PRELIMINARY
NOTES
Internalization of neuronal plasma membrane ricin receptors into the Golgi apparatus N. K. GONATAS, ANNA STEIBER, S. U. KIM, D. I. GRAHAM and S. AVRAMEAS, Divisiun of
Neuropathology, Depurtment of Pathology. University of Pennsylvanin School of Medicine, Philadelphia, PA 19174, and Unit& d’lmmunocytochimie, DPpartment du Biologic Moltculaire, lnstitut Pasteur, Paris, France
We have reported that immunoglobulins of the plasma membrane of plasma cells labeled with bivalent or monovalent antiimmunoglobulin antibodies undergo internalization (endocytosis) into membranes of smooth vesicles located in the area of the Golgi apparatus [ 11. In this communication we report that plasma membranes of sympathetic neurons grown in vitro, labeled with conjugates of the lectin ricin which binds to D-g&CtOSecontaining moieties [2] and the marker enzyme horseradish peroxidase, internalize in the vesicles and cisterns of the Golgi apparatus. These observations indicate that component(s) of the plasma membrane of certain cells exposed to monovalent or multivalent ligands undergo internalization into the membranes of the Golgi apparatus. In view of the role of the Golgi apparatus on the synthesis of plasma membrane glycoproteins [3, 41, it is quite possible that the internalization of the complexes of the ricinperoxidase with the plasma membrane Dcontaining polysaccharides galactose (glycoproteins), and perhaps with other plasma membrane ricin receptors, may reflect recycling of the component(s) of plasma membrane into the Golgi apparatus [5Exptl Cell Res 94 (1975)
81. This plasma membrane recycling into the Golgi apparatus could serve control functions of plasma membrane biosynthesis by the Golgi apparatus; in addition, other cellular functions (such as surface-tosurface specificities, contact inhibition, response to antibody or hormone) which may depend on interactions between receptors of plasma membrane and their specific ligands could be mediated through the internalization of the ligand-receptor complexes into the Golgi apparatus. Materials
and Methods
Superior cervical ganglia from newborn Swiss albino mice were dissected and collected in Ca- and Me-free Hanks balanced salt solution (BSS). The ganglia-were transferred into 0.25% trvosin (Difco 1 : 250) in Caand Mg-free Hanks BSS &r a !+Omin incubation at 36°C. After incubation, the ganglia were washed gently in 3 changes of Eagle’s minimum essential medium (MEM); the loosened ganglia were transferred to the nutrient medium (comp&edof 30 parts of horse serum, 58 parts Eagle’s MEM and supplementary glucose to a final concentration of I % and dissociated into single cells by gentle pipetting. One drop of cell suspension containing 1X 105-5X lo5 cells/l ml was placed on a collagen-coated coverslip and placed in a 60 mm Falcon PeGi dish or sealed in a Maxcmow slide. Cultures were incubated at 36°C in 5 % CO,, 95 % air mixture. Tissue (cell) culture conditions have been described in detail [9, 101. By 1-2 weeks in vitro, sympathetic neuron; were-easily identifiable by their large globular perikarya, prominent nuclei and nucleoli. Axonal networks, although visible, were not as extensive as in explants of sympathetic neurons [lo]. Cultures containing many isolated neurons were selected for staining with ricin-peroxidase. Ricin was purified and ricin-peroxidase (HRP) conjugates were prepared according to previously described methods [2]. In order to prevent binding of ricin with serum saccharides or elvcomoteins. the cultures. nrior to incubation with-&in:HRP, were thoroughly ‘washed in Eagle’s BSS buffered at pH 7.35 with freshly made 0.1 M carbonate buffer. Cultures were incubated with 100 &ml of ricin-HRP coniugates (1 mg of ricin was con&ated with 10 mg of HkPj or with 1%mg HRP/ml (control) for 1 h at 4°C. The solution of ricin-HRP or of HRP was made in buffered Eagle’s BSS, and one to two drops of the solution were used per culture. The cultures were subsequently washed in buffered Eagle’s
Preliminary at 4°C and transferred to the full culture medium. For internalization of the labeled plasma membranes, thoroughly washed cultures were incubated in full culture medium at 36°C for 30 minor 3 h. After the incubation, the cultures were fixed for 30 min at 22°C in 2.5 %-5 % glutaraldehyde and 1% p-formaldehyde in a 0.1 M cacodylate buffer, nH 7.35. Additional control cultures were-incubated for 3 h at 36°C in full culture medium containing 10 mg/ml of HRF’ (Sigma type VI), washed in buffered Eagle’s BSS and fixed in the above fixative. Activity for peroxidase was revealed with the method of Graham & Karnovskv with diaminobenzidine (DAB) tetrahydrochloride-as substrate (pH 7.3) [2, 111. The staining reaction was carried out at 22”Cfor lb min. After the cytochemical reaction for peroxidase, cells were washed in 0.1 cacodylate buffer, pH 7.35 and fixed in 1% osmium textroxide in 0.2 cacodylate buffer and embedded in Araldite.
