The Effect of lsoproterenol upon the Chemical Composition of Plasma Membranes in the Mouse Parotid Gland R. LOPEZ and N. GALANTI Unidad de Biologia Celular Departamento de Biologia Celular y Genetica Universidad de Chile Sede Santiago Norte Zafiartu 1042, Casilla 6556 Santiago 4, Chile
A single intraperitoneal injection of isoproterenol induces resting cells from the acini of the mouse parotid gland to enter the proliferative cycle. Parotid plasma membrane from non-stimulated and isoproterenol-treated mice were prepared by differential centrifugation of the homogenates. Comparing the chemical composition of plasma membranes from non-stimulated and isoproterenol-treated mice, no variation in the phospholipid/protein ratio was observed. However, the levels of neutral sugars, hexosamines and sialic acid falls drastically in the early prereplicative phase. The decrease in neutral sugars and hexosamines in plasma membranes caused by isoproterenol is imitated by pilocarpine, which induces secretion but little or no increase in DNA synthesis. However, pilocarpine does not mobilize sialic acid from the plasma membrane. Moreover, dosis of isoproterenol that elicits secretion but not mitosis in the acinar cells, does not induce the movement of sialic acid from the plasma membrane, The mobilization of sialic acid from plasma membranes caused by isoproterenol was also demonstrated in an in vitro system. Treatment of the plasma membrane with chloroform/methanol shows that around 60% of the sialic acid is present in the less polar phase. We conclude that the separation of sialic acid from the plasma membrane is one of the early steps in the sequence of events leading to DNA synthesis and cell division in the isoproterenol-stimulatedparotid gland of mice.
Introduction
A single intraperitoneal injection of isoproterenol causes, after a lag period of 20 h, a marked stimulation in DNA synthesis in the salivary glands of both rats [ 11 and mice 121. The increase in DNA synthesis reaches a peak at 28 to 30 h, followed a few hours later by a wave of mitosis. Changes in protein synthesis [3-51, RNA synthesis [61, glycolipid synthesis [71, glycogen concentration [81, cyclic nucleotides levels [91, the activity and intracellular distribution of several enzymes [ 10- 141 and the activation of chromatin template activity 1151 have been described in mouse salivary glands in the prereplicative period between the administration of isoproterenol and the onset of DNA synthesis. Components of cell membranes may be of interest when studying the prereplicative phase of the isoprotereno1 stimulated salivary glands. Changes in membrane components such as glycoproteins, glycolipids and Differentiation 5, 155-160 (1976) - 0 by Springer-Verlag 1976
phospholipids have been reported in virus-transformed cells 116-221, in tumor cells [21-241 and in cells in various phases of the cell cycle 121, 22-25, 261. Changes in these components of the cell membrane may play a key role in the sequence of events leading to DNA synthesis and cell division. However, most of the data found in the literature is connected with changes that appear within the cell membrane after the activation of the chromatin. These changes seem to be directed by the “high” active chromatin in order to have the cell in the best situation to synthesize DNA and divide. A different situation is that of resting cells that are stimulated to proliferate. In this case, it is possible to investigate any change at the cell membrane level prior to the activation of the chromatin. We have, therefore, investigated whether changes in the chemical composition of plasma membranes may occur in the mouse parotid gland after a single injection
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of isoproterenol. Our present results suggest that a change in the chemical composition of the parotid cell plasma membrane is one of the early steps in the series of events leading to DNA synthesis. Methods Animals. C3H/B1O Snell mice, bred in our laboratory, were used when about 4 months old and weighing 26-28 g. The animals were kept on a natural light-and-dark schedule and fed ad libitum until 2 h before the experiment, when their food, but not their water, was withdrawn. The mice were killed by cervical dislocation. Parotid glands were quickly dissected free from adhering fat and lymph nodes and chilled on ice. The pooled glands from 10 mice were minced with a razor blade and homogenised with 8 strokes at 1000 rpm in 7 vols (w/v) cold Medium A (0.32 M sucrose in 0.05 M TrisHCI pH 7.4 at 20' C, 0.025 M KCI, 0.003 M MgCI,, 0.002 M CaCI,) in a wide clearance Teflon Glass homogeniser (Potter Elvejhem). Preparation of Plasma Membranes. The preparation of plasma membranes was essentially achieved as previously described [ 14, 271. The above homogenate was passed through two layers of cheesecloth, centrifuged at 650 x g for 10 min and the supernatant retained. The sediment (650 x g pellet) was resuspended in Medium A and recentrifuged at 650 x g for 10 min. This step was repeated twice. The supernatants were combined, centrifuged at 15,000 x g for 10 min and the supernatant recovered. The sediment (15,000 x g pellet) was resuspended in Medium A and recentrifuged at 15,000 x g for 10 min. The supernatants were combined and centrifuged at 38,000 x g for 60 min. The sediment (pellet 38,000 x g). was washed twice by resuspending in 0.05 M Tris-HC1 pH 7.4 at 20" C and centrifuging at 38,000 x g for 60 min. The resulting pellet contains the plasma membranes [ 14, 271. Sediments were hand-resuspended in the centrifuge tubes using loose glass