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Glycogen content and activities of enzymes involved in the carbohydrate metabolism of the salivary glands of rats during postnatal development
Archives of Oral Biology (2003) 48, 101—109
Glycogen content and activities of enzymes involved in the carbohydrate metabolism of the salivary glands of rats during postnatal development ´ Nicolau*, Emily Ganzerla, Douglas Nesadal de Souza Jose ˜o Paulo, Oral Biology Research Center, Faculty of Dentistry, University of Sa ˜o Paulo, Brazil Av. Prof. Lineu Prestes, 2227, 05508-900 Sa Accepted 12 September 2002
KEYWORDS Salivary glands; Development; Glycolytic enzymes
Summary Carbohydrate metabolism was examined in the developing rat salivary glands by analysing enzymatic activity and glycogen content in the postnatal parotid and submandibular glands. The following enzymes of the carbohydrate metabolism, hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), glucose-6phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) as well as the content of glycogen were determined in the salivary glands of rats aged 2, 7, 14, 21, 30 and 60 days. The specific activity of HK increased from days 2 to 21 and then it decreased up to 60 days old. The values found for the submandibular glands were from 2.5 to 4.9 times higher than those found for the parotid gland, except for rats aged 60 days. PFK-1 showed a different pattern of variation between the glands. In the submandibular gland there was a statistically significant increase in PFK-1 specific activity from 2 to 30 days of age and then, in the 60 days old group a return to level of the rats aged 2 days. In parotid gland, the specific activity of PFK-1 decreased between 2 and 7 days of age, from 7 to 14 days the specific activity increased markedly and from 14 to 60 days old it gradually decreased. The specific activity of PK followed the same pattern of variation in the submandibular and parotid glands, showing no great variation. The specific activity of LDH decreased from 2 to 60 days old in the submandibular glands. In the parotid glands the mean values for this enzyme were higher for the 2 days old group, and then decreased to remained more or less constant. The potential capacity of the pentose phosphate pathway was greater than that of glycolysis at early ages. The glycogen content showed similar variation in both glands. It was initially high and then decreased. In conclusion, our results on the activities of enzymes involved in carbohydrate metabolism in submandibular and parotid glands may be relevant to the initiation of saliva secretion in these animals. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction At birth, the salivary gland of rats are not developed and undergo progressive development into mature
Corresponding author. Tel.: þ11-3818-7842. E-mail address: [email protected] (J. Nicolau).
organs during the first weeks of life.1—3 Both the submandibular and parotid glands originate as ingrowths of cells from the epithelium on the 13—14th days of foetal life.4,5. While differentiation of the submandibular gland occurs at 18—19 days of gestation,3 cytodifferentiation of the parotid gland occurs postnatally, the acinar cells mature gradually
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(02)00165-6
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and at 25 days of age they resemble the adult acinar cells.6,7 At birth there are two main cell types in the submandibular gland, proacinar or type III cells and terminal tubule or type I cells. Type III cells differentiate into the seromucous acinar cells, and type I cells participate in the formation of the intercalated ducts.8 It has been reported that between 25 and 30 days after birth, the number of type I cells decreases by about 70%.9 At birth the parotid gland is primarily composed of terminal clusters of undifferentiated acinar cells and small ducts. Based upon morphological criteria and protein expression, parotid gland development is divided into three stages after birth, neonatal (from 1 to 9 days), transitional (from 9 to 25 days) and adult (from 25 days onwards).10 Functional development of rodent salivary glands involves changes in the content or activity of various enzymes;11—13 changes in secretory components;14,15 increases in autonomic receptors16 and in the ability to secrete the fluid portion of saliva.17,18 At birth, in the submandibular salivary glands, muscarinic receptors are present, responsive and increase during glandular maturation.19—21 In immature glands, adenylate cyclase activity reaches adult levels at 18 days of gestation12 and becomes progressively responsive to b-adrenergic agonists between 1 and 6 days after birth.22 In 1 day old rats the muscarinic cholinergic receptor density is low and reaches adult levels by 3 weeks of age.16 However, in an in vivo experiment no secretion of fluid was obtained during a 1 h collection period in rats, until approximately 2 weeks after birth.18 In the parotid gland, there is no secretory activity in response to stimulation of cholinergic receptors during the first 2 weeks after birth13 correlating with a lack of responsiveness to b-adrenergic stimuli.13,23,24 It has been found by histochemical techniques that neonatal canine salivary glands contain a large quantity of glycogen, but most of this disappears as the gland differentiates.25 Glycogen or phosphorylase, the enzyme that degrades the polysaccharide, has been detected in acini, striated ducts and myoepithelium cells.26—29 Considering that all the functions carried out by cells or organs are energy-dependent the purpose of the present study was to examine the glycogen content and the activities of some enzymes of the carbohydrate metabolism during the postnatal development of salivary glands.
