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The spatial relationship between type I glomus cells and arteriolar myocytes in the mouse carotid body
ANNALS OF ANATOMY
The spatial relationship between type I glomus cells and arteriolar myocytes in the mouse carotid body Amina B. EI-Fadaly 1'2 and Wolfgang Kummer 1 1Institute for Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, 35385 Giessen, Germany, 2Anatomy Department, Faculty of Medicine, Cairo University, Egypt
Correspondence to: A. B. E1-Fadaly
(Nunn 1993). The arterio-venous pO 2 difference is, therefore, small in accordance with its rapid response within 1-3 seconds (Ponte and Purves 1974). It is evident that modulation of the arterial vascular tone plays an essential role in perfusion rate control and, in turn, chemosensory activity (Wang et al. 1994 a; Wang et al. 1995; Lahiri and Buerk 1998; Buerk and Lahiri 2000; Iturriaga et al. 2000). The specialized vascular system of the carotid body is controlled by an extensive sympathetic, parasympathetic and sensory innervation (O'Regan 1981; McDonald and Larue 1983; Kummer 1990). Indeed, most of the sympathetic effects on chemoreception are exerted via vascular actions (Eyzaguirre and Lewin 1961; O'Regan 1981). In addition to perivascular nerve fibres, endothelial cells regulate arteriolar tone, e.g. by release of nitric oxide (NO) (Wang et al. 1994b). Several vasoactive substances are also produced and released by type I glomus cells. These include catecholamines (B6ck 1973; B6ck and Gorgas 1976; Wang et al. 1991a), adenine nucleotides (BOck 1980 a, b; Wang et al. 1991 b; Chen et al. 2000), NO (Prabhakar et al. 1993), and endothelin (He et al. 1996). It still remains an open question whether and to what extent glomus cells play a role in the regulation of vascular tone in the carotid body. As a structural basis for such a regulation, an intimate relationship between type I glomus cells and arterioles shall be expected. Despite the vast amount of structural investigations on the carotid body, there is very little information concerning this issue. In the mouse, Gorgas and B6ck (1977) have referred to the presence of cytoplasmic processes of type I glomus cells in contact with sensory nerve terminals in the tunica adventitia of some arterioles, but their relationship to the vascular myocytes was not reported. The present investigation was carried out to address this issue and to morphologically define a possible connection between type I glomus cells and the vascular smooth muscle in the mouse carotid body.
Ann Anat (2003) 185:507-515
0940-9602/03/185/6-507 $15.00/0
Summary: The structural relationship between type I glomus cells and the vascular smooth muscle was investigated by electron microscopy in the mouse carotid body. A close apposition (<0.1 pm) between the glomus parcnchyma and the neighbouring arterioles was regularly present. Profiles of type I glomus cells were found to be exposed to the vascular smooth muscle without any supporting cell investment. In circumscribed areas of these profiles, type I glomus cells and the vascular smooth muscle cells made contact by fusion of their basal laminae. These glomus-cell-myocyte junctions structurally resemble vascular neuromuscular junctions of sympathetic nerve terminals. In addition to the occurrence of such glomus cell-myocyte contacts, myoendothelial junctions also appeared frequently. On the basis of these observations, it is suggested that type I glomus cells play a role in the regulation of the vascular tone in the carotid body and that a physiological interaction between the endothelial cells, the vascular smooth muscle cells and the type I glomus cells exists. Key words: Carotid body - Type I glomus cells - Vascular smooth muscle cells - Ultrastructure - Mouse
Introduction The carotid body is a chemoreceptive organ acting as a rapid-responding monitor of arterial blood gases. It is provided with a highly specialised vascular system characterised by a high perfusion rate reaching ten times the level which would be proportional to its metabolic rate
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Material and m e t h o d s Four male adult 129SvEvx CF1 mice and two adult female FVB mice were sacrificed by inhalation of sevoflurane (Abbott, Wiesbaden, Germany). Immediately upon arrest of respiration, the thorax was opened and a cannula was inserted into the ascending aorta via the left ventricle. The vascular system was flushed with heparin and lidocaine containing rinsing solution (Forssmann et al. 1977) and the animals were fixed by perfusion with 1.5% glutardialdehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The carotid bifurcations were dissected, stored for an additional 3 h in the same fixative, and then washed in 0.1 M TRIS-HCI buffer, osmicated for i h in aqueous 1% OsO4, washed 3x15 min in 0.05 M maleate buffer (pH 5.2), stained en bloc for 1 h in 1% uranylacetate in maleate buffer at pH 6.0, washed again 3 x 15 min in 0.05 M maleate buffer (pH 5.2), and then routinely dehydrated in ethanol/propylene oxide and embedded in Epon. Semithin sections (0.75 gm) and ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E, Leica, Bensheim Germany). Semithin sections were stained with Rfideberg-solution (1 g/1 methylene blue, l g/1 thionine, 17.8g/1 Na2HPO4x2H20, in distilled water). Thin sections were stained with alkaline lead citrate and viewed in a EM 902 transmission electron microscope (Zeiss, Jena, Germany).
