Nitric oxide synthase containing nerves in the atrioventricular node of the guinea pig heart

Journal of the

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Journal of the Autonomic Nervous System 51 (1995) 245-253

Autonomic Nervous System

Nitric oxide synthase containing nerves in the atrioventricular node of the guinea pig heart Koichi Tanaka *, Tanemichi Chiba The Third Department of Anatomy, Chiba University School of Medicine, 1-8-1 Inohana, Cyuoku, Chiba 260, Japan

Received 14 June 1994; revision received and accepted 21 July 1994

Abstract Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in the guinea-pig atrioventricular node. Many small ganglia were found in the interatrial septum near the atrioventricular node. A small number of neurons in these ganglia were immunoreactive. NOS immunoreactive axons were observed in nerve bundles near or within the node. Very thick immunoreactive axons, 8-10 tzm in diameter, were found between the conductive tissue and the ordinary cardiac muscles. Electron-microscopic examination confirmed that these axons were unmyelinated. Nerve bundles in this region contained both myelinated and unmyelinated axons; however, immunoreactivity was detected exclusively in unmyelinated axons. Fine NOS-immunoreactive nerve fibers with terminal varicosities were often seen in the atrioventricular node. Close contact between NOS-immunoreactive axon varicosity and specialized cardiac muscle cell was observed. Intranodal ganglia were observed among the specialized cardiac muscles. They were surrounded by numerous axons, some of them were immunoreactive. Direct axo-somatic synapses from NOS-immunoreactive terminals to the intranodal ganglion cells were observed. The present results indicate a possibility that nitric oxide plays a role in the neural control of the conductive tissuc in the heart through direct neuromuscular contact. Keywords: Nitric oxide synthase; Cardiac ganglion; Guinea pig; Atrioventricular node; Immunohistochemistry

I. Introduction The specialized structures of the atrioventricular n o d e o f m a m m a l i a n h e a r t s a r e richly i n n e r v a t e d [2,9,30,32,43]. A c c o r d i n g to p h y s i o l o g i c a l a n d m i c r o a n a t o m i c a l studies, t h e n e r v e fibers innervating the atrioventricular node are derived f r o m left p a r a v e r t e b r a l s y m p a t h e t i c g a n g l i a

* Corresponding author. Tel.: (81-43) 226-2024; Fax: (81-43) 226-2025.

[17,24,40] a n d i n t r a c a r d i a c g a n g l i a [20,27,37]. Hist o c h e m i c a l l y , they w e r e c h a r a c t e r i z e d by a s t r o n g a c e t y l c h o l i n e e s t e r a s e r e a c t i o n [7,20,22,47] a n d occasionally, by vasoactive i n t e s t i n a l p e p t i d e imm u n o r e a c t i v i t y [45]. Nitric oxide ( N O ) is g e n e r a t e d by several diff e r e n t cell types, i n c l u d i n g n e u r o n a l a n d e n d o t h e lial cells, a n d it has b e e n shown to act as a m e s s e n g e r m o l e c u l e in m a n y systems [10,11,29,39]. N O is f o r m e d f r o m L-arginine by the action of nitric oxide s y n t h a s e ( N O S ) , a n d t h e p r e s e n c e of this e n z y m e a n d its m R N A has b e e n u s e d to

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Fig. 1. (A) A low-power micrograph of a resin-embedded section (50-~m thick) through the atrioventricular node (enclosed by dots). NOS-immunoreactive perikarya (arrows) are seen in the ganglia near the atrioventricular node. In the nerve bundles, very thick positive fibers (large arrow-heads) and fine varicosities (small arrowheads) are observed. Bar, 100 /zm. (B) A 1-/xm-thick semi-thin section of the region B in panel A (toluidine blue stain). Ordinary striated cardiac muscles are seen. Bar, 50/zm. (C) A semi-thin section of the region C in Panel A. Conductive tissue of the atrioventricular node is distinguishable from the adjacent myocardium by their lower content of myofibrils. Bar, 50/~m.

K. Tanaka, T. Chiba /Journal of the Autonomic Nervous System 51 (1995) 245-253

identify cells that synthesize NO [3]. In addition, there is evidence that N A D P H - d i a p h o r a s e (NADPH-d) activity can be used as a marker for NOS in many cell types [5,19]. The presence of NOS has also been proved in the nervous elements of the heart [16,23,41,42]. Klimaschewski et al. [23] described a dense distribution of NOS-immunoreactive (NOS-ir) fiber plexus in the atrioventricular node, but, they could not identify the target structure of these NOS-positive nerve fibers. The present study was designed to clarify (1) the distribution of NOS-ir nerve fibers in the atrioventricular node and (2) whether NOS-ir axons have direct contacts with specialized cardiac muscle ceils.

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Fig. 2. A micrograph of a resin-embedded section near the atrioventricular node. Three NOS-immunoreactiveperikarya and their varicose processes (arrowheads) surrounding adjacent non-immunoreactiveneurons (stars) are seen in a small ganglion. Bar, 100 ~m.