Materials and methods Wistar rats aged 2, 7, 14, 21, 30 and 60 days were used in the present investigation. The animals were killed by a blow to the head, always in the morning
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(9:00—11:00 a.m.). To ensure that the parotid samples from rats of 2 and 7 days old were representative of this gland they were removed under a stereoscopic microscope. The submandibular glands were separated from the sublingual glands. Once removed the glands were immediately clamped between aluminium tongs, pre-cooled in dry ice and maintained at 80 8C until analysis. The frozen glands were homogenised in 9 their volume of 50 mmol/l imidazol buffer pH 7.0, containing 2 mmol/l EDTA and 1 mmol/l 2-mercaptoethanol, in a glass homogeniser with a Teflon pestle. After centrifugation of the homogenate at 12,100 g for 20 min, the supernatant was used for enzymatic and protein determinations. For animals aged 2 and 7 days several glands had to be pooled to obtain adequate samples. Enzyme activities of the various supernatants were assayed by measuring changes in absorption at 340 nm due to the reduction of NADP or oxidation of NADH. The reaction was initiated by adding portions of the homogenate supernatant to the reaction mixture in the cuvette and absorbance was monitored for 10 min at 30 8C in a Beckman DU-68 spectrophotometer, using a 1 ml cuvette with 1 cm path length. One unit of enzyme activity corresponds to the amount of enzyme that converts 1 mmol of substrate per min; specific activity is expressed in U/mg of protein. Enzyme activities were measured as follows: hexokinase (HK) in a reaction mixture containing 50 mmol/l imidazol buffer pH 7.2, 5 mmol/l MgCl2, 5 mmol/l ATP, 1 mmol/l glucose, 0.4 mmol/l NADP and 0.3 U glucose-6-phosphate dehydrogenase;30 phosphofructokinase-1 (PFK-1) in a medium containing 50 mmol/l triethanolamine (TEA) buffer pH 7.0, 2 mmol/l EDTA, 0.5 mmol/l fructose-6phosphate, 0.65 mmol/l NADH, 0.4 U aldolase, 0.08 U glycerophosphate dehydrogenase and 0.08 U triosephosphate isomerase;31 pyruvate kinase (PK), in a medium containing 50 mmol/l imidazol buffer pH 7.2, 5 mmol/l MgCl2, 0.1 mol/l KCl, 1 mmol/l ADP, 0.15 mmol/l NADH, 2 mmol/l phosphoenolpyruvate and 0.9 U lactate dehydrogenase;32 lactate dehydrogenase (LDH), in a reaction mixture containing 48 mmol/l phosphate buffer pH 7.5, 0.6 mmol/l pyruvate and 0.18 mmol/l NADH;33 glucose-6-phosphate dehydrogenase (G6PD) in a reaction medium containing 50 mmol/l glycylglycine buffer pH 7.6, 2.3 mol/l MgCl2, 0.1 mmol/l NADP and 1.33 mmol/l glucose-6-phosphate.34 Protein was estimated in the supernatant fraction by the method of Lowry et al.35 with bovine serum albumin as standard. Statistical analysis: Data are presented as mean S:D. Statistical significance of experimental
Activities of hexokinase (HK) and phosphofructokinase-1 (PFK-1) in the submandibular and parotid glands of rats at different ages.
Age (days)
Submandibular
Parotid
HK
2 7 14 21 30 60
PFK 1
HK
PFK 1
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
1.628 (12) a 2.478 (16) 3.431 (9) b 5.002 (6) 3.168 (21) b 1.010 (12) a
0.801 0.193 (12) a 1.251 0.318 (16) ab 1.290 0.176(7) ac 1.067 0.550 (7) ad 2.010 0.763 (17) 1.522 0.180 (18) bcd
0.580 (7) a 1.028 (10) b 1.311 (8) bcd 1.680 (6) c 1.705 (18) cd 0.755 (12) a
0.477 (7) 1.025 (10) a 1.290 (7) ab 2.238 (4) c 1.724 (19) bc 1.182 (12) ab
0.540 (18) a 0.945 (12) ab 1.387 (21) c 1.015 (16) b 0.709 (8) ab 1.430 (12) c
0.294 (18) a 0.443 (12) ab 0.718 (22) b 1.488 (17) 0.672 (12) b 0.814 (12) b
0.534 (18) a 0.135 (6) a 4.290 (16) 2.720 (12) 1.730 (9) 1.282 (10) a
0.268 (18) a 0.138 (6) a 4.508 (16) b 4.310 (12) b 2.173 (12) 0.753 (10)
0.451 0.899 0.619 0.670 0.503 0.407
0.223 0.144 0.337 0.361 0.550 0.264
0.029 0.257 0.428 0.434 0.405 0.330
0.160 0.477 0.274 0.343 0.278 0.568
0.058 0.141 0.151 0.771 0.259 0.205
0.176 0.031 1.185 0.905 0.444 0.420
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
0.089 0.033 0.981 1.120 0.539 0.253
Glycogen content and activities of enzymes involved in the carbohydrate metabolism
Table 1
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Table 2 Activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the submandibular and parotid glands of rats at different ages. Age (days)
Submandibular
Parotid
PK
2 7 14 21 30 60
LDH
LDH
PK
U/mg protein
U/g tissue
U/mg protein
U/g tissue
U/mg protein
U/g tissue
U/mg protein
U/g tissue
0.236 (10) a 0.211 (10) b 0.313 (10) a 0.330 (9) a 0.263 (7) a 0.315 (12) a
9.15 (10) a 11.85 (10) ab 14.26 (10) bc 19.87 (9) cd 18.78 (7) d 11.20 (12) ab
0.710 (10) 0.392 (9) a 0.365 (10) a 0.309 (10) a 0.195 (16) 0.069 (12)
18.12 (12) a 13.80 (16) a 19.11 (10) a 19.28 (9) a 17.65 (19) a 8.02 (9) b
0.231 (17) a 0.132 (12) b 0.203 (6) ab 0.217 (7) a 0.137 (11) b 0.310 (12)
11.53 (17) a 6.55 (12) b 10.74 (6) ac 13.90 (11) ac 8.20 (11) b 11.24 (12)
0.521 (18) 0.174 (12) a 0.352 (17) b 0.152 (10) a 0.348 (11) b 0.299 (12) b
25.8 8.86 (18) a 8.30 1.97 (12) 18.47 4.22 (17) b 14.82 4.04 (12) b 21.12 3.51 (11) ab 18.03 1.84 (12) b
0.016 0.014 0.178 0.024 0.047 0.110
1.22 2.24 2.40 1.59 3.40 3.33
0.12 0.08 0.08 0.10 0.06 0.025
8.96 8.10 2.90 4.42 3.86 1.18
0.05 0.03 0.05 0.04 0.01 0.11
