Cell Tiss. Res. 168, 55-63 (1976)
Cell and Tissue Research 9 by Springer-Veriag 1976
Fine Structure of Chemosensitive Cells (Glomus caroticum) in Tissue Culture* ** F. Pietruschka and D. Sch/ifer with technical assistance of S. Gattermann and H. Poloczek Max-Planck-Institut f/Jr Systemphysiologie, Dortmund, Germany
Summary. Cells of the carotid body of both embryonic and 1 to 2-d-old rabbits were cultured in monolayer in primary tissue culture. The cells grew in their original association of type I and type II cells. The fine structure of both cell types was similar to that in vivo. Even after one week in culture their morphological characteristics, such as the dense-cored vesicles, were preserved. The prolonged synthesis of catecholamines in culture and the formation of new granular vesicles is discussed.
Key words: Glomus caroticum (rabbit) - Primary tissue culture - Densecored vesicles - Fine structure.
The carotid body is an arterial, chemoreceptive organ which is stimulated by oxygen deprivation and rise of CO2 pressure in the arterial blood (Heymans et al., 1930; Hornbein et al., 1961; Acker et al., 1973). Sensory nerve endings around the cells of the carotid body are thought to be involved in the process of chemoreception (de Castro, 1928; Biscoe and Pallot, 1972; Verna, 1973; McDonald and Mitchell, 1975), though little is known about the origin of the stimulus. To obtain further information as to the function of the carotid body cells, we cultured them as monolayer (Pietruschka, 1974). This allowed us to perform physiological experiments on single cells (Acker et al., 1975). The aim of this work is to show that ultrastructural cell differentiation is distinctly preserved in vitro. Send offprint requests to: Dr. F. Pietruschka, Max-Planck-Institut for Systemphysiologie, Rheinlanddamm 201, D-4600 Dortmund, Federal Republic of Germany * The results were reported in part at "17. Tagung fiir Elektronenmikroskopie" held on September 21-26, 1975 in Berlin ** The authors want to thank Prof. Dr. D.W. Lfibbers for his advice and helpful suggestions
56
F. Pietruschka and D. Schfifer
Materials and Methods The carotid bodies were prepared as described previously (Pietruschka et al., 1973) from embryos (28 to 30 d of gestation) or l- to 2-d-old rabbits. They were carefully reduced to small pieces and enzymatically dissociated (Pietruschka, 1974) in trypsin, collagenase and hyaluronidase in Minimum Essential Medium (Flow Laboratories, Bonn) for 15 min at 37 ~ C. pH was adjusted to 6.6 with HCI. The cells of 12 carotid bodies were cultured on polystyrene coverslips (Lux Scientific Corp., Thousand Oaks, Calif.), coated with reconstituted rat-tail collagen (Bornstein, 1958), in 1.5 ml Medium 199 (Flow Laboratories, Bonn) +500 mg-% glucose + 15% fetal calf serum + 10% glutamine in Leighton tubes at 37 ~ C. The pH of the culture medium was adjusted to 6.9 by bubbling with CO2. For electronmicroscopic examination the cells were fixed in 0.2 M glutaraldehyde in 0.1 M cacodylate buffer +0.2 M saccharose (480 mosm) for 15 min at 4 ~ (Hiindgen et al., 197t; Weissenfels et al., 1971) either immediately after enzymatic dissociation or 6 to 25 h later, and collected by centrifugation. Usually the cells were cultured for a period of several days to two weeks and then, after removing the coverslips from the Leighton tubes, fixed in situ by dipping them into the fixative ( + 4 ~ C). After washing (0.24 M saccharose in 0.1 M cacodylate buffer solution, pH 7.4, 4 ~ C, 320 mosm; 6 times for 10 min) and postfixation (1% OSO4+0.24 saccharose in 0.1 M cacodylate buffer solution, pH 7.4, 20 ~ C, 320 mosm) for 1 h, the cells were dehydrated in ethanol series from 15% to absolute alcohol in seven steps at 20~ for 21/2 h and embedded in Epon 812 (Luft, 1961). Propylene oxide as an intermedium was omitted (Rubin, personal communication) because it dissolves the plastic coverslip on which the cells are being cultured. So it was necessary to take the specimens from absolute alcohol directly in Epon (changed 3 times, for a total of 15 h). Contrasting was carried out with 1% phosphotungstic acid + 1% uranyl acetate in 70% alcohol (bloc). After mechanical removal of the coverslips from the hardened Epon bloc sections were made with ultramicrotomes in the usual manner. The examination was carried out with the Elmiscope 101 (Siemens, Berlin).