pestles from Dounce homogenisers. Centrifugations were done in the B-20 International Centrifuge using the 873 rotor and in the Beckman Mdlo. L Centrifuge using rotor 40. Further purification of the plasma membrane fraction was achieved by overlaying the membranes on 2 M sucrose and centrifuging at 22,500 rpm for 3 h in the SW 25.1 rotor of the Beckman Mdlo. L Centrifuge. Electron microscopy and marker enzymes of the various fractions were performed as previously described [ 14, 271. Chemicals. Isoproteremol-HC1 (1-(3,4-dihydroxiphenyl)-2-isopropylaminoethanol hydrochloride) was purchased from Wintrop Laboratories, New York and pilocarpine-HCI from Mann Research. Isoproterenol (0.67 pmoles/g, body wt) and pilocarpine (0.27 pmoles/g, body wt) dissolved in 0.2 ml sterile saline, were injected intraperitoneally. AU other compounds were purchased from Sigma, and were of reagent grade. Analytical Determinations. Nucleic acids were extracted from the plasma membranes according to the method of Schneider [281. DNA and RNA were determined using the diphenylamine reaction as modified by Burton [291 and the orcinol reaction [301, respectively. Lipids were extracted using the method of Folch et al. [311 as modified by Peterson and Rubin [321. Total lipids, cholesterol and phospholipids phosphorus were analysed in the air dried chloroform/methanol extract following the methods of Johnson [ 331, Clarck et al. [341 and Bartlett [351 respectively. The amount of phospholipids was calculated by assuming 25 pg of phospholipids per pg
R. Lopez and N. Galanti:
of phosphorous. Sialic acids and hexosamines were extracted after the method of Molnar [361, and determined as described by Warren (371 and Gatt and Berman [381 respectively. Total carbohydrates were determined by using the anthrone reaction [391. Prior to the estimation of the total sugars, plasma membranes were washed in distilled water until negative reaction of the supernatant resulted with the anthrone reagent. Uronic acids were determined using the carbazol reaction as described by Dische 1401. Proteins were measured by the procedure of Lowry et al. 1411.
Results
Chemical Composition. The chemical composition of the isolated plasma membranes from the control and IPR-stimulated parotid glands is listed in Table 1. No important variation in the phospholipid/protein ratio was observed. However, small changes in the cholesterol/protein ratio seem to be present at the time when DNA synthesis begins. A clear decrease in the neutral carbohydrates, hexosamines and sialic acid is shown in early times after isoproterenol administration. The values return to control levels by 20 h. These results suggest that isoproterenol induces changes in the carbohydrate content of the parotid plasma membrane. Effect of Pilocarpine on the Carbohydrate Content of Plasma Membranes. Pilocarpine is known to induce secretion but little or no increase in DNA synthesis [ 1, 91. Figs. 1, 2 and 3 show the effect of isoproterenol and pilocarpine on the total carbohydrates, hexosamines and Table 1. Chemical composition of parotid plasma membranes in the pre-replicativeand replicativephases Hours after isoproterenol
0
2
20
28
Total lipids Phospholipids Cholesterol Total carbohydrates Hexosamines Sialic acid Uronic acid
669 435 195 86 18.7 8.4 0.0
680 462 218 45 12.6 4.4 n.d.
655 386 260 88 n.d. 7.5 n.d.
672 421 246 93 n.d. 8.4 n.d.
Groups of 50 mice received an intraperitoneal injection of isoprotereno1 (0.67 umoleslg body wt). The animals were killed at 2, 20 and 28 h after isoproterenol. Control mice represent animals not injected or injected with sterile saline. Membranes were prepared as described under Methods and divided in several aliquots after measuring total proteins. Total lipids, phospholipids and cholesterol were determined from the chloroform/methanol extract from one aliquot. Total carbohydrates and uronic acids were determined from another aliquot, previously washed until anthrone negative reaction was obtained from the 38,000 x g supernatant. Hexosamines and sialic acid were extracted and determined on a third aliquot, as described under Methods. Each value is the average of two experiments expressed in pg/mg protein.
157
Plasma Membranes in Mouse Parotid Gland
1
2 Hours after injection
3
L
Fig. 1. Total carbohydrate content of parotid plasma membranes prepared from isoproterenol or pilocarpine treated mice. Isoprotereno1 (0.67 pmoles/g body wt) or pilocarpine (0.27 pmoles/g body wt) were injected intraperitoneally to groups of 8 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area corresponds to control values, plus or minus the standard deviation. U , isoproterenol; G 0, pilocarpine
1
2
3
L
Hours after injection.
Fig. 2. Hexosamine content of parotid plasma membrane prepared from isoproterenol or pilocarpine treated mice. Isoproterenol (0.67 ymoles/g body wt) or pilocarpine (0.27 moles/g body wt) were injected intraperitoneally to groups of 16 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area correspond to control values plus or minus the standard deviation. C 4,isoproterenol; 0 4, pilocarpine
sialic acid content of the plasma membrane, respectively. Fig. 1 shows that 30 min after an isoproterenol or pilocarpine injection there is a marked decrease in the total carbohydrate content of the plasma membranes. This decrease is maximal at 2 h after isoproterenol, when the values for pilocarpine are returning to normal levels. Four hours after isoproterenol, there are still approximately 25% less carbohydrates in the plasma membrane. At the same time, parotid plasma mem-
1
2
3
Hours after injection.