Results and Discussion
All observations were made on the principal ganglion cells characterized by well-formed Nissl bodies, several Golgi complexes, and large nuclei with prominent nucleoli [lo]. Small neurons with numerous dense-core cytoplasmic vesicles were not frequent in our material; these small neurons are considered to be interneurons with intense fluorescent granules [ 12, 133. In cells fixed and stained for peroxidase activity, immediately after their incubation in ricin-HRP at 4°C only a peripheral rim (plasma membrane) of oxidized diaminobenzidine (DAR)-osmium black precipitate was seen (fig. 1). Cytoplasmic or intranuclear staining was not seen. Occasionally, neurons incubated in HRP for 3 h at 36°C (controls) showed only peripheral (plasma membrane) staining, which is probably due to adsorption of HRP on the plasma membrane; intracytoplasmic staining was not seen. Neurons incubated at 36°C in full medium after the initial incubation in ricinHRP at 4°C showed varying degrees of cytoplasmic staining (figs 2, 3). The staining was in the form of round, oval or elongated vesicles which appeared in clusters (fig. 2); the clusters of these stained vesicles were adjacent to the elongated 28-751812
notes
427
cisterns of the Golgi apparatus; the relationship of these clusters of vesicles in relation to the elongated cisterns of the Golgi apparatus varied, but the vesicles were usually located near the forming aspect of the Golgi cisterns or at the edges of the cisterns of the Golgi apparatus. When neurons, after the initial labeling with ricin-HRP, were incubated for 3 h at 36”C, staining was observed in one or two of the parallel cisterns of the Golgi apparatus (fig. 3). In neurons, fixed 30 min after incubation at 36°C staining was predominantly in vesicles. Plasma membrane staining was diminished or absent after 30 min or 3 h incubations at 36°C (fig. 4). ‘Cap’-like staining was not observed, but some small patches of peroxidase staining were observed after 30 min or 3 h at 36°C (fig. 4). The degree of the disappearance of the surface staining after incubation at 36°C for 30 min or 3 h was similar. In neurons which were completely dissociated and the entire plasma membrane was exposed to the nutrient fluids, the disappearance of the surface staining after 30 min or 3 h at 36°C was more extensive. Surface staining persisted in neurons covered by satellite cells, and incubated at 36°C for 30 min or 3 h. The same cells showed internal peroxidase staining. The vesicular staining probably corresponds to the membranes of the vesicular component of the Golgi apparatus while the elongated stained profiles probably belong to the Golgi cisterns (fig. 3). Peroxidase activity was not present in large dense bodies (probably neuronal lysosomes) (fig. 2). In preparations stained for peroxidase, and counterstained only with uranyl acetate, dense bodies of dark core vesicles were barely visible (fig. 2). Since during the incubation of the neurons at 36°C there was no ricin-HRP in the medium, the vesicular and cisternal E.xpt/ Cell Res 94 (1975)
428
Preliminary
n0te.c
1. Mouse sympathetic neuron in culture. Incubated 1 h, 4°C with kin-horseradish peroxidase (HRP), washed, fixed and, stained for HRP activity. Counterstained with uranyl acetate. Note peripheral stain of plasma membrane and absence of internal staining. N, nucleus. X 7000.
Fig.
Exprl Cd Res 94 (1975)
Fig. 2. Same as in fig. I, except that after in cub: ition in kin-HRP, neurons were incubated for 3 h a:t 36°C. Note extensive vesicular (V), and limite d ci sternal staining near the Golgi (G). Counterstained with uranyi acetate. X 27 000.