Results Since in an adult animal the contribution of connective tissue to the whole carotid body tissue is about 50% (Seidl, personal communication), we prepared carotid bodies from embryos or a few days old rabbits. In the 28- to 30-days-old embryo the morphological organization of the carotid body is similar to that of an adult animal (de Kock, 1966; Verna, 1971). It is also composed of functional units, the glomoids (Seidl, 1973), each of which is surrounded by connective tissue. The glomoids contain two types of cells (Fig. 1), the chromaffin type I cells (main cells, I in Fig. 1) which are enveloped by elongate processes of the type II cells (sustentacular cells, II in Fig. 1). The type I cells have round or oval nuclei with peripheral aggregations of chromatin. Sometimes nucleoli are observed. In the cytoplasm we find mitochondria and a well-developed Golgi apparatus. Besides the cisternae of the rough endoplasmatic reticulum the cytoplasm contains a great number of free ribosomes and polysomes and, Fig. 1. Electron micrograph from the carotid body of a 29d old rabbit embryo. The morphological organization is similar to that in an adult animal. Note the well developed nervous supply. 1, type I cell, II, type II cell, A, axon, bar: 1 lam. x 8,000 Fig. 2. Cells of the carotid body derived from a 30d old embryo fixed immediately after enzymatic treatment. N, nucleus of a type I cell, ~ processus of a type II cell, ~ dense-cored vesicles with an enlarged halo stained with lead citrate for 2 min, bar : 1 lam. x 20,000
Glomus caroticum in Tissue Culture
57
58
F. Pietruschka and D. Sch~ifer
as a most characteristic feature, numerous dense-cored vesicles. Small nerves and nerve endings are found on the membrane of the cell. To preserve differentiation of the carotid body cells in culture, we tried to maintain the functional units consisting of type I and type II cells. As we intended to culture the carotid body cells in their original cluster, we interrupted the enzymatic dissociation before the cell isolation was achieved. Figure 2 shows cells which had been fixed immediately after enzymatic treatment. We find an association of type I cells and a small, elongate processes of a type II cell ( ~ in Fig. 2). The main feature of the type I cells, the numerous dense-cored vesicles (--. in Fig. 2) have been preserved throughout the procedure of dissociation and centrifugation. The granules are not evenly distributed over the whole cytoplasm, but largely restricted to the periphery and, in some cells, aligned along the outer membrane ( ~ in Fig. 2). In many vesicles the usually narrow, clear halo is enlarged. The diameter of these vesicles reaches 200 nm. Their dense cores assume an eccentric position and, as a consequence of the thin sections (500 A), sometimes even clear vesicles are found. Presumably water has been taken up during isolation of the cells. However, widening of the vesicles has also been observed in untreated carotid bodies (B6ck, 1973). During the first 2 to 3 days of culturing the cells remained undisturbed. After this time they had settled down and attached to the dish. Aside from single cells, clusters of various size were found. The cells had flattened but still exhibited the morphology of type I cells (Fig. 3) with their round or oval nuclei (N1) and cloudy chromatin network, and type II cells with reniform nuclei (N2) and largely condensed chromatin. With the flattening of the cytoplasm the mitochondria became elongate, their diameters varied between 0.15 to 0.5 pm, their lengths reached 2.5 pm. In contrast to the shape observed immediately after enzymatic treatment, the dense-cored vesicles (arrows in Fig. 3) had now a normal appearance. Like in vivo, they were mostly spherical with a more or less dense core and a