Results The mouse carotid body was provided with a well-developed arterial system. An average of ten arterioles was
found per semithin cross section through the middle portion of the organ (measuring approximately 250 x 100 ~tm). The arterioles were embedded in the interstitial connective tissue between glomeruli formed by groups of glomus cells (Fig. 1). In some semithin sections, cytoplasmic processes of type I glomus cells were also seen emerging from the cell bodies and approaching the arterioles (Fig. 2). At the electron microscopic level, type I glomus cells facing the interstice were mostly enveloped by lamellar extensions of supporting cells formed by type II glomus cells or Schwann cells. These were usually covered by a continuous, regularly structured basal lamina. In some circumscribed areas where type I glomus cells were not enveloped by a supporting cell, the basal lamina directly covered type I glomus cells continuous with that of the surrounding supporting cells (Fig. 3). In most cases, the interstice between the glomus parenchyma and the neighbouring arteriole was reduced to a very narrow space reaching less than 0.1 gm in width. In these regions, type I glomus cells, together with their supporting cell investment, came into close spatial relationship with the adjacent arteriole; being separated from the surface of the vascular smooth muscle only by one or two extremely attenuated processes of adventitial cells (Fig. 3). Occasionally, type I glomus cells showed direct contact with the arteriolar myocyte (Fig. 4). At such sites, the cell membranes were separated by a regularly spaced cleft. This cleft was occupied by a single basal lamina that appeared to represent a fusion between the basal lamina of the type I glomus cell and that of the smooth muscle cell. In very rare occasions, omega profiles suggestive of vesicle exo- or endocytosis could be observed at the surface of type I glomus cells facing the myocyte.
Fig. 1. Semithin section through the middle portion of the carotid body showing one arteriole (A) located within the interstitial connective tissue between the glomeruli (G). (Riideberg staining; Bar = 10 pro) Fig. 2. A scmithin section showing processes (arrows) of type I glomus cells extending away from their cell body (I) to reach the wall of the neighboufing arterioles (A). (Bar = 10 gin)
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Fig. 3. a: Low power electron micrograph shows part of the glomus parenchyma and a nearby arteriole. Towards the interstitial tis-
sue, type I glomus cells (I) are seen enveloped by lamellar extensions (arrows) of supporting cells. Higher magnifications of regions indicated by rectangles are shown in b and c. b: illustrates a regularly constructed basal lamina (arrowheads) continuously covering the supporting cell investment of type I glomus cells, c: shows a process of type I glomus cell (I) with no supporting cell covering towards the interstitial space. The arrows in c point to the regions where the type II glomus cell envelope (II) ends and the basal laminae of the two cell types become continuous with each other. (Bar = 1 gm) Fig. 4. A contact area between a type I glomus cell (I) and a myocyte (M) of the neighbouring arteriole. A narrow, regularly spaced junctional cleft occupied by a single basal lamina is prominent. Arrow points to a pit at the surface of the type I glomus cell facing the myocyte indicative of a vesicle from an endo- or exocytosis process. E = endothelial cell (Bar = 1 gm) S l e n d e r processes of type I glomus cells with a diam e t e r ranging from 0.5 to 1.5 g m were m o r e frequently seen in relation to the adventitial connective tissue of the arterioles. In favourable sections, long p o r t i o n s of these processes were f o u n d to be enclosed within the l a m e l l a r processes of the adventitia a n d e x t e n d e d over a considerable distance of the circumference of the arteriole (Fig. 6a). I n such cases, wide areas of these processes 509
m a d e contact with the myocyte. In contact regions, the c y t o p l a s m of the processes c o n t a i n e d a b u n d a n t free ribosomes and s o m e t i m e s clusters of m i t o c h o n d r i a , while m i c r o t u b u l e s were only occasionally found (Fig. 6b, c). D e n s e - c o r e d vesicles with a m e a n d i a m e t e r of 121 + 22 n m (range: 88-167 nm), identical to those found in the cell body, were r e g u l a r constituents along the entire length of the process.
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Fig. 7. Among the numerous caveolae and clear vesicles in the cytoplasm of the vascular smooth muscle cell (M), prominent densecored vesicles (arrows) are observed close to the cell surface (a) and in the cytoplasm adjacent to the smooth endoplasmic reticulum (SER) (b). These vesicles have a distinct limiting membrane and display a central core of mild to moderate electron-dense matrix. N = nucleus; E = endothelial cell (Bar = 0,5 lam)
A r t e r i o l a r myocytes, on the o t h e r hand, showed no specific m o r p h o l o g i c a l characteristics at the site of contact with type I glomus cells. H o w e v e r , a m o n g the ordinarily clear vesicles n o r m a l l y found in the c y t o p l a s m of the myocytes, one or two p r o m i n e n t d e n s e - c o r e d vesicles p e r a r t e r i o l a r cross section could b e identified in a considerable n u m b e r of cases. These vesicles were mostly spherical and m e a s u r e d 91 + 10 n m (range: 75-110 nm) in their greatest diameter. T h e y h a d a distinct limiting m e m b r a n e and displayed a central core of mild to m o d e r a t e elect r o n - d e n s e matrix (Fig. 7). T h e central core was s e p a r a t e d from the limiting m e m b r a n e by a clear halo measuring 14 + 2 n m (range: 10-17 nm). In vesicles with faint electron-density, the matrix showed g r a n u l a r substructures with a d i a m e t e r of 8 + i n m (range: 6-12 nm). T h e vesi-
cles were f o u n d to be p r e d o m i n a n t l y l o c a t e d in areas rich in s m o o t h e n d o p l a s m i c reticulum. I n addition, it was also noticed that in most a r t e r i o l a r sections, the e n d o t h e l i a l cells and the underlying myocytes m a d e close contacts with each o t h e r t h r o u g h finger- or tongue-like processes. A t the contact areas, the basal l a m i n a e of t h e two cell types were continuous with each o t h e r without entering into the junctional cleft (Fig. 8). In circumscribed areas of the contact zones, some caveolae might be seen o p e n i n g at the surface of the m y o c y t e o p p o s i t e the intercellular junctional cleft (Fig. 8 c). A schematic drawing of the relationship b e t w e e n e n d o t h e l i a l cells, vascular myocytes, and type I glomus cell processes is p r e s e n t e d in Figure 9. Unexpectedly, W e i b e l - P a l a d e bodies were n o t o b s e r v e d in the a r t e r i o l a r e n d o t h e l i a l cells.
