Archs oral Biol. Vol. 35, No. 8, pp. 677-619, 1990 Printedin Great Britain. All rights reserved
WO3-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
SHORT COMMUNICATION CHANGES IN MICROFILAMENT DISTRIBUTION DURING AMYLASE EXOCYTOSIS IN RAT PAROTID SALIVARY GLANDS IN VITRO K. OKUMURA,’Y. TOJYO~and M. KANAZAWA’ Departments of ‘Oral Surgery (1st division) and ‘Dental Pharmacology, School of Dentistry, Higashi-Nippon-Gakuen University, Ishikari-Tobetsu, Hokkaido 061-02, Japan (Received
13 July 1989; accepted 26 February 1990)
Summary-In parotid acini, microfilaments were predominantly localized at the luminal border when visualized with the fluorescent probe, rhodamine-phalloidin. There was no fluorescence in the cytoplasm. When acini were stimulated by isoprenaline, the membranes of the secretory granules appeared to become undercoated. by microfilaments immediately after fusion with the luminal membrane. The intensity of fluorescence was correlated with the extent of enlargement of the lumina. These findings indicate that the microlilament system (actin-myosin contractile system) may play a role in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen. Key words: microfilaments, actin, parotid glands, excqtosis, amylase.
Actincontaining +microfilaments in parotid acinar cells have been shown, by immunofluorescence, to be predominantly localized at the luminal border (Drenckhahn and Mannherz, 1983). These microfilaments are generally thought to play an important but as yet unspecified role in the exocytosis of parotid amylase. When exocytosis is induced by stimulation of /3sadrenergic receptors, the acinar lumen is dramatlcally enlarged by fusion of the secretory granules with the luminal membrane; it then rapidly returns to its original size (Amsterdam, Ohad and Schramm, 1969). It is therefore of interest to examine changes in the distribution of microfilaments during amylase exocytosis. We have now visualized the microfilaments in rat parotid acini with rhodamine-phalloiam, which selectively binds F-actin (Barak et al., 1980). Parotid acini were prepared according to the method of Takuma and Ichida (1986). The parotid glands from one rat (about 300 g body weight) were excised, minced filnely and incubated for 60 min at 37°C in lOm1 of Hanks’ balanced salt solution buffered with 20 mM HEPES-NaOH, pH 7.4, containing collagenase (CLS II, Cooper Biomedical, Malvem, PA, U.S.A., 130 U/ml) and bovine testicular hyaluronidase (type I-S, Sigma Chemical Co., St Louis, MO, U.S.A., 0.25mg/ml) under 95% O,-5% COZ. At 20&n intervals, the minced tissue was gently pipetted and gassed. Thereafter, the cells were filtered through one layer of gauze, washed twice with the buffered Hanks’ solution containing 0.1% bovine
Abbreviation:
PBS, phosphate-buffered saline.
serum albumin (Sigma Chemical Co.) and suspended in 20-30 ml of the same medium. A 1.0 ml sample of cell suspension was incubated with or without 1 PM isoprenaline at 37°C for various periods. After incubation, the cell pellets were fixed with 4% formaldehyde in PBS, pH 7.4, for 30 min at 4”C, and then quickly frozen. Sections were cut at 6 pm thickness on a cryotome (Tissue-Tek-II, Miles Laboratories, International Equipment Company, MA, U.S.A.). They were then incubated with rhodamine-phalloidin (Molecular Probe Inc., OR, U.S.A., diluted 1: 20 in PBS) for 30 min at room temperature, washed 3 times in PBS and examined with a fluorescence microscope. Control sections were incubated with rhodaminephalloidin plus unlabelled phalloidin (100 p g/ml; Sigma), instead of incubation with rhodaminephalloidin alone. Other cell pellets were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (PH 7.4) and post-fixed with 1% 0~0, solution. After dehydration in graded acetone, the pellets were embedded in low-viscosity Spurr resin. Sections of 1 pm were stained with toluidine blue and examined under a light microscope. In unstimulated acini. the lumina were very narrow and the numerous secretory granules were localized in the apical cytoplasm (Fig. 1). There was intense rhodamine-phalloidin labelling at the luminal border, but only weak fluorescence at the basolateral border (Fig. 3). No fluorescence was seen in the cytoplasm, suggesting that the microfilament system does not associate there with the secretory granules. In the control sections, there was no labelling (Fig. 2). On stimulation with isoprenaline, dilation of the 677
678
K. OKUMURA
acinar lumina as a result of exocytosis was elicited within 1 min (Fig. 4); the dilation was also recognized by the rhodamine-phalloidin stain (Figs 5 and 6). Occasionally, the dilated lumina were seen as small fluorescent rings on the luminal surface (Fig. 6). The granule membrane appeared to become undercoated by microfilaments immediately after fusion with the luminal membrane. At 15 min, luminal enlargement was greatest, and many lumina appeared as holelike vacuoles (Fig. 7). The structural changes were correlated with increasing intensity of fluorescence at the luminal part (Fig. 8). This suggests that the polymerization of microfilaments may be accelerated by the activation of exocytosis. After incubation for 30mit-1, the number of secretory granules in the cytoplasm was markedly reduced and the acinar lumina had returned to their original size (Fig. 9). The fluorescence pattern (Fig. 10) was rather similar to that of unstimulated acini. We believe this is the first report of the distribution of microfilaments during exocytosis in parotid acini using rhodamine-phalloidin staining. We have shown that microfilaments were more numerous at the dilated luminal border. This suggests that actin polymerization was accelerated and was concomitant with enlargement of the lumen. Such transformation of actin pools has been demonstrated in rat pancreatic p-cells after glucose stimulation (Swanston-Flatt, Carlsson and Gylfe, 1980) and in platelets after exposure to thrombin (Carlsson et al., 1979) using the DNase 1 inhibition assay. We have earlier reported that treatment with cytochalasin D, which is known to disrupt microfilaments, does not prevent membrane fusion during the exocytosis of rat parotid amylase, although it causes marked enlargement of acinar lumina (Tojyo
et al.