narrow halo between the core and the membrane. Their diameters averaged 50 to 150 nm. After one week of culturing the cells had grown without losing the intimate contact to each other. Type I and type II cells were well discernable by the different form of their nuclei (N1 and N 2 in Fig. 4). The sustentacular cells still enveloped the compact glomus cells (=~ in Fig. 4). In the type I cells the proportion of the endoplasmatic reticulum and numerous rosettes of free ribosomes varied. The number of lysosomes, which were already present in vivo, seemed to increase with prolonged time of culture, and this was also true for the lipid droplets (L). As in vivo, the cells were rich in mitochondria (Sch~ifer et al., 1973), some of which had electron-dense granules and osmiophilic bodies of heterogenous density (B in Fig. 5) and often double limited membranes which had the size of the mitochondria ( ~ in Fig. 6). The dictyosomes of the Golgi Fig. 3. Cells after 3 d of culturing. They still exhibit the morphology of type I cells with round or oval nuclei with cloudy chromatin network, and type II cells with reniform nuclei and largely condensed chromatin. The dense-cored vesicles are normal in appearance ( ~ ) . N1, nucleus of a type I cell, N2, nucleus of a type II cell, M, mitochondria, bar I ~tm. • 20,000
G l o m u s caroticum in Tissue Culture
59
Fig. 4. Cells after 6 d of culturing. The sustentacular cells still envelop the compact glomus cells. N1, nucleus of a type I cell, N2, nucleus of a type II cell, L, lipid droplet, ~ processes of a type II cell, bar: 1 tim. x 12,000
60
F. Pietruschka and D. Sch/ifer
Fig. 5. Cells after 6 d of culturing. Few mitochondria show electron-dense granules (--,). B, osmiophilic bodies of heterogenous density, D, dictyosomes of the Golgi apparatus, bar: 1 lam. • 30,000
Glomus caroticum in Tissue Culture
61
apparatus were well developed (D in Fig. 6), and the vesicular component of the dictyosomes often contained electron-dense material ( ~ in Fig. 6). Densecored vesicles were found in their immediate vicinity (---, Fig. 6). Occasionally parts of a cilium were found (C in Fig. 6) in the ultrathin sections of cultured type I cells that had been observed also in vivo (Biscoe, 1971; B6ck, 1973).
Discus~on
From physiological experiments on new-born lambs (Biscoe and Purves, 1967) it is known that the carotid bodies are mature at birth. The chemoreceptor response to both hypoxia and rise of CO2 in the inspired air is similar to that observed in adult animals. Even in a far younger state of development of the midterm human fetus, Hervonen and Korkala (1972a, b) found carotid bodies of mature appearance as to fine structure and catecholamine fluorescence. In full agreement with these findings we could not detect conspicuous morphological differences between carotid bodies of rabbit embryos 2 to 4d before birth and those of adult rabbits investigated by M611mann et al. (1973). As there is evidence that the carotid body is of neural crest origin and belongs to the A P U D (Amine Precursor Uptake and Decarboxylation) series (Pearse et al., 1973), we cultured the carotid body cells in a medium which is of high concentrations of serum and glucose and low pH value (Lumsden, 1968; H6sli and H6sli, 1970) usually used for nerve cells in vitro. Indeed, we were not able to maintain the dense-cored vesicles in the cultured cells unless we lowered the pH of the enzyme solution to 6.61 and that of the culture medium from 7.4 to 6.9. Like neurons in culture, aging carotid body cells also show an increased amount of lipofuscin bodies (Fig. 5). Spoerri and Glees (1973) showed that they originate from degenerating mitochondria. Lipofuscin bodies were also found in the carotid bodies of adult human beings and animals in situ (Grimly