1.73 0.66 2.87 4.32 0.60 2.79
0.188 0.072 0.083 0.024 0.069 0.086
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
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Glycogen content and activities of enzymes involved in the carbohydrate metabolism
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activity from 2 to 14 days although it was not statistically significant. The values found for rats aged 30 and 60 days were higher than in the preceding groups, although from 30 to 60 days there was a decrease in activity. In the parotid glands the specific activity of this enzyme also showed a tendency to increase from 2 to 14 days old, then decrease from 21 to 30 days old; whilst the value for the 60 days old group was statistically greater than in the other groups. Comparing the specific activity of hexokinase obtained in the two glands, the values for the submandibular were from 2.5 to 4.9 times higher than those found in the parotid gland for the 2 to 30 days old groups. However, in the 60 days old rats the mean value for the submandibular was lower than the mean value for the parotid glands. In terms of the activity per g of tissue, the mean value in the 21 days old group was higher than that in the other groups. The values for the submandibular glands were still higher than those in the parotid glands when compared as activity per gram of tissue, but the differences were smaller. Phosphofructokinase-1 showed a different
observations was determined by analysis of variance with the process of multiple comparison of Tukey. The level of significance was set at P < 0:05.
Results In the present work we did not separate results from male from female animals. It has been reported that no differences in the development of the salivary glands were observed until day 60 after birth,36 and data on cell cycle, labelling and mitotic indexes show no significant differences between sexes.37 The results of our investigation are expressed in Tables 1—5. Table 1 shows the data for hexokinase and phosphofructokinase-1 in the submandibular and parotid glands of rats aged 2—60 days. The specific activity of hexokinase in submandibular glands increased from days 2 to 21 then decreased up to 60 days; there was no difference between the values at this age and those in 2 days old rats. Concerning the results expressed as activity per g of tissue, there is a tendency for an increase in
Table 3 Activity of glucose-6-phosphate dehydrogenase (G6PD) in submandibular and parotid glands of rats at different ages. Age (days)
G6PD Submandibular
Parotid 2
U/mg protein, 10 2 7 14 21 30 60
1.757 2.497 2.933 2.892 2.616 2.130
0.316 0.672 0.288 0.630 0.245 0.768
(9) a (10) b (13) b (13) b (16) b (12) ab
U/mg protein, 102
U/g tissue 0.666 1.778 1.244 1.190 2.021 0.56
0.106 0.129 0.261 0.300 0.335 0.158
(9) (10) a (10) a (13) a (21) (12)
0.957 1.178 2.511 2.190 0.828 1.000
0.271 0.559 0.805 0.705 0.230 0.370
(18) a (12) ab (8) c (16) bc (12) a (12) a
U/g tissue 0.478 0.560 1.336 1.429 0.592 0.596
0.108 0.158 0.389 0.317 0.114 0.152
(18) a (12) a (8) b (23) b (12) a (12) a
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05). Table 4 Data of the activities of phosphofructokinase-1 (PFK-1) and glucose-6-phosphate dehydrogenase (G6PD) to show the ratio between the two enzymes which give the potential capacity of the pentose-phosphate pathway and glycolysis. Age (days)
2 7 14 21 30 60
Submandibular
Parotid
PFK1
G6PD
U/mg protein, 102
U/mg protein, 102
0.58 1.03 1.31 1.68 1.70 0.75
1.76 2.50 2.93 2.90 2.62 2.13
PFK1
G6PD
G6PD/PFK-1
U/mg protein, 102
U/mg protein, 102
G6PD/PFK-1
3.0 2.4 2.2 1.7 1.5 2.8
0.53 0.13 4.29 2.72 1.73 1.28
0.96 1.17 2.51 2.19 0.83 1.00
1.8 9.0 0.6 0.8 0.5 0.8
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Table 5 The content of glycogen in the submandibular and parotid glands of rats at different ages. Age (days)
Submandibular (mg glycogen/mg tissue)
2 7 14 21 30 60
(10) (43) (21) (22) (20) (15)
2.747 2.369 1.649 1.020 0.772 0.349
0.761 0.645 0.451 0.360 0.202 0.051
a a
Parotid (mg glycogen/mg tissue) (12) (12) (10) (12) (14) (9)
b bc c
0.974 0.964 0.459 0.470 0.397 0.361
0.251 0.196 0.071 0.154 0.107 0.140
a a b b b b
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
pattern of variation in the two glands. In submandibular glands the specific activity increased from 2nd to 30th day after birth. Although the value of the 60 days group was higher than that of 2 days, the difference was not statistically significant. In the parotid gland the specific activity of phosphofructokinase-1 decreased between 2 and 7 days of age, increased in the 14 days old group and then decreased until 60 days of age. The specific activities and activities per gram of tissue of pyruvate kinase and lactate dehydrogenase from the submandibular and parotid glands are shown in Table 2. In the submandibular glands, except in the 7 days old rats, the specific activity of pyruvate kinase was similar in all the age groups. The specific activity of lactate dehydrogenase in submandibular glands increased from 2 to 7 days old rats and decreased again in the 30 and 60 days old groups. In the parotid glands the specific activity of pyruvate kinase remained similar in the 2, 14 and 21 days old