et al., 1989). Those and the present findings together suggest that the microfilament system (actin-myosin contractile system) may play a part in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen.
REFERENCES
Amsterdam A., Ohad I. and Schramm M. (1969) Dynamic changes in the ultrastructure of the acinar cell of the rat parotid gland during the secretory cycle. J. Cell Eiol. 41, 753-773. Barak L. S., Yoccum R. R., Nothnagel E. A. and Webb W. W. (1980) Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa1,3-diazole-phallacidin. Proc. natn. Acad. Sci. V.S.A. 77, 980-984.
Carlsson L., Markey F., Bhkstad I., Lindberg U. (1979) Reorganization of stimulated by thrombin as measured inhibition assay. Proc. natn. Acad.
Persson T. and actin in platelets by the DNase I Sci.
U.S.A.
76,
637M380.
Drenckhahn D. and Mannherz H. G. (1983) Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin and villin in rat and bovine exocrine-glands. Eur. J. Cell Biol. 30, 167-176. Swanston-Flatt S. K.. Carlsson L. and Gvlfe F. E. (1980) Actin filament formation in pancreatic B-cells during gluccse stimulation of insulin secretion. FEBS Leff. 117, 299-302. Takuma T. and Ichida T. (1986) Does cyclic AMP mobilize Ca’+ for amylase secretion from rat parotid cell? Biochim. biophys. Acfa 887, 113-I 17. Tojyo Y., Okumura K., Kanazawa M. and Matsumoto Y. (1989) Effect of cytochalasin D on the acinar cell structure and secretion in rat parotid salivary glands in u&o. Archs oral Biol. 34, 847-855.
Plate 1 Figs 1, 4, 7, 9. Light micrographs of dispersed rat parotid acini. Spurr resin section, toluidine-blue staining. x 700 Figs 2, 3, 5, 6, 8, 10. Fluorescence staining of F-actin in dispersed rhodamine-phalloidin. x 1300
rat parotid
acini using
Fig. I. Resting acini incubated for 30 min without isoprenaline; the arrows show a narrow lumen with discrete granules. Fig. 2. Control section of resting parotid acini; no specific fluorescence is visible. Fig. 3. Resting acini incubated for 30 min without isoprenaline; the bright fluorescence delineates the luminal border (arrows). Fig. 4. Acini stimulated for 1min with 1FM isoprenahne; luminal dilation is visible (arrows). Fig. 5. Acini stimulated for 1 min with 1FM isoprenaline; the luminal dilation (arrow) as a result of exocytosis is also recognized by rhodamine-phalloidin staining. Fig. 6. Acini stimulated for 1 min with 1PM isoprenaline; note the small fluorescent rings on the luminal border (arrow). Fig. 7. Acini stimulated for 15 min with I PM isoprenaline; there is a marked increase in the number and size of vacuoles. Fig. 8. Acini stimulated for 15 min with 1 nM isoprenaline; the intensity of the fluorescence is markedly increased in the dilated lumina. Fig. 9. Acini stimulated for 30 min with 1 PM isoprenahne; the lumina (arrows) have returned to their original size. Fig. 10. Acini stimulated for 30 min with 1 PM isoprenaline; the bright fluorescence (arrow) is similar to that seen in unstimulated acini.
