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β-adrenergic stimulation of mouse parotid gland: Amylase activity and secretory granules during isoproterenol-induced mitosis
Gen. Pharmac. Vol. 16, No. 4, pp. 419-421, 1985 Printed in Great Britain. All rights reserved
0306-3623/85 $3.00+0.00 Copyright © 1985 Pergamon Press Ltd
fl-ADRENERGIC STIMULATION OF MOUSE PAROTID GLAND: AMYLASE ACTIVITY A N D SECRETORY GRANULES D U R I N G ISOPROTERENOL-INDUCED MITOSIS ANTONIO CHAPOLA, I NELSON VILLA, 1 LU1Z B. S. VALLE, 2 RICARDO M. OLIVEIRA-FILHO2 and CONCEI~.O A. S. A. MINETTI2 Departamentos de IHistologia e Embriologia and 2Farmacologia, Instituto de Cirncias Biomrdicas, Universidade de S~o Paulo, Cidade Universithria, 05508 S~o Paulo (SP), Brasil (Received 15 October 1984)
Abstract--1. This paper examines the mitotic activity of parotid glands of groups of mice sacrificed at 1 hr-intervals between 26 and 36 hr after being injected with dl-isoproterenol (IPR, 1/~mol/g, single dose, i.p.). 2. Maximal mitotic activity occurs 35 hr after IPR injection; there is a concomitant rise of amylase activity (64~ over control). 3. Mitotic cells showed increased number of secretory granules (N) with regard to interphasic cells. However, the increment of N is paralleled by an increment of the cell section area (.4); the N/A ratio is thus maintained around 0.45 throughout. 4. These findings suggest that during IPR-induced mitosis of mice parotid cells there is not a blockade of the biosynthetic pathways which lead to the appearance of secretory granules in the cell cytoplasm.
INTRODUCTION
METHODS
Stimulation of fl-adrenergic receptors initiates the process of exocrine secretion in salivary gland cells (Butcher and Putney, 1980). Besides increased secretion, a number of other cellular events take place, including D N A synthesis and cell growth (Tsang et aL, 1980), and stimulation of several metabolic steps in these glands (Herman and Rossignol, 1975; Nicolau and Sassaki, 1983). Electron microscopy o f isoproterenol (IPR)-stimulated salivary glands shows that acinar cells of rat parotid and submaxillary glands discharge virtually all of their membranebound secretory granules within 1 hr after (Bogart, 1975). On the other hand, it is well known that 30 hr after I P R an intense hyperplasia of mice parotid acini occurs (Barka, 1965; Baserga et al., 1969). Although some authors have reported that differentiated cells such as condrocytes and pancreatic acinar cells were not capable of synthesizing secretion granules while the mitotic process develops (Holtzer, 1961; Wessels, 1964), results by others did not confirm this point of view (Cahn and Lasher, 1967; Sesso, 1968; A b r a h a m sohn, 1971). The interest on the point remained in that no further data were available concerning this approach in mice salivary glands. Therefore the purpose of this paper is to study mice parotid acinar cells undergoing mitosis after IPR stimulation.
Animals Adult male mice of the Swiss strain, 60-day old (30-35 g body weight) were used. The animals were fed Purine diet and water ad libitum and maintained at 20-25°C in a 12 hr light-dark cycle. Animal groupings were as follows. Experimental group. Animals injected i.p. with i/~mol/g body wt of dl-isoproterenol (Boehringer) and, 2 hr before sacrifice, with 1/~g/g body wt of colchicine (Merck). Control group. Similarly treated animals except that IPR solution was replaced by physiological saline (0.85~o NaCI). Mice were fasted 10 hr before being sacrificed by cervical dislocation, always between 1000 and 1200 hr.