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2. Materials and methods Ten young guinea pigs of the Hartley strain weighing 250-350 g of either sex were used. Under general anesthesia (Nembutal, 30 mg/kg), the animals were fixed on a cork board. After laparotomy, an 18-gauge needle was introduced into the abdominal aorta, pointing towards the heart. The hearts were perfused retrogradely with 20 ml aqueous 0.9% NaC1, and then fixed by perfusion with 200 ml fixative composed of 4.0% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The blocks of the tissue containing the interatrial septum and the atrioventricular junction were then dissected and fixed again for 2 h in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C. They were then rinsed several times in 0.1 M phosphate-buffered saline (PBS), and stored in 20% sucrose in PBS for 12 h. Pre-embedding immunohistochemistry was performed on 20-~m-thick cryostat sections using the NOS antibody (a gift of Dr. H. Esumi, National Cancer Research Institute, East). The specificity of NOS antibody used in the present study was tested and found to react only with neuronal NOS but neither with endothelial nor macrophage NOS as described previously [35]. Free-floating sections of the interatrial septum and the atrioventricular junction were preincubated in PBS containing 10% normal goat serum, 0.2% bovine serum albumin and 0.1% Triton X-100 for 60 min at room temperature. The antiserum against NOS was applied at a dilution of 1:200 in PBS containing 0.2% bovine serum albumin, 0.1% Triton X-100 for 2 days at 4°C. After rinsing with PBS, sections were incubated with biotinylated anti-rabbit IgG produced in goat (Vector) diluted 1:100 in PBS containing 0.2% bovine serum albumin, 0.1% Triton X-100 for 60 min at room temperature. After rinsing with PBS, sections were incubated with avidin-biotin complex (Vector) at 1 : 100 dilution in PBS for 60 min at room temperature. They were then transferred to 0.05 M Tris-HC1 buffer (pH 7.2) and incubated with 0.02% diaminobenzidine, 0.002% H 2 0 2 in 0.05 M Tris-HCl buffer for 10 min. The sections were fixed with 2.5% glutaraldehyde in 0.1 M

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phosphate buffer for 1 h and 1% osmium tetroxide in phosphate buffer for 1 h. They were then dehydrated with ethanol and flatly embedded in Spurr's resin. These sections were observed and photographed with light microscope. A nomarsky interference microscope (Nikon) was used to distinguish specialized cardiac muscle from ordinary cardiac muscle cell. Toluidine-blue-stained semithin sections were used to identify the tissue adjacent to immunoreactive nerve fibers. Ultrathin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate and observed with a J E O L 1200EX electron microscope.

3. Results

Light-microscopic examination of the flatly embedded cryostat sections of the septal portion of the atrium revealed the presence of abundant NOS-ir nervous tissue. Many small ganglia occurred in the interatrial septum. Neurons in the ganglia were round in shape and closely packed

together. Less than 10% of ganglionic perikarya were NOS-ir (Fig. 1A). Some non-immunoreactive perikarya were surrounded by processes of neighboring NOS-ir neurons (Fig. 2). The atrioventricular node was differentiated from the adjacent ordinary cardiac muscle cells by use of a Nomarsky interference microscope or toluidine blue stained semi-thin sections (Fig. 1B,D). In the vicinity of interatrial ganglia and atrioventricular node, many nerve bundles containing NOS-ir axons were observed (Fig. 1A). The processes of NOS-ir ganglionic cells joined the adjacent nerve bundles. Very thick axons, 8 - 1 0 / x m in diameter, were seen between the specialized cardiac muscle cells and the ordinary cardiac muscle cells (Figs. 1 and 3). Fine NOS-ir fibers with varicosities were often derived from the nerve bundles and invaded the atrioventricular node (Figs. 1A and 3). Electron microscopy confirmed the above light-microscopic observation and showed better cytological features of the NOS-ir nervous tissue. The very thick axons in nerve bundles observed near the specialized cardiac muscles, were un-

Fig. 3. A micrograph of a nerve bundle between the conductive tissue (right upper part) and ordinary myocardium (left bottom part). Very thick fibers (large arrowheads) and fine varicosities(small arrowheads) are NOS-immunoreactive.Bar, 100/zm.

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myelinated (Fig. 4A). T h e s e nerve bundles contained both myelinated and u n m y e l i n a t e d fibers; however, N O S immunoreactivity was f o u n d exclusively in unmyelinated axons. Very fine nerve bundles were also observed a m o n g the specialized muscle cells (Fig. 4B). A few axons were NOS-ir. In m a n y cases, b o t h negative and positive axons were s u r r o u n d e d by a c o m m o n S c h w a n n cell sheath. Direct contacts between NOS-ir axons and specialized cardiac muscle cells were observed (Fig. 5). N O S - i r densities were observed in the axoplasm and large granules. Postsynaptic density was not seen. In the vicinity of the atrioventricular node, solitary ganglion cell was occasionally observed (Fig. 6). It was situated near the

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conductive tissue and was associated with n u m e r ous axons. This intranodal n e u r o n itself was NOS-negative, but, some axons surrounding it were NOS-ir. Axo-somatic synapses were observed b e t w e e n NOS-ir axons and the intranodal neuron. NOS-ir axon varicosities were filled with synaptic vesicles and postsynaptic density was clearly observed in the ganglionic cell body.

