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Am J Physiol Heart Circ Physiol 294: H829-H838, 2008. First published November 21, 2007; doi:10.1152/ajpheart.01141.2007
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Cholinergic location of {delta}-opioid receptors in canine atria and SA node

Shekhar H. Deo, Matthew A. Barlow, Leticia Gonzalez, Darice Yoshishige, and James L. Caffrey

Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas

Submitted 2 October 2007 ; accepted in final form 15 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{delta}-Opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75–85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75–85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.

opioids; vagal function

ENKEPHALINS, the presumed endogenous agonists for the {delta}-opioid receptor (DOR), are abundant in the heart (20, 26, 39). Although locally administered enkephalin produces robust physiological responses in heart (10–13, 33), the location of the DORs mediating these responses remains a matter of discussion (5, 21, 27). Myocardial enkephalin content is relatively low (20, 39), and the enzymes that degrade enkephalin are both aggressive and pervasive (19, 36). Enkephalin-mediated changes in cardiac function are difficult to demonstrate in the absence of nerve traffic (5, 18, 33). These observations suggest that cardiac enkephalins are primarily paracrine in character and the DORs mediating their effects do so by moderating local neurotransmission. When neurotransmitters were administered locally, the myocardial effects of enkephalin were eliminated, suggesting that the opioid receptors mediating these responses were located prejunctionally on the nerve terminals innervating the heart (5, 10, 18, 33).

DORs belong to the superfamily of G protein-coupled seven transmembrane receptors (34). The identification of the sequence of the receptor protein has made immunochemical quantification and localization of the DOR possible. Despite evidence for a single DOR amino acid sequence (16), physiological and biochemical studies support the existence of two functional subtypes of the DOR. Farias et al. (13) demonstrated subtype-specific bimodal actions for DORs in canine sinoatrial (SA) node. The DOR-1 phenotype improved the vagal transmission in the SA node, while the DOR-2 phenotype impaired it. The predicted net effect would then depend on the concentration of enkephalin locally and the relative proportion of receptors assuming each receptor phenotype.

A working hypothesis based on cultured cell systems suggests that the DOR-1 phenotype couples to Gs{alpha} and DOR-2 to Gi{alpha} (8). DOR-1-Gs{alpha} coupling thus increases adenylyl cyclase activity and intraneuronal calcium and facilitates acetylcholine release to improve vagal transmission. In contrast, DOR-2 coupling reduces cyclase activity and impairs vagal transmission. Excitatory Gs{alpha}-coupled receptors were also more efficient and were active at lower opioid doses (8). The local membrane environment appeared to determine which of the opposing receptor phenotypes predominated.

Cardiac DORs participate in the cardioprotection mediated by ischemic preconditioning (31, 32). DOR stimulation also reduced arrhythmias and preserved the viability of isolated cells and organs (25, 35). The location of the responsible receptors remains undefined, but studies in vitro (21, 27, 35, 39) support the widely held presumption that postjunctional DORs are involved. Intrinsic adrenergic cells, cardiomyocytes, adipocytes, fibroblasts, and resident leukocytes are all potential sites for nonneural DORs (21, 27, 29, 35).

Opioids, however, traditionally function as neuromodulators that moderate neurotransmitter release, and considerable support for this view is evident in other parts of the nervous system (1, 14, 24, 25, 40). Acute functional responses in heart are consistent with the neural thesis and suggest that a substantial proportion of myocardial DORs are located prejunctionally on parasympathetic nerves. However, a discrete location for DOR in heart remains unsettled. The present study combines immunofluorescent, cytochemical, and biochemical methods to demonstrate that DORs concentrated in the canine SA node and atria are strongly associated with cholinergic nerve fibers and isolated synaptosomes and not with nearby adrenergic structures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Canine right atrial and sinus node tissues were obtained from mixed-gender adult (15–25 kg) mongrel dogs. The Institutional Animal Care and Use Committee, in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, approved all procedures. Animals were anesthetized with pentobarbital sodium (32.5 mg/kg), intubated, and mechanically ventilated initially at 225 ml·min–1·kg–1 with room air. A right thoracotomy exposed the right heart between ribs three and four. The heart was briefly fibrillated with a surface electrode, and the cardiac tissues were excised and either fixed in paraformaldehyde or frozen at –20°C.

SA node from three dogs was fixed in 4% paraformaldehyde for 2 h and progressively equilibrated in 15% and 30% sucrose solutions. The fixed nodal tissue was embedded in OCT-4583 (Sakura Fine Technical, Tokyo, Japan) and stored frozen at –90°C.