and Glenner, 1968; Edwards and Heath, 1972). With the methods employed we are able to culture carotid body cells up to 14d in their original association of type I and type II cells. Furthermore, the type I cells show their morphological characteristics, such as the compact shape, round or oval nuclei, many mitochondria, rough endoplasmatic reticulum, numerous free ribosomes, a well-developed Golgi apparatus and a great number of dense-cored vesicles. The electron-dense material in the vesicles of dictyosomes and in the granular vesicles near the Golgi apparatus indicate that the synthesis of catecholamines continues in culture (see also Erfink6 et al., 1972). This assumption agrees with previous findings (Pietruschka, 1974) of an increase in the catecholamine fluorescence of carotid body cells after one week of culture. 1
The authors are indebted to Dr. H. Starlinger for giving this advice
Fig. 6. Cells after 3d of culturing. The dictyosomes of the Golgi apparatus are well developed and the vesicular component of the apparatus often contains electron dense material ( ~ ) and dense-cored vesicles are found in their immediate vicinity (-,). ~ osmiophilic body with double limiting membrane, D, dictyosomes of the Golgi apparatus, C, cilium, bar: 1 ~tm. x 30,000
62
F. Pietruschka and D. Schiller
References Acker, H., Keller, H.-P., Lfibbers, D.W. : The relationship between neuronal activity of chemoreceptor fibers and tissue PO 2 of the carotid body of the cat during changes in arterial PO 2 and blood pressure. Pflfigers Arch. 343, 287-296 (1973) Acker, H., Pietruschka, F., Lfibbers, D.W. : First measurements of membrane potentials of cultivated carotid body chemoreceptor cells of rabbit and cat under controlled PO2 and pH conditions. Pflfigers Arch. (Europ. J. Physiol. Suppl.) 359, R 128 (1975) Biscoe, T.J. : Carotid body: structure and function. Physiol. Rev. 51, 437495 (1971) Biscoe, T.J., Pallot, D. : Serial reconstruction with the electron microscope of carotid body tissue. The type I cell nerve supply. Experientia (Basel) 28, 33-34 (1972) Biscoe, T.J., Purves, M.J. : Carotid body chemoreceptor activity in the new-born lamb. J. Physiol. (Lond.) 190, 443~153 (1967) B6ck, P.: Das Glomus caroticum der Maus. Adv. Anatom., Embryol. Cell. Biol. 48, 7-84 (1973) Bornstein, M.B.: Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes. Lab. Invest. 7, 134-137 (1958) De Castro, F. : Sur la structure et l'innervation du sinus carotidien de l'homme et des mammif+res. Nouveaux faits sur l'innervation et la fonction du glomus caroticum. Trav. Lab. Rech. Biol. Univ. Madrid 25, 330-380 (1928) Edwards, C., Heath, D., Harris, P. : Ultrastructure of the carotid body in high-altitude guinea-pigs (Plates LXVIII-LXXIII) J. Path. 107, 131 136 (1972) Erank6, G., Heath, J., Er~nk6, L.: Effect of hydrocortisone on the ultrastructure of the small, intensely fluorescent, granule-containing cells in culture of sympathetic ganglia of new-born rats. Z. Zellforsch. 134, 297 310 (1972) Grimley, P.M., Glenner, G.G.: Ultrastructure of the human carotid body. A perspective on the mode of chemoreception. Circulation 37, 648~665 (1968) Hervonen, A., Korkala, O.: Fine structure of the carotid body of the midterm human fetus. Z. Anat. Entwickl.-Gesch. 138, 135 144 (1972) Hervonen, A., Korkala, O. : The histochemically demonstrable monoamines of human fetal carotid body. Experientia (Basel) 28, 449~,50 (1972) Heymans, C., Bouckaert, J.J., Dautrebande, L.: Sinus carotidien et r6flexes respiratoires. II. Influences respiratoires r6flexes de l'acidose, de l'alcalose, de l'anhydride carbonique, de l'hydrog~ne et de l'anox6mie. Sinus carotidiens et 6changes respiratoires dans les poumons et au del6 des poumons. Arch. int. Pharmocodyn. 