which ANP acting on GC-A receptors should not cross-react with GC-B (17). In that case, accumulation occurred in the presence of EHNA, an inhibitor of PDE2 (Fig. 4C). Because EHNA has no measurable effect on cGMP accumulation due to CNP in myocytes, this suggests that CNP induction through GC-A in myocytes was not occurring in our experiments.
CNP Does Not Influence ERK Phosporylation in Cardiac Fibroblasts
Because myocardial fibroblasts contain significant levels of GC-B, it seemed likely that these cells might exhibit a physiological response to CNP. Therefore, we investigated whether CNP interacted with ERK-1 and -2 phosphorylation in cardiac fibroblasts expressing native levels of GC-B. As expected, ERK phosphorylation was dose dependently increased by FBS readdition to serum-starved cells (Fig. 5A). However, preincubation with CNP did not reduce the phosphorylation of ERK-1 or -2 in response to serum. Even when cGMP levels were elevated farther by incubation in CNP plus IBMX, no effect on ERK-1 and -2 phosphorylation was found nor did serum inhibit CNP-induced cGMP accumulation in these cells (Fig. 5B). These results do not support the notion that activation of GC-B exerts any influence on the proliferative state of myocardial fibroblasts.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of the present study was to investigate the
possibility that CNP exerts effects in the adult heart and to ascertain which cell type(s) is the likely recipient of these effects. Whereas ANP is a well-characterized endocrine peptide in the myocardium serving
to modulate cardiomyocyte function during development and also during
cardiac hypertrophy (7), the function of CNP is less
understood. In rat neonatal ventricle myocytes, a 200-fold higher level
of CNP-induced cGMP accumulation is observed than in our adult myocytes
(16). Although GC-B mRNA in adult cardiac myocytes is
detected equivalent to that in adult cardiac fibroblasts by
RT-PCR (9), our immunofluorescence microscopy results
suggest that the CNP receptor protein resides at substantially higher concentration in ventricular nonmyocytes (interstitial ventricular fibroblasts and cells of the vasculature, possibly including smooth muscle cells and/or fibroblasts) than in ventricular myocytes. Likewise, the protein is barely detectable by immunoblotting of proteins from isolated cardiomyocytes. This does not appear to be a
limitation of the antibody at our disposal, as this antibody detects
GC-B in other tissues (3) and reacts with samples from the
rat brain and cardiac fibroblasts in the present work. Our measurement
of very small changes in cGMP levels in response to CNP addition to
acutely isolated cardiomyocytes is in agreement with these results.
Failure to elicit appreciable levels of cGMP accumulation in these
cells is unlikely to be due to cell damage, because identical
preparations could respond to ANP with cGMP elevations and to
activation of the 
-adrenergic receptor with cAMP production.
Assuming that the cGMP production detected in neonatal cells is not due
to nonmyocyte contamination, our results suggest that there may well be
a developmental decline in this receptor in the myocyte population.
Our results fail to support the conclusions of another study in which 1 µM CNP was reported to induce a response in nominally purified myocytes (1). At this concentration, it is known that CNP can evoke responses in GC-A as well as GC-B receptors (17). Because the cell preparation was "myocyte enriched" but not further purified by Percoll gradient centrifugation, a minor contamination with GC-A receptors from cells, such as fibroblasts, could well have led to the observed increases in cGMP. Instead we find, in agreement with previous observations (9), that CNP receptors are concentrated in myocardial fibroblasts rather than in cardiomyocytes. This result is supported by immunofluorescent staining in sections of the rat ventricle and biochemical observations of GC-B protein in fibroblast extracts as well as substantial cGMP accumulation in response to CNP in purified fibroblast cultures. In addition, we measured an intermediate accumulation of cGMP in A7r5 cells and detected GC-B microscopically in vascular cross sections of the rat ventricle, although in cell extracts, the GC-B signal is below the level of detection. This result is consistent with reports of vasodilatory responses to CNP present in smooth muscle cells of the cardiac vasculature (2, 28).
For each of the three types of cells observed, a single, unique PDE appears to predominantly, if not exclusively, modulate CNP-induced cGMP accumulation. In our observations, PDE1 was predominant in heart fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes. This may result from a cell specificity of the PDEs in rat heart or a compartmentalization of the GC-B/PDE interaction that dictates this selectivity. Of interest in the present context is the question of whether the physiological signal seen in our myocyte-enriched preparation is localized to myocytes, despite the absence of immunological detection. The selectivity of the PDE response we observed in the three cell types allows us to infer that the small CNP-generated signal detected in myocytes is likely specific and not due to contamination by other cell types.
Previously, we detected ANP bound to the extracellular surface of rat atrial myocyte caveolae by immunoelectron microscopy (18) and GC-B in surface sarcolemma of rat atrial myocytes by confocal immunofluorescence microscopy (4). We interpreted our results to suggest that GC-B resided in caveolin-3 containing caveolae of those membranes, although we did not present biochemical evidence to support this conclusion. Our present observations in ventricular fibroblasts that GC-B does not comigrate with caveolin-1 in sucrose density gradients clearly suggests that GC-B is not in caveolin-1-containing caveolae in these cells.
Previously, no direct observations have been made with respect to the role of fibroblasts in response to CNP in the heart. In BALB/3T3 fibroblasts over-expressing GC-B, a reciprocal antagonism between CNP and mitogen-activated pathways has been reported (3). However, in rat cardiac fibroblasts, only agents that activate cAMP-kinase, but not cGMP-kinase, inhibited fibroblast growth (12). It may be that overexpression of GC-B with the attendant ability to generate very high levels of intracellular cGMP can influence other pathways related to mitogenesis, but this does not appear to be the case in cardiac fibroblasts expressing native amounts of the NP-B receptor. Our results, in agreement with those of others (12), suggest that some or all fibroblasts that we isolate and culture from the heart do not share the property of other, nonmyocardial fibroblasts (3) for which there is a reciprocal antagonism between CNP and serum growth factors. Thus some other function for CNP receptors in adult myocardium must be sought.
In conclusion, we have investigated the localization and function of the CNP receptor NPR-B (or GC-B) in the adult rat heart ventricle. We found the expression of GC-B is predominantly in nonmyocytes. The receptor protein is detected by immunofluorescence microscopy in interstitial cells and vascular cells. By immunoblot analysis, receptor protein is detected in extracts of fibroblasts, but not A7r5 cells or myocytes. In functional studies, CNP-induced cGMP accumulation was greater by an order of magnitude in fibroblasts than A7r5 cells and, in turn, greater by an order of magnitude in A7r5 cells than in myocytes. Modulation of cGMP accumulation by PDEs was cell-specific PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes. There did not appear to be an interactive relationship between NPR-B responsiveness to CNP and proliferation-related mitogen-activated signal transduction pathway in these cardiac fibroblasts.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ernest Page, University of Chicago, for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-54302.
Address for reprint requests and other correspondence: D. D. Doyle, Dept. of Neurobiology, Pharmacology, and Physiology MC0926, 947 E. 58th St., Chicago IL 60637 (E-mail: ddoyle{at}drugs.bsd.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 31, 2002;10.1152/ajpheart.00988.2001
Received 13 November 2001; accepted in final form 4 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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