Correspondence should be addressed to: Professor Ricardo M. Oliveira-Filho, Dept. Farmacologia, Instituto de Ci~ncias Biomrdicas, Universidade de Silo Paulo, Cidade Universithria, 05508 S. Paulo (SP), Brazil. G,P. 16/4~H
419
Mitotic peak In order to determine the glandular mitotic peak, the animals were sacrificed at 1 hr intervals between the 26th and the 36th hr after isoproterenol injection. Sampling Immediately after sacrifice, parotid glands were removed, freed from all non-glandular adherent tissue, fixed in Bouin's solution and routinely processed as to the obtention of 5#m-thick paraffin sections, further stained with haematoxylin-eosin. Drug action assessment Ten areas from every paraffin section were selected at random and mitotic counts were performed in 100 acinar cells (i.e. 1000 cells/gland). Electron microscopy For the ultrastructural analysis, parotid gland fragments were routinely processed according to Harven (1967) and Luft (1961) (see Kulay et al., 1981). Tissue sections were obtained with glass or diamond knives on a Porter Blum ultramicrotome (Sorvall MT-I). Thick sections (0.5/~m) were stained with a 1:1 mixture of 1~ Azur II and 1~o
420
ANTONIO CHAPOLA et al.
methylene blue (Merck). Thin sections (150-200~.) were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined with a EM-9S2 Zeiss electron microscope.
Table 1. Cell section area and number of secretory granules as assessed by electron microscopic examination of mice parotid glands 2hr after injection of colchicine (l#g/g body wt). Interphase (control group) and mitotic cells (IPR-treated group) were examined
Secretory granule counting
Interphase
The electron micrographs of mitotic acinar cells (experimental group) and of interphase cells (control group) were set together in order to make montages of several typical cells of each kind. Over every resulting picture the cell section area and the number of secretory granules were then determined with a MAHO-380 planimeter and a magnifying glass, respectively.
Mitosis "
Area of cell section (#m 2) 58 + 7.1 208 + 25.6* No. of secretory granules 26 _ 6.3 102 + 18.0" No of secretory granules per 1 #m2-unit of cell section 0.42 + 0.07 0.48 + 0.04 Each group consisted of 21 animals; results are mean + SD of 1000 cells/gland countings. Asterisk indicates significant difference with regard to interphase (control) cells (*P < 0.01).
Amylase assay The amylase activity was determined according to Summer and Howeel (1955), using starch in phosphate buffer pH 6.9; incubations were carried out during 15 min at 37°C and the final colour developed was measured at 520 nm in a DU-2 Beckman Spectrophotometer.
Table 2. Amylase activity of mice parotid glands, control and 35 hr after a single i.p. injection of IPR (1 #mol/g body wt) Amylase activity Treatment (mg glucose released/mg fresh tissue) Control (10)
RESULTS Figure 1 shows the d e t e r m i n a t i o n o f m a x i m a l mitotic activity o f acinar cells o f mice p a r o t i d gland after I P R stimulation. This activity reaches a peak 35 hr after d r u g a d m i n i s t r a t i o n . As s h o w n in T a b l e 1, 35 h r after I P R there was a significant increase in the cell area a n d in the total c o n t e n t o f secretory granules. However, the n u m b e r o f secretion granules per unit o f cell section area r e m a i n e d u n c h a n g e d either in mitotic ( I P R group) or in i n t e r p h a s e cells (control group). A paired analysis o f the n u m b e r o f secretory granules as a f u n c t i o n o f cell section area o f b o t h mitotic a n d interphase cells s h o w e d good fitting to the following regression e q u a t i o n (Fig. 2). N = - 9 . 4 7 + 0.55A
(1)
where N = n u m b e r o f granules a n d A = cell section area (#m2). E q u a t i o n 1 gives a good a p p r o x i m a t i o n between the n u m b e r of granules per section area a n d the n u m b e r o f granules theoretically estimated to the same area, with a c o r r e l a t i o n coefficient o f 0.94
1.81 + 0.10
IPR (10) 2.96 + 0.29* Results are mean + SD of duplicate determinations for every animal. Number of animals is given in parentheses. *P < 0.01.