groups. The values in the 60 days old group were higher than those in younger groups. There was a biphasic pattern of lactate dehydrogenase expression with higher values at 2 days of age, and then decrease to a subsequent plateau up to 60 days of age. Table 3 shows the specific activity and the activity per gram of tissue of glucose-6-phosphate dehydrogenase. In the submandibular glands the specific activity of this enzyme showed a significant increase from the 2nd to the 14th day but thereafter showed no significant differences between ages. In the parotid glands no difference in glucose-6-phosphate dehydrogenase activities were observed at 2, 7, 30 and 60 days of age. The potential capacity of the pentose phosphate and glycolytic pathways as indicated by the ratio between the activities of glucose-6-phosphate dehydrogenase and phosphofructokinase-1 is shown in Table 4. In the submandibular glands the highest mean values were found in glands from 2 days old rats. After this age there was a decrease in the ratio until 30 days of age. There was, however,
an increase in the 60 days age group. In the parotid glands there was a peak in the ratio at 7 days suggesting a predominance of the pentose phosphate pathway over glycolysis. The glycogen content of both glands is shown in Table 5. The pattern was similar for both glands, the content being higher in the 2 days old rats and then decreasing up to 60 days of age.
Discussion The data from this study must be considered in the context of the changes in metabolism that occurs between the perinatal and postnatal period. The rat foetus has a plentiful supply of glucose from the maternal blood via the placenta. Hence, in the rat foetus the main source of energy is carbohydrate. For at least the first weeks after birth, the rat appears to require a liquid diet rich in fat and protein and relatively poor in carbohydrate.38 In fact, the reported milk composition gives a concentration for lactose that varies from 2.5 to 3.8%39,40 and for lipids a concentration between 16.8 and 20.7%.40 Milk consumption by the pups increases progressively to 18 days, then declines until weaning.41 In our experiments, weaning occurred at 21 days of age, so that there were four suckling groups (2, 7, 14 and 21 days after birth) and two groups on solid food (30 and 60 days after birth). According to Thomas and Fell,42 for a change in activity of any enzyme to cause a change in metabolic flux, there has to be a consequent change in concentration of one or more metabolites in the pathways and any enzyme will become rate-limiting if it is inhibited sufficiently. According to these authors, this is a mechanism by which the signal (change in enzyme activity), is propagated to cause a change in flux. The specific activity of hexokinase was higher in the submandibular glands than that in parotid glands. In muscle, according to Fell43 this enzyme is not considered as part of the glycolytic pathway,
Glycogen content and activities of enzymes involved in the carbohydrate metabolism
because the substrate of muscle glycolysis is glycogen, and hexokinase activity is related to glycogen synthesis. By analogy, if we consider this in the salivary glands, it is possible that the specific activity of hexokinase in the submandibular glands being higher than in the parotid glands was related to the higher glycogen content in the submandibular glands. The specific activity of phosphofructokinase-1 showed some interesting variations. Although the submandibular and parotid glands of 2 days old rats are in different stages of development no difference was observed in the specific activity of this enzyme. In the submandibular glands at this age the majority of cells belong to two categories, type III and type I whilst in the parotid glands, the terminal cluster form typical acini and their cells undergo cytodifferentiation.10 However, the mean values found for the parotid glands in 7 days old were approximately eight times lower than the mean values found for the submandibular glands, and at this age the glands are still in the neonatal period.10 Despite this difference for the 7 days old animals, from 14 days old onwards the mean values are higher for the parotid glands. The differences observed between the submandibular and parotid glands are understandable, because they are in different stages of maturation. Further, whilst in submandibular glands anaerobic metabolism is predominant, in the parotid gland aerobic metabolism predominates.44 Although the maturation of the acinar cells of the submandibular gland precedes that in the parotid, no measurable secretory response through a ductal cannula was observed earlier than 2 weeks of age with pilocarpine or 3 weeks of age with isoproterenol.18 Looking at the several enzymes involved in a pathway, mainly in the 7 and 14 days old rats, we observed that, despite the increase in the specific activities of hexokinase and phosphofructokinase-1 in the submandibular glands of the 7 days old rats, the specific activities of phosphofructokinase and lactate dehydrogenase decreased. In 14 days old rats changes in pyruvate kinase activity were similar to those in hexokinase and phosphofructokinase-1 activity. On the other hand, although the specific activity of hexokinase had increased in the parotid glands of 7 days old rats, the specific activity of phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase decreased. In these glands, all the enzymes studied showed increased specific activities in 14 days old animals. However, in interpreting the results of our work we must consider that at birth the suckling rat turns to a diet of milk. During this early postnatal period the specific activities of hexokinase and phosphofructokinase-1 in the sub-