Preparation of Cardiac Synaptosomes

SA nodal and right atrial tissues were dissected free from fat and connective tissue. Tissues were minced in iced 0.32 M sucrose and digested with collagenase in a shaking water bath for 60–90 min at 37°C. The collagenase (Worthington, 200 U/g wet wt) was prepared in HEPES-buffered salt solution containing (mM) 50 HEPES (pH 7.4), 144 NaCl, 5 KCl, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose (38). Partially digested tissues were collected by low-speed centrifugation (1,000 g) for 5 min at 4°C. The tissue pellet was suspended in 4 vols of 0.32 M sucrose solution and homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 650 g for 10 min at 4°C, and the supernatant was collected. The pellet was suspended again in 4 vols of 0.32 M sucrose, homogenized, and centrifuged again at 650 g for 10 min at 4°C. The two supernatants were combined, and the constituent synaptosomes were separated by centrifugation at 20,000 g for 20 min at 4°C. The enriched synaptosome pellet was suspended in 500 µl/g of HEPES-buffered salt solution per original wet tissue weight and purified further by discontinuous gradient centrifugation. The synaptosomal suspension was layered on 1.2 M sucrose and centrifuged at 20,000 g for 120 min at 4°C. Purified synaptosomes were retrieved by pipette from the 0.32 M–1.2 M sucrose interface. The synaptosomes were either stored at –20°C or fixed by mixing with equal volumes of 4% paraformaldehyde.

Immunocytochemistry

Fixed synaptosomes obtained from SA node or right atrial sources were centrifugally dispersed with a cytocentrifuge (CytoPro 7620; Wescor, Logan, UT) onto precharged glass slides at 1,000 rpm. Each slide contained 60 µl of synaptosomes (13–35 µg protein). Specific antisera for DOR (rabbit anti-DOR), vesicular acetylcholine transporter (goat anti-VAChT) (2), and tyrosine hydroxylase (mouse anti-TH) (4) were obtained commercially from Chemicon Intl. (Chicago, IL) and for synapsin I (goat anti-Syn) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Synaptosomes were reacted with specific antibodies at the dilutions indicated in Table 1 for 12–18 h at 4°C. After incubating with primary antibodies the sections were rinsed in phosphate-buffered saline (PBS) and then incubated with the secondary antibodies (listed in Table 1) for 60 min in the dark. The slides were rinsed in PBS and sealed under coverslips with Fluoromount-G reagent (Electron Microscopy Sciences, Hatfield, PA). Slides were examined by fluorescent microscopy.


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  		Table 1. Immunocytochemical labeling of synaptosomes

 
Immunohistochemistry

Fixed SA node tissues from three dogs were sectioned by cryostat (12 µm, Vibratome UltraPro 5000, St. Louis, MO) perpendicular to the SA node artery, and the slices were mounted on charged slides. The dilutions for the primary and secondary antibodies are listed in Table 2. Sections were examined with fluorescent microscopy at x10–40 magnification or with confocal microscopy at x40 magnification.


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  		Table 2. Immunohistochemical labeling for nodal tissue sections

 
Control

Control slides for synaptosomes as well as tissue sections were incubated with equal protein concentrations of isotype-specific immunoglobulin (IgG) matched with the IgG class of the respective primary antisera (Vector Laboratories, Burlingame, CA). In addition, the exposure time required to acquire images for the experimental slides under the fluorescent microscope was matched for the corresponding control slides.

Fluorescent Microscopy

Specimens were examined with the Olympus AX70 Fluorescence Imaging System with automatic photomicrography (Olympus America, Center Valley, PA). Alexa Fluor 488 was imaged with the fluorescent filter using 482-nm excitation and 536-nm emission wavelengths, while Alexa Fluor 594 was imaged with the filter using 562-nm excitation and 624-nm emission wavelengths. Images were acquired at the surface of the specimens in two dimensions.

Quantification of Dual Immunolabeling

Synaptosomes. The numbers of dual-labeled (e.g., DOR-VAChT and DOR-TH) synaptosomes were analyzed and quantified with ImagePro Plus 5.1 software (Media Cybernetics, Silver Spring, MD). All counts were performed in the fluorescent microscope with the x40 objective. Two mounted synaptosome slides were prepared from each animal for each treatment (antibody pairing). Five microscopic fields were analyzed from each slide. The resulting 10 analyses were averaged and treated statistically as 1 value. Colocalization of DOR with selected synaptosomal targets of interest (Syn, VAChT, and TH) was calculated as a (yellow/red) percentage.