39, 400~448 (1930) H6sli, E., H6sli, L.: The presence of acetylcholinesterase in cultures of cerebellum and brain stem. Brain Res. 19, 494496 (1970) Hornbein, T.F., Griffo, Z.J., Roos, A.: Quantitation of chemoreceptor activity: Interrelation of hypoxia and hypercapnia. J. Neurophysiol. 24, 561 568 (1961) Hiindgen, M., Schgfer, D., Weissenfels, N. : Der FixierungseinfluB acht verschiedener Aldehyde auf die Ultrastruktur kultivierter Zellen. II. Der Strukturzustand des Cytoplasmas. Cytobiologie 3, 202 214 (1971) Kock, L.L. de, Dunn, A.E.G.: An electron microscope study for the carotid body. Acta anat. (Basel) 64, 163-178 (1966) Luft, H.J.: Improvements du Epoxy Resin. J. biophys, biochem. Cytol. 9, 409-414 (1961) Lumsden, L.E.: Nervous tissue in culture. In: The structure and function of nervous tissue (G.H. Bourne, ed.), pp. 67 140. New York-London: Academic Press 1968 McDonald, D., Mitchell, R.A. : The innervation of glomus cells, ganglion cells and blood vessels in the rat carotid body: a quantitative ultrastructural study. J. Neurocytol. 4, 177-230 (1975) M611mann, H., Knoche, H., Niemeyer, D.H., Alfes, H., Kienecker, E.W., Decker, S. : Experimenteller Beitrag zur Kenntnis der biogenen Amine im Glomus caroticum des Kaninchens. Elektronenund fluoreszenzmikroskopische Untersuchungen nach Reserpin- und PCPA-Applikation. Z. Zellforsch. 124, 238 246 (1972) Pearse, A.C.E., Polak, J.M., Rost, F.W.D., Fontaine, J., Le Li6vre, C., Le Douarin, N. : Demonstration of the neural crest origin of type I (APUD) cells in the avian carotid body using a cytochemical marker system. Histochemie 34, 191-203 (1973) Pietruschka, F.: Cytochemical demonstration of catecholamines in cells of the carotid body in primary tissue culture. Cell Tiss. Res. 151, 317-321 (1974)
Glomus caroticum in Tissue Culture
63
Pietruschka, F., Acker, H., Gattermann, S., Seidl, E., Lfibbers, D.W.: Cells of the carotid body in primary tissue cultures. Arzneimittel-Forsch. (Drug Res.) 23, 1610 (1973) Sch~ifer, D., Sch~ifer, S., Lfibbers, D.W. : On the population of type I glomus cells of the cat and in cells of other organs. Arzneimittel-Forsch. (Drug Res.) 23, 1612 (1973) Seidl, E. : Die Anwendung des Interferenzkontrast-Verfahrens am gef'~.rbten Schnitt als Hilfsmittel bei der Rekonstruktion yon Organellen. Leitz-Mitteil. Wiss. Tech. 6, 57 58 (1973) Spoerri, P.E., Glees, P.: Neuronal aging in cultures: an electronmicroscopic study. Exp. Geront. 8, 259-263 (1973) Verna, A.: Infrastructure des divers types de terminaisons nerveuses dans le glomus carotidien du lapin. J. Microscopie 10, 59~6 (1971) Verna, A.: Terminaisons nerveuses aff6rentes et eff6rentes dans le glomus carotidien du lapin. Microscopie 16, 299-308 (1973) Weissenfels, N., Sch~ifer, D., Hfindgen, M. : Der FixierungseinfluB acht verschiedener Aldehyde auf die Ultrastruktur kultivierter Zellen. I. Der Strukturzustand des Zellkerns. Cytobiologie 3, 188 201 (1971)
Received December 19, 1975
Cell and Tissue Research 9 by Springer-Veriag 1976
Fine Structure of Chemosensitive Cells (Glomus caroticum) in Tissue Culture* ** F. Pietruschka and D. Sch/ifer with technical assistance of S. Gattermann and H. Poloczek Max-Planck-Institut f/Jr Systemphysiologie, Dortmund, Germany
Summary. Cells of the carotid body of both embryonic and 1 to 2-d-old rabbits were cultured in monolayer in primary tissue culture. The cells grew in their original association of type I and type II cells. The fine structure of both cell types was similar to that in vivo. Even after one week in culture their morphological characteristics, such as the dense-cored vesicles, were preserved. The prolonged synthesis of catecholamines in culture and the formation of new granular vesicles is discussed.
Key words: Glomus caroticum (rabbit) - Primary tissue culture - Densecored vesicles - Fine structure.