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mandibular glands increased up to 21 days of age. At this age the values were higher than those preceding, and this coincides with the end of lactation and the switch to solid food. It is interesting that in the parotid glands the higher values for the specific activities of hexokinase and phosphofructokinase1 appeared at 14 days of age, during suckling period. However, at weaning (21 days of age) the specific activity of hexokinase was higher than at 2 days but lower than at 14 days. On the other hand, an eightfold increase in the activity of phosphofructokinase1 was noted in the parotid glands of 14 days old group, and five-fold increase in the 21 days old group as compared with the group 2 days after birth. Between 8 and 15 days of age the prolongation of the rat parotid acinar cell cycle is reported to be primarily due to an increase in the duration of the G-1 phase.45 During G-1 phase there is considerable synthesis of enzymes involved in nucleotide metabolism46 which may explain our findings, as synthetic processes are energy-dependent. The product of the action of hexokinase, glucose6-phosphate, is an intermediate in three pathways– —namely glycolysis, synthesis of glycogen and the pentose phosphate pathway. According to Fell43 hexokinase does not change in concert with the glycolytic enzymes, and in muscle it changes activity in the opposite direction to the other enzymes.47 In the present work, hexokinase activity changed in the same direction as phosphofructokinase-1 and glucose-6-phosphate dehydrogenase in the submandibular glands, but in the parotid glands this occurred only with glucose-6-phosphate dehydrogenase and not with phosphofructokinase-1. The treatment of Shonk and Boxer48 for the potential capacity of the pentose phosphate and glycolytic pathways, demonstrated by the ratio of glucose-6-phosphate dehydrogenase and phosphfructokinase-1 activities, give us interesting information. While phosphofructokinase-1 is the key enzyme in glycolysis, glucose-6-phosphate dehydrogenase is the first enzyme in the pentosephosphate-pathway. This enzyme supplies NADPH, which is important in the reducing processes of biosynthesis of ribose-5-phosphate constituent of nucleotides. For the submandibular gland at 2 days old, the ratio (3.0) favoured the pentose phosphate pathway, but as the age of the animals increased this ratio became smaller, indicating increased glycolytic activity. In the parotid glands, it is noticeable that at 7 days of age the ratio (9.0) is markedly in favour of the pentose-phosphate pathway. However, from 14 days on the ratio favours glycolysis. It should be point out that the glands are at different stages of development at the time when the animals were killed, and that no secretion of saliva has been
108 observed before day 14.18,24 Considering that all functions of a cell are energy dependent, we may speculate, mainly in the relation to the parotid gland, that up to 7 days after birth the metabolism of the cells are particularly directed towards biosynthetic processes. The glycogen content in the submandibular glands was greater than that in the parotid glands, and in both glands it decreased with increasing age. This decrease in animals from 2 to 60 days old seems to agree with the reported for the liver.49 They showed that at birth the foetal rat liver accumulates large amounts of glycogen. It is suggested that the accumulated polysaccharides are utilised after birth to sustain the animal until feeding is established.50,51 This suggestion is understandable, as the liver is an homeostatic organ. Our results on glycogen concentration agree with those reported for dog salivary glands.25 It is possible that the glycogen concentration in the glands might vary during the 2 h period over which different animals were killed. However, preliminary results from our laboratory showed no differences in glandular polysaccharide concentration between rats killed at 9:00 a.m. and those killed at 11:00 a.m. We believe that the high content of glycogen in the glands during the first 2 weeks after birth provides a reserve to enable the glands to mature fully. In conclusion, this study shows differences in the specific activity of some enzymes involved in carbohydrate metabolism and in glycogen concentrations in developing salivary glands. It is possible that these differences may have some influence on the process of salivary secretion in baby rats.
Acknowledgements This work was supported by a grant from Fundac¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP). JN is a recipient of a fellowship from Conselho Nacional de Cie ˆncia e Tecnologia (CNPq).