Nodal tissue sections. The numbers for colocalization of DOR-VAChT and DOR-TH in neural varicosities were analyzed with Image J 1.35 software (developed by Dr. Wayne Rasband at Research Services Branch, National Institute of Mental Health, Bethesda, MD). In each tissue slice, colocalization was specifically quantified in the wall of the nodal artery, in the adjacent nodal tissue, and among the nearby atrial muscle cells. The colocalization counts in each field were quantified digitally at x10. Two adjacent sections from three different animals were analyzed for colocalization of DOR and VAChT and DOR and TH. The DOR overlap with cholinergic and adrenergic structures was again expressed as a percentage.

Western Blot

Cardiac synaptosomes obtained as described above were analyzed for protein content (Lowry), and 20 µg of each preparation was separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary anti-DOR and anti-Syn antibodies (1:1,000 rabbit polyclonal antibodies, Chemicon Intl.). The membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit gamma globulin (1:10,000; Amersham Biosciences). The blots were developed with an enhanced chemiluminescence kit (Super Signal West Dura Extended Duration Substrate, Pierce Biotechnology, Rockford, IL). Densitometric analysis of the bands was performed with image analysis software (Scion, National Institutes of Health, Bethesda, MD). The relative band intensities were expressed as a DOR-to-Syn ratio as means ± SE for the three individual experiments.

Estimation of Synaptosomal Acetylcholine

Synaptosomal acetylcholine content was estimated by fluorospectrometry with Amplex Red (Invitrogen-Molecular Probes, Eugene, OR). Synaptosomal acetylcholine was hydrolyzed with added acetylcholine esterase (Sigma-Aldrich, St. Louis, MO), and the choline was converted to betaine and hydrogen peroxide with choline oxidase (Sigma-Aldrich). The peroxide was quantified by reaction with added HRP (Sigma-Aldrich) and Amplex Red to generate the highly fluorescent product resorufin. The final assay was conducted in 50 mM Tris pH 8.0 with 10,000 U/ml acetylcholine esterase, 4 U/ml HRP, 0.4 U/ml choline oxidase, and 1 U/ml Amplex Red. The fluorescence was recorded at excitation and emission wavelengths of 563 and 587 nm, respectively, at 500 V with AB2 software in an Aminco-Bowman Series 2 Luminescence Spectrometer (SLM-Aminco, Urbana, IL). Values were compared with choline standards.

Estimation of Synaptosomal Norepinephrine

Atrial and SA nodal synaptosomes (11–30 µg of protein) were extracted in 1 N acetic acid and 0.02 N HCl, boiled, and neutralized with 1.5 M Tris at pH 8.6. The catecholamines were extracted with alumina (Bioanalytical System, West Lafayette, IN), eluted with 0.1 M perchloric acid (Fischer Scientific, Fair Lawn, NJ), and separated by HPLC on a reverse-phase C18 analytical column (150 mm x 3.9 mm, 5 µm, Waters, Milford, MA). Norepinephrine was eluted with 5 mM lithium acetate (Sigma-Aldrich), 2.0 mM sodium 1-heptanesulfonate (Sigma-Aldrich), 100 µM EDTA (Sigma-Aldrich), and 40% methanol (Fisher Scientific) and quantified coulometrically with an ESA Coulchem III detection system. 3,4-Dihydroxybenzylamine (DHBA) was used as the internal standard.

Statistical Methods

All data are means and SEs. Differences were evaluated with ANOVA, and post hoc analysis was performed with Tukey's test for multiple cross comparisons and Dunnett's test for multiple comparisons to control. Differences determined to occur by chance with a probability <0.05 were deemed statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Tissue Sections

Nodal arterial sections.
MORPHOLOGICAL DESCRIPTION. The SA node artery traverses the long axis of the SA node and serves as a reliable marker for orientation within the node. The junction of tunica media and tunica adventitia in the wall of the nodal artery is also a reliable location in which to demonstrate autonomic nerves. The arterial wall served as a representative site in which to probe the relationship between DORs and autonomic nerve terminals. Intense green fluorescent labeling for DOR concentrated on punctuate varicose processes within the wall of the nodal artery. These strings of beaded fluorescence appeared as predicted at the junction of tunica media and tunica adventitia encircling the arterial lumen. Colocalizing neural peptide targets (Syn, VAChT, and TH) appeared as red filamentous profiles in the same area as that of the DOR.

