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(Circulation Research. 1997;81:86-91.)
© 1997 American Heart Association, Inc.

Articles

Type B Atrial Natriuretic Peptide Receptor in Cardiac Myocyte Caveolae

Donald D. Doyle, S. Kelly Ambler, Judy Upshaw-Earley, Amir Bastawrous, Gwendolyn E. Goings, , Ernest Page

From the Departments of Medicine and Pharmacological and Physiological Sciences, University of Chicago (Ill).

Correspondence to Dr Ernest Page, Department of Medicine (Cardiology), University of Chicago, 5841 S Maryland Ave, MC5085, Chicago, IL 60637. E-mail page{at}hearts.bsd.uchicago.edu

	Abstract

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Abstract We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein, caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca²⁺ channel activity and contribute to a negative inotropic effect of ANP.

Key Words: atrial natriuretic peptide B receptor • atrial myocyte • guanylyl cyclase • heart

	Introduction

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We have recently reported that atrial natriuretic peptide (ANP), a hormone secreted by mammalian atria and ventricles, is present in plasma membrane caveolae of rat atrial myocytes, as determined by immunoelectron microscopy.¹ Caveolae are non–clathrin-coated plasma membrane–associated vesicles that contain or interact with multiple signal-transducing proteins.² The finding of ANP in caveolae suggests that at least part of its peptide sequence may be bound within caveolae to one of the known isoforms of ANP receptors.^{3 4 5} In this regard, the plasma membrane-associated type B ANP receptor (ANP-RB) is of particular interest. ANP-RB is a single-pass integral membrane protein whose guanylyl cyclase activity is encoded entirely by amino acid sequences of its catalytic domain and that is located in noncardiac tissues, on the cytoplasmic side of the plasma membrane.^{3 4 5} Like other guanylyl cyclases, this plasma membrane–associated enzyme converts GMP to the second messenger cGMP, which can phosphorylate target proteins. Such ANP- and cGMP-mediated phosphorylations are required for the effects of ANP on noncardiac target tissues like vascular smooth muscle, renal and endocrine cells involved in natriuresis, and multiple other tissues mediating diverse functions. Thus, although in mammals most of secreted ANP originates in atrial and ventricular myocytes, the targets for this endocrine secretion are, with at least two important exceptions, multiple noncardiac peripheral tissues.⁶ The two well-documented exceptions are that ANP also modulates ventricular myocyte volume by regulating the plasma membrane cotransport of Na⁺, K⁺, and Cl^- ions⁷ and that this hormone mediates inhibition of cardiac L-type Ca²⁺ channels through the intracellular production of cGMP, with the consequent activation of protein kinase G.^{8 9}

Since ANP is unequivocally present in atrial myocyte caveolae, we thought it probable that a fraction of atrial myocyte ANP might exist bound within caveolae to ANP-RB. In the present study, we report the immunocytological localization of ANP-RB in two preparations of myocytes derived from atria of adult rats: primary cultures of atrial myocytes and purified freshly dispersed (but not cultured) atrial myocytes, both studied by conventional and confocal immunofluorescence microscopy. For these experiments, antibody against caveolin-3, the muscle-specific isoform of the caveolar coating protein family of caveolins,^{10 11} was used to establish colocalization with ANP-RB by confocal fluorescence microscopy.

	Materials and Methods

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Preparations
Primary cultures of atrial myocytes from the hearts of adult rats were prepared from ether-anesthetized 300- to 350-g male Sprague-Dawley rats by procedures described in this laboratory.^{12 13} The myocytes were grown on Falcon No. 4108 tissue culture–treated glass slides in an eight-chamber polystyrene vessel (Becton Dickinson Labware) and used on days 8 to 9 of culture. Five separate cultures, each made from one or two rats, were studied.