Fig. 5. Profiles of three slender processes (1-3) of type I glomus cells approaching the wall of an arteriole can be identified in a single section (a). One of these processes (1) is completely free of any supporting cell envelope (b). It penetrates through cytoplasmic processes (asterisks) of the adventitial cells to reach the myocyte (M). A single basal lamina (arrowheads in b) is shown interposed between this process and the myocyte. In e, another process (2) shows no contact with the vascular smooth muscle and is still separated from the myocyte by a single layer of lameltar processes of adventitiat cells. It is enclosed together with a vascular nerve terminal (N) by a common Schwann cell (S), A continuous basal lamina (arrowheads in c) covers the Schwann cell and the nerve terminal and continues over the free portions of the glomus cell process. (Bar = 1 gm) Fig. 6. a: This figure illustrates a longitudinally sectioned process of type I glomus cell (I) located within the lamellar processes of the adventitial connective tissue of one arteriole. In a stage section of the same process (b, e), one of its ends (b) comes into contact with the myocyte with only a single basal lamina (arrowheads) interposed. At this region, the process is characteristically free of any supporting cell investment. Its cytoplasm appears rich in free ribosomes and mitochondria but without microtubules. In contrast, the other end of the process (e) shows no contact with the myocyte. It is enveloped by cytoplasmic processes of supporting cells and contains abundant prominent microtubules (MT), but very few ribosomes. Dense-cored vesicles (DCV), characteristic of type I glomus cells, are shown along the entire length of the process. (Bar = 1 Bm in a, b; 0,5 pin in c) 511
Fig. 8. Profiles of myoendothelial junctions in different arteriolar sections. Figures a, b show processes (arrows) emerging from the endothelial cells (E) to reach the underlying myocytes (M) while in c the process (arrow) projects from the myocyte to reach the overlying endothelial cell. The cell membranes of the two cell types come into close apposition with no basal lamina in between. The basal laminae (arrowheads) of the two cells are reflected at the sides of the contact zones to be continuous with each other. Two caveolae open at the surface of the myocyte opposite to the intercellular junctional cleft (small arrows in b). (Bar = 1 ~m in a, c; 0,5 gm in b)
Fig. 9. Schematic drawing showing the structural relationship between an arteriolar endothelial cell (E), a vascular smooth muscle cell (M) and a type I glomus cell process (I). BL = Basal lamina.
Discussion The p r e s e n t study d e m o n s t r a t e s an intimate structural relationship b e t w e e n type I glomus cells and the vascular s m o o t h muscle. In a considerable n u m b e r of cases, the interstitial space b e t w e e n the glomus p a r e n c h y m a and the neighbouring arterioles was r e d u c e d to a very n a r r o w cleft reaching less than 0.1 g m in width. A t such sites, the glomus p a r e n c h y m a was s e p a r a t e d from the vascular s m o o t h muscle by only one or two e x t r e m e l y a t t e n u a t e d processes of adventitial cells, and even cell bodies of t y p e ! glomus cells c a m e into direct contact with the art e r i o l a r myocytes. A r e a s of type I glomus cells e x p o s e d to the a r t e r i o l a r wall have also b e e n d e s c r i b e d in the rat carotid b o d y ( M c D o n a l d 1983; M c D o n a l d and L a r u e 1983) but direct contacts as o b s e r v e d h e r e in the m o u s e have not b e e n r e p o r t e d for that species. L o n g slender cytoplasmic processes of type I glomus cells, often free of a supporting cell investment, were found to be m o r e frequently r e l a t e d to the adventitia of the a r t e r i o l a r walls than their cell somata. G o r g a s and B6ck (1977) and B6ck (1982) have also r e f e r r e d to the existence of similar processes in the tunica adventitia of some arterioles in the m o u s e carotid body. T h e y focussed 512
upon the relationship between these processes and lanceolate nerve endings which they interpreted as representing baroreceptors (Gorgas and B6ck 1977). Our present study focussed on the relationship between type I glomus cell processes and the arteriolar myocytes. As a consistent finding, a junctional cleft was found in the contact areas which characteristically contained a single basal lamina formed by fusion of the basal lamina of the myocyte with that of the type I glomus cell. The cytoplasm of type I glomus cells in these regions typically contained abundant free ribosomes and might show clusters of mitochondria with occasional microtubules, unlike the ordinary processes which were characterised by abundant microtubules but few ribosomes and mitochondria. Besides these organelles, dense-cored vesicles, identical to those found in the cell body, were present along the entire length of the processes. The morphological criteria of the junctional area between type I glomus cells and arteriolar myocytes resemble sympathetic vascular neuromuscular junctions described in various vascular beds (Luff et al. 1987, 1991, 1995; Luff and McLachlan 1988, 1989). These investigators, using serial section analysis of individual varicosities, found a high proportion of the varicosities forming neuromuscular junctions with the vascular smooth muscle. They also reported a junctional cleft occupied by a single basal lamina formed