Figure 1, A and B, illustrate the same nodal arterial cross section immunolabeled for DOR and Syn, respectively. The merged image in Fig. 1C illustrates significant areas of overlap between Syn and DOR, particularly at the junction of tunica media and tunica adventitia. There were two other areas of concentration and colocalization, the endothelium and the presumed pacemaker area. Figure 1D is an example of an identical control merged image of a serial section in which isotype-specific immunoglobulin was substituted for the two primary antibodies. Figure 2, A and B, represent similar images for another section localizing DOR and the cholinergic marker VAChT in the same general regions of the section. The merged image in Fig. 2C illustrates a significant degree of overlap of the DOR signal with VAChT, presumably representing cholinergic varicosities. Figure 3A presents a higher magnification (x40) of the vessel wall in Fig. 2C observed under the confocal microscope. The image clearly demonstrates intense discrete areas of colocalization between DOR and VAChT both in the outer vessel wall and within the endothelium. Progressive confocal images confirmed the colocalization of the two labels in the same visual volumes. Figure 4 illustrates another representative section through the nodal artery immunostained in this case for DOR and the adrenergic marker TH. The labeling for DOR and TH was similar to that described above. Both labels were again concentrated in the outer vessel wall and in the endothelium. While there appears to be continued signal overlap within the endothelium, the merged image in Fig. 4C clearly suggests that the DOR and TH are labeling different structures within the outer wall. These tissue images suggest that DOR preferentially localizes with cholinergic fibers within the nodal artery wall and not with nearby adrenergic structures.


Figure 1
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  		Fig. 1. Fluorescent images illustrate {delta}-opioid receptor (DOR; green, A)- and synapsin (Syn; red, B)-positive nerve terminals at the junction of tunica media and tunica adventitia in the wall of the sinoatrial (SA) nodal artery. Merged image in C superimposes the fluorescence to illustrate areas of colocalization. D: identical control merged image of a serial section in which isotype-specific immunoglobulin was substituted for primary antibodies. Fluorescent images were acquired at x10 magnification in a standard fluorescent microscope. Scale bars = 50 µm. TM, tunica media; TA, tunica adventitia; ET, endothelium; PM, pacemaker region; M, atrial muscle.

 

Figure 2
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  		Fig. 2. Fluorescent images illustrate DOR (green, A)- and vesicular acetylcholine transporter (VAChT; red, B)-positive nerve terminals at the junction of tunica media and tunica adventitia in the wall of the SA nodal artery and in the adjacent nodal parenchyma (bottom left). Merged image in C superimposes the fluorescence to illustrate areas of colocalization. Fluorescent images were acquired at x10 magnification in a standard fluorescent microscope. Scale bars = 50 µm.

 

Figure 3
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  		Fig. 3. A: higher-magnification (x40; scale bar = 10 µm) confocal image of part of the same vessel wall shown in Fig. 2C. Image illustrates dense areas of DOR-positive cholinergic nerve terminals. B: nearby section of right atrial muscle that demonstrates colocalization of DOR-positive nerve fibers with cholinergic varicosities running parallel to the long axis of the atrial muscle fibers; merged 2-dimensional image was acquired at x40 (scale bar = 10 µm). CN, cholinergic nerves.

 

Figure 4
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  		Fig. 4. Fluorescent images illustrate DOR (green, A)- and tyrosine hydroxylase (TH; red, B)-positive labeling near the SA nodal artery and in the adjacent nodal parenchyma. The majority of the TH labeling near the periphery in B is autofluorescence illustrated to demonstrate the absence of TH labeling in the arterial wall. Merged image in C superimposes the fluorescence and reinforces the absence of TH colocalization with DOR positive nerve tracts within the vessel wall. Fluorescent images were acquired at x10 magnification in a standard fluorescent microscope. Scale bars = 50 µm.

 

QUANTIFICATION. DOR labeling in these autonomic nerve terminals was quantified with image analysis software and expressed as a relative fraction for each arterial cross section. Figure 5 illustrates percent colocalization for DOR relative to Syn, VAChT, and TH in the nodal artery wall. About 75% of the Syn-positive nerve terminals in the arterial wall were DOR-positive. The majority of those DOR-positive (85%) structures are also positive for the cholinergic marker VAChT. In contrast, DOR localization with TH was near 8%. Thus a significant proportion of DORs were associated with nerves in the arterial wall, and the vast majority of those nerves were cholinergic rather than adrenergic in character. The antibody controls for these slides were consistently nonreactive when analyzed for the same structural areas and at the same time exposures.