Isolation of Myocytes and Nonmyocytes
For isolation of dispersed rat atrial myocytes and nonmyocytes (not cultured), we applied the following procedure to five separate preparations (one rat per preparation). Briefly, rat atria, excised under ether anesthesia, were minced and incubated with digestive enzyme (2 mg/mL of collagenase, type B, Boehringer-Mannheim) to disperse the cells. All incubations were performed at 37°C. After the first application of proteases and mild trituration of the digested tissues, clumps of cells were transferred to fresh digestion media. The supernatant, which contained damaged myocytes as well as nonmyocytes and red blood cells, was discarded. The tissue clumps were subjected to three more digestions, each of which was followed by more vigorous trituration and collection of fractions partially enriched in myocytes by brief centrifugation (1 minute at 1000g). This atrial myocyte–enriched product (in which atrial myocytes could readily be identified by light microscopy) was used for confocal immunofluorescence microscopy of antibodies against caveolin-3, ANP-RB, and ANP (see below). In all double-labeling experiments, a monoclonal antibody to one primary antigen was paired with a polyclonal antibody to the other antigen.

Fluorescent Labeling of Dissociated Rat Atrial Cells
Atrial myocyte–enriched fractions produced as described above were fixed overnight at 4°C with 4% paraformaldehyde in sodium phosphate buffer, pH 7.4. The cells were pelleted for 1 minute at 100g, and the supernatant was discarded. After resuspension in 10 mL of 100 mmol/L sodium phosphate buffer (pH 7.4) for 10 minutes at room temperature, the pellet was resuspended and pelleted three times, as described previously, and then resuspended for 30 minutes at room temperature in 10 mL of 100 mmol/L NH₄Cl in sodium phosphate buffer. After repelleting and resuspending the pellet in phosphate buffer for 10 minutes, the pellet was resuspended in 5 mL of 0.1% Triton X-100 in the above phosphate buffer at room temperature, pelleted, and resuspended in 10 mL phosphate buffer for 5 minutes. Next, the pellet was resuspended in a microcentrifuge tube containing the selected primary antibody (eg, primary antibody to caveolin-3, ANP-RB, or ANP) in 10% goat serum and 0.02% Tween 20 in sodium phosphate buffer for 1.5 hours at 4°C, pelleted for 2 minutes at 2000 rpm in a microcentrifuge at 4°C, resuspended in 1 mL of sodium phosphate buffer for 10 minutes at 4°C, resuspended in fluorescein and/or rhodamine-labeled secondary antibody in 10% goat serum and 0.02% Tween 20 in sodium phosphate buffer, and resuspended and repelleted twice in 1 mL phosphate buffer for 10 minutes at 4°C. The product thus obtained was resuspended in a small volume and mounted on poly-L-lysine–coated slides in glycerol/phosphate buffer medium. The coverslips were sealed with fingernail polish, allowed to dry, and stored at -20°C for viewing in the confocal microscope.

For these studies, we used five preparations of cultures from each of five animals, with each preparation distributed onto multiple coverslips. For freshly dissociated cells, one or more rats were used per preparation. Five such preparations, each distributed onto multiple coverslips, were studied. One experiment with the antibodies used required at least two coverslips, because a control was run with each experiment. A coverslip routinely contained 100 myocytes.

Confocal Microscopy
Confocal microscopy of primary cultures of atrial myocytes, immunostained as described previously,^{12 13} was carried out by imaging the cells using an Odyssey XL laser scanning confocal microscope (Noran Instruments) and a Carl Zeiss Axioskop 1335 HD/TV, attached to a Silicon Graphics Indy workstation running Noran Instruments InterVision software. The same protocol was also used for confocal microscopy of freshly dispersed (but not cultured) rat atrial myocytes. The advantages of being able to study cultured or dissociated noncultured cardiac myocytes with this instrumentation were twofold: First, this approach made it possible to make "optical sections" through the tissue and to rotate the plane of such sections; second, it permitted identification of sites of colocalization of the respective antibodies to two different proteins, eg, caveolin-3 and ANP-RB.

Using this confocal microscope and the fluorescent antibodies of the preceding paragraph, we had no difficulty finding labeled cells. Not all cells were (or could be) favorably oriented, but all cells that were or could be so oriented were, in our experience, similarly labeled; specifically, no cells were found to be labeled in such a way as to suggest a different or anomalous labeling pattern.