by fusion of the basal laminae of the sympathetic varicosity with the myocyte. In support of the concept of equivalence of type I glomus cell-myocyte junctions with neuromuscular junction, McDonald and Michell (1975) have also reported that the neural crest-derived glomus cells have general structural characteristics common to neurones, being enveloped by glial-like sheath ceils, having dendritic processes arising from the cell bodies, and forming synaptic junctions with one another or with nerve terminals. Type I glomus cells and their processes contain catecholamines and a variety of other transmitters and neuromodulators (B/Sck 1973, 1982; BOck and Gorgas 1976; Heym and Kummer 1989; Kummer 1990). Consequently, it could be assumed that type I glomus cell processes forming junctions on the arteriolar myocytes might release transmitters or modulators as, for example, catecholamines. However, the presence of membrane specializations indicating a site of attachment of the vesicles for exocytosis or vesicle release, as in morphologically typical synapses and skeletal neuromuscular junctions, could not be detected. Similarly, prejunctional membrane specializations are almost absent at the vascular neuromuscular junctions and have been reported only occasionally (Luff 1996). This author has attributed the presence or absence of such membrane specializations to different postfixation times used during tissue preparation. The present results also demonstrated that junctional regions between type I glomus cells and the vascular smooth muscle did not localise at any specific region of the myocyte and were not accompanied by any obvious postjunctional membrane specializations such as dense bands or accumulations of caveolae at the cell membrane. Similar data were also re-
ported by Luff (1996) concerning sympathetic nerve terminals forming junctions with the vascular smooth muscle. This author proposed that postjunctional membrane characteristics are not essential for a neuromuscular junction to operate since neuromuscular junctions lacking postmembrane specialisation are present in skeletal muscles of non-mammalian vertebrate and invertebrate species. Moreover, in case of type I glomus cells, it is generally accepted that morphologically typical synapses are not the only site at which transmitters or other secretory products are released (McDonald and Michell 1975; B6ck 1973, 1982). The morphological data obtained from the present investigation are strongly indicative of an intimate physiological relationship between type I glomus cells and the vascular smooth muscle. There are several lines of evidence suggesting that type I glomus cells may play a role in modifying the vascular tone in the carotid body. 1) Type I glomus cells and arteriolar myocytes establish direct contacts. 2) These contacts resemble sympathetic vascular neuromuscular junctions. 3) Type I glomus cells contain and release substances that are known to influence vascular smooth muscle tone such as catecholamines (B6ck 1973), acetylcholine (Wang et al. 1989), endothelin (Chen et al. 2000), adenine nucleotides (B6ck 1980a, b; Wang et al. 1991b; Chen et al. 2000), and possibly nitric oxide (Prabhakar et al. 1993). In turn, McDonald (1983) has also posed the question as to whether areas of glomus cells exposed to the perivascular space might be regions of high sensitivity to neurotransmitters or humoral substances. In this respect, the present observation of the occurrence of dense-cored vesicles, morphologically resembling secretory peptide containing vesicles, in a considerable number of arteriolar myocytes is striking. In the human carotid body, immunoreactivity to the neuropeptide bombesin has indeed been described in arteriolar myocytes (Smith et al. 1990). However, the exact chemical nature of the material contained in the vesicles observed here in the murine carotid body arterioles and their functional significance remains to be clarified. In addition to the assumed interaction between type I glomus cells and vascular smooth muscle, it is likely that the activity of the myocytes is influenced by the endothelial cells in direct contact with the blood stream. The frequent occurrence of close contacts between the arteriolar endothelial cells and the underlying myocytes reported in the present study as well as in that of B6ck (1973) provides morphological support to this view. These myoendothelial junctions are commonly observed in various vascular beds and are generally accepted as sites for transmission of signals from the vascular lumen through the layers of the arteriolar wall (Yamamoto et al. 1998; Emerson et al. 2002; Goto et al. 2002). Surprisingly, Weibel-Palade bodies that are considered to be storage organelles for the endothelial vasoconstrictor endothelin (van Mourik et al. 2002) have not been observed in the present study. This finding is in line, however, with the absence of
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endothelin-l-immunoreactivity in arteriolar endothelial cells of the rat carotid body both under normoxia and after chronic exposure to hypoxia (Chen et al. 2002). In conclusion, vascular smooth muscle cells of murine carotid body arterioles are sandwiched between endothelial cells and type I glomus cells, and these three diverse cell types can be considered to be an important functional unit in the carotid body.
Acknowledgements.The authors thank Ms. K. Michael, Mr. G. Kripp and Mr. G. Magdowski for skillful technical assistance.