The carotid body is an arterial, chemoreceptive organ which is stimulated by oxygen deprivation and rise of CO2 pressure in the arterial blood (Heymans et al., 1930; Hornbein et al., 1961; Acker et al., 1973). Sensory nerve endings around the cells of the carotid body are thought to be involved in the process of chemoreception (de Castro, 1928; Biscoe and Pallot, 1972; Verna, 1973; McDonald and Mitchell, 1975), though little is known about the origin of the stimulus. To obtain further information as to the function of the carotid body cells, we cultured them as monolayer (Pietruschka, 1974). This allowed us to perform physiological experiments on single cells (Acker et al., 1975). The aim of this work is to show that ultrastructural cell differentiation is distinctly preserved in vitro. Send offprint requests to: Dr. F. Pietruschka, Max-Planck-Institut for Systemphysiologie, Rheinlanddamm 201, D-4600 Dortmund, Federal Republic of Germany * The results were reported in part at "17. Tagung fiir Elektronenmikroskopie" held on September 21-26, 1975 in Berlin ** The authors want to thank Prof. Dr. D.W. Lfibbers for his advice and helpful suggestions
56
F. Pietruschka and D. Schfifer
Materials and Methods The carotid bodies were prepared as described previously (Pietruschka et al., 1973) from embryos (28 to 30 d of gestation) or l- to 2-d-old rabbits. They were carefully reduced to small pieces and enzymatically dissociated (Pietruschka, 1974) in trypsin, collagenase and hyaluronidase in Minimum Essential Medium (Flow Laboratories, Bonn) for 15 min at 37 ~ C. pH was adjusted to 6.6 with HCI. The cells of 12 carotid bodies were cultured on polystyrene coverslips (Lux Scientific Corp., Thousand Oaks, Calif.), coated with reconstituted rat-tail collagen (Bornstein, 1958), in 1.5 ml Medium 199 (Flow Laboratories, Bonn) +500 mg-% glucose + 15% fetal calf serum + 10% glutamine in Leighton tubes at 37 ~ C. The pH of the culture medium was adjusted to 6.9 by bubbling with CO2. For electronmicroscopic examination the cells were fixed in 0.2 M glutaraldehyde in 0.1 M cacodylate buffer +0.2 M saccharose (480 mosm) for 15 min at 4 ~ (Hiindgen et al., 197t; Weissenfels et al., 1971) either immediately after enzymatic dissociation or 6 to 25 h later, and collected by centrifugation. Usually the cells were cultured for a period of several days to two weeks and then, after removing the coverslips from the Leighton tubes, fixed in situ by dipping them into the fixative ( + 4 ~ C). After washing (0.24 M saccharose in 0.1 M cacodylate buffer solution, pH 7.4, 4 ~ C, 320 mosm; 6 times for 10 min) and postfixation (1% OSO4+0.24 saccharose in 0.1 M cacodylate buffer solution, pH 7.4, 20 ~ C, 320 mosm) for 1 h, the cells were dehydrated in ethanol series from 15% to absolute alcohol in seven steps at 20~ for 21/2 h and embedded in Epon 812 (Luft, 1961). Propylene oxide as an intermedium was omitted (Rubin, personal communication) because it dissolves the plastic coverslip on which the cells are being cultured. So it was necessary to take the specimens from absolute alcohol directly in Epon (changed 3 times, for a total of 15 h). Contrasting was carried out with 1% phosphotungstic acid + 1% uranyl acetate in 70% alcohol (bloc). After mechanical removal of the coverslips from the hardened Epon bloc sections were made with ultramicrotomes in the usual manner. The examination was carried out with the Elmiscope 101 (Siemens, Berlin).
Results Since in an adult animal the contribution of connective tissue to the whole carotid body tissue is about 50% (Seidl, personal communication), we prepared carotid bodies from embryos or a few days old rabbits. In the 28- to 30-days-old embryo the morphological organization of the carotid body is similar to that of an adult animal (de Kock, 1966; Verna, 1971). It is also composed of functional units, the glomoids (Seidl, 1973), each of which is surrounded by connective tissue. The glomoids contain two types of cells (Fig. 1), the chromaffin type I cells (main cells, I in Fig. 1) which are enveloped by elongate processes of the type II cells (sustentacular cells, II in Fig. 1). The type I cells have round or oval nuclei with peripheral aggregations of chromatin. Sometimes nucleoli are observed. In the cytoplasm we find mitochondria and a well-developed Golgi apparatus. Besides the cisternae of the rough endoplasmatic reticulum the cytoplasm contains a great number of free ribosomes and polysomes and, Fig. 1. Electron micrograph from the carotid body of a 29d old rabbit embryo. The morphological organization is similar to that in an adult animal. Note the well developed nervous supply. 1, type I cell, II, type II cell, A, axon, bar: 1 lam. x 8,000 Fig. 2. Cells of the carotid body derived from a 30d old embryo fixed immediately after enzymatic treatment. N, nucleus of a type I cell, ~ processus of a type II cell, ~ dense-cored vesicles with an enlarged halo stained with lead citrate for 2 min, bar : 1 lam. x 20,000