Antibodies
Rabbit polyclonal antibody against ANP-RB was provided by Dr D.L. Garbers, and monoclonal antibody against ANP-RC ("clearance" receptor) was provided by Dr T. Maack. Antibody against ANP was from Peninsula Laboratories. Monoclonal (mouse) and polyclonal (rabbit) antibodies against the muscle-specific isoform, caveolin-3, were obtained from Transduction Laboratories. For confocal microscopy, secondary antibodies were labeled with fluorescein or rhodamine.

Immunoelectron Microscopy
Immunoelectron microscopy of semithin sections, used for localizing caveolin-3 antibody in caveolae of intact rat atria, was performed as described by Chang et al.¹⁴

	Results

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Experiments on Dispersed Single-Cell Preparations of Atrial Myocytes
Preliminary experiments using conventional immunofluorescence microscopy indicated that primary cultures of atrial myocytes could be readily immunostained with antiserum against the high-affinity ANP receptor ANP-RB (Fig 1a), but were not detectably stained by antibody against the low-affinity ANP receptor ANP-RC (data not shown). Therefore, we confined further studies to ANP-RB. We first used conventional immunofluorescence microscopy of primary cultures for exploratory experiments to compare the localization of antibody to ANP-RB with the localizations of antibodies to the muscle-specific isoform caveolin-3^{10 11} (data not shown). Thereafter, we used confocal immunofluorescence microscopy to examine the localization of ANP-RB relative to caveolin-3 in the same section of the same cell. Fig 1b is an electron micrograph of a semithin section of intact rat atrium that has been immunostained with antibody to caveolin-3. This figure documents that in atrial myocytes, as in other mammalian cardiac cell types,^{10 11} this muscle-specific antibody specifically stains rat atrial myocyte caveolae.

View larger version (116K): [in this window] [in a new window]   Figure 1. a, Photomicrograph of primary culture of atrial myocytes from adult rats. The myocytes were stained with antiserum to type B atrial natriuretic peptide receptor and secondary rhodamine–coupled fluorescent antibody. b, Electron micrograph of semithin section of rat atrium immunostained with primary antibody against caveolin-3 and colloidal gold–conjugated secondary antibody. Note the presence of gold particles on cardiac myocyte caveolae and the absence of label on endothelial cell caveolae.

Since ANP is present in caveolae of in situ rat atrial myocytes,¹ we also wanted to test, in primary cultures (a related but somewhat different experimental system), whether antibodies to the caveolar marker protein, caveolin-3, colocalize in caveolae with antibodies to ANP-RB. For this purpose it was useful to examine optical sections collected with the confocal microscope from two regions of the cells: from the cytosol, where caveolin-3 would not normally be expected to be abundant, and from a cell surface region that includes the plasma membrane, where caveolae, and therefore caveolin-3, would be expected to be concentrated (see Fig 1 of Reference 15¹⁵ ).

In Fig 2, panels a and b are confocal micrographs of an optical section of primary cultures. The optical section includes the plasma membrane and is oriented roughly parallel to the cell surface. The cell in Fig 2a has been labeled with caveolin-3 antibody, and the same cell in Fig 2b has been labeled with ANP-RB antibody. Because the ANP-RB antibody was much less potent than the caveolin-3 antibody, the ANP-RB signal was much less intense and yielded fewer punctate densities than that for caveolin-3. The ANP-RB signal was therefore electronically amplified in order to identify areas of colocalization. The images of the two antibodies thus became superimposed to a limited extent (Fig 2c), as indicated by yellow patches at the cell envelope, reflecting the superposition of red and green pseudocolors to yield a yellow pseudostain. However, although in primary cultures the ANP-RB signal was unequivocal (Fig 2b), the confocal microscopic evidence for colocalization of ANP-RB with caveolin-3 at the plasma membrane, while present, was relatively weak.

View larger version (47K): [in this window] [in a new window]   Figure 2. Photomicrographs of optical sections through primary cultured atrial myocytes obtained with the confocal microscope. The section is oriented roughly parallel to and includes the plasma membrane. a, Distribution of antibody to caveolin-3. b, Distribution of antibody to type B atrial natriuretic peptide receptor (ANP-RB). c, Superimposition of panel a on panel b, with caveolin-3 distribution pseudocolored red, ANP-RB distribution pseudocolored green, and areas where caveolin-3 and ANP-RB colocalize pseudocolored yellow. Note that colocalization (yellow) is present but relatively sparse.