Microfilaments
in parotid
Plate
1
glands
619
WO3-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
SHORT COMMUNICATION CHANGES IN MICROFILAMENT DISTRIBUTION DURING AMYLASE EXOCYTOSIS IN RAT PAROTID SALIVARY GLANDS IN VITRO K. OKUMURA,’Y. TOJYO~and M. KANAZAWA’ Departments of ‘Oral Surgery (1st division) and ‘Dental Pharmacology, School of Dentistry, Higashi-Nippon-Gakuen University, Ishikari-Tobetsu, Hokkaido 061-02, Japan (Received
13 July 1989; accepted 26 February 1990)
Summary-In parotid acini, microfilaments were predominantly localized at the luminal border when visualized with the fluorescent probe, rhodamine-phalloidin. There was no fluorescence in the cytoplasm. When acini were stimulated by isoprenaline, the membranes of the secretory granules appeared to become undercoated. by microfilaments immediately after fusion with the luminal membrane. The intensity of fluorescence was correlated with the extent of enlargement of the lumina. These findings indicate that the microlilament system (actin-myosin contractile system) may play a role in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen. Key words: microfilaments, actin, parotid glands, excqtosis, amylase.
Actincontaining +microfilaments in parotid acinar cells have been shown, by immunofluorescence, to be predominantly localized at the luminal border (Drenckhahn and Mannherz, 1983). These microfilaments are generally thought to play an important but as yet unspecified role in the exocytosis of parotid amylase. When exocytosis is induced by stimulation of /3sadrenergic receptors, the acinar lumen is dramatlcally enlarged by fusion of the secretory granules with the luminal membrane; it then rapidly returns to its original size (Amsterdam, Ohad and Schramm, 1969). It is therefore of interest to examine changes in the distribution of microfilaments during amylase exocytosis. We have now visualized the microfilaments in rat parotid acini with rhodamine-phalloiam, which selectively binds F-actin (Barak et al., 1980). Parotid acini were prepared according to the method of Takuma and Ichida (1986). The parotid glands from one rat (about 300 g body weight) were excised, minced filnely and incubated for 60 min at 37°C in lOm1 of Hanks’ balanced salt solution buffered with 20 mM HEPES-NaOH, pH 7.4, containing collagenase (CLS II, Cooper Biomedical, Malvem, PA, U.S.A., 130 U/ml) and bovine testicular hyaluronidase (type I-S, Sigma Chemical Co., St Louis, MO, U.S.A., 0.25mg/ml) under 95% O,-5% COZ. At 20&n intervals, the minced tissue was gently pipetted and gassed. Thereafter, the cells were filtered through one layer of gauze, washed twice with the buffered Hanks’ solution containing 0.1% bovine
Abbreviation:
PBS, phosphate-buffered saline.
serum albumin (Sigma Chemical Co.) and suspended in 20-30 ml of the same medium. A 1.0 ml sample of cell suspension was incubated with or without 1 PM isoprenaline at 37°C for various periods. After incubation, the cell pellets were fixed with 4% formaldehyde in PBS, pH 7.4, for 30 min at 4”C, and then quickly frozen. Sections were cut at 6 pm thickness on a cryotome (Tissue-Tek-II, Miles Laboratories, International Equipment Company, MA, U.S.A.). They were then incubated with rhodamine-phalloidin (Molecular Probe Inc., OR, U.S.A., diluted 1: 20 in PBS) for 30 min at room temperature, washed 3 times in PBS and examined with a fluorescence microscope. Control sections were incubated with rhodaminephalloidin plus unlabelled phalloidin (100 p g/ml; Sigma), instead of incubation with rhodaminephalloidin alone. Other cell pellets were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (PH 7.4) and post-fixed with 1% 0~0, solution. After dehydration in graded acetone, the pellets were embedded in low-viscosity Spurr resin. Sections of 1 pm were stained with toluidine blue and examined under a light microscope. In unstimulated acini. the lumina were very narrow and the numerous secretory granules were localized in the apical cytoplasm (Fig. 1). There was intense rhodamine-phalloidin labelling at the luminal border, but only weak fluorescence at the basolateral border (Fig. 3). No fluorescence was seen in the cytoplasm, suggesting that the microfilament system does not associate there with the secretory granules. In the control sections, there was no labelling (Fig. 2). On stimulation with isoprenaline, dilation of the 677
678
K. OKUMURA
acinar lumina as a result of exocytosis was elicited within 1 min (Fig. 4); the dilation was also recognized by the rhodamine-phalloidin stain (Figs 5 and 6). Occasionally, the dilated lumina were seen as small fluorescent rings on the luminal surface (Fig. 6). The granule membrane appeared to become undercoated by microfilaments immediately after fusion with the luminal membrane. At 15 min, luminal enlargement was greatest, and many lumina appeared as holelike vacuoles (Fig. 7). The structural changes were correlated with increasing intensity of fluorescence at the luminal part (Fig. 8). This suggests that the polymerization of microfilaments may be accelerated by the activation of exocytosis. After incubation for 30mit-1, the number of secretory granules in the cytoplasm was markedly reduced and the acinar lumina had returned to their original size (Fig. 9). The fluorescence pattern (Fig. 10) was rather similar to that of unstimulated acini. We believe this is the first report of the distribution of microfilaments during exocytosis in parotid acini using rhodamine-phalloidin staining. We have shown that microfilaments were more numerous at the dilated luminal border. This suggests that actin polymerization was accelerated and was concomitant with enlargement of the lumen. Such transformation of actin pools has been demonstrated in rat pancreatic p-cells after glucose stimulation (Swanston-Flatt, Carlsson and Gylfe, 1980) and in platelets after exposure to thrombin (Carlsson et al., 1979) using the DNase 1 inhibition assay. We have earlier reported that treatment with cytochalasin D, which is known to disrupt microfilaments, does not prevent membrane fusion during the exocytosis of rat parotid amylase, although it causes marked enlargement of acinar lumina (Tojyo
et al.