Figure 5
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  		Fig. 5. Quantification in tissue sections: estimation of proportions of DORs on nodal artery, nodal tissue, and right atrial muscle based on colocalization percentages. Graph is quantitative representation of DOR-positive areas in total (Syn), cholinergic (VAChT), and adrenergic (TH) nerve terminals. Values illustrated are DOR% in the respective nerve terminal. Note significantly higher percentage of DOR on cholinergic nerve terminals in arterial wall, nodal tissue, and atrial muscle than on corresponding adrenergic structures. Values are means and SE for 3 subjects. **Number of colocalizations in cholinergic nerves was significantly different from that in adrenergic nerves (P < 0.01). SAN, SA node.

 
Nodal tissue.
MORPHOLOGICAL DESCRIPTION. The SA node is composed of loose connective tissue, pacemaker cells near the artery, and atrial myocytes that are progressively better organized structurally as one moves from the node into the atria (23). The presumed pacemaker tissues are outside the vessel wall in the lower left quadrant of Figs. 1–4. Although not illustrated in the images, 4',6-diamidino-2-phenylindole (DAPI)-stained nuclear profiles indicated that the area was densely populated with noncontractile cells. DOR-positive and VAChT-positive structures in this quadrant distributed in percentages similar to those above among the Syn-positive nerve fibers. Once again, 80% of the nerves were DOR-positive. The number of apparent colocalizations between DOR and TH were by comparison equally few in number at 10%.


QUANTIFICATION. As illustrated in Fig. 5, a DOR-positive signal was consistently recorded from 80% of the Syn-positive nerve terminals. The DOR signal was also similarly colocalized 81% of the time with VAChT and only 10% of the time with TH. Once again, a large proportion of DORs are closely associated with cholinergic nerve terminals in the SA node and much less so if at all with adrenergic nerve terminals.

Right atrial muscle.
MORPHOLOGICAL DESCRIPTION. Figure 3B illustrates an atrial muscle section at higher magnification (x40) not far from the SA node artery. The cells sectioned parallel to their long axes provide a clear illustration of a network of DOR-positive nerve fibers enveloping the atrial myocytes. The DOR signals appeared to be organized as intermittent varicosities parallel to the surface of the myocytes. The tissue section was also stained for VAChT, which colocalized with DOR 76% of the time. The image illustrates occasional intense VAChT-positive, DOR-negative cholinergic fibers mixed with the DOR-positive fibers. Staining for DOR and TH among the atrial myocytes in serial sections from each of the same animals provided much lower rates of colocalization.


QUANTIFICATION. Figure 5 illustrates numerically equal percentages for DOR-Syn and DOR-VAChT colocalizations of 76% in among the atrial myocytes. DOR-TH localizations were recorded only 17% of the time. The somewhat larger colocalization of DOR with TH in areas populated by myocytes suggests potential differences in the degree of opioid influence over pacemaker and contractile activities.

Synaptosomes

Morphological description. Synaptosomes are spheres of nerve membranes that form after disruption of the terminal nerve fibers. Such spheres are generally devoid of nuclear material and thus do not stain with DAPI. However, synaptosomes do contain neurotransmitter vesicles and, presumably during formation, may enclose other buoyant cell particulates and cytoplasmic constituents. Synaptosomes were prepared from SA node and right atrium and were immunolabeled for the target peptides DOR, Syn, VAChT, and TH. When centrifugally dispersed on charged slides in appropriate concentrations, immunolabeled synaptosomes in the microscope field appear as fluorescent stars in a dark field. Fluorescent signals and their respective quantitative colocalizations were based on software analysis of the digital images.

Quantification.
SA NODE SYNAPTOSOMES. Syn labeling verified the synaptosomal character of the dispersed particulate and served to normalize the relative numbers of DOR-, VAChT-, and TH-positive synaptosomes. As illustrated in Fig. 6, right, 80% of the synaptosomes were cholinergic and <10% were adrenergic. Even higher percentages (85%) of the synaptosomes had expressed DORs. In addition to the DOR-Syn colocalization profiles, limited numbers of DOR-negative but Syn-positive and DOR-positive and Syn-negative synaptosomes were also evident on the synaptosomal slides. The Syn-positive but DOR-negative synaptosomes likely derive from adrenergic nerves and other nonautonomic nerves that do not bear axon terminal DORs.