Therefore, we reexamined this issue in freshly dissociated (but not cultured) atrial myocytes from adult rats. In Fig 3, panels a, b, and c are images of such myocytes pseudocolored and recorded as described above for primary cultures. Unlike primary cultures of atrial myocytes, which are highly flattened in the dimension perpendicular to the plane of the image, freshly dissociated atrial myocytes retain this dimension and, therefore, their in situ appearance of depth. For optimal interpretability of these confocal images, it was essential to vary and optimize the distance and rotation so as to gain an understanding of the orientation and three-dimensional shape, as well as of the area or volume imaged. In this way, immunostaining of the cell interior could be better differentiated from that of the cell surface region. This advantage is exemplified by the left lower part of Fig 3c (the composite of Fig 3a superimposed on 3b), which shows three regions: a region including the plasma membrane (colored red for caveolin), a subplasmalemmal region (colored green for ANP-RB), and multiple yellow patches denoting colocalization of caveolin-3 and ANP-RB, apparently at the interface between the red- and green-stained regions. Since the plane of the optical section passes obliquely from the cell surface region just described into a region remote from this surface, the confocal image also reveals this interior region of the myocyte. That region appears to be unstained by antibody to either caveolin-3 or ANP-RB, indicating that most of ANP-RB is, like caveolin-3, predominantly associated with and just below the plasma membrane.

View larger version (37K): [in this window] [in a new window]   Figure 3. Photomicrographs of optical sections through freshly dispersed atrial myocytes, which were not cultured. a, Distribution of antibody to caveolin-3. b, Distribution of antibody to type B atrial natriuretic peptide receptor (ANP-RB). c, Superimposition of panel a on panel b, with caveolin-3 and ANP-RB pseudocolored red and green, respectively. Compare with Fig 2, and note more extensive colocalization (yellow) in panel c, as well as a three-dimensional effect, permitting comparison of dense pseudostaining at the cell perimeter with paucity or absence of staining of the cell interior.

Our previously published immunoelectron microscopic finding that in situ rat atrial myocyte caveolae contain ANP¹ and our present hypothesis that this intracaveolar ANP is bound to ANP-RB also predict that antibodies against ANP and ANP-RB should colocalize at the cell surface by confocal microscopy, if caveolae of freshly dissociated atrial myocytes behave, in this respect, like caveolae of in situ atrial myocytes. In Fig 4, panels a, b, and c illustrate the localization of ANP (panel a) and ANP-RB (panel b) in freshly dissociated atrial myocytes. Thus, the confocal image (panel c) is produced by superimposing panel a on panel b, with ANP pseudocolored red, ANP-RB pseudocolored green, and the superposition of panel a on panel b pseudocolored yellow. The distribution of ANP, including large aggregates of atrial granules at the nuclear poles, corresponds well with that in previous electron microscopic studies on rodent atria¹⁵ ; the distribution of ANP-RB overlaps that for caveolin-3 (Fig 3c), produced by Fig 3e superimposed on Fig 3d. It is also apparent that colocalization of ANP with caveolin-3 is readily observable as spots of yellow pseudocolor, which are prominent at the cell periphery of Fig 4f.