Preliminary
notes
429
Fig. 3. Same as in fig. 2. Counterstained with uranyl acetate and lead citrate. Note stain in vesicles (V) and
cisterns of the Golgi apparatus (G). (Arrow) peroxidase in elongated cistern of the Golgi. x 3 1000.
staining is due to peroxidase activity on the membrane itself rather than to the presence of r-kin-HRP in the contents of the vesicle. Neurons incubated for 3 h at 36°C in the presence of HRP in the medium (10 mg/ml) have failed to pinocytoze the enzyme. Since the initial labeling of the plasma membrane with t-kin-HRP conjugate occurred at 4°C and under these conditions no internal staining was observed (fig. l), the vesicular and cisternal staining probably represents actively translocated plasma membrane labeled components rather than a passive diffusion of the label through tortuous channels in continuity with the extracellular space. There was no evidence of fusion of the labeled membranes with preformed secondary lysosomes and in that aspect our observations are probably analogous to the observations of Edelson &
Cohn on ConA-treated peritoneal macrophages [ 141, although in the peritoneal macrophages, ConA-HRP was not observed in the Golgi apparatus [14]. In human lymphocytes, internalization of plasma membranes labeled with ConA occurred in cytoplasmic vesicles which appeared at one pole of the cell and subsequently fused in larger vesicles [ 151. Thus in four different cell types (neurons, lymphocytes, plasma cells, and macrophages), plasma membrane labeled with lectins internalizes, but the pattern of the internal staining varies [ 1, 14, 151;only in neurons and in plasma cells fairly typical Golgi cisterns and vesicles are labeled. These differences may reflect the prominence of the Golgi apparatus in nerve cells or, possibly, different functional implications of the observed phenomenon. The observed internalization of neuronal Exprl Cell Res 94 (1975)
430
Preliminary notes
Fig. 4. Mouse sympathetic neuron in culture incubated 1 h, 4°C with kin-HRP, washed, incubated 3 h at 36°C. Fixed and stained with DAB. (Arrmhends)
Exprl Cell Rcs 94 (1975)
‘patchy’ plasma membrane stain; (arrows ) internalized rickHRP, x8000. Itzsrf: Magnification from fig. 4. ‘patches’ of stain. x 19000. (Arrm*head.~)
Preliminary notes plasma membrane ricin receptors is consistent with the observed mobility of ConA receptors of synaptic membranes [ 161. Since horseradish peroxidase was covalently bound with glutaraldehyde to ricin, it is unlikely that the observed intracellular HRP activity is due to a dissociated HRP. Also the high affinity of lectins to their sugar receptors is consistent with our interpretation that the internal labeling represents translocated plasma membranericin-HRP complexes rather than intracellular transport of unattached molecules of ricin-HRP [17]. Recently Wood et al. have demonstrated, in fixed Purkinje cells, an extensive cisternal system including the Golgi apparatus, rich in carbohydrate-binding ConA [ 181.We have demonstrated in unfixed sympathetic neurons that part of this system, i.e., the Golgi apparatus, is in a dynamic relationship with the plasma membrane. Although the present experiments have not shown that ricin-HRP binds exclusively with D-galactose-containing moieties, previous experiments with this conjugate suggest that ricin has a high affinity with D-galactose [2]. We are currently investigating (a) binding patterns of other lectin-HRP conjugates on normal neurons, glia and murine neuroblastoma cells in culture, and (b) the dependence of the process responsible for the endocytosis of neuronal plasma membrane ricin receptors on energy, protein synthesis, and integrity of microtubules.
4. 5. 6. 7. 8.
431
Dauwalder, M, Whaley, W G & Kephart, J E, Sub-cell biochem I (1972) 225. Palade, G E, Subcellular particles (ed T Hayashi) p. 64. The Ronald Press Co, New York (1959). Fawcett, D W, Circulation 26 (1962) 1105. Hokin, L E, Int rev cytol 23 (1968) 187. Jamieson, J D & Palade, G E, J cell biol 48 (1971) 503.
9.
Kim, S U & Wenger, E L, J neurobiol 4 (1972-73) 513.
14.
Kim, S U & Munkacsi, I, Exp neural 45 (1974)94. Graham, R & Karnovsky, M J, J histochem cytochem 14(1966)291. Eranko, 0 & Eranko, L, Brain res 34 (1971) 39. Eranko, 0 L & Eranko, C, Hill, E & Burnstock, C, Histochemistry 4 (1972) 49. Edelson, P J & Cohn, A Z, J exp med 140 (1974)
15.