L
Fig. 3. Sialic acid content of parotid plasma membranes prepared from isoproterenol or pilocarpine treated mice. Isoproterenol (0.67 pmoles/g body wt) or pilocarpine (0.27 pmoles/g body wt) were injected intraperitoneally to groups of 16 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area corresponds to control values, plus or , isoproterenol; 0- --0, minus the standard deviation. U pilocarpine
branes prepared from pilocarpine treated mice show a normal content of total carbohydrates. Fig. 2 shows a drastic decrease in the hexosamine content of parotid plasma membranes prepared from both isoproterenolor pilocarpine-treated mice. The decrease is within the same range at 30 min and 2 h7returning to normal levels by 4 h. Fig. 3 shows that 30 min after isoproterenol there is a dramatic decrease in the sialic acid content of the parotid plasma membranes, which was not observed with pilocarpine. This decrease is maximal at 2 h and it is still down at 4 h after isoproterenol. On the contrary, only slight movements of sialic acid from the plasma membranes were seen at 2 and 4 h after pilocarpine. However, it is important to take into consideration that the pellet of membranes obtained from the parotid gland during secretion is always bigger than those obtained from non-stimulated glands. This is possibly due to the increase in the surface of the acinar cells during secretion. Table 2 shows that in spite of this fact, there is an actual mobilisation of sialic acid from the parotid plasma membranes after isoproterenol, which is not seen after pilocarpine. From the same Table it is possible to conclude that concomitantly with the separation of sialic acid there is an entrance of proteins into the plasma membranes after isoproterenol. With pilocarpine the same effect can be seen, but in this case the entrance of proteins into the plasma membranes is concurrent with the entrance of sialic acid. The Effect of Isoproterenol in vitro. Isoproterenol provokes mobilisation of sialic acid from the parotid
158
R. Lopez and N. Galanti:
Table 2. Effect of isoproterenol and pilocarpine on the sialic acid content of parotid plasma membranes Factor
Table 4. The effect of two different doses of isoproterenol on the mobilisation of sialic acid from plasma membranes and on the induction of mitosis in acinar cells
Total protein of treated membranes =
~~
Total protein of control membranes
% mitosis
Condition
pg sialic
acid/mg protein
Corrected values = Specific activity x factor pg Sialic acid
pg Sialic acid ~
.___
mg Protein Control Isoproterenol Pilocarpine
8.39 f 0.4 4.10 f 0.6 7.17 f 0.2
Factor
1 1.68 1.83
pg protein (Corrected) 8.39 6.9 13.1
Isoproterenol(O.67 pmolesig body wt) or pilocarpine (0.27 pmolesig body wt) were injected intraperitoneally and the animals killed 2 h later. Plasma membranes were prepared and sialic acid extracted and determined as described under Methods. The values are the average of 7 experiments.
Table 3. The effect of isoproterenol on the sialic acid content of parotid plasma membranes in vitro Condition
pH 4.2
pH 7.0
Control Isoproterenol
7.61 f 0.04 5.97 f 0.12
7.21 f 0.16 5.63 f 0.15
Plasma membranes were prepared as described under Methods. Incubation of the membranes with or without isoproterenol was carried out at two different pH for 75 min at 37" C. After incubation, membranes were isolated at 38,000 x g for 60 min. Sialic acid was extracted and determined as described under Methods. The values are expressed as pg sialic acid per mg of protein. Buffer pH 4.2; Sodium acetate-acetic acid 0.075 M. Buffer pH 7.0: tris-HC10.080 M-ascorbic acid 0.003 M-CaCl, 0.001 M. Membranes without incubation: 7.92 pg sialic acid/mg protein.
plasma membranes when injected to the animal, in v i v a This effect is exposed to problems derived from the catabolism of isoproterenol and other changes produced by the drug in the parotid. To avoid this problem, in vitro experiments were performed. Table 3 shows the effect of isoproterenol on isolated plasma membranes from mouse parotid glands using two different pH conditions. In both, it is evident that a separation of sialic acid results from the plasma membranes. Interestingly enough, this effect is of the same order as the one observed in vivo after isoproterenol (corrected values). The Efect of Low Doses of Isoproterenol.Very low doses of isoproterenol injected intraperitoneally to the mouse provoke secretion as measured by the disappearance of alpha-amylase from the parotid gland, without inducing DNA synthesis [421.
Control Isoproterenol (0.0015 pmoles/g body wt) Isoproterenol (0.67 umoleslg body wt)
0.238 & 0.065 0.443 f 0.156 26.52 f 2.64
7.32 f 0.23 7.85 f 0.32 3.78 f 0.02
Groups of 16 mice received an intraperitoneal injection of isoprotereno1 (0.67 pmoles/g body wt or 0.0015 pmoles/g body wt). Control animals represent non-injected mice. The animals were killed 2 h later. Membranes were prepared and the sialic acid extracted and determined as described under Methods. Simultaneously, groups of 3 mice received the same treatment with the two different doses of isoproterenol. Twenty five and 34 h later the mice received an inlraperitoneal injection of colchicine (2 ug/g body wt). The animals were killed 37 h after isoproterenol, the parotid glands quickly fixed in Duboq-Brazil and included in paraffin. Five microns sections were stained with hematoxylin-eosin and mitosis counted in 5000 cells.