View larger version (89K): [in this window] [in a new window]   Figure 4. Photomicrographs of optical sections through freshly dissociated atrial myocytes. a, Atrial natriuretic peptide (ANP). b, Type B ANP receptor (ANP-RB). c, Confocal image (yellow) produced by superposition of panel b (green) on panel a (red). Panel a shows perinuclear aggregates of ANP-containing atrial granules; panel b, distribution of ANP-RB; and panel c, multiple punctate yellow foci of colocalization of ANP and ANP-RB (yellow), most readily seen at the cell periphery. d, ANP (red). e, Caveolin-3 (green). f, Confocal image of panel d superimposed on panel e, showing colocalization of ANP and caveolin-3 (yellow).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Is ANP-RB Localized in Atrial Myocyte Caveolae?
Our previous finding that ANP is present in atrial myocyte caveolae of intact rat atria¹ suggests that the ANP is bound to one of the known receptor types for this peptide. In the present study, we have predominantly studied primary cultures of atrial myocytes and dispersed (but not cultured) atrial myocytes rather than in situ myocytes of intact atria, because both the application of confocal immunofluorescence microscopy and the interpretation of the results of these experimental systems are more straightforward with these well-studied systems consisting of multiple dispersed single cells of the same cell type. The findings of the present study indicate unequivocally that a protein immunostaining with antiserum to ANP-RB colocalizes by confocal fluorescence microscopy with caveolin-3-immunoreactive sites. Such sites are cell surface–associated, presumably at the plasma membrane. Most of the remaining immunostaining for ANP-RB appears to occupy the cytosolic domain immediately subjacent to the plasma membrane. It seems probable that some portion of the immunoreactive ANP-RB detected by fluorescence microscopy in this subplasmalemmal domain may be within vesicles too small to be resolved with the techniques used here. Because the antigenicity of the ANP-RB antibody available to us did not survive the fixation procedures necessary for immunoelectron microscopy, we could not confirm this conjecture at the ultrastructural level. Nevertheless, although not a completely closed argument, the finding (by confocal fluorescence microscopy) that both ANP and ANP-RB colocalize in freshly dissociated atrial myocytes both with each other and with caveolin-3, taken together with our previous immunoelectron microscopic documentation that ANP is present within caveolae of in situ rat atrial myocytes,¹ strongly supports an intracaveolar localization of ANP-RB in rat atrial myocytes, both in situ and in cultured or freshly dissociated myocytes.

Functional Implications of Intracaveolar ANP-RB
It is of interest to consider the potential functional implications of intracaveolar ANP bound to ANP-RB. Since the guanylate cyclase activity is expressed entirely by amino acid sequences on the cytoplasmic surface of the caveolar membrane, this discussion focuses on the significance of guanylate cyclase activity in the cytoplasmic microdomain immediately subjacent to the caveolar membrane. This microdomain is already known to be the locus for the cardiac plasma membrane Ca²⁺ pump ATPase¹⁶ and for an isoform of the inositol triphosphate receptor.¹⁷

Although the cGMP generated in the subcaveolar cytoplasmic domain of cardiac myocytes by ANP-RB may ultimately be implicated in multiple and diverse reactions, two physiologically important ANP- and cGMP-dependent processes are already supported by substantial experimental evidence: (1) the modulation of myocyte volume by regulation of Na⁺, K⁺, and Cl^- ion cotransport,⁷ as well as the cGMP-mediated inhibition of L-type Ca²⁺ channel activity in response to the binding of ANP to ANP-RB, and (2) the associated intracellular activation of cGMP-dependent protein kinase G.^{4 8 9} Tohse et al⁸ have suggested that this ANP-induced inhibition of Ca²⁺ channel activity contributes to the negative inotropic effect of ANP. The plurality of cGMP-mediated processes, taken together with the plurality of caveolar and noncaveolar membrane microdomains, suggests the possibility that the functional effects of ANP-RB may be compartmentalized.

It is also plausible that the presence of ANP-RB in caveolae of atrial myocytes confers on the myocyte the ability to sense the concentration of ANP in the interstitial space by monitoring the degree of saturation with ANP of the binding sites in the intracaveolar ANP binding domain of the receptor. The myocyte could then respond to this information by appropriately adjusting the guanylate cyclase activity in the cytoplasmic domain of the molecule.⁵ In addition to monitoring the concentration of endocrine ANP impinging on the atrial myocyte caveolae from the circulating blood, variants of this scheme also suggest that caveolar proteins like ANP-RB may engage in paracrine interactions with neighboring nonmuscle cells, as proposed by Anderson.²

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-54302 and HL-105503. We thank Drs D.L. Garbers and T.L. Maack for providing us with antibodies to ANP-RB and ANP-RC, respectively, and Dr Dorothy A. Hanck for a critique of the manuscript. Drs Eugene Chang and T.A. Brasitus generously gave us access to the confocal microscopy facility in the Gastroenterology Section of the Department of Medicine.

Received January 16, 1997; accepted April 7, 1997.

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