Glomus caroticum in Tissue Culture
57
58
F. Pietruschka and D. Sch~ifer
as a most characteristic feature, numerous dense-cored vesicles. Small nerves and nerve endings are found on the membrane of the cell. To preserve differentiation of the carotid body cells in culture, we tried to maintain the functional units consisting of type I and type II cells. As we intended to culture the carotid body cells in their original cluster, we interrupted the enzymatic dissociation before the cell isolation was achieved. Figure 2 shows cells which had been fixed immediately after enzymatic treatment. We find an association of type I cells and a small, elongate processes of a type II cell ( ~ in Fig. 2). The main feature of the type I cells, the numerous dense-cored vesicles (--. in Fig. 2) have been preserved throughout the procedure of dissociation and centrifugation. The granules are not evenly distributed over the whole cytoplasm, but largely restricted to the periphery and, in some cells, aligned along the outer membrane ( ~ in Fig. 2). In many vesicles the usually narrow, clear halo is enlarged. The diameter of these vesicles reaches 200 nm. Their dense cores assume an eccentric position and, as a consequence of the thin sections (500 A), sometimes even clear vesicles are found. Presumably water has been taken up during isolation of the cells. However, widening of the vesicles has also been observed in untreated carotid bodies (B6ck, 1973). During the first 2 to 3 days of culturing the cells remained undisturbed. After this time they had settled down and attached to the dish. Aside from single cells, clusters of various size were found. The cells had flattened but still exhibited the morphology of type I cells (Fig. 3) with their round or oval nuclei (N1) and cloudy chromatin network, and type II cells with reniform nuclei (N2) and largely condensed chromatin. With the flattening of the cytoplasm the mitochondria became elongate, their diameters varied between 0.15 to 0.5 pm, their lengths reached 2.5 pm. In contrast to the shape observed immediately after enzymatic treatment, the dense-cored vesicles (arrows in Fig. 3) had now a normal appearance. Like in vivo, they were mostly spherical with a more or less dense core and a narrow halo between the core and the membrane. Their diameters averaged 50 to 150 nm. After one week of culturing the cells had grown without losing the intimate contact to each other. Type I and type II cells were well discernable by the different form of their nuclei (N1 and N 2 in Fig. 4). The sustentacular cells still enveloped the compact glomus cells (=~ in Fig. 4). In the type I cells the proportion of the endoplasmatic reticulum and numerous rosettes of free ribosomes varied. The number of lysosomes, which were already present in vivo, seemed to increase with prolonged time of culture, and this was also true for the lipid droplets (L). As in vivo, the cells were rich in mitochondria (Sch~ifer et al., 1973), some of which had electron-dense granules and osmiophilic bodies of heterogenous density (B in Fig. 5) and often double limited membranes which had the size of the mitochondria ( ~ in Fig. 6). The dictyosomes of the Golgi Fig. 3. Cells after 3 d of culturing. They still exhibit the morphology of type I cells with round or oval nuclei with cloudy chromatin network, and type II cells with reniform nuclei and largely condensed chromatin. The dense-cored vesicles are normal in appearance ( ~ ) . N1, nucleus of a type I cell, N2, nucleus of a type II cell, M, mitochondria, bar I ~tm. • 20,000
G l o m u s caroticum in Tissue Culture
59
Fig. 4. Cells after 6 d of culturing. The sustentacular cells still envelop the compact glomus cells. N1, nucleus of a type I cell, N2, nucleus of a type II cell, L, lipid droplet, ~ processes of a type II cell, bar: 1 tim. x 12,000
60
F. Pietruschka and D. Sch/ifer
Fig. 5. Cells after 6 d of culturing. Few mitochondria show electron-dense granules (--,). B, osmiophilic bodies of heterogenous density, D, dictyosomes of the Golgi apparatus, bar: 1 lam. • 30,000
Glomus caroticum in Tissue Culture
61
apparatus were well developed (D in Fig. 6), and the vesicular component of the dictyosomes often contained electron-dense material ( ~ in Fig. 6). Densecored vesicles were found in their immediate vicinity (---, Fig. 6). Occasionally parts of a cilium were found (C in Fig. 6) in the ultrathin sections of cultured type I cells that had been observed also in vivo (Biscoe, 1971; B6ck, 1973).