Figure 6
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  		Fig. 6. Quantification in synaptosomes: estimation of proportions of DORs on SA nodal and right atrial synaptosomes based on colocalization percentages. Graph is quantitative representation of DOR-positive synaptosomes from total (Syn), cholinergic (VAChT), and adrenergic (TH) nerve terminals. In addition, figure illustrates % of cholinergic (VAChT-Syn) and adrenergic (TH-Syn) synaptosomes. Values illustrated are DOR% in the respective nerve terminal in the first 3 groups of bars. The fourth and fifth groups represent % of cholinergic and adrenergic synaptosomes. Note significantly higher % of DOR on cholinergic nerve terminals in nodal as well as atrial synaptosomes. Values are means and SE for 3 subjects. **Number of colocalizations in cholinergic nerves was significantly different from that in adrenergic nerves (P < 0.01).

 
A direct comparison between cholinergic and adrenergic synaptosomes regarding the distribution of DORs indicates that 82% of the cholinergic synaptosomes from the SA node express DORs while only 8% of adrenergic synaptosomes do so. DORs appear to be preferentially associated with cholinergic nerve terminals by a wide margin in the SA node. There were small percentages of synaptosomes observed outside of DOR-VAChT colocalization profiles. The VAChT-positive but DOR-negative profiles may represent a subset of DOR agonist-resistant cholinergic fibers. The VAChT-negative but DOR-positive profiles may be DORs derived from contaminating nonsynaptosomal membranes derived from atrial myocytes, fibroblasts, or adipocytes, all of which reportedly express DOR. The 8% TH-DOR colocalization represents a low fractional distribution on an already much smaller number of TH/Syn-positive adrenergic synaptosomes.

The control slides in which the isotype-specific IgG was substituted for the primary target antibody of interest were uniformly unreactive with the secondary antibodies.


RIGHT ATRIAL SYNAPTOSOMES. The fractional ~10:1 distribution of VAChT-positive relative to TH-positive synaptosomes from atria was remarkably similar to the estimates described above for synaptosomes isolated from the SA node. Fractional estimates for DOR-positive profiles among the total atrial synaptosomes were significantly lower at 57%. Once more, expressed DORs were preferentially concentrated on VAChT-positive, cholinergic synaptosomes compared with TH-positive, adrenergic synaptosomes at a now familiar ~8-to-1 ratio. As illustrated in Fig. 6, anti-DOR antibodies immunolabeled 79% of the cholinergic synaptosomes and only 11% of the adrenergic synaptosomes. These results confirm the consistent preferential association of DOR with presumed prejunctional cholinergic nerve terminals.

Western blot. Western blots were conducted with synaptosomal proteins to provide additional support for the identity of synaptosomal DOR. Figure 7A illustrates immunoreactive bands obtained with rabbit anti-DOR and rabbit anti-Syn antibodies on nodal and atrial synaptosomes. Equal amounts of proteins were loaded in each well. The image illustrates distribution of synaptosomal proteins in six lanes, with the first three lanes for synaptosomes from the SA node of three different dogs and the next three lanes for right atrial synaptosomes from the same animals. Two immunoreactive bands were evident for DOR corresponding to molecular masses of ~75 and 50 kDa. The estimated molecular masses were consistent with those reported for monomers and dimers of the DOR in other species. Parallel gels were immunostained for Syn, and an appropriate molecular mass band was quantified by densitometry at ~100 kDa. The density for each DOR band was normalized to the density for the total Syn in the same sample.


Figure 7
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  		Fig. 7. Verification of the presence of DOR on synaptosomes by Western blot analysis. A: Western blots with rabbit anti-DOR and rabbit anti-Syn antibodies. Proteins were derived from synaptosomes obtained from canine SA node and right atrium (n = 3). First 3 lanes represent proteins from SA node of dogs 1, 2, and 3, while second 3 lanes represent proteins from right atrium of dogs 1, 2, and 3. Thus first and fourth lanes represent nodal and atrial synaptosomal proteins, respectively, from dog 1. Protein loading was 20 µg/lane. As illustrated, 2 immunoreactive bands were obtained for DOR at 75 and 50 kDa. B: densitometric analysis for the Western blots in A. DOR intensities were normalized to Syn from the same extract. Open bars illustrate the total DOR-to-Syn intensity ratio, and filled and gray bars indicate similar ratios for monomers and dimers, respectively. Graph illustrates significantly higher DOR protein content in SA node than in right atrium. Values are means and SE for 3 subjects. **Expression of total and monomeric DOR in SA node was significantly different from that in right atrium (P < 0.01). OD, optical density.