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Accepted July 2, 2003
The spatial relationship between type I glomus cells and arteriolar myocytes in the mouse carotid body Amina B. EI-Fadaly 1'2 and Wolfgang Kummer 1 1Institute for Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, 35385 Giessen, Germany, 2Anatomy Department, Faculty of Medicine, Cairo University, Egypt
Correspondence to: A. B. E1-Fadaly
(Nunn 1993). The arterio-venous pO 2 difference is, therefore, small in accordance with its rapid response within 1-3 seconds (Ponte and Purves 1974). It is evident that modulation of the arterial vascular tone plays an essential role in perfusion rate control and, in turn, chemosensory activity (Wang et al. 1994 a; Wang et al. 1995; Lahiri and Buerk 1998; Buerk and Lahiri 2000; Iturriaga et al. 2000). The specialized vascular system of the carotid body is controlled by an extensive sympathetic, parasympathetic and sensory innervation (O'Regan 1981; McDonald and Larue 1983; Kummer 1990). Indeed, most of the sympathetic effects on chemoreception are exerted via vascular actions (Eyzaguirre and Lewin 1961; O'Regan 1981). In addition to perivascular nerve fibres, endothelial cells regulate arteriolar tone, e.g. by release of nitric oxide (NO) (Wang et al. 1994b). Several vasoactive substances are also produced and released by type I glomus cells. These include catecholamines (B6ck 1973; B6ck and Gorgas 1976; Wang et al. 1991a), adenine nucleotides (BOck 1980 a, b; Wang et al. 1991 b; Chen et al. 2000), NO (Prabhakar et al. 1993), and endothelin (He et al. 1996). It still remains an open question whether and to what extent glomus cells play a role in the regulation of vascular tone in the carotid body. As a structural basis for such a regulation, an intimate relationship between type I glomus cells and arterioles shall be expected. Despite the vast amount of structural investigations on the carotid body, there is very little information concerning this issue. In the mouse, Gorgas and B6ck (1977) have referred to the presence of cytoplasmic processes of type I glomus cells in contact with sensory nerve terminals in the tunica adventitia of some arterioles, but their relationship to the vascular myocytes was not reported. The present investigation was carried out to address this issue and to morphologically define a possible connection between type I glomus cells and the vascular smooth muscle in the mouse carotid body.
Ann Anat (2003) 185:507-515
0940-9602/03/185/6-507 $15.00/0
Summary: The structural relationship between type I glomus cells and the vascular smooth muscle was investigated by electron microscopy in the mouse carotid body. A close apposition (<0.1 pm) between the glomus parcnchyma and the neighbouring arterioles was regularly present. Profiles of type I glomus cells were found to be exposed to the vascular smooth muscle without any supporting cell investment. In circumscribed areas of these profiles, type I glomus cells and the vascular smooth muscle cells made contact by fusion of their basal laminae. These glomus-cell-myocyte junctions structurally resemble vascular neuromuscular junctions of sympathetic nerve terminals. In addition to the occurrence of such glomus cell-myocyte contacts, myoendothelial junctions also appeared frequently. On the basis of these observations, it is suggested that type I glomus cells play a role in the regulation of the vascular tone in the carotid body and that a physiological interaction between the endothelial cells, the vascular smooth muscle cells and the type I glomus cells exists. Key words: Carotid body - Type I glomus cells - Vascular smooth muscle cells - Ultrastructure - Mouse
Introduction The carotid body is a chemoreceptive organ acting as a rapid-responding monitor of arterial blood gases. It is provided with a highly specialised vascular system characterised by a high perfusion rate reaching ten times the level which would be proportional to its metabolic rate
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Material and m e t h o d s Four male adult 129SvEvx CF1 mice and two adult female FVB mice were sacrificed by inhalation of sevoflurane (Abbott, Wiesbaden, Germany). Immediately upon arrest of respiration, the thorax was opened and a cannula was inserted into the ascending aorta via the left ventricle. The vascular system was flushed with heparin and lidocaine containing rinsing solution (Forssmann et al. 1977) and the animals were fixed by perfusion with 1.5% glutardialdehyde, 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The carotid bifurcations were dissected, stored for an additional 3 h in the same fixative, and then washed in 0.1 M TRIS-HCI buffer, osmicated for i h in aqueous 1% OsO4, washed 3x15 min in 0.05 M maleate buffer (pH 5.2), stained en bloc for 1 h in 1% uranylacetate in maleate buffer at pH 6.0, washed again 3 x 15 min in 0.05 M maleate buffer (pH 5.2), and then routinely dehydrated in ethanol/propylene oxide and embedded in Epon. Semithin sections (0.75 gm) and ultrathin sections were cut with an ultramicrotome (Reichert Ultracut E, Leica, Bensheim Germany). Semithin sections were stained with Rfideberg-solution (1 g/1 methylene blue, l g/1 thionine, 17.8g/1 Na2HPO4x2H20, in distilled water). Thin sections were stained with alkaline lead citrate and viewed in a EM 902 transmission electron microscope (Zeiss, Jena, Germany).