DISCUSSION
O u r results showed a peak o f mitotic activity in mice p a r o t i d acinar cells 35 hr after fl-adrenergic s t i m u l a t i o n by IPR. A c c o r d i n g to M a l a m u d a n d Baserga (1968), there is peak D N A c o n c e n t r a t i o n a r o u n d the 30th h r after I P R injection; observation o f o u r material in this period revealed t h a t the nuclei o f acinar cells were polyploid, whereas mitotic figures were r a t h e r rare. Since n o great deviations were detected between the c o n t e n t o f secretory material a n d the c o r r e s p o n d ing cytoplasm section area o f either interphase or dividing cells, a good c o r r e l a t i o n between these p a r a m e t e r s was found. In addition, if in e q u a t i o n (1) one m a k e s N = 0 then A has t h a t cell section area where no granules exist ( a b o u t 17 #m2); such a n area correlates well with the area o f the nucleus.
(P < 0.01). T h e amylase activity o f mice p a r o t i d g l a n d showed to be significantly increased 35 hr after I P R (Table 2).
00[ 1601IN=-9A~
500
400
o
g 120 o 300
•
o E
200
z 100
time after IPR injection
(hi
Fig. l. Mitotic activity in mice parotid gland after a single i.p. injection of IPR, 1/~mol/g body wt. Two hours before sacrifice the animals were given colchicine, 1 #g/g body wt. Each point is the mean _ SD of 6 determinations.
80tt
4o
0
0 o.//o~, 70
0 0 ol o 140
I
i
|
210
280
350
A (cell area, pm2)
Fig. 2. Correlation of cell section area to the number of secretory granules therein. The 6 points from 35 up to 8 7 # m 2 represent data of interphase cells; the 8 points between 130 and 340 # m 2 represent mitotic cells. See the text for details.
IPR-stimulated mouse parotid acini The higher amylase activity found in the experimental group is most presumably due to the dividing rather than to the few interphase cells. Since no attempt was made to compare the salivary flow a m o n g the groups, thus the hypothesis that the enhanced number of secretory granules in IPRtreated animals could be due to impaired extrusion of exportable material cannot be discarded. D a t a by other authors suggest that mitotic cells have increased secretory activity (Byrt, 1966; T a k a h a m a and Barka, 1967). The goal of the present paper has been the study of salivary exocrine material during cell mitosis. Several authors (Holtzer, 1961; Wessels, 1964) admit that differentiated cells undergoing division "turn off" the synthesis of secretion products. Others agree in that during cell mitosis the secretion would be just diminished (Cahn and Lasher, 1967; Sesso et al., 1968; Robbins and Morrill, 1970) or not altered at all (Kazanev, 1966; Abrahmsohn, 1971). Our results favour the view that during cell division the synthesis of secretable material is not blocked. Instead, if one assumes the membrane extrusion processes to be unaltered, the biosynthetic pathways might be unchanged or even slightly enhanced. Acknowledgements--This investigation was partially supported by Grants from FAPESP (76/0129 and 78/0954) and FINEP (54-83-0503-00). C.A.S.A. Minetti is recipient of FAPESP 82/0495-0 fellowship.
REFERENCES
Abrahamsohn P. A. (1971) [SH]Leucine uptake and cytoplasmic RNA content during mitosis in the acinar cell o f rat pancreas. Ph.D. Thesis, Instituto de Ci~ncias Biom6dicas, Universidade de Silo Paulo (Silo Paulo, Brazil). Barka T. (1965) Induced cell proliferation: the effect of isoproterenol. Expl. Cell Res. 37, 662-679. Baserga R., Sasaki T. and Whitlock Jr J. P. (1969) The prereplicative phase of isoproterenol-stimulated DNA synthesis. In Biochemistry of Cell Division (Edited by Baserga R.) p. 77. Thomas, Springfield. Bogart B. I. (1975) Secretory dynamics of the rat submandibular gland. An ultrastructural and cytochemical study of the isoproterenol-induced secretory cycle. J. Ultrastruct. Res. 52, 139-155. Butcher F. R. and Putney J. W. (1980) Regulation of parotid gland function by cyclic nucleotides and calcium. Adv. cyclic Nucleotide Res. 13, 215-249. Byrt P. (1966) Secretion and synthesis of amylase in the rat parotid gland after isoprenaline. Nature (Lond.) 212, 1212-1215.