Barat, N & Avrameas, S, Exp cell res 76 (1973)
IO. 11. 12. 13.
1364. 451. 16. 17. 18.
Matus, A, DePetris, S & Raff, M C, Nature new bio1244 (1973) 278. Lis, H & Sharon, N, Ann rev bioch 42 (1973) 541. Wood, J G, McLaughlin, B J & Barber, R P, J cell biol 63 (1974) 541.
Received March 3, 1975 Revised version received April 29, 1975
Induction of DNA synthesis by dichloroisoproterenol without initial rise of the CAMP level in the parotid gland of mouse I. FURUNO and H. MATSUDAIRA, Division ofBiology, National Institute of Radiological Sciences, Anagawa, Chiha-shi, 280, Japan The increase in the CAMP concentration in the parotid gland of mouse, observed shortly after administration of isoproterenol, was not detected after dichloroisoproterenol, a P-adrenergic blocking agent. The latter compound, however, could induce DNA synthesis which was comparable to that induced by IPR in several respects. The results strongly suggest that the initial rise of CAMP concentration in the parotid gland after IPR injection is not related directly to the initiation of the stimulated DNA synthesis.
Summary.
This work is supported by USPHS Grants NS 0557211, NS 10648-3 and by research grant DGRST No.
Administration of isoproterenol (IPR) induces DNA synthesis and cell division in the salivary gland of the rat [l] and of the References mouse [2]. Biochemical changes associated 1. Antoine, J C, Avrameas, S, Gonatas, N K, with the stimulated DNA synthesis were Stieber, A & Gonatas, J 0, J cell biol63 (1974) 12. 2. Gonatas, N K & Avrameas, S, J cell biol59 (1973) reviewed by Baserga [3]. A marked increase 436. in CAMP concentration [4] and a moderate 3. Begs, H W & Kessel, R G, Int rev cytol23 (1968) increase in adenylate cyclase activity [5] 72-70298.
Exprl Cell Res 94 (1975)
NOTES
Internalization of neuronal plasma membrane ricin receptors into the Golgi apparatus N. K. GONATAS, ANNA STEIBER, S. U. KIM, D. I. GRAHAM and S. AVRAMEAS, Divisiun of
Neuropathology, Depurtment of Pathology. University of Pennsylvanin School of Medicine, Philadelphia, PA 19174, and Unit& d’lmmunocytochimie, DPpartment du Biologic Moltculaire, lnstitut Pasteur, Paris, France
We have reported that immunoglobulins of the plasma membrane of plasma cells labeled with bivalent or monovalent antiimmunoglobulin antibodies undergo internalization (endocytosis) into membranes of smooth vesicles located in the area of the Golgi apparatus [ 11. In this communication we report that plasma membranes of sympathetic neurons grown in vitro, labeled with conjugates of the lectin ricin which binds to D-g&CtOSecontaining moieties [2] and the marker enzyme horseradish peroxidase, internalize in the vesicles and cisterns of the Golgi apparatus. These observations indicate that component(s) of the plasma membrane of certain cells exposed to monovalent or multivalent ligands undergo internalization into the membranes of the Golgi apparatus. In view of the role of the Golgi apparatus on the synthesis of plasma membrane glycoproteins [3, 41, it is quite possible that the internalization of the complexes of the ricinperoxidase with the plasma membrane Dcontaining polysaccharides galactose (glycoproteins), and perhaps with other plasma membrane ricin receptors, may reflect recycling of the component(s) of plasma membrane into the Golgi apparatus [5Exptl Cell Res 94 (1975)
81. This plasma membrane recycling into the Golgi apparatus could serve control functions of plasma membrane biosynthesis by the Golgi apparatus; in addition, other cellular functions (such as surface-tosurface specificities, contact inhibition, response to antibody or hormone) which may depend on interactions between receptors of plasma membrane and their specific ligands could be mediated through the internalization of the ligand-receptor complexes into the Golgi apparatus. Materials
and Methods