 
Figure 7B graphically presents the mean DOR-to-Syn ratios for both the SA node and the right atrial synaptosomes: open bars illustrate the total DOR-to-Syn ratio for both tissues, and black and gray bars illustrate the ratios for the monomers and dimers, respectively. DOR abundance was significantly greater in SA node synaptosomes than in companion atrial synaptosomes. The difference was largely attributable to a greater abundance of monomers in the nodal synaptosomes. These immunoblot analyses corroborate that the immunoreactivity for the synaptosomal DOR is consistent with its reported molecular mass. Finally, DOR may be more concentrated in the SA node.

Table 3 illustrates the mixed neurotransmitter content of the synaptosome preparations. Acetylcholine content was greater in nodal synaptosomes compared with the right atrium. Acetylcholine content was likewise much greater than that for norepinephrine in both SA node and right atrium, supporting the predominantly cholinergic character of the synaptosomes as isolated from both SA node and right atrium. In addition, norepinephrine content in the nodal adrenergic synaptosomes was greater than in the atrial synaptosomes.


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  		Table 3. Acetylcholine and NE content from SA nodal and right atrial synaptosomes

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The observations presented here support the suggestion that a large proportion of DORs in heart are on postganglionic prejunctional parasympathetic nerve terminals. Both tissue sections and isolated synaptosomes were internally consistent, with 80% of the immunolabeled DORs associated with VAChT-labeled cholinergic membranes. More specifically, 85% of nodal synaptosomes were DOR-positive and 82% of cholinergic synaptosomes were DOR-positive. If these two populations are the same, then 100% of the DOR-positive nodal synaptosomes may be cholinergic. That estimate would represent an upper limit for DOR expression on nodal cholinergic synaptosomes. If the DOR/VAChT-positive synaptosomes were not all coincident, the minimum overlap predicted would approximate 68%. The DOR-positive synaptosomes from atrial muscle were a smaller proportion (57%) of the total synaptosomes, suggesting greater numbers of nonautonomic nerve terminals in the atria. The remaining 43% Syn-positive membranes were DOR-negative. Of the total synaptosomes, 8% were adrenergic. About 79% of the atrial synaptosomes were cholinergic, and 79% of those were DOR-positive. Thus the estimated upper limit of the total DOR positive synaptosomes in atria would be 62%. Since two estimates of total DOR-positive synaptosomes were similar (57% and 62%), all of the atrial DORs may be associated with cholinergic nerve endings. The DOR-VAChT relationships were not evaluated in ventricular tissue, and whether the same atrial pattern persists in the ventricle remains unclear.

The presence of DORs on the adrenergic synaptosomes was significantly lower for synaptosomes from both SA node and atrium. Few (6–8%) of the total synaptosomes were adrenergic, and a small percentage of the adrenergic synaptosomes were DOR-positive. Thus <1% of the total synaptosomes were dual DOR/TH labeled. Although opioids can modify adrenergic function and norepinephrine secretion in the heart, the receptor is most likely {kappa}-opioid in character (3, 18, 33). Whether the DOR/TH-positive membranes are physiologically significant or represent a methodological background cannot be determined from the present data. Contamination of cholinergic synaptosomes during isolation with soluble TH might contribute to false positive results of this small magnitude.

Intact cultured ventricular cardiomyocytes and resident adrenergic cells isolated from myocardial digests both convincingly immunostain positive for DOR (21, 27). Membranes from both of these cell types might contaminate the synaptosomal preparation. Resident adrenergic cells do not seem sufficiently abundant to account for the very high DOR-Syn staining. However, small numbers of chromaffin-like cells might easily account for the much lower DOR-TH colocalization. Contamination with DOR associated with the potentially far more abundant cardiomyocyte membranes is less easily ruled out but could again represent an atrial-ventricular or species difference in the origin of the cells. Cell surface DORs are commonly associated with lipid rafts and routinely exchange with intracellular DOR pools. The receptor exchange may involve caveolar membranes, and DOR protein was reportedly isolated in rat caveolin-rich cardiomyocyte membrane fractions (25). The isolation of cardiac caveolar membranes and synaptosomes involve similar density gradient centrifugation systems, and some degree of cross contamination might be expected. The DOR observed in tissue slices was colocalized 80% of the time with nerves, and DOR staining near myocytes appeared to be discretely associated with neural structures. Thus the evidence for a neural association is significant. Despite the absence of convincing DOR-myocyte localizations, 20% of DOR-positive staining was quantified morphometrically as Syn-negative and thus remained unaccounted for. DORs actively traffic between the cell surface and substantive pools of intracellular receptor. The specific identification of DORs in cultured cardiomyocytes and cultured myocardial chromaffin cells suggests perhaps that those DORs were available on the surface of the cultured cells but may have been otherwise sequestered in intact tissues and thus unavailable to interact with labeling antibodies.