421
Cahn R. D. and Lasher R. (1967) Simultaneous synthesis of DNA and specialized cellular products by differentiating cartilage cells in vitro. Proc. natn. Acad. Sci. 58, 11311138. Harven E. (1967) Methods in electron microscopic cytology. Meth. Cancer Res. 1, 37-38. Herman G. and Rossignol B. (1975) Regulation of protein secretion and metabolism in rat salivary glands. Eur. J. Biochem. 55, 105-110. Holtzer H. (1961) Aspects of chondrogenesis and myogenesis. In Synthesis o f Molecular and Cellular Structures (Edited by Rudnick D.). Ronald Press, New York. Kazanev V. V. (1966) Isutschemie sekretomoy aktivnosti v deliaschtschichsia kletkach podsheludotschnoy shelezy. Byull. Eksp. Biol. Med. 62, 94-96. Kulay Jr L., Sim6es M. J., Oliveira-Filho R. M., Egami M. I., Nascimento H. M., Pacheco I. P. and Kulay M. N. C. (1981) Effects of magnesium sulphate on maternal and fetal rat liver. Gen. Pharmac. 12, 25-29. Luft J. H. (1961) Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 409-414. Malamud D. and Baserga R. (1968) Glycogen concentration and DNA synthesis in isoproterenol-stimulated salivary glands. Expl. Cell Res. 50, 581-588. Nicolau J. and Sassaki K. T. (1983) Metabolism of carbohydrate in vitro of the submandibular salivary glands from mice injected with isoproterenol. Gen. Pharmac. 14, 705-708. Reynolds E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. Robbins E. and Morrill G. A. (1970) Oxygen uptake during the HeLa cell life cycle and its correlation with macromolecular synthesis. J. Cell Biol. 44, 400-416. Sesso A. (1968) Secretory activity of acinar cells o f the rat pancreas during DNA synthesis and mitosis. Thesis, Faculdade de Medicina, Universidade de Silo Paulo (Ribeirio Preto, Brazil). Sesso A., Abrahamsohn P. A., Freymuller E. and Val6ri V. (1968) Incorporation d'amino acides triti6s, par les cellules acineuses du pancreas, observ~e aux microscopique optique et 61ectronique, avant et pendant la mitose chez le rat jeune. C.R. acad. Sci. (Paris) 266, 1668-1670. Summer J. B. and Howeel S. F. (1955) Amylases alfa and beta. In Methods in Enzymology, Vol. 1 (Edited by Colowick S. P. and Kaplan N. O.) p. 149. Academic Press, New York. Takahama M. and Barka T. (1967) Electron microscopic alterations of submaxillary glands produced by isoproterenoI. J. Ultrastruct. Res. 17, 452-474. Tsang B. K., Rixon R. H. and Whitfield J. F. (1980) A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells. J. cell. Physiol. 102, 19-26. Wessels N. K. (1964) DNA synthesis, mitosis, and differentiation in pancreatic acinar cells in vitro. J. Cell Biol. 20, 415-433.
