Distribution of the neuropeptide galanin in the cat heart and coexistence with vasoactive intestinal peptide, substance P and neuropeptide Y

J Mol Cell Cardiol

24, 35-41 (1992)

Distribution Coexistence

of the Neuropeptide Gala&u in the Cat Heart and with Vasoactive Intesthal Peptide, Substance P and Neuropeptide Y Weimin

Department

ofAnatomy,

West Virginia

Zhu and Richard

D. Dey*t

Uniuersity, Health Sciences North, Morgantown,

WV26506,

USA

(Received 29 April 1991, accepted in revisedform 4 September 1991) WEIMIN ZHU AND R. D. DEY. Distribution of the Neuropeptide Galanin in the Cat Heartand Coexistence with Vasoactive Intestinal Peptide, Substance P and Neuropeptide Y. Journal ofMolecular and Cellular Cardiolop (1992) 24, 35-41. The neuropeptide galanin (GAL) has been detected in the peripheral and central nervous systems. However, little is known about its distribution and localization in heart, and the possible coexistence of GAL with other neuropeptides in the heart is not established. The present immunocytochemical study describes the distribution of GAL in nerves of the feline heart and its colocalization with vasoactive intestinal peptide (VIP), substance P (SP), and neuropeptide Y (NPY). GAL-like immunoreactivity was widely distributed in the atrial and ventricular myocardium and around coronary arteries. &localization of GAL with VIP, SP and NYP was observed in many nerve fibers. Further, GAL and NPY were colocalized in nerve cell bodies of intracardiac ganglia. Since these neuropeptides have been found to be associated with sensory and autonomic innervation in the heart, the present findings provide evidence that GAL is shared by functionally different neuronal populations in the heart and that GAL may participate in controlling cardiac function by combined action with other neuropeptides. KEY WORDS: Colocalization;

Galanin; Heart Neuropeptides.

innervation;

Vasoactive

Introduction Nerve fibers innervating the mammalian heart contain not only the classical neurotransmitters, but also recently described neuropeptides, such as neuropeptide Y (NPY; Lundberg et al., 1982; Sternini and Brecha, 1985), vasoactive intestinal polypeptide (VIP; Weihe and Renecke, 1981; Della et al., 1983; Rechardt et al., 1986) and substance P (SP; Wharton et al., 1981). A newly discovered neuropeptide, galanin (GAL), is present in the nerves of the mudpuppy heart (Parsons et al., 1989). GAL is a 29 amino acid peptide originally isolated from porcine upper small intestine (Tatemoto et al., 1983). Galanin-like immunoreactivity (GAL-IR) is present in many regions of both the peripheral and central nervous system (Rokaeus, 1987). GAL-containing nerves are involved-in the functional regulation in various tissues, including the contraction of the smooth muscles in the gut and the trachea (Ekblad et *To whom

correspondence

tR. D. Dey is the recipient 0022-2828/92/010035

intestinal

polypeptide;

Substance

P; Neuropeptide

Y:

al., 1985; Rokaeus, 1987), the endocrine pancreatic secretion (Ahren et al., 1988), inhibition of acetylcholine and dopamine release in central nervous system (Fisone et al., 1987; Nordstrom et al., 1987), and stimulation of cell growth (Sethi and Rozengurt, 1991). In the heart, GAL causes a strong inhibition of cardiac vagal action (Revington et al., 1990) and hyperpolarizes neuron cell membranes (Konopka et al., 1989). Since it usually coexists with classical neurotransmitters and other neuropeptides (Cheung et al., 1985; Furness et al., 1987; Grunditz et al., 1987; Kummer, 1987; Luts et al., 1989; Crowley, 1990), GAL may exert effects by interacting with coexisting transmitters. The coexistence of GAL with other neuropeptides in mammalian heart has not been studied, but would contribute to understanding of the mechanism of its action. For this purpose, we studied the distribution of GAL-IR in nerves of the cat heart and its coexistence with the neuropeptides, VIP-, SP-

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36

Weimin

Zhu and R. D. Dey

and NPY-IR by applying standard immunocytochemical procedures and recently developed colocalization techniques.