References 1. Jacoby F, Leeson CR. The postnatal development of the rat submaxillary gland. J Anat 1959;93:201—216. 2. Kim SK, Han SS, Nasjleti CE. The fine structure of secretory granules in submandibular gland of the rat during early postnatal development. Anat Rec 1970;168:463—476. 3. Cutler LS, Chaudhy AP. Cytodiferentiation of the striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland. Am J Anat 1975;143:201—217. 4. Redman RS. Development of the salivary glands. In: Sreebny LM, editor. The salivary system. Boca Raton: CRC Press; 1987. p. 1—20.
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5. Redman RS, Sreebny LM. Prenatal phase of morphosis of the rat parotid gland. Anat Rec 1970;168:127—138. 6. Redman RS, Sreebny LM. Morphologic and biochemical observations on the development of the rat parotid gland. Dev Biol 1971;25:248—279. 7. Taga R, Sesso A. Ultrastructural studies on developing parotid gland of the rat at early postnatal periods. Arch Histol Jpn 1979;42:427—444. 8. Denny PC, Ball WD, Redman RS. Salivary glands: a paradigm for diversity of gland development. Crit Rev Oral Biol Med 1997;8:51—75. 9. Hecht R, Connelly M, Marchetti L, Ball WD, Hand AR. Cell death during development of intercalated ducts in the rat submandibular gland. Anat Rec 2000;258:349—358. 10. Sivakumar S, Mirels L, Miranda AJ, Hand AR. Secretory protein expression patterns during rat parotid gland development. Anat Rec 1998;252:485—497. 11. Barka T, Van der Noen H. Adenylate cyclase activity in rat submandibular gland during postnatal development. Life Sci 1974;14:267—280. 12. Cutler LS, Rodan SB. Biochemical and cytochemical studies on adenylate cyclase activity in the developing rat submandibular gland: differentiation of the acinar secretory compartment. J Embryol Exp Morphol 1976;36:291—303. 13. Grand RJ, Schay MI. Development of secretory function in rat parotid gland. Pediatr Res 1978;12:100—104. 14. Cutler LS, Chaudhry AP. Cytodifferentiation of the acinar cells of the rat submandibular gland. Dev Biol 1974;41:31—41. 15. Fava-de-Moraes F, Nicolau J. Aging effects on sialic acid concentration of submandibular-sublingual glands of rats. Naturwissenschaften 1966;53:711—712. 16. Bylund DB, Martinez JR, Camden J, Jones SB. Autonomic receptors in the developing submandibular glands of neonatal rats. Arch Oral Biol 1982;27:945—950. 17. Schneyer CA, Schneyer HD. Secretion by salivary glands deficient in acini. Am J Physiol 1961;201:939—942. 18. Martinez JR, Camden J. Volume and composition of pilocarpine and isoproterenol stimulated submandibular saliva of early postnatal rats. J Dent Res 1983;62:543—547. 19. Martinez JR, Camden J. In vitro release of Kþ from the developing submandibular gland of early postnatal rats. Arch Oral Biol 1982;27:939—944. 20. Martinez JR, Camden J. 36Cl fluxes in dispersed cell aggregates of the submandibular gland of early postnatal rats. Arch Oral Biol 1987;32:659—663. 21. Martinez JR, Camden J. C2þ mobilization and Cl efflux in submandibular salivary cells of adult and newborn rats. Arch Oral Biol 1989;34:147—152. 22. Cutler LS, Christian CP, Boltaro B. Development of stimulus—secretion coupling in salivary glands. In: Hand AR, Oliver C, editors. Methods of cell biology. New York: Academic Press; 1981. p. 23—51. 23. Grand RJ, Chong DA, Ryan SJ. Postnatal development of adenylate cyclase in rat salivary glands: patterns of hormonal sensitivity. Am J Physiol 1975;228:608—612. 24. Ludford JM, Talamo BR. Independent regulation of badrenergic receptor and nucleotide binding proteins of adenylate cyclase. J Biol Chem 1983;258:4831—4838. 25. Reifel CW, Travill AA. Structure and carbohydrate histochemistry of postnatal canine salivary glands. Am J Anat 1972;134:377—393. 26. Bolande RP, Towler WF. Terminal autonomic nervous system in cystic fibrosis. Arch Pathol 1973;95:172—177. 27. Compton JS, Denniss AR, Hung P, Kennerson AR, van Lennep ´. The effect of sodium deficient diet on the EW, Young JA striated ducts on the rabbit mandibular gland. Proc Aust Physiol Pharmacol Soc 1975;6:132—133.