Table 5. Differential distribution of sialic acid in polar and nonpolar fractions from parotid plasma membrane Fractions
pg sialic acidimg protein Control Isoproterenol
Corrected values for isoproterenol results Factor A Factor B (2.45) (3.7)
Protein Upper phase Lower phase Total
1.01 f 0.52 1.37 f 0.49 4.38 & 0.18 6.76
0.93 1.47 1.32 3.72
0.38 f 0.05 0.60 2 0.04 0.54 & 0.04 1.52
1.41 2.22 2.0 5.63
Groups of 33 mice received an intraperitoneal injection of isoprotereno1 (0.67 pmoles/g body wt). The animals were killed 2 h later. Control mice represent non-injected animals. Membranes were prepared and lipids extracted as described under Methods. Three fractions were obtained. A protein fraction, retained in the filter; an upper phase containing mainly gangliosides (7,44) and a lower phase containing the less polar glycolipids (cerebrosides, glyceride glycolipids and the less polar gangliosides) (3 1). Each fraction was dried and the sialic acid extracted and determined as described under Methods. Factor A is thc average of the ratios control/isoproterenol for protein and lower phase fractions. Factor B is the ratio total protein of treated membranes/total protein of control membranes.
Table 4 shows the effect of two different doses of isoproterenol on the mobilisation of sialic acid from the parotid plasma membrane and on the percentage of mitosis in acinar cells. From the data presented, it appears that there is a correlation between the effect of the drug on the plasma membrane and the appearance of mitosis in the acinar cells of the parotid gland.
Plasma Membranes in Mouse Parotid Gland
Distribution of Sialic Acid in Polar and Nonpolar Fractions from the Plasma Membrane. It was interesting to investigate the localisation of the sialic acid containing molecule in the plasma membrane. One approach was to fractionate the membranes in polar and non polar fractions in order to localise this molecule as a periphereal or an integral part of the plasma membrane 1431. Table 5 shows that most of the sialic acid (65%) is present in the lower phase (less polar) of the Folch Extract. This result suggest that the molecule is inserted in the non-polar region of the plasma membrane. Upon stimulation with isoproterenol most of the sialic acid attached to this non-polar molecule is mobilised (see corrected values).
Discussion
It has been suggested that the control of replication and transcription may involve an interaction between two macromolecular aggregates, this is, the chromatin and cell membranes. In this hypothesis, it is postulated that an incoming molecule interacting with any one of these components will induce a conformational change leading to the expression of new abilities of the macromolecular aggregates [451. This idea has become increasingly interesting in light of the evidence that cell membranes are mostly aggregates of amphipatic molecules arranged in an interphase, within which movements of constituent macromolecules may occur [22, 431. This movement of molecules is mostly in the plane of the membrane. However, it is also possible that movement of molecules occurs through the membranes or through changes in the conformation of the molecules in a zone of the interphase. thus changing the relationships in the macromolecular aggregate. Either mechanism, or both, may be involved in the transmission of outside signals to the chromatin aggregates with the cell membrane acting as a modulator of this activity [22, 431. In recent years, it has been observed that changes occur in the cell surface of virus-transformed, and spontaneously transformed tumorigenic mouse cell lines, or tumoral cells. Of particular interest is the stimulation of cell division caused by neuraminidase [461 and the synthesis of the sialic acid during a limited time in the late G2 phase of the cell cycle of cultured human lymphoid cells t471. A summary of surface characteristics of interphase, mitotic and transformed cells is shown in the work of Mannino and Burger [481 indicating the striking similarity between the surfaces of mitotic and transformed cells.
159 The purpose of our study was to compare the chemical composition of the plasma membrane of the mouse parotid gland in a resting state, and when stimulated to synthesise DNA and divide. After the administration of isoproterenol a considerable amount of sialic acid is removed from the plasma membrane of the parotid gland cells. The importance of this effect is emphasised by our failure to separate the mobilisation of sialic acid from the plasma membrane with the stimulation of DNA synthesis that occurs 20 h later. Pilocarpine, for instance, induces secretion but very little or no increase in DNA synthesis, and is incapable of removing sialic acid from the plasma membranes. Moreover, doses of isoproterenol known to induce secretion but to have no effect on DNA synthesis are unable to separate sialic acid from the plasma membrane. Even more interesting, the same effect of isoproterenol has been demonstrated in an in vitro system. This result clearly points out the direct effect of the drug on the cell membrane. It was of great importance to locate the sialic acid that is removed by the effect of the isoproterenol. It is known that the sialic acid moiety is located on the outside of the plasma membrane. Our results show that the sialic acid is attached mainly to non-polar molecules, probably immersed in the non-polar region of the plasma membrane. The removal of sialic acid from this molecule will produce a change, not only in the molecule but in its relationship with neighbouring molecules. That is, it will induce a conformational change in the cell membrane that may translate the outside signal to citoplasmic information. This change in the plasma membrane is prior to the activation of chromatin that it is known to occur 6 h after the administration of isoproterenol [ 151. In conclusion, our results point to a direct effect of isoproterenol on the plasma membrane of the parotid gland as one of the first steps in the sequence of events leading to DNA synthesis and cell &vision. As a result of this action, part of the sialic acid is removed from the plasma membrane. This result is in agreement with our previous finding that an early increase in the synthesis of a glycolipid occurs upon stimulation of the parotid gland with isoproterenol [71. The change at the cell membrane is followed some time later by the activation of chromatin. At the present time we are working on the relationship between the removal of the sialic acid from the plasma membrane, and both its connection with other molecules in the membrane and molecules or structures in the cytoplasm adjacent to the plasma membrane,
160 Acknowledgements: The authors would like to thank the following: Mr. Jorge Sans for assistance in preparing sections and recording the mitotic indices; Dr. John Durham for his comments; Mrs. Xenia Tchernitchin for correcting the manuscript and Miss Lydia Corail for the final typing. This work was supported by Grants No. 634 and No. 1006 from the Oficina Tknica de Desarrollo Cientifico y Creacion Artistica, Universidad de Chile.