Materials and Methods Three cats were anaesthetized with sodium pentobarbital (45mg/kg body wt i.p.) and intracardially perfused with ice-cold fixative consisting of 2% paraformaldehyde, 15% saturated picric acid and 0.15 M phosphate buffer (Stefanini et al., 1967). Hearts were removed carefully and stored in the same fixative at 4% for 3 h. Specimens were rinsed with 0.1 M phosphate buffered saline containing 0.3 % Triton X 100 (PBS-TX) at 4°C overnight. Tissues were frozen in isopentane cooled with the liquid nitrogen. For the localization of GAL-IR, cryostat sections were cut 12 pm thick and processed for indirect immunocytochemistry. Each section was incubated at 37% for 30 min with a rabbit antisera against GAL (Peninsula, Inc., Belmont, CA) diluted 1: 100 in PBS-TX. After rinsing three times for 5 min each, the sections were incubated at 37% for 30min with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ICN Biomedicals, Inc., Costa Mesa, CA) diluted 1: 100 in PBS-TX. For colocalization of GAL with VIP and SP, simultaneous immunocytochemistry was performed on the same section using the procedures described by Wessendorf and Elde (1985). Briefly, combined primary antisera consisted of rabbit anti-GAL and either mouse monoclonal anti-VIP (1: 100, from Dr John Porter, Southwestern Medical School, Dallas, TX) or rat monoclonal anti-SP (1: 50, Accurate Chemical and Scientific Corp., Westbury, NY). The second combined secondary antiserum consisted of FITC-labelled goat anti-rabbit IgG to label anti-GAL, and either rhodamine isothiocyanate (RITC)-conjugated goat anti-mouse IgG (1: 100, Southern Biotechnologies, Inc., Birmingham, AL) to label anti-VIP or RITCconjugated goat anti-rat (1: 100; Southern Biotechnologies, Inc., Birmingham, AL). Characteristics of the primary and secondary antisera are provided in previous publications (Dey et al., 1988, 1990). Since antisera against NPY and GAL raised in different animals are not available, the

elution-restaining technique of Tramu et al. (1978) was conducted to determined the possible colocalization of NPY and GAL. Incubation was first performed with GAL antiserum as described above. After photography, the anti-GAL/FITC-anti-rabbit complex was eluted with a mixture of 0.15~ KMn04 and 0.01 N H$04. Then, the same section was incubated with rabbit anti-NPY (1 : 100, Peninsula, Inc., Belmont, CA) followed by FITC-labelled goat anti-rabbit IgG as above. In addition, colocalization of GAL and NPY in nerve cell bodies was accomplished by immunocytochemical staining on consecutive serial sections. All sections were examined and photographed using an Olympus BHPT florescence microscope equipped with specific rhodamine and fluorescein filters. Photography was accomplished using an automatic exposure camera with Kodak Tri X (ASA 400). Control experiments were performed as follows: substitution of the primary antiserum with the antiserum which had been preabsorbed with an excess amount of the antigen (5O~g pure natural peptide/ml diluted antiserum) overnight at 4%. For the colocalization staining, additional controls, as described previously (Dey et al., 1988, 1990), were conducted to exclude possible cross-reactions between inappropriate antibodies.

Results In sections of both atria and ventricles, a dense network of GAL-IR nerve fibers were distributed in the myocardium throughout the cat heart. These varicose single nerve fibers ran parallel to and between the muscle bundles (Fig. 1). The density was greater in atria than in ventricles. In both atria and ventricles, a high density of varicose GAL-IR fibers were associated with coronary vessels, being most prominent in medium-sized arterial branches. Here, the GAL-IR fibers were confined to the adventitia-media junction (Fig. 2). GAL-IR was also found in some of the nerve cell bodies of intracardiac ganglia (Fig. 3) and in fibers of the endocardium [Fig. 4(c)] and epicardium [Not shown]. Comparison of the double-stained sections revealed colocalization of GAL-IR in the same nerve terminals both with VIP-IR and with

Galanin

FIGURE 1. GAL-IR nerve fibers in immunoreactive nerve fibers (arrows) run lumen) is present in the atrial myocardium. tissue are present in unlabelled sections as

Nerves

atrial (a) and parallel to and The punctate well, and thus

FIGURE 2. GAL-IR nerve fibers associated (arrows) are at the junction between the tunica

in Heart

ventricular (b) myocardium (my). The dense network of between bundles of cardiac muscle. A coronary artery (* in fluorescent objects evenly distributed throughout the muscle represent nonspecific endogenous fluorescence. x 200.

with a coronary artery (‘) m the atrial myocardium media (M) and the tunica adventitia (A). x 200.

(my).

Nerve

fibers

38

FIGURE 3. GAL-immunoreactive in the connective tissue (ct) beneath cell bodies surrounds a non-fluorescent

Weimin

Zhu and R. D. Dey

neurons in a cardiac ganglion the epicardium (Ep). In several cell nucleus. x 200.

in the wall of the left atrium. neurons (arrows), fluorescence

The ganglion is located in the GAL-IR nerve

FIGURE 4. Colocalization of GAL -IR (a,c) with VIP-IR (b) or SP-IR (d) by simultaneous labelling in the same section. (a), (b) A single nerve fiber (arrow) closely associated with the myocardium (my) contains both GALand VIPIR. Close examination of individual varicosities suggest that this is a single fiber containing both peptides. In contrast, the nerve fiber associated with the vessel wall (double arrow) contains only GAL-IR. The double arrow in (b) points to the in the subendocardium (S) contains both approximate location of the nerve fiber. (c), (d) A small nerve fiber located GAL-(c), and SP-IR (arrows). Notice the exact correspondence of varicosities indicating that both peptides are in the same nerve fiber. x ZOO.