Results The mouse carotid body was provided with a well-developed arterial system. An average of ten arterioles was
found per semithin cross section through the middle portion of the organ (measuring approximately 250 x 100 ~tm). The arterioles were embedded in the interstitial connective tissue between glomeruli formed by groups of glomus cells (Fig. 1). In some semithin sections, cytoplasmic processes of type I glomus cells were also seen emerging from the cell bodies and approaching the arterioles (Fig. 2). At the electron microscopic level, type I glomus cells facing the interstice were mostly enveloped by lamellar extensions of supporting cells formed by type II glomus cells or Schwann cells. These were usually covered by a continuous, regularly structured basal lamina. In some circumscribed areas where type I glomus cells were not enveloped by a supporting cell, the basal lamina directly covered type I glomus cells continuous with that of the surrounding supporting cells (Fig. 3). In most cases, the interstice between the glomus parenchyma and the neighbouring arteriole was reduced to a very narrow space reaching less than 0.1 gm in width. In these regions, type I glomus cells, together with their supporting cell investment, came into close spatial relationship with the adjacent arteriole; being separated from the surface of the vascular smooth muscle only by one or two extremely attenuated processes of adventitial cells (Fig. 3). Occasionally, type I glomus cells showed direct contact with the arteriolar myocyte (Fig. 4). At such sites, the cell membranes were separated by a regularly spaced cleft. This cleft was occupied by a single basal lamina that appeared to represent a fusion between the basal lamina of the type I glomus cell and that of the smooth muscle cell. In very rare occasions, omega profiles suggestive of vesicle exo- or endocytosis could be observed at the surface of type I glomus cells facing the myocyte.
Fig. 1. Semithin section through the middle portion of the carotid body showing one arteriole (A) located within the interstitial connective tissue between the glomeruli (G). (Riideberg staining; Bar = 10 pro) Fig. 2. A scmithin section showing processes (arrows) of type I glomus cells extending away from their cell body (I) to reach the wall of the neighboufing arterioles (A). (Bar = 10 gin)
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Fig. 3. a: Low power electron micrograph shows part of the glomus parenchyma and a nearby arteriole. Towards the interstitial tis-
sue, type I glomus cells (I) are seen enveloped by lamellar extensions (arrows) of supporting cells. Higher magnifications of regions indicated by rectangles are shown in b and c. b: illustrates a regularly constructed basal lamina (arrowheads) continuously covering the supporting cell investment of type I glomus cells, c: shows a process of type I glomus cell (I) with no supporting cell covering towards the interstitial space. The arrows in c point to the regions where the type II glomus cell envelope (II) ends and the basal laminae of the two cell types become continuous with each other. (Bar = 1 gm) Fig. 4. A contact area between a type I glomus cell (I) and a myocyte (M) of the neighbouring arteriole. A narrow, regularly spaced junctional cleft occupied by a single basal lamina is prominent. Arrow points to a pit at the surface of the type I glomus cell facing the myocyte indicative of a vesicle from an endo- or exocytosis process. E = endothelial cell (Bar = 1 gm) S l e n d e r processes of type I glomus cells with a diam e t e r ranging from 0.5 to 1.5 g m were m o r e frequently seen in relation to the adventitial connective tissue of the arterioles. In favourable sections, long p o r t i o n s of these processes were f o u n d to be enclosed within the l a m e l l a r processes of the adventitia a n d e x t e n d e d over a considerable distance of the circumference of the arteriole (Fig. 6a). I n such cases, wide areas of these processes 509
m a d e contact with the myocyte. In contact regions, the c y t o p l a s m of the processes c o n t a i n e d a b u n d a n t free ribosomes and s o m e t i m e s clusters of m i t o c h o n d r i a , while m i c r o t u b u l e s were only occasionally found (Fig. 6b, c). D e n s e - c o r e d vesicles with a m e a n d i a m e t e r of 121 + 22 n m (range: 88-167 nm), identical to those found in the cell body, were r e g u l a r constituents along the entire length of the process.
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Fig. 7. Among the numerous caveolae and clear vesicles in the cytoplasm of the vascular smooth muscle cell (M), prominent densecored vesicles (arrows) are observed close to the cell surface (a) and in the cytoplasm adjacent to the smooth endoplasmic reticulum (SER) (b). These vesicles have a distinct limiting membrane and display a central core of mild to moderate electron-dense matrix. N = nucleus; E = endothelial cell (Bar = 0,5 lam)
A r t e r i o l a r myocytes, on the o t h e r hand, showed no specific m o r p h o l o g i c a l characteristics at the site of contact with type I glomus cells. H o w e v e r , a m o n g the ordinarily clear vesicles n o r m a l l y found in the c y t o p l a s m of the myocytes, one or two p r o m i n e n t d e n s e - c o r e d vesicles p e r a r t e r i o l a r cross section could b e identified in a considerable n u m b e r of cases. These vesicles were mostly spherical and m e a s u r e d 91 + 10 n m (range: 75-110 nm) in their greatest diameter. T h e y h a d a distinct limiting m e m b r a n e and displayed a central core of mild to m o d e r a t e elect r o n - d e n s e matrix (Fig. 7). T h e central core was s e p a r a t e d from the limiting m e m b r a n e by a clear halo measuring 14 + 2 n m (range: 10-17 nm). In vesicles with faint electron-density, the matrix showed g r a n u l a r substructures with a d i a m e t e r of 8 + i n m (range: 6-12 nm). T h e vesi-
cles were f o u n d to be p r e d o m i n a n t l y l o c a t e d in areas rich in s m o o t h e n d o p l a s m i c reticulum. I n addition, it was also noticed that in most a r t e r i o l a r sections, the e n d o t h e l i a l cells and the underlying myocytes m a d e close contacts with each o t h e r t h r o u g h finger- or tongue-like processes. A t the contact areas, the basal l a m i n a e of t h e two cell types were continuous with each o t h e r without entering into the junctional cleft (Fig. 8). In circumscribed areas of the contact zones, some caveolae might be seen o p e n i n g at the surface of the m y o c y t e o p p o s i t e the intercellular junctional cleft (Fig. 8 c). A schematic drawing of the relationship b e t w e e n e n d o t h e l i a l cells, vascular myocytes, and type I glomus cell processes is p r e s e n t e d in Figure 9. Unexpectedly, W e i b e l - P a l a d e bodies were n o t o b s e r v e d in the a r t e r i o l a r e n d o t h e l i a l cells.