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A single intraperitoneal injection of isoproterenol induces resting cells from the acini of the mouse parotid gland to enter the proliferative cycle. Parotid plasma membrane from non-stimulated and isoproterenol-treated mice were prepared by differential centrifugation of the homogenates. Comparing the chemical composition of plasma membranes from non-stimulated and isoproterenol-treated mice, no variation in the phospholipid/protein ratio was observed. However, the levels of neutral sugars, hexosamines and sialic acid falls drastically in the early prereplicative phase. The decrease in neutral sugars and hexosamines in plasma membranes caused by isoproterenol is imitated by pilocarpine, which induces secretion but little or no increase in DNA synthesis. However, pilocarpine does not mobilize sialic acid from the plasma membrane. Moreover, dosis of isoproterenol that elicits secretion but not mitosis in the acinar cells, does not induce the movement of sialic acid from the plasma membrane, The mobilization of sialic acid from plasma membranes caused by isoproterenol was also demonstrated in an in vitro system. Treatment of the plasma membrane with chloroform/methanol shows that around 60% of the sialic acid is present in the less polar phase. We conclude that the separation of sialic acid from the plasma membrane is one of the early steps in the sequence of events leading to DNA synthesis and cell division in the isoproterenol-stimulatedparotid gland of mice.
Introduction
A single intraperitoneal injection of isoproterenol causes, after a lag period of 20 h, a marked stimulation in DNA synthesis in the salivary glands of both rats [ 11 and mice 121. The increase in DNA synthesis reaches a peak at 28 to 30 h, followed a few hours later by a wave of mitosis. Changes in protein synthesis [3-51, RNA synthesis [61, glycolipid synthesis [71, glycogen concentration [81, cyclic nucleotides levels [91, the activity and intracellular distribution of several enzymes [ 10- 141 and the activation of chromatin template activity 1151 have been described in mouse salivary glands in the prereplicative period between the administration of isoproterenol and the onset of DNA synthesis. Components of cell membranes may be of interest when studying the prereplicative phase of the isoprotereno1 stimulated salivary glands. Changes in membrane components such as glycoproteins, glycolipids and Differentiation 5, 155-160 (1976) - 0 by Springer-Verlag 1976
phospholipids have been reported in virus-transformed cells 116-221, in tumor cells [21-241 and in cells in various phases of the cell cycle 121, 22-25, 261. Changes in these components of the cell membrane may play a key role in the sequence of events leading to DNA synthesis and cell division. However, most of the data found in the literature is connected with changes that appear within the cell membrane after the activation of the chromatin. These changes seem to be directed by the “high” active chromatin in order to have the cell in the best situation to synthesize DNA and divide. A different situation is that of resting cells that are stimulated to proliferate. In this case, it is possible to investigate any change at the cell membrane level prior to the activation of the chromatin. We have, therefore, investigated whether changes in the chemical composition of plasma membranes may occur in the mouse parotid gland after a single injection
156
of isoproterenol. Our present results suggest that a change in the chemical composition of the parotid cell plasma membrane is one of the early steps in the series of events leading to DNA synthesis. Methods Animals. C3H/B1O Snell mice, bred in our laboratory, were used when about 4 months old and weighing 26-28 g. The animals were kept on a natural light-and-dark schedule and fed ad libitum until 2 h before the experiment, when their food, but not their water, was withdrawn. The mice were killed by cervical dislocation. Parotid glands were quickly dissected free from adhering fat and lymph nodes and chilled on ice. The pooled glands from 10 mice were minced with a razor blade and homogenised with 8 strokes at 1000 rpm in 7 vols (w/v) cold Medium A (0.32 M sucrose in 0.05 M TrisHCI pH 7.4 at 20' C, 0.025 M KCI, 0.003 M MgCI,, 0.002 M CaCI,) in a wide clearance Teflon Glass homogeniser (Potter Elvejhem). Preparation of Plasma Membranes. The preparation of plasma membranes was essentially achieved as previously described [ 14, 271. The above homogenate was passed through two layers of cheesecloth, centrifuged at 650 x g for 10 min and the supernatant retained. The sediment (650 x g pellet) was resuspended in Medium A and recentrifuged at 650 x g for 10 min. This step was repeated twice. The supernatants were combined, centrifuged at 15,000 x g for 10 min and the supernatant recovered. The sediment (15,000 x g pellet) was resuspended in Medium A and recentrifuged at 15,000 x g for 10 min. The supernatants were combined and centrifuged at 38,000 x g for 60 min. The sediment (pellet 38,000 x g). was washed twice by resuspending in 0.05 M Tris-HC1 pH 7.4 at 20" C and centrifuging at 38,000 x g for 60 min. The resulting pellet contains the plasma membranes [ 14, 271. Sediments were hand-resuspended in the centrifuge tubes using loose glass pestles from Dounce homogenisers. Centrifugations were done in the B-20 International Centrifuge using the 873 rotor and in the Beckman Mdlo. L Centrifuge using rotor 40. Further purification of the plasma membrane fraction was achieved by overlaying the membranes on 2 M sucrose and centrifuging at 22,500 rpm for 3 h in the SW 25.1 rotor of the Beckman Mdlo. L Centrifuge. Electron microscopy and marker enzymes of the various fractions were performed as previously described [ 14, 271. Chemicals. Isoproteremol-HC1 (1-(3,4-dihydroxiphenyl)-2-isopropylaminoethanol hydrochloride) was purchased from Wintrop Laboratories, New York and pilocarpine-HCI from Mann Research. Isoproterenol (0.67 pmoles/g, body wt) and pilocarpine (0.27 pmoles/g, body wt) dissolved in 0.2 ml sterile saline, were injected intraperitoneally. AU other compounds were purchased from Sigma, and were of reagent grade. Analytical Determinations. Nucleic acids were extracted from the plasma membranes according to the method of Schneider [281. DNA and RNA were determined using the diphenylamine reaction as modified by Burton [291 and the orcinol reaction [301, respectively. Lipids were extracted using the method of Folch et al. [311 as modified by Peterson and Rubin [321. Total lipids, cholesterol and phospholipids phosphorus were analysed in the air dried chloroform/methanol extract following the methods of Johnson [ 331, Clarck et al. [341 and Bartlett [351 respectively. The amount of phospholipids was calculated by assuming 25 pg of phospholipids per pg
R. Lopez and N. Galanti:
of phosphorous. Sialic acids and hexosamines were extracted after the method of Molnar [361, and determined as described by Warren (371 and Gatt and Berman [381 respectively. Total carbohydrates were determined by using the anthrone reaction [391. Prior to the estimation of the total sugars, plasma membranes were washed in distilled water until negative reaction of the supernatant resulted with the anthrone reagent. Uronic acids were determined using the carbazol reaction as described by Dische 1401. Proteins were measured by the procedure of Lowry et al. 1411.