Galanin

Nerves

39

in Heart

FIGURE 5. Colocalization of GAL- and NPY-IR in the same section of ventricle by labelling first for GAL-IR (a), followed by elution, and then relabelling for NPY-IR (b). (a) V aricose GAL-IR nerve fibers (arrows) are located in close association with the myocardium (my). (b) After elution and relabelling, NPY-IR fibers are seen with varicosities in identical positions (arrows). The labelling is faint due to the elution procedure. The nonspecific fluorescence is quenched by elution as well. x ZOO.

FIGURE 6. Two consecutive sections of a small intrinsic ganglion located in the myocardium The nerve cell bodies comprising the ganglion contain both GAL- and (inset) NPY-IR. Numbers nerve cell bodies. GAL-IR nerve fibers (arrows) are seen in myocardium.

SP-IR (Fig. 4). More GAL-IR nerves were observed than either VIP- or SP-IR. This phenomenon is particularly evident at the region surrounding blood vessels, as shown in Figure 4(a) and (b) where a nerve fiber contains GAL-IR but lacks VIP-IR even though both peptides are present in a nerve fiber located in the myocardium. All nerve fascicles displaying VIP-IR in the atrial myocardium also reacted with GAL an&serum, but

(my) of the left atrium. correspond to identical

only a few GAL-IR nerve fibers showed VIPIR. Fibers exhibiting both GAL- and SP-IR occurred

in

both

atria1

and

ventricular

myocardium as well as in the endocardium [Fig. 4(c) and (d)]. None of the sections we examined for colocalization of SP- or VIP-IR included neurons of intracardiac ganglia. NPY-IR nerves were seen in abundance in the myocardium, mainly running between cardiac muscle fibers. Using the elution proce-

40

Weimin

Zhu and RD.

dure, many were found to contain both GALand NPY-IR (Fig. 5). Further, consecutive sections of atria showed many neurons of intracardiac ganglia displaying both GAL- and NPY-IR, although some contained only one peptide (Fig. 6). Immunoreactivity was not seen in sections incubated in anti-GAL antiserum absorbed with GAL peptide. No immunoreactivity was observed in tissues incubated in GAL antiserum followed by incubation with goat anti-mouse or goat anti-rat IgG. No immunoreactivity was observed on the sections incubated in SP or VIP antisera and sequentially exposed to goat anti-rabbit IgG. The control experiments ruled out the possibility of nonspecific cross-staining between secondary antisera.

Discussion The presence of GAL-IR in heart was first reported in the mudpuppy (Parsons et al., 1989). The present study has shown an extensive distribution of GAL-IR in the cat heart as well. GAL-IR nerve fibers were present in both atrial and ventricular myocardium, in epicardial and endocardial layers, and around coronary arteries. Some cell bodies of intracardiac neurons also contained GAL-IR. Therefore, some of GAL-IR nerve fibers in the cat heart probably originate from neurons in the intracardiac ganglia. The present investigation also examined the coexistence of GAL with several neuropeptides. We demonstrated

Dey

the coexistence of GAL-/VIP-, GAL-/SPand GAL-/NPY-IR in nerve fibers and GAL-/NPY-IR in a nerve cell bodies of cardiac ganglia. Colocalization of several neuropeptides in the nerve fibers of the mammalian heart have been reported previousIy. For example, calcitonin gene-related peptide-IR and SP-IR are colocalized in sensory nerve fibers (Gibbins et al., 1985; Dalsgaard et al., 1986), and VIP-IR is colocalized with NPY-IR in the nerve fibers and nerve cell bodies of the bovine heart (Forsgren, 1989). Previous studies have also demonstrated that neurons of intracardiac ganglia contain different subsets of neuropeptides, i.e. VIP (Weihe and Renecke, 1981; Reinecke and Forssmann, 1984), VIP/NPY (Forsgren, 1989), and NPY (Lundberg et al., 1983; Uddman et al., 1985). Our findings add two subsets of neurons in cat hearts: those that contain immunoreactive GAL, and those that contain both GAL- and NPY-IR. In conclusion, we have described the distribution of GAL in the feline heart and obtained evidence for the coexistence of GAL with several other neuropeptides. Our findings suggest that some GAL-containing fibers originate from neurons of intracardiac ganglia.

Acknowledgements R. D Dey is the recipient of a Research Development Award from NIH-NHLBI, KO4 HL-02125.

Career No.

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