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28. Ohanian C. Histochemical studies on phosphorylase activity in the tissues of the albino rat under normal and experimental conditions. Acta Anat 1973;86:15—22. 29. Redman RS. Myoepithelium of salivary glands. Microsc Res Tech 1994;27:25—45. 30. Uyeda K, Racker E. Regulatory mechanisms in carbohydrate metabolism. VII. Hexokinase and phosphofructokinase. J Biol Chem 1965;240:4682—4688. 31. Mansour TE. Studies on heart phosphofructokinase: purification, inhibition and activation. J Biol Chem 1963;238:2285— 2292. 32. Llorente P, Marco R, Sols A. Regulation of liver pyruvate kinase and phosphoenol pyruvate cross roads. Eur J Biochem 1970;13:45—54. 33. Bergmeyer HU, Bernt E. Lactate dehydrogenase. In: Bermeyer HU, editor. Methods of enzymatic analysis. New York: Verlag-Chemie; 1974. p. 579—582. 34. Eggleston LV, Krebs HA. Regulation of the pentose phosphate cycle. Biochem J 1974;138:425—435. 35. Lowry OH, Rosebrough NS, Farr AL, Randall R. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:265—275. 36. Schneyer CA, Hall DH. Growth pattern of postnatally developing rat parotid gland. Proc Soc Exp Biol Med 1969;130:603—607. 37. Klein RM, Harrington DB. Acinar cell cycle of developing rat parotid gland: comparison of males and females. J Dent Res 1976;55:712. 38. Dymsza HA, Czajka DM, Miller SA. Influence of artificial diet on weight gain and body composition of the neonatal rat. J Nutr 1964;84:100—106. 39. Passos MCF, Ramos CF, Dutra SCP, Mouc¸o T, Moura EG. Long term effects of malnutrition during lactation on the thyroid function of offspring. Horm Metab Res 2002;34:40—43.
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40. Grigor MR, Allan J, Carne A, Carrington JM, Geursen A. Milk composition of rats feeding restricted litters. Biochem J 1986;233:917—919. 41. Redman RS, Sweney LR. Changes in diet and patterns of feeding activity of developing rats. J Nutr 1976;106:615— 626. 42. Thomas S, Fell DA. The role of multiple enzyme activation in metabolic flux control. Adv Enzyme Regul 1998;38:65—85. 43. Fell DA. Signal transduction and the control of expression of enzyme activity. Adv Enzyme Regul 2000;40:35—46. 44. Nicolau J, Sassaki KT. Metabolism of carbohydrate in the major salivary glands of rats. Arch Oral Biol 1976;21: 659—661. 45. Klein RM. Alteration of neonatal rat parotid gland acinar cell proliferation by guanethidine-induced sympathectomy. Cell Tissue Kinet 1979;12:411—423. 46. Cory JG. Purine and pyrimidine nucleotide metabolism. In: Devlin TM, editor. Textbook of biochemistry with clinical correlations. New York: Wiley; 1986. p. 489—529. 47. Henriksson J, Chi MMY, Hintz CS, Young DA, Kaiser KK, Salmons S, et al. Chronic stimulation of mammalian muscle. Changes in enzymes of six metabolic pathways. Am J Physiol 1986;251:C614—C632. 48. Shonk CE, Boxer GE. Enzyme pattern in human tissues. I. Methods for the determination of glycolytic enzymes. Cancer Res 1964;24:709—721. 49. Snell K, Walker DG. Glucose metabolism in the newborn rat. Temporal studies in vivo. Biochem J 1973;132:739— 752. 50. Shelley HJ. Glycogen reserves and their changes at birth and in anoxia. Br Med Bull 1961;17:137—143. 51. Greengard O, Dewey HK. The premature deposition or lysis of glycogen in livers of foetal rats injected with hydrocortisone or glucagon. Dev Biol 1970;21:452—461.
Glycogen content and activities of enzymes involved in the carbohydrate metabolism of the salivary glands of rats during postnatal development ´ Nicolau*, Emily Ganzerla, Douglas Nesadal de Souza Jose ˜o Paulo, Oral Biology Research Center, Faculty of Dentistry, University of Sa ˜o Paulo, Brazil Av. Prof. Lineu Prestes, 2227, 05508-900 Sa Accepted 12 September 2002
KEYWORDS Salivary glands; Development; Glycolytic enzymes
Summary Carbohydrate metabolism was examined in the developing rat salivary glands by analysing enzymatic activity and glycogen content in the postnatal parotid and submandibular glands. The following enzymes of the carbohydrate metabolism, hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), glucose-6phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) as well as the content of glycogen were determined in the salivary glands of rats aged 2, 7, 14, 21, 30 and 60 days. The specific activity of HK increased from days 2 to 21 and then it decreased up to 60 days old. The values found for the submandibular glands were from 2.5 to 4.9 times higher than those found for the parotid gland, except for rats aged 60 days. PFK-1 showed a different pattern of variation between the glands. In the submandibular gland there was a statistically significant increase in PFK-1 specific activity from 2 to 30 days of age and then, in the 60 days old group a return to level of the rats aged 2 days. In parotid gland, the specific activity of PFK-1 decreased between 2 and 7 days of age, from 7 to 14 days the specific activity increased markedly and from 14 to 60 days old it gradually decreased. The specific activity of PK followed the same pattern of variation in the submandibular and parotid glands, showing no great variation. The specific activity of LDH decreased from 2 to 60 days old in the submandibular glands. In the parotid glands the mean values for this enzyme were higher for the 2 days old group, and then decreased to remained more or less constant. The potential capacity of the pentose phosphate pathway was greater than that of glycolysis at early ages. The glycogen content showed similar variation in both glands. It was initially high and then decreased. In conclusion, our results on the activities of enzymes involved in carbohydrate metabolism in submandibular and parotid glands may be relevant to the initiation of saliva secretion in these animals. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction At birth, the salivary gland of rats are not developed and undergo progressive development into mature
Corresponding author. Tel.: þ11-3818-7842. E-mail address: [email protected] (J. Nicolau).