Superior cervical ganglia from newborn Swiss albino mice were dissected and collected in Ca- and Me-free Hanks balanced salt solution (BSS). The ganglia-were transferred into 0.25% trvosin (Difco 1 : 250) in Caand Mg-free Hanks BSS &r a !+Omin incubation at 36°C. After incubation, the ganglia were washed gently in 3 changes of Eagle’s minimum essential medium (MEM); the loosened ganglia were transferred to the nutrient medium (comp&edof 30 parts of horse serum, 58 parts Eagle’s MEM and supplementary glucose to a final concentration of I % and dissociated into single cells by gentle pipetting. One drop of cell suspension containing 1X 105-5X lo5 cells/l ml was placed on a collagen-coated coverslip and placed in a 60 mm Falcon PeGi dish or sealed in a Maxcmow slide. Cultures were incubated at 36°C in 5 % CO,, 95 % air mixture. Tissue (cell) culture conditions have been described in detail [9, 101. By 1-2 weeks in vitro, sympathetic neuron; were-easily identifiable by their large globular perikarya, prominent nuclei and nucleoli. Axonal networks, although visible, were not as extensive as in explants of sympathetic neurons [lo]. Cultures containing many isolated neurons were selected for staining with ricin-peroxidase. Ricin was purified and ricin-peroxidase (HRP) conjugates were prepared according to previously described methods [2]. In order to prevent binding of ricin with serum saccharides or elvcomoteins. the cultures. nrior to incubation with-&in:HRP, were thoroughly ‘washed in Eagle’s BSS buffered at pH 7.35 with freshly made 0.1 M carbonate buffer. Cultures were incubated with 100 &ml of ricin-HRP coniugates (1 mg of ricin was con&ated with 10 mg of HkPj or with 1%mg HRP/ml (control) for 1 h at 4°C. The solution of ricin-HRP or of HRP was made in buffered Eagle’s BSS, and one to two drops of the solution were used per culture. The cultures were subsequently washed in buffered Eagle’s
Preliminary at 4°C and transferred to the full culture medium. For internalization of the labeled plasma membranes, thoroughly washed cultures were incubated in full culture medium at 36°C for 30 minor 3 h. After the incubation, the cultures were fixed for 30 min at 22°C in 2.5 %-5 % glutaraldehyde and 1% p-formaldehyde in a 0.1 M cacodylate buffer, nH 7.35. Additional control cultures were-incubated for 3 h at 36°C in full culture medium containing 10 mg/ml of HRF’ (Sigma type VI), washed in buffered Eagle’s BSS and fixed in the above fixative. Activity for peroxidase was revealed with the method of Graham & Karnovskv with diaminobenzidine (DAB) tetrahydrochloride-as substrate (pH 7.3) [2, 111. The staining reaction was carried out at 22”Cfor lb min. After the cytochemical reaction for peroxidase, cells were washed in 0.1 cacodylate buffer, pH 7.35 and fixed in 1% osmium textroxide in 0.2 cacodylate buffer and embedded in Araldite.
Results and Discussion
All observations were made on the principal ganglion cells characterized by well-formed Nissl bodies, several Golgi complexes, and large nuclei with prominent nucleoli [lo]. Small neurons with numerous dense-core cytoplasmic vesicles were not frequent in our material; these small neurons are considered to be interneurons with intense fluorescent granules [ 12, 133. In cells fixed and stained for peroxidase activity, immediately after their incubation in ricin-HRP at 4°C only a peripheral rim (plasma membrane) of oxidized diaminobenzidine (DAR)-osmium black precipitate was seen (fig. 1). Cytoplasmic or intranuclear staining was not seen. Occasionally, neurons incubated in HRP for 3 h at 36°C (controls) showed only peripheral (plasma membrane) staining, which is probably due to adsorption of HRP on the plasma membrane; intracytoplasmic staining was not seen. Neurons incubated at 36°C in full medium after the initial incubation in ricinHRP at 4°C showed varying degrees of cytoplasmic staining (figs 2, 3). The staining was in the form of round, oval or elongated vesicles which appeared in clusters (fig. 2); the clusters of these stained vesicles were adjacent to the elongated 28-751812
notes
427
cisterns of the Golgi apparatus; the relationship of these clusters of vesicles in relation to the elongated cisterns of the Golgi apparatus varied, but the vesicles were usually located near the forming aspect of the Golgi cisterns or at the edges of the cisterns of the Golgi apparatus. When neurons, after the initial labeling with ricin-HRP, were incubated for 3 h at 36”C, staining was observed in one or two of the parallel cisterns of the Golgi apparatus (fig. 3). In neurons, fixed 30 min after incubation at 36°C staining was predominantly in vesicles. Plasma membrane staining was diminished or absent after 30 min or 3 h incubations at 36°C (fig. 4). ‘Cap’-like staining was not observed, but some small patches of peroxidase staining were observed after 30 min or 3 h at 36°C (fig. 4). The degree of the disappearance of the surface staining after incubation at 36°C for 30 min or 3 h was similar. In neurons which were completely dissociated and the entire plasma membrane was exposed to the nutrient fluids, the disappearance of the surface staining after 30 min or 3 h at 36°C was more extensive. Surface staining persisted in neurons covered by satellite cells, and incubated at 36°C for 30 min or 3 h. The same cells showed internal peroxidase staining. The vesicular staining probably corresponds to the membranes of the vesicular component of the Golgi apparatus while the elongated stained profiles probably belong to the Golgi cisterns (fig. 3). Peroxidase activity was not present in large dense bodies (probably neuronal lysosomes) (fig. 2). In preparations stained for peroxidase, and counterstained only with uranyl acetate, dense bodies of dark core vesicles were barely visible (fig. 2). Since during the incubation of the neurons at 36°C there was no ricin-HRP in the medium, the vesicular and cisternal E.xpt/ Cell Res 94 (1975)
428
Preliminary
n0te.c
1. Mouse sympathetic neuron in culture. Incubated 1 h, 4°C with kin-horseradish peroxidase (HRP), washed, fixed and, stained for HRP activity. Counterstained with uranyl acetate. Note peripheral stain of plasma membrane and absence of internal staining. N, nucleus. X 7000.
Fig.
Exprl Cd Res 94 (1975)
Fig. 2. Same as in fig. I, except that after in cub: ition in kin-HRP, neurons were incubated for 3 h a:t 36°C. Note extensive vesicular (V), and limite d ci sternal staining near the Golgi (G). Counterstained with uranyi acetate. X 27 000.
Preliminary
notes
429
Fig. 3. Same as in fig. 2. Counterstained with uranyl acetate and lead citrate. Note stain in vesicles (V) and
cisterns of the Golgi apparatus (G). (Arrow) peroxidase in elongated cistern of the Golgi. x 3 1000.
staining is due to peroxidase activity on the membrane itself rather than to the presence of r-kin-HRP in the contents of the vesicle. Neurons incubated for 3 h at 36°C in the presence of HRP in the medium (10 mg/ml) have failed to pinocytoze the enzyme. Since the initial labeling of the plasma membrane with t-kin-HRP conjugate occurred at 4°C and under these conditions no internal staining was observed (fig. l), the vesicular and cisternal staining probably represents actively translocated plasma membrane labeled components rather than a passive diffusion of the label through tortuous channels in continuity with the extracellular space. There was no evidence of fusion of the labeled membranes with preformed secondary lysosomes and in that aspect our observations are probably analogous to the observations of Edelson &
Cohn on ConA-treated peritoneal macrophages [ 141, although in the peritoneal macrophages, ConA-HRP was not observed in the Golgi apparatus [14]. In human lymphocytes, internalization of plasma membranes labeled with ConA occurred in cytoplasmic vesicles which appeared at one pole of the cell and subsequently fused in larger vesicles [ 151. Thus in four different cell types (neurons, lymphocytes, plasma cells, and macrophages), plasma membrane labeled with lectins internalizes, but the pattern of the internal staining varies [ 1, 14, 151;only in neurons and in plasma cells fairly typical Golgi cisterns and vesicles are labeled. These differences may reflect the prominence of the Golgi apparatus in nerve cells or, possibly, different functional implications of the observed phenomenon. The observed internalization of neuronal Exprl Cell Res 94 (1975)
430
Preliminary notes
Fig. 4. Mouse sympathetic neuron in culture incubated 1 h, 4°C with kin-HRP, washed, incubated 3 h at 36°C. Fixed and stained with DAB. (Arrmhends)
Exprl Cell Rcs 94 (1975)
‘patchy’ plasma membrane stain; (arrows ) internalized rickHRP, x8000. Itzsrf: Magnification from fig. 4. ‘patches’ of stain. x 19000. (Arrm*head.~)
Preliminary notes plasma membrane ricin receptors is consistent with the observed mobility of ConA receptors of synaptic membranes [ 161. Since horseradish peroxidase was covalently bound with glutaraldehyde to ricin, it is unlikely that the observed intracellular HRP activity is due to a dissociated HRP. Also the high affinity of lectins to their sugar receptors is consistent with our interpretation that the internal labeling represents translocated plasma membranericin-HRP complexes rather than intracellular transport of unattached molecules of ricin-HRP [17]. Recently Wood et al. have demonstrated, in fixed Purkinje cells, an extensive cisternal system including the Golgi apparatus, rich in carbohydrate-binding ConA [ 181.We have demonstrated in unfixed sympathetic neurons that part of this system, i.e., the Golgi apparatus, is in a dynamic relationship with the plasma membrane. Although the present experiments have not shown that ricin-HRP binds exclusively with D-galactose-containing moieties, previous experiments with this conjugate suggest that ricin has a high affinity with D-galactose [2]. We are currently investigating (a) binding patterns of other lectin-HRP conjugates on normal neurons, glia and murine neuroblastoma cells in culture, and (b) the dependence of the process responsible for the endocytosis of neuronal plasma membrane ricin receptors on energy, protein synthesis, and integrity of microtubules.
4. 5. 6. 7. 8.
431
Dauwalder, M, Whaley, W G & Kephart, J E, Sub-cell biochem I (1972) 225. Palade, G E, Subcellular particles (ed T Hayashi) p. 64. The Ronald Press Co, New York (1959). Fawcett, D W, Circulation 26 (1962) 1105. Hokin, L E, Int rev cytol 23 (1968) 187. Jamieson, J D & Palade, G E, J cell biol 48 (1971) 503.
9.
Kim, S U & Wenger, E L, J neurobiol 4 (1972-73) 513.
14.
Kim, S U & Munkacsi, I, Exp neural 45 (1974)94. Graham, R & Karnovsky, M J, J histochem cytochem 14(1966)291. Eranko, 0 & Eranko, L, Brain res 34 (1971) 39. Eranko, 0 L & Eranko, C, Hill, E & Burnstock, C, Histochemistry 4 (1972) 49. Edelson, P J & Cohn, A Z, J exp med 140 (1974)
15.
Barat, N & Avrameas, S, Exp cell res 76 (1973)
IO. 11. 12. 13.
1364. 451. 16. 17. 18.
Matus, A, DePetris, S & Raff, M C, Nature new bio1244 (1973) 278. Lis, H & Sharon, N, Ann rev bioch 42 (1973) 541. Wood, J G, McLaughlin, B J & Barber, R P, J cell biol 63 (1974) 541.
Received March 3, 1975 Revised version received April 29, 1975
Induction of DNA synthesis by dichloroisoproterenol without initial rise of the CAMP level in the parotid gland of mouse I. FURUNO and H. MATSUDAIRA, Division ofBiology, National Institute of Radiological Sciences, Anagawa, Chiha-shi, 280, Japan The increase in the CAMP concentration in the parotid gland of mouse, observed shortly after administration of isoproterenol, was not detected after dichloroisoproterenol, a P-adrenergic blocking agent. The latter compound, however, could induce DNA synthesis which was comparable to that induced by IPR in several respects. The results strongly suggest that the initial rise of CAMP concentration in the parotid gland after IPR injection is not related directly to the initiation of the stimulated DNA synthesis.
Summary.
This work is supported by USPHS Grants NS 0557211, NS 10648-3 and by research grant DGRST No.
Administration of isoproterenol (IPR) induces DNA synthesis and cell division in the salivary gland of the rat [l] and of the References mouse [2]. Biochemical changes associated 1. Antoine, J C, Avrameas, S, Gonatas, N K, with the stimulated DNA synthesis were Stieber, A & Gonatas, J 0, J cell biol63 (1974) 12. 2. Gonatas, N K & Avrameas, S, J cell biol59 (1973) reviewed by Baserga [3]. A marked increase 436. in CAMP concentration [4] and a moderate 3. Begs, H W & Kessel, R G, Int rev cytol23 (1968) increase in adenylate cyclase activity [5] 72-70298.
Exprl Cell Res 94 (1975)