0306-3623/85 $3.00+0.00 Copyright © 1985 Pergamon Press Ltd
fl-ADRENERGIC STIMULATION OF MOUSE PAROTID GLAND: AMYLASE ACTIVITY A N D SECRETORY GRANULES D U R I N G ISOPROTERENOL-INDUCED MITOSIS ANTONIO CHAPOLA, I NELSON VILLA, 1 LU1Z B. S. VALLE, 2 RICARDO M. OLIVEIRA-FILHO2 and CONCEI~.O A. S. A. MINETTI2 Departamentos de IHistologia e Embriologia and 2Farmacologia, Instituto de Cirncias Biomrdicas, Universidade de S~o Paulo, Cidade Universithria, 05508 S~o Paulo (SP), Brasil (Received 15 October 1984)
Abstract--1. This paper examines the mitotic activity of parotid glands of groups of mice sacrificed at 1 hr-intervals between 26 and 36 hr after being injected with dl-isoproterenol (IPR, 1/~mol/g, single dose, i.p.). 2. Maximal mitotic activity occurs 35 hr after IPR injection; there is a concomitant rise of amylase activity (64~ over control). 3. Mitotic cells showed increased number of secretory granules (N) with regard to interphasic cells. However, the increment of N is paralleled by an increment of the cell section area (.4); the N/A ratio is thus maintained around 0.45 throughout. 4. These findings suggest that during IPR-induced mitosis of mice parotid cells there is not a blockade of the biosynthetic pathways which lead to the appearance of secretory granules in the cell cytoplasm.
INTRODUCTION
METHODS
Stimulation of fl-adrenergic receptors initiates the process of exocrine secretion in salivary gland cells (Butcher and Putney, 1980). Besides increased secretion, a number of other cellular events take place, including D N A synthesis and cell growth (Tsang et aL, 1980), and stimulation of several metabolic steps in these glands (Herman and Rossignol, 1975; Nicolau and Sassaki, 1983). Electron microscopy o f isoproterenol (IPR)-stimulated salivary glands shows that acinar cells of rat parotid and submaxillary glands discharge virtually all of their membranebound secretory granules within 1 hr after (Bogart, 1975). On the other hand, it is well known that 30 hr after I P R an intense hyperplasia of mice parotid acini occurs (Barka, 1965; Baserga et al., 1969). Although some authors have reported that differentiated cells such as condrocytes and pancreatic acinar cells were not capable of synthesizing secretion granules while the mitotic process develops (Holtzer, 1961; Wessels, 1964), results by others did not confirm this point of view (Cahn and Lasher, 1967; Sesso, 1968; A b r a h a m sohn, 1971). The interest on the point remained in that no further data were available concerning this approach in mice salivary glands. Therefore the purpose of this paper is to study mice parotid acinar cells undergoing mitosis after IPR stimulation.
Animals Adult male mice of the Swiss strain, 60-day old (30-35 g body weight) were used. The animals were fed Purine diet and water ad libitum and maintained at 20-25°C in a 12 hr light-dark cycle. Animal groupings were as follows. Experimental group. Animals injected i.p. with i/~mol/g body wt of dl-isoproterenol (Boehringer) and, 2 hr before sacrifice, with 1/~g/g body wt of colchicine (Merck). Control group. Similarly treated animals except that IPR solution was replaced by physiological saline (0.85~o NaCI). Mice were fasted 10 hr before being sacrificed by cervical dislocation, always between 1000 and 1200 hr.
Correspondence should be addressed to: Professor Ricardo M. Oliveira-Filho, Dept. Farmacologia, Instituto de Ci~ncias Biomrdicas, Universidade de Silo Paulo, Cidade Universithria, 05508 S. Paulo (SP), Brazil. G,P. 16/4~H
419
Mitotic peak In order to determine the glandular mitotic peak, the animals were sacrificed at 1 hr intervals between the 26th and the 36th hr after isoproterenol injection. Sampling Immediately after sacrifice, parotid glands were removed, freed from all non-glandular adherent tissue, fixed in Bouin's solution and routinely processed as to the obtention of 5#m-thick paraffin sections, further stained with haematoxylin-eosin. Drug action assessment Ten areas from every paraffin section were selected at random and mitotic counts were performed in 100 acinar cells (i.e. 1000 cells/gland). Electron microscopy For the ultrastructural analysis, parotid gland fragments were routinely processed according to Harven (1967) and Luft (1961) (see Kulay et al., 1981). Tissue sections were obtained with glass or diamond knives on a Porter Blum ultramicrotome (Sorvall MT-I). Thick sections (0.5/~m) were stained with a 1:1 mixture of 1~ Azur II and 1~o
420
ANTONIO CHAPOLA et al.