Results
Chemical Composition. The chemical composition of the isolated plasma membranes from the control and IPR-stimulated parotid glands is listed in Table 1. No important variation in the phospholipid/protein ratio was observed. However, small changes in the cholesterol/protein ratio seem to be present at the time when DNA synthesis begins. A clear decrease in the neutral carbohydrates, hexosamines and sialic acid is shown in early times after isoproterenol administration. The values return to control levels by 20 h. These results suggest that isoproterenol induces changes in the carbohydrate content of the parotid plasma membrane. Effect of Pilocarpine on the Carbohydrate Content of Plasma Membranes. Pilocarpine is known to induce secretion but little or no increase in DNA synthesis [ 1, 91. Figs. 1, 2 and 3 show the effect of isoproterenol and pilocarpine on the total carbohydrates, hexosamines and Table 1. Chemical composition of parotid plasma membranes in the pre-replicativeand replicativephases Hours after isoproterenol
0
2
20
28
Total lipids Phospholipids Cholesterol Total carbohydrates Hexosamines Sialic acid Uronic acid
669 435 195 86 18.7 8.4 0.0
680 462 218 45 12.6 4.4 n.d.
655 386 260 88 n.d. 7.5 n.d.
672 421 246 93 n.d. 8.4 n.d.
Groups of 50 mice received an intraperitoneal injection of isoprotereno1 (0.67 umoleslg body wt). The animals were killed at 2, 20 and 28 h after isoproterenol. Control mice represent animals not injected or injected with sterile saline. Membranes were prepared as described under Methods and divided in several aliquots after measuring total proteins. Total lipids, phospholipids and cholesterol were determined from the chloroform/methanol extract from one aliquot. Total carbohydrates and uronic acids were determined from another aliquot, previously washed until anthrone negative reaction was obtained from the 38,000 x g supernatant. Hexosamines and sialic acid were extracted and determined on a third aliquot, as described under Methods. Each value is the average of two experiments expressed in pg/mg protein.
157
Plasma Membranes in Mouse Parotid Gland
1
2 Hours after injection
3
L
Fig. 1. Total carbohydrate content of parotid plasma membranes prepared from isoproterenol or pilocarpine treated mice. Isoprotereno1 (0.67 pmoles/g body wt) or pilocarpine (0.27 pmoles/g body wt) were injected intraperitoneally to groups of 8 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area corresponds to control values, plus or minus the standard deviation. U , isoproterenol; G 0, pilocarpine
1
2
3
L
Hours after injection.
Fig. 2. Hexosamine content of parotid plasma membrane prepared from isoproterenol or pilocarpine treated mice. Isoproterenol (0.67 ymoles/g body wt) or pilocarpine (0.27 moles/g body wt) were injected intraperitoneally to groups of 16 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area correspond to control values plus or minus the standard deviation. C 4,isoproterenol; 0 4, pilocarpine
sialic acid content of the plasma membrane, respectively. Fig. 1 shows that 30 min after an isoproterenol or pilocarpine injection there is a marked decrease in the total carbohydrate content of the plasma membranes. This decrease is maximal at 2 h after isoproterenol, when the values for pilocarpine are returning to normal levels. Four hours after isoproterenol, there are still approximately 25% less carbohydrates in the plasma membrane. At the same time, parotid plasma mem-
1
2
3
Hours after injection.