The high acetylcholine content relative to norepinephrine combined with the VAChT and TH labeling supports the supposition that the synaptosomes isolated in the present study were predominantly cholinergic in character. The greater acetylcholine content in the nodal synaptosomes relative to atrial synaptosomes suggests that the cholinergic innervation of the node may be better equipped to make and release acetylcholine than its atrial counterpart.

Western blot analysis confirmed that the DOR immunoreactivity extracted from the synaptosomes was consistent with the expected molecular mass of the receptor based on reports in other animals (1, 16, 28). The proportionately greater density of the DOR bands from nodal synaptosomes relative to atrial synaptosomes was consistent with the parallel results from immunostaining of the synaptosomes and their quantification under the fluorescent microscope. Western analysis also indicated that DOR monomers were proportionately greater in the nodal synaptosomes. Both electrophoresis and fluorescent microscopy suggested that nodal synaptosomes expressed ~50% more receptor than atrial synaptosomes. The Western analysis suggested that much of the difference resulted from greater numbers of DOR monomers in the SA node. Monomers and dimers could represent the functional DOR-1 and DOR-2 phenotypes of the receptor. The formation of DOR monomers also appears to precede their internalization and presumed inactivation (9). Although opioids regulate the vagal control of both heart rate and atrial contraction, it remains unclear whether the greater numbers of DORs or greater numbers of monomers in the SA node translate into a difference in function or sensitivity.

Syn is a membrane protein situated specifically on synaptic vesicles and nerve terminals. VAChT is a membrane-associated transporter that moves acetylcholine into the synaptic vesicles in cholinergic nerves (2). The close association of DORs, Syn, and VAChT strongly suggests that the majority of DORs in the atria and SA node are on postganglionic prejunctional parasympathetic nerve terminals.

The strong colocalization of DOR and VAChT within fibers innervating the nodal artery wall and endothelium was surprising. Images from the confocal microscope confirmed the discrete neural tract-like appearance of these signals, suggesting that opioids may moderate coronary blood flow as well as heart rate and contractility.

The heart receives qualitatively different parasympathetic innervations from the dorsal motor nucleus of vagus and nucleus ambiguus (6, 7). The nucleus ambiguus projects faster type B fibers, and the dorsal motor nucleus projects slower type C fibers. Two-thirds of the fibers to the right heart originate from the nucleus ambiguus, and these faster fibers are likely responsible for the fast phasic regulation of heart rate and atrial contraction associated with vagal stimulation. Similar percentages of atrial cholinergic fibers are DOR-positive, and approximately two-thirds of the vagal response in vivo is routinely susceptible to blockade by administered enkephalin. These observations suggest the hypothesis that the DOR-positive vagal fibers originate in the nucleus ambiguus and opioids moderate their acute vagal responses. Alternatively, the enkephalin-resistant, DOR-negative fibers would then be responsible for slower vegetative or background control of heart rhythms.

The low incidence of DOR/TH-positive nerve terminals suggests the probable absence of DOR on adrenergic nerve terminals consistent with the failure of DOR stimulation to alter sympathetically mediated tachycardia (3, 18, 33). The apparent low-level DOR-TH colocalization may in fact represent the failure of the method to resolve DOR signals from nearby cholinergic nerve terminal membranes that are near the adrenergic synaptosomes.

In conclusion, the present study supports the hypotheses that DORs are located on the postganglionic prejunctional parasympathetic nerve terminals and that the expression of DOR is significantly greater on the cholinergic nerve terminals than on the adrenergic nerve terminals in SA node and right atrium.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by the American Heart Association Texas Affiliate.


   ACKNOWLEDGMENTS
 
The authors acknowledge the expert advice and assistance of Harlan Jones (Microbiology and Immunology) with the quantitative immunofluorescent analysis, Vidhya R. Rao (Pharmacology and Neuroscience) with the Western blot analysis, Robert Wordinger (Anatomy and Cell Biology) with the immunofluorescence, and Ignacy Gryczynski (Center for Commercialization of Fluorescence Technology) with the confocal analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Caffrey, Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (e-mail: caffreyj{at}hsc.unt.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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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