Discus~on
From physiological experiments on new-born lambs (Biscoe and Purves, 1967) it is known that the carotid bodies are mature at birth. The chemoreceptor response to both hypoxia and rise of CO2 in the inspired air is similar to that observed in adult animals. Even in a far younger state of development of the midterm human fetus, Hervonen and Korkala (1972a, b) found carotid bodies of mature appearance as to fine structure and catecholamine fluorescence. In full agreement with these findings we could not detect conspicuous morphological differences between carotid bodies of rabbit embryos 2 to 4d before birth and those of adult rabbits investigated by M611mann et al. (1973). As there is evidence that the carotid body is of neural crest origin and belongs to the A P U D (Amine Precursor Uptake and Decarboxylation) series (Pearse et al., 1973), we cultured the carotid body cells in a medium which is of high concentrations of serum and glucose and low pH value (Lumsden, 1968; H6sli and H6sli, 1970) usually used for nerve cells in vitro. Indeed, we were not able to maintain the dense-cored vesicles in the cultured cells unless we lowered the pH of the enzyme solution to 6.61 and that of the culture medium from 7.4 to 6.9. Like neurons in culture, aging carotid body cells also show an increased amount of lipofuscin bodies (Fig. 5). Spoerri and Glees (1973) showed that they originate from degenerating mitochondria. Lipofuscin bodies were also found in the carotid bodies of adult human beings and animals in situ (Grimly and Glenner, 1968; Edwards and Heath, 1972). With the methods employed we are able to culture carotid body cells up to 14d in their original association of type I and type II cells. Furthermore, the type I cells show their morphological characteristics, such as the compact shape, round or oval nuclei, many mitochondria, rough endoplasmatic reticulum, numerous free ribosomes, a well-developed Golgi apparatus and a great number of dense-cored vesicles. The electron-dense material in the vesicles of dictyosomes and in the granular vesicles near the Golgi apparatus indicate that the synthesis of catecholamines continues in culture (see also Erfink6 et al., 1972). This assumption agrees with previous findings (Pietruschka, 1974) of an increase in the catecholamine fluorescence of carotid body cells after one week of culture. 1
The authors are indebted to Dr. H. Starlinger for giving this advice
Fig. 6. Cells after 3d of culturing. The dictyosomes of the Golgi apparatus are well developed and the vesicular component of the apparatus often contains electron dense material ( ~ ) and dense-cored vesicles are found in their immediate vicinity (-,). ~ osmiophilic body with double limiting membrane, D, dictyosomes of the Golgi apparatus, C, cilium, bar: 1 ~tm. x 30,000
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F. Pietruschka and D. Schiller
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Pietruschka, F., Acker, H., Gattermann, S., Seidl, E., Lfibbers, D.W.: Cells of the carotid body in primary tissue cultures. Arzneimittel-Forsch. (Drug Res.) 23, 1610 (1973) Sch~ifer, D., Sch~ifer, S., Lfibbers, D.W. : On the population of type I glomus cells of the cat and in cells of other organs. Arzneimittel-Forsch. (Drug Res.) 23, 1612 (1973) Seidl, E. : Die Anwendung des Interferenzkontrast-Verfahrens am gef'~.rbten Schnitt als Hilfsmittel bei der Rekonstruktion yon Organellen. Leitz-Mitteil. Wiss. Tech. 6, 57 58 (1973) Spoerri, P.E., Glees, P.: Neuronal aging in cultures: an electronmicroscopic study. Exp. Geront. 8, 259-263 (1973) Verna, A.: Infrastructure des divers types de terminaisons nerveuses dans le glomus carotidien du lapin. J. Microscopie 10, 59~6 (1971) Verna, A.: Terminaisons nerveuses aff6rentes et eff6rentes dans le glomus carotidien du lapin. Microscopie 16, 299-308 (1973) Weissenfels, N., Sch~ifer, D., Hfindgen, M. : Der FixierungseinfluB acht verschiedener Aldehyde auf die Ultrastruktur kultivierter Zellen. I. Der Strukturzustand des Zellkerns. Cytobiologie 3, 188 201 (1971)
Received December 19, 1975