Fig. 5. Profiles of three slender processes (1-3) of type I glomus cells approaching the wall of an arteriole can be identified in a single section (a). One of these processes (1) is completely free of any supporting cell envelope (b). It penetrates through cytoplasmic processes (asterisks) of the adventitial cells to reach the myocyte (M). A single basal lamina (arrowheads in b) is shown interposed between this process and the myocyte. In e, another process (2) shows no contact with the vascular smooth muscle and is still separated from the myocyte by a single layer of lameltar processes of adventitiat cells. It is enclosed together with a vascular nerve terminal (N) by a common Schwann cell (S), A continuous basal lamina (arrowheads in c) covers the Schwann cell and the nerve terminal and continues over the free portions of the glomus cell process. (Bar = 1 gm) Fig. 6. a: This figure illustrates a longitudinally sectioned process of type I glomus cell (I) located within the lamellar processes of the adventitial connective tissue of one arteriole. In a stage section of the same process (b, e), one of its ends (b) comes into contact with the myocyte with only a single basal lamina (arrowheads) interposed. At this region, the process is characteristically free of any supporting cell investment. Its cytoplasm appears rich in free ribosomes and mitochondria but without microtubules. In contrast, the other end of the process (e) shows no contact with the myocyte. It is enveloped by cytoplasmic processes of supporting cells and contains abundant prominent microtubules (MT), but very few ribosomes. Dense-cored vesicles (DCV), characteristic of type I glomus cells, are shown along the entire length of the process. (Bar = 1 Bm in a, b; 0,5 pin in c) 511
Fig. 8. Profiles of myoendothelial junctions in different arteriolar sections. Figures a, b show processes (arrows) emerging from the endothelial cells (E) to reach the underlying myocytes (M) while in c the process (arrow) projects from the myocyte to reach the overlying endothelial cell. The cell membranes of the two cell types come into close apposition with no basal lamina in between. The basal laminae (arrowheads) of the two cells are reflected at the sides of the contact zones to be continuous with each other. Two caveolae open at the surface of the myocyte opposite to the intercellular junctional cleft (small arrows in b). (Bar = 1 ~m in a, c; 0,5 gm in b)
Fig. 9. Schematic drawing showing the structural relationship between an arteriolar endothelial cell (E), a vascular smooth muscle cell (M) and a type I glomus cell process (I). BL = Basal lamina.
Discussion The p r e s e n t study d e m o n s t r a t e s an intimate structural relationship b e t w e e n type I glomus cells and the vascular s m o o t h muscle. In a considerable n u m b e r of cases, the interstitial space b e t w e e n the glomus p a r e n c h y m a and the neighbouring arterioles was r e d u c e d to a very n a r r o w cleft reaching less than 0.1 g m in width. A t such sites, the glomus p a r e n c h y m a was s e p a r a t e d from the vascular s m o o t h muscle by only one or two e x t r e m e l y a t t e n u a t e d processes of adventitial cells, and even cell bodies of t y p e ! glomus cells c a m e into direct contact with the art e r i o l a r myocytes. A r e a s of type I glomus cells e x p o s e d to the a r t e r i o l a r wall have also b e e n d e s c r i b e d in the rat carotid b o d y ( M c D o n a l d 1983; M c D o n a l d and L a r u e 1983) but direct contacts as o b s e r v e d h e r e in the m o u s e have not b e e n r e p o r t e d for that species. L o n g slender cytoplasmic processes of type I glomus cells, often free of a supporting cell investment, were found to be m o r e frequently r e l a t e d to the adventitia of the a r t e r i o l a r walls than their cell somata. G o r g a s and B6ck (1977) and B6ck (1982) have also r e f e r r e d to the existence of similar processes in the tunica adventitia of some arterioles in the m o u s e carotid body. T h e y focussed 512
upon the relationship between these processes and lanceolate nerve endings which they interpreted as representing baroreceptors (Gorgas and B6ck 1977). Our present study focussed on the relationship between type I glomus cell processes and the arteriolar myocytes. As a consistent finding, a junctional cleft was found in the contact areas which characteristically contained a single basal lamina formed by fusion of the basal lamina of the myocyte with that of the type I glomus cell. The cytoplasm of type I glomus cells in these regions typically contained abundant free ribosomes and might show clusters of mitochondria with occasional microtubules, unlike the ordinary processes which were characterised by abundant microtubules but few ribosomes and mitochondria. Besides these organelles, dense-cored vesicles, identical to those found in the cell body, were present along the entire length of the processes. The morphological criteria of the junctional area between type I glomus cells and arteriolar myocytes resemble sympathetic vascular neuromuscular junctions described in various vascular beds (Luff et al. 1987, 1991, 1995; Luff and McLachlan 1988, 1989). These investigators, using serial section analysis of individual varicosities, found a high proportion of the varicosities forming neuromuscular junctions with the vascular smooth muscle. They also reported a junctional cleft occupied by a single basal lamina formed by fusion of the basal laminae of the