L
Fig. 3. Sialic acid content of parotid plasma membranes prepared from isoproterenol or pilocarpine treated mice. Isoproterenol (0.67 pmoles/g body wt) or pilocarpine (0.27 pmoles/g body wt) were injected intraperitoneally to groups of 16 mice and the animals killed at the times indicated. Other conditions were as described under Methods. Shadowed area corresponds to control values, plus or , isoproterenol; 0- --0, minus the standard deviation. U pilocarpine
branes prepared from pilocarpine treated mice show a normal content of total carbohydrates. Fig. 2 shows a drastic decrease in the hexosamine content of parotid plasma membranes prepared from both isoproterenolor pilocarpine-treated mice. The decrease is within the same range at 30 min and 2 h7returning to normal levels by 4 h. Fig. 3 shows that 30 min after isoproterenol there is a dramatic decrease in the sialic acid content of the parotid plasma membranes, which was not observed with pilocarpine. This decrease is maximal at 2 h and it is still down at 4 h after isoproterenol. On the contrary, only slight movements of sialic acid from the plasma membranes were seen at 2 and 4 h after pilocarpine. However, it is important to take into consideration that the pellet of membranes obtained from the parotid gland during secretion is always bigger than those obtained from non-stimulated glands. This is possibly due to the increase in the surface of the acinar cells during secretion. Table 2 shows that in spite of this fact, there is an actual mobilisation of sialic acid from the parotid plasma membranes after isoproterenol, which is not seen after pilocarpine. From the same Table it is possible to conclude that concomitantly with the separation of sialic acid there is an entrance of proteins into the plasma membranes after isoproterenol. With pilocarpine the same effect can be seen, but in this case the entrance of proteins into the plasma membranes is concurrent with the entrance of sialic acid. The Effect of Isoproterenol in vitro. Isoproterenol provokes mobilisation of sialic acid from the parotid
158
R. Lopez and N. Galanti:
Table 2. Effect of isoproterenol and pilocarpine on the sialic acid content of parotid plasma membranes Factor
Table 4. The effect of two different doses of isoproterenol on the mobilisation of sialic acid from plasma membranes and on the induction of mitosis in acinar cells
Total protein of treated membranes =
~~
Total protein of control membranes
% mitosis
Condition
pg sialic
acid/mg protein
Corrected values = Specific activity x factor pg Sialic acid
pg Sialic acid ~
.___
mg Protein Control Isoproterenol Pilocarpine
8.39 f 0.4 4.10 f 0.6 7.17 f 0.2
Factor
1 1.68 1.83
pg protein (Corrected) 8.39 6.9 13.1
Isoproterenol(O.67 pmolesig body wt) or pilocarpine (0.27 pmolesig body wt) were injected intraperitoneally and the animals killed 2 h later. Plasma membranes were prepared and sialic acid extracted and determined as described under Methods. The values are the average of 7 experiments.
Table 3. The effect of isoproterenol on the sialic acid content of parotid plasma membranes in vitro Condition
pH 4.2
pH 7.0
Control Isoproterenol
7.61 f 0.04 5.97 f 0.12
7.21 f 0.16 5.63 f 0.15
Plasma membranes were prepared as described under Methods. Incubation of the membranes with or without isoproterenol was carried out at two different pH for 75 min at 37" C. After incubation, membranes were isolated at 38,000 x g for 60 min. Sialic acid was extracted and determined as described under Methods. The values are expressed as pg sialic acid per mg of protein. Buffer pH 4.2; Sodium acetate-acetic acid 0.075 M. Buffer pH 7.0: tris-HC10.080 M-ascorbic acid 0.003 M-CaCl, 0.001 M. Membranes without incubation: 7.92 pg sialic acid/mg protein.
plasma membranes when injected to the animal, in v i v a This effect is exposed to problems derived from the catabolism of isoproterenol and other changes produced by the drug in the parotid. To avoid this problem, in vitro experiments were performed. Table 3 shows the effect of isoproterenol on isolated plasma membranes from mouse parotid glands using two different pH conditions. In both, it is evident that a separation of sialic acid results from the plasma membranes. Interestingly enough, this effect is of the same order as the one observed in vivo after isoproterenol (corrected values). The Efect of Low Doses of Isoproterenol.Very low doses of isoproterenol injected intraperitoneally to the mouse provoke secretion as measured by the disappearance of alpha-amylase from the parotid gland, without inducing DNA synthesis [421.
Control Isoproterenol (0.0015 pmoles/g body wt) Isoproterenol (0.67 umoleslg body wt)
0.238 & 0.065 0.443 f 0.156 26.52 f 2.64
7.32 f 0.23 7.85 f 0.32 3.78 f 0.02
Groups of 16 mice received an intraperitoneal injection of isoprotereno1 (0.67 pmoles/g body wt or 0.0015 pmoles/g body wt). Control animals represent non-injected mice. The animals were killed 2 h later. Membranes were prepared and the sialic acid extracted and determined as described under Methods. Simultaneously, groups of 3 mice received the same treatment with the two different doses of isoproterenol. Twenty five and 34 h later the mice received an inlraperitoneal injection of colchicine (2 ug/g body wt). The animals were killed 37 h after isoproterenol, the parotid glands quickly fixed in Duboq-Brazil and included in paraffin. Five microns sections were stained with hematoxylin-eosin and mitosis counted in 5000 cells.
Table 5. Differential distribution of sialic acid in polar and nonpolar fractions from parotid plasma membrane Fractions
pg sialic acidimg protein Control Isoproterenol
Corrected values for isoproterenol results Factor A Factor B (2.45) (3.7)
Protein Upper phase Lower phase Total
1.01 f 0.52 1.37 f 0.49 4.38 & 0.18 6.76
0.93 1.47 1.32 3.72
0.38 f 0.05 0.60 2 0.04 0.54 & 0.04 1.52
1.41 2.22 2.0 5.63
Groups of 33 mice received an intraperitoneal injection of isoprotereno1 (0.67 pmoles/g body wt). The animals were killed 2 h later. Control mice represent non-injected animals. Membranes were prepared and lipids extracted as described under Methods. Three fractions were obtained. A protein fraction, retained in the filter; an upper phase containing mainly gangliosides (7,44) and a lower phase containing the less polar glycolipids (cerebrosides, glyceride glycolipids and the less polar gangliosides) (3 1). Each fraction was dried and the sialic acid extracted and determined as described under Methods. Factor A is thc average of the ratios control/isoproterenol for protein and lower phase fractions. Factor B is the ratio total protein of treated membranes/total protein of control membranes.