organs during the first weeks of life.1—3 Both the submandibular and parotid glands originate as ingrowths of cells from the epithelium on the 13—14th days of foetal life.4,5. While differentiation of the submandibular gland occurs at 18—19 days of gestation,3 cytodifferentiation of the parotid gland occurs postnatally, the acinar cells mature gradually
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(02)00165-6
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and at 25 days of age they resemble the adult acinar cells.6,7 At birth there are two main cell types in the submandibular gland, proacinar or type III cells and terminal tubule or type I cells. Type III cells differentiate into the seromucous acinar cells, and type I cells participate in the formation of the intercalated ducts.8 It has been reported that between 25 and 30 days after birth, the number of type I cells decreases by about 70%.9 At birth the parotid gland is primarily composed of terminal clusters of undifferentiated acinar cells and small ducts. Based upon morphological criteria and protein expression, parotid gland development is divided into three stages after birth, neonatal (from 1 to 9 days), transitional (from 9 to 25 days) and adult (from 25 days onwards).10 Functional development of rodent salivary glands involves changes in the content or activity of various enzymes;11—13 changes in secretory components;14,15 increases in autonomic receptors16 and in the ability to secrete the fluid portion of saliva.17,18 At birth, in the submandibular salivary glands, muscarinic receptors are present, responsive and increase during glandular maturation.19—21 In immature glands, adenylate cyclase activity reaches adult levels at 18 days of gestation12 and becomes progressively responsive to b-adrenergic agonists between 1 and 6 days after birth.22 In 1 day old rats the muscarinic cholinergic receptor density is low and reaches adult levels by 3 weeks of age.16 However, in an in vivo experiment no secretion of fluid was obtained during a 1 h collection period in rats, until approximately 2 weeks after birth.18 In the parotid gland, there is no secretory activity in response to stimulation of cholinergic receptors during the first 2 weeks after birth13 correlating with a lack of responsiveness to b-adrenergic stimuli.13,23,24 It has been found by histochemical techniques that neonatal canine salivary glands contain a large quantity of glycogen, but most of this disappears as the gland differentiates.25 Glycogen or phosphorylase, the enzyme that degrades the polysaccharide, has been detected in acini, striated ducts and myoepithelium cells.26—29 Considering that all the functions carried out by cells or organs are energy-dependent the purpose of the present study was to examine the glycogen content and the activities of some enzymes of the carbohydrate metabolism during the postnatal development of salivary glands.
Materials and methods Wistar rats aged 2, 7, 14, 21, 30 and 60 days were used in the present investigation. The animals were killed by a blow to the head, always in the morning
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(9:00—11:00 a.m.). To ensure that the parotid samples from rats of 2 and 7 days old were representative of this gland they were removed under a stereoscopic microscope. The submandibular glands were separated from the sublingual glands. Once removed the glands were immediately clamped between aluminium tongs, pre-cooled in dry ice and maintained at 80 8C until analysis. The frozen glands were homogenised in 9 their volume of 50 mmol/l imidazol buffer pH 7.0, containing 2 mmol/l EDTA and 1 mmol/l 2-mercaptoethanol, in a glass homogeniser with a Teflon pestle. After centrifugation of the homogenate at 12,100 g for 20 min, the supernatant was used for enzymatic and protein determinations. For animals aged 2 and 7 days several glands had to be pooled to obtain adequate samples. Enzyme activities of the various supernatants were assayed by measuring changes in absorption at 340 nm due to the reduction of NADP or oxidation of NADH. The reaction was initiated by adding portions of the homogenate supernatant to the reaction mixture in the cuvette and absorbance was monitored for 10 min at 30 8C in a Beckman DU-68 spectrophotometer, using a 1 ml cuvette with 1 cm path length. One unit of enzyme activity corresponds to the amount of enzyme that converts 1 mmol of substrate per min; specific activity is expressed in U/mg of protein. Enzyme activities were measured as follows: hexokinase (HK) in a reaction mixture containing 50 mmol/l imidazol buffer pH 7.2, 5 mmol/l MgCl2, 5 mmol/l ATP, 1 mmol/l glucose, 0.4 mmol/l NADP and 0.3 U glucose-6-phosphate dehydrogenase;30 phosphofructokinase-1 (PFK-1) in a medium containing 50 mmol/l triethanolamine (TEA) buffer pH 7.0, 2 mmol/l EDTA, 0.5 mmol/l fructose-6phosphate, 0.65 mmol/l NADH, 0.4 U aldolase, 0.08 U glycerophosphate dehydrogenase and 0.08 U triosephosphate isomerase;31 pyruvate kinase (PK), in a medium containing 50 mmol/l imidazol buffer pH 7.2, 5 mmol/l MgCl2, 0.1 mol/l KCl, 1 mmol/l ADP, 0.15 mmol/l NADH, 2 mmol/l phosphoenolpyruvate and 0.9 U lactate