4. Discussion T h e existence of a preferential innervation of the atrioventricular n o d e has actually b e e n known for a long time [4,8,28,34,46]. M o r e recently,

Fig. 4. Electron micrographs of the same material as shown in Fig. 3. (A) The thick NOS-immunoreactive fibers are confirmed as unmyelinated axons. Myelinated axons in the same bundle are NOS-negative. Bar, 2~m. (B) A NOS-positive axon (arrowhead) is present in a small nerve bundle between the specialized muscle cells (S). Bar, 1/xm.

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physiological studies have determined the functional hierarchy of the nervous system [12,18,25]. According to these studies, the intracardiac nerve plexus could be considered as a terminal portion of the extrinsic nerves to the heart [38]. However, with respect to the nerve fibers directed to both specialized structures of the atrioventricular node and the bundle of His, at least some of them may be provided by intracardiac sources. This has been suggested by denervation studies of Jacobowitz et al. [21], Potter et al. [36] and D r akeHolland et al. [6], who found, after surgical sympathectomy, the persistence of varicose nerve fibers in the heart. Using serial sections of rat atrioventricular junction, Moravec [31] described that a chain-like accumulation of ganglionic cells was observed in this region and their axons were distributed in the atrioventricular node and the bundle of His. In the present study, we observed NOS-ir gan-

glionic cells in the atrioventricular junction. The processes of these neurons joined in thick nerve bundles running toward the atrioventricular node. Fine axons with terminal varicosities were derived from these bundles and terminated in the node. Accordingly, these results show that the atrioventricular node receives NOS-containing inputs from intracardiac ganglia. Neurons in these ganglia were round in shape and closely packed together. Less than 10% of ganglionic perikarya were NOS-ir. Some non-immunoreactive perikarya were surrounded by NOS-ir processes of adjacent neurons. We also found NOS-immunoreactive axo-somatic synapses to a intranodal neuron. These findings suggest that NOS is also present in the interneurons as observed in the left atrium [42]. The most striking result in this study was the direct contact of NOS-ir axons with the nodal muscle cells. Immunoreactivity was observed in

Fig. 5. (A) A low-power micrograph through the atrioventricular node. A NOS-positive terminal (arrow-head) has close contacts to nodal muscle cells. Bar, 1 /.~m. (B) Higher magnification of (A). Large granules, as well as axoplasm are NOS-immunoreactive. Postsynaptic specialization is not seen. Bar, 500 nm.

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the axoplasm and large granules. There was an accumulation of synaptic vesicles towards the regions of close apposition. The presence of NOS-ir varicosities in close proximity to smooth muscle cells was reported [15,26]. The present results indicate that NO is a transmitter not only to smooth muscles but also specialized cardiac muscle cells of the atrioventricular node. NO is known as a neurotransmitter which affects an intracellular target by transmembrane diffusion and is effective on a long distance and in a limited time coarse [3,11]. In this respect, close apposition of NOS-ir axon varicosities with nodal cells suggests co-existence of another transmitter in these axons. It may well be possible that some of the NOS-ir neuronal processes observed here were of extrinsic origin since they were fairly numerous and, some N A D P H - d labeled nerve fibers were ob-

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served in the vagus nerve before it reached the heart [41]. Extrinsic NOS-ir fibers may have several different sources; for example, they may represent sensory afferent neurons, preganglionic parasympathetic fibers, or postganglionic sympathetic nerves. Parasympathetic preganglionic neurons in the dorsal motor nucleus of vagus or ambiguous, however, were N A D P H - d negative [13,44]. Sympathetic ganglionic neurons of the superior cervical or stellate ganglia were also N A D P H - d negative in the rat (our observation) and mouse [14]. On the other hand, there is evidence that subpopulations of sensory neurons in the nodose and dorsal root ganglia contain a high level of N A D P H - d activity [1,33]. Moravec [31] reported the occurrence of sensory nerve fibers in the atrioventricular junction in the rat. Accordingly, some of these NOS-ir nerve profiles shown here may be sensory fibers.

Fig. 6. Electron micrographs of an intranodal neuron. (A) This neuron is surrounded by n u m e r o u s axons. Most of them are NOS negative (asterisks), however, one of them contain N O S (arrow-head). Bar, 1 /zm. (B) In higher magnification, postsynaptic density (arrows) is seen. Bar, 500 nm.

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Acknowledgements The authors thank Dr. Hiroyasu Esumi (National Cancer Research Institute, East) for providing the NOS antibody, and Dr. Hidehisa Torikai for excellent technical assistance. This work was supported by Research grant 05680651 from the Japanese Ministry of Education, Science and Culture.

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