Table 4 shows the effect of two different doses of isoproterenol on the mobilisation of sialic acid from the parotid plasma membrane and on the percentage of mitosis in acinar cells. From the data presented, it appears that there is a correlation between the effect of the drug on the plasma membrane and the appearance of mitosis in the acinar cells of the parotid gland.
Plasma Membranes in Mouse Parotid Gland
Distribution of Sialic Acid in Polar and Nonpolar Fractions from the Plasma Membrane. It was interesting to investigate the localisation of the sialic acid containing molecule in the plasma membrane. One approach was to fractionate the membranes in polar and non polar fractions in order to localise this molecule as a periphereal or an integral part of the plasma membrane 1431. Table 5 shows that most of the sialic acid (65%) is present in the lower phase (less polar) of the Folch Extract. This result suggest that the molecule is inserted in the non-polar region of the plasma membrane. Upon stimulation with isoproterenol most of the sialic acid attached to this non-polar molecule is mobilised (see corrected values).
Discussion
It has been suggested that the control of replication and transcription may involve an interaction between two macromolecular aggregates, this is, the chromatin and cell membranes. In this hypothesis, it is postulated that an incoming molecule interacting with any one of these components will induce a conformational change leading to the expression of new abilities of the macromolecular aggregates [451. This idea has become increasingly interesting in light of the evidence that cell membranes are mostly aggregates of amphipatic molecules arranged in an interphase, within which movements of constituent macromolecules may occur [22, 431. This movement of molecules is mostly in the plane of the membrane. However, it is also possible that movement of molecules occurs through the membranes or through changes in the conformation of the molecules in a zone of the interphase. thus changing the relationships in the macromolecular aggregate. Either mechanism, or both, may be involved in the transmission of outside signals to the chromatin aggregates with the cell membrane acting as a modulator of this activity [22, 431. In recent years, it has been observed that changes occur in the cell surface of virus-transformed, and spontaneously transformed tumorigenic mouse cell lines, or tumoral cells. Of particular interest is the stimulation of cell division caused by neuraminidase [461 and the synthesis of the sialic acid during a limited time in the late G2 phase of the cell cycle of cultured human lymphoid cells t471. A summary of surface characteristics of interphase, mitotic and transformed cells is shown in the work of Mannino and Burger [481 indicating the striking similarity between the surfaces of mitotic and transformed cells.
159 The purpose of our study was to compare the chemical composition of the plasma membrane of the mouse parotid gland in a resting state, and when stimulated to synthesise DNA and divide. After the administration of isoproterenol a considerable amount of sialic acid is removed from the plasma membrane of the parotid gland cells. The importance of this effect is emphasised by our failure to separate the mobilisation of sialic acid from the plasma membrane with the stimulation of DNA synthesis that occurs 20 h later. Pilocarpine, for instance, induces secretion but very little or no increase in DNA synthesis, and is incapable of removing sialic acid from the plasma membranes. Moreover, doses of isoproterenol known to induce secretion but to have no effect on DNA synthesis are unable to separate sialic acid from the plasma membrane. Even more interesting, the same effect of isoproterenol has been demonstrated in an in vitro system. This result clearly points out the direct effect of the drug on the cell membrane. It was of great importance to locate the sialic acid that is removed by the effect of the isoproterenol. It is known that the sialic acid moiety is located on the outside of the plasma membrane. Our results show that the sialic acid is attached mainly to non-polar molecules, probably immersed in the non-polar region of the plasma membrane. The removal of sialic acid from this molecule will produce a change, not only in the molecule but in its relationship with neighbouring molecules. That is, it will induce a conformational change in the cell membrane that may translate the outside signal to citoplasmic information. This change in the plasma membrane is prior to the activation of chromatin that it is known to occur 6 h after the administration of isoproterenol [ 151. In conclusion, our results point to a direct effect of isoproterenol on the plasma membrane of the parotid gland as one of the first steps in the sequence of events leading to DNA synthesis and cell &vision. As a result of this action, part of the sialic acid is removed from the plasma membrane. This result is in agreement with our previous finding that an early increase in the synthesis of a glycolipid occurs upon stimulation of the parotid gland with isoproterenol [71. The change at the cell membrane is followed some time later by the activation of chromatin. At the present time we are working on the relationship between the removal of the sialic acid from the plasma membrane, and both its connection with other molecules in the membrane and molecules or structures in the cytoplasm adjacent to the plasma membrane,
160 Acknowledgements: The authors would like to thank the following: Mr. Jorge Sans for assistance in preparing sections and recording the mitotic indices; Dr. John Durham for his comments; Mrs. Xenia Tchernitchin for correcting the manuscript and Miss Lydia Corail for the final typing. This work was supported by Grants No. 634 and No. 1006 from the Oficina Tknica de Desarrollo Cientifico y Creacion Artistica, Universidad de Chile.
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