sympathetic varicosity with the myocyte. In support of the concept of equivalence of type I glomus cell-myocyte junctions with neuromuscular junction, McDonald and Michell (1975) have also reported that the neural crest-derived glomus cells have general structural characteristics common to neurones, being enveloped by glial-like sheath ceils, having dendritic processes arising from the cell bodies, and forming synaptic junctions with one another or with nerve terminals. Type I glomus cells and their processes contain catecholamines and a variety of other transmitters and neuromodulators (B/Sck 1973, 1982; BOck and Gorgas 1976; Heym and Kummer 1989; Kummer 1990). Consequently, it could be assumed that type I glomus cell processes forming junctions on the arteriolar myocytes might release transmitters or modulators as, for example, catecholamines. However, the presence of membrane specializations indicating a site of attachment of the vesicles for exocytosis or vesicle release, as in morphologically typical synapses and skeletal neuromuscular junctions, could not be detected. Similarly, prejunctional membrane specializations are almost absent at the vascular neuromuscular junctions and have been reported only occasionally (Luff 1996). This author has attributed the presence or absence of such membrane specializations to different postfixation times used during tissue preparation. The present results also demonstrated that junctional regions between type I glomus cells and the vascular smooth muscle did not localise at any specific region of the myocyte and were not accompanied by any obvious postjunctional membrane specializations such as dense bands or accumulations of caveolae at the cell membrane. Similar data were also re-
ported by Luff (1996) concerning sympathetic nerve terminals forming junctions with the vascular smooth muscle. This author proposed that postjunctional membrane characteristics are not essential for a neuromuscular junction to operate since neuromuscular junctions lacking postmembrane specialisation are present in skeletal muscles of non-mammalian vertebrate and invertebrate species. Moreover, in case of type I glomus cells, it is generally accepted that morphologically typical synapses are not the only site at which transmitters or other secretory products are released (McDonald and Michell 1975; B6ck 1973, 1982). The morphological data obtained from the present investigation are strongly indicative of an intimate physiological relationship between type I glomus cells and the vascular smooth muscle. There are several lines of evidence suggesting that type I glomus cells may play a role in modifying the vascular tone in the carotid body. 1) Type I glomus cells and arteriolar myocytes establish direct contacts. 2) These contacts resemble sympathetic vascular neuromuscular junctions. 3) Type I glomus cells contain and release substances that are known to influence vascular smooth muscle tone such as catecholamines (B6ck 1973), acetylcholine (Wang et al. 1989), endothelin (Chen et al. 2000), adenine nucleotides (B6ck 1980a, b; Wang et al. 1991b; Chen et al. 2000), and possibly nitric oxide (Prabhakar et al. 1993). In turn, McDonald (1983) has also posed the question as to whether areas of glomus cells exposed to the perivascular space might be regions of high sensitivity to neurotransmitters or humoral substances. In this respect, the present observation of the occurrence of dense-cored vesicles, morphologically resembling secretory peptide containing vesicles, in a considerable number of arteriolar myocytes is striking. In the human carotid body, immunoreactivity to the neuropeptide bombesin has indeed been described in arteriolar myocytes (Smith et al. 1990). However, the exact chemical nature of the material contained in the vesicles observed here in the murine carotid body arterioles and their functional significance remains to be clarified. In addition to the assumed interaction between type I glomus cells and vascular smooth muscle, it is likely that the activity of the myocytes is influenced by the endothelial cells in direct contact with the blood stream. The frequent occurrence of close contacts between the arteriolar endothelial cells and the underlying myocytes reported in the present study as well as in that of B6ck (1973) provides morphological support to this view. These myoendothelial junctions are commonly observed in various vascular beds and are generally accepted as sites for transmission of signals from the vascular lumen through the layers of the arteriolar wall (Yamamoto et al. 1998; Emerson et al. 2002; Goto et al. 2002). Surprisingly, Weibel-Palade bodies that are considered to be storage organelles for the endothelial vasoconstrictor endothelin (van Mourik et al. 2002) have not been observed in the present study. This finding is in line, however, with the absence of
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endothelin-l-immunoreactivity in arteriolar endothelial cells of the rat carotid body both under normoxia and after chronic exposure to hypoxia (Chen et al. 2002). In conclusion, vascular smooth muscle cells of murine carotid body arterioles are sandwiched between endothelial cells and type I glomus cells, and these three diverse cell types can be considered to be an important functional unit in the carotid body.
Acknowledgements.The authors thank Ms. K. Michael, Mr. G. Kripp and Mr. G. Magdowski for skillful technical assistance.
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Accepted July 2, 2003