et al., 1989). Those and the present findings together suggest that the microfilament system (actin-myosin contractile system) may play a part in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen.
REFERENCES
Amsterdam A., Ohad I. and Schramm M. (1969) Dynamic changes in the ultrastructure of the acinar cell of the rat parotid gland during the secretory cycle. J. Cell Eiol. 41, 753-773. Barak L. S., Yoccum R. R., Nothnagel E. A. and Webb W. W. (1980) Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa1,3-diazole-phallacidin. Proc. natn. Acad. Sci. V.S.A. 77, 980-984.
Carlsson L., Markey F., Bhkstad I., Lindberg U. (1979) Reorganization of stimulated by thrombin as measured inhibition assay. Proc. natn. Acad.
Persson T. and actin in platelets by the DNase I Sci.
U.S.A.
76,
637M380.
Drenckhahn D. and Mannherz H. G. (1983) Distribution of actin and the actin-associated proteins myosin, tropomyosin, alpha-actinin, vinculin and villin in rat and bovine exocrine-glands. Eur. J. Cell Biol. 30, 167-176. Swanston-Flatt S. K.. Carlsson L. and Gvlfe F. E. (1980) Actin filament formation in pancreatic B-cells during gluccse stimulation of insulin secretion. FEBS Leff. 117, 299-302. Takuma T. and Ichida T. (1986) Does cyclic AMP mobilize Ca’+ for amylase secretion from rat parotid cell? Biochim. biophys. Acfa 887, 113-I 17. Tojyo Y., Okumura K., Kanazawa M. and Matsumoto Y. (1989) Effect of cytochalasin D on the acinar cell structure and secretion in rat parotid salivary glands in u&o. Archs oral Biol. 34, 847-855.
Plate 1 Figs 1, 4, 7, 9. Light micrographs of dispersed rat parotid acini. Spurr resin section, toluidine-blue staining. x 700 Figs 2, 3, 5, 6, 8, 10. Fluorescence staining of F-actin in dispersed rhodamine-phalloidin. x 1300
rat parotid
acini using
Fig. I. Resting acini incubated for 30 min without isoprenaline; the arrows show a narrow lumen with discrete granules. Fig. 2. Control section of resting parotid acini; no specific fluorescence is visible. Fig. 3. Resting acini incubated for 30 min without isoprenaline; the bright fluorescence delineates the luminal border (arrows). Fig. 4. Acini stimulated for 1min with 1FM isoprenahne; luminal dilation is visible (arrows). Fig. 5. Acini stimulated for 1 min with 1FM isoprenaline; the luminal dilation (arrow) as a result of exocytosis is also recognized by rhodamine-phalloidin staining. Fig. 6. Acini stimulated for 1 min with 1PM isoprenaline; note the small fluorescent rings on the luminal border (arrow). Fig. 7. Acini stimulated for 15 min with I PM isoprenaline; there is a marked increase in the number and size of vacuoles. Fig. 8. Acini stimulated for 15 min with 1 nM isoprenaline; the intensity of the fluorescence is markedly increased in the dilated lumina. Fig. 9. Acini stimulated for 30 min with 1 PM isoprenahne; the lumina (arrows) have returned to their original size. Fig. 10. Acini stimulated for 30 min with 1 PM isoprenaline; the bright fluorescence (arrow) is similar to that seen in unstimulated acini.
Microfilaments
in parotid
Plate
1
glands
619