methylene blue (Merck). Thin sections (150-200~.) were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined with a EM-9S2 Zeiss electron microscope.
Table 1. Cell section area and number of secretory granules as assessed by electron microscopic examination of mice parotid glands 2hr after injection of colchicine (l#g/g body wt). Interphase (control group) and mitotic cells (IPR-treated group) were examined
Secretory granule counting
Interphase
The electron micrographs of mitotic acinar cells (experimental group) and of interphase cells (control group) were set together in order to make montages of several typical cells of each kind. Over every resulting picture the cell section area and the number of secretory granules were then determined with a MAHO-380 planimeter and a magnifying glass, respectively.
Mitosis "
Area of cell section (#m 2) 58 + 7.1 208 + 25.6* No. of secretory granules 26 _ 6.3 102 + 18.0" No of secretory granules per 1 #m2-unit of cell section 0.42 + 0.07 0.48 + 0.04 Each group consisted of 21 animals; results are mean + SD of 1000 cells/gland countings. Asterisk indicates significant difference with regard to interphase (control) cells (*P < 0.01).
Amylase assay The amylase activity was determined according to Summer and Howeel (1955), using starch in phosphate buffer pH 6.9; incubations were carried out during 15 min at 37°C and the final colour developed was measured at 520 nm in a DU-2 Beckman Spectrophotometer.
Table 2. Amylase activity of mice parotid glands, control and 35 hr after a single i.p. injection of IPR (1 #mol/g body wt) Amylase activity Treatment (mg glucose released/mg fresh tissue) Control (10)
RESULTS Figure 1 shows the d e t e r m i n a t i o n o f m a x i m a l mitotic activity o f acinar cells o f mice p a r o t i d gland after I P R stimulation. This activity reaches a peak 35 hr after d r u g a d m i n i s t r a t i o n . As s h o w n in T a b l e 1, 35 h r after I P R there was a significant increase in the cell area a n d in the total c o n t e n t o f secretory granules. However, the n u m b e r o f secretion granules per unit o f cell section area r e m a i n e d u n c h a n g e d either in mitotic ( I P R group) or in i n t e r p h a s e cells (control group). A paired analysis o f the n u m b e r o f secretory granules as a f u n c t i o n o f cell section area o f b o t h mitotic a n d interphase cells s h o w e d good fitting to the following regression e q u a t i o n (Fig. 2). N = - 9 . 4 7 + 0.55A
(1)
where N = n u m b e r o f granules a n d A = cell section area (#m2). E q u a t i o n 1 gives a good a p p r o x i m a t i o n between the n u m b e r of granules per section area a n d the n u m b e r o f granules theoretically estimated to the same area, with a c o r r e l a t i o n coefficient o f 0.94
1.81 + 0.10
IPR (10) 2.96 + 0.29* Results are mean + SD of duplicate determinations for every animal. Number of animals is given in parentheses. *P < 0.01.
DISCUSSION
O u r results showed a peak o f mitotic activity in mice p a r o t i d acinar cells 35 hr after fl-adrenergic s t i m u l a t i o n by IPR. A c c o r d i n g to M a l a m u d a n d Baserga (1968), there is peak D N A c o n c e n t r a t i o n a r o u n d the 30th h r after I P R injection; observation o f o u r material in this period revealed t h a t the nuclei o f acinar cells were polyploid, whereas mitotic figures were r a t h e r rare. Since n o great deviations were detected between the c o n t e n t o f secretory material a n d the c o r r e s p o n d ing cytoplasm section area o f either interphase or dividing cells, a good c o r r e l a t i o n between these p a r a m e t e r s was found. In addition, if in e q u a t i o n (1) one m a k e s N = 0 then A has t h a t cell section area where no granules exist ( a b o u t 17 #m2); such a n area correlates well with the area o f the nucleus.
(P < 0.01). T h e amylase activity o f mice p a r o t i d g l a n d showed to be significantly increased 35 hr after I P R (Table 2).
00[ 1601IN=-9A~
500
400
o
g 120 o 300
•
o E
200
z 100
time after IPR injection
(hi
Fig. l. Mitotic activity in mice parotid gland after a single i.p. injection of IPR, 1/~mol/g body wt. Two hours before sacrifice the animals were given colchicine, 1 #g/g body wt. Each point is the mean _ SD of 6 determinations.
80tt
4o
0
0 o.//o~, 70
0 0 ol o 140
I
i
|
210
280
350
A (cell area, pm2)
Fig. 2. Correlation of cell section area to the number of secretory granules therein. The 6 points from 35 up to 8 7 # m 2 represent data of interphase cells; the 8 points between 130 and 340 # m 2 represent mitotic cells. See the text for details.
IPR-stimulated mouse parotid acini The higher amylase activity found in the experimental group is most presumably due to the dividing rather than to the few interphase cells. Since no attempt was made to compare the salivary flow a m o n g the groups, thus the hypothesis that the enhanced number of secretory granules in IPRtreated animals could be due to impaired extrusion of exportable material cannot be discarded. D a t a by other authors suggest that mitotic cells have increased secretory activity (Byrt, 1966; T a k a h a m a and Barka, 1967). The goal of the present paper has been the study of salivary exocrine material during cell mitosis. Several authors (Holtzer, 1961; Wessels, 1964) admit that differentiated cells undergoing division "turn off" the synthesis of secretion products. Others agree in that during cell mitosis the secretion would be just diminished (Cahn and Lasher, 1967; Sesso et al., 1968; Robbins and Morrill, 1970) or not altered at all (Kazanev, 1966; Abrahmsohn, 1971). Our results favour the view that during cell division the synthesis of secretable material is not blocked. Instead, if one assumes the membrane extrusion processes to be unaltered, the biosynthetic pathways might be unchanged or even slightly enhanced. Acknowledgements--This investigation was partially supported by Grants from FAPESP (76/0129 and 78/0954) and FINEP (54-83-0503-00). C.A.S.A. Minetti is recipient of FAPESP 82/0495-0 fellowship.
REFERENCES
Abrahamsohn P. A. (1971) [SH]Leucine uptake and cytoplasmic RNA content during mitosis in the acinar cell o f rat pancreas. Ph.D. Thesis, Instituto de Ci~ncias Biom6dicas, Universidade de Silo Paulo (Silo Paulo, Brazil). Barka T. (1965) Induced cell proliferation: the effect of isoproterenol. Expl. Cell Res. 37, 662-679. Baserga R., Sasaki T. and Whitlock Jr J. P. (1969) The prereplicative phase of isoproterenol-stimulated DNA synthesis. In Biochemistry of Cell Division (Edited by Baserga R.) p. 77. Thomas, Springfield. Bogart B. I. (1975) Secretory dynamics of the rat submandibular gland. An ultrastructural and cytochemical study of the isoproterenol-induced secretory cycle. J. Ultrastruct. Res. 52, 139-155. Butcher F. R. and Putney J. W. (1980) Regulation of parotid gland function by cyclic nucleotides and calcium. Adv. cyclic Nucleotide Res. 13, 215-249. Byrt P. (1966) Secretion and synthesis of amylase in the rat parotid gland after isoprenaline. Nature (Lond.) 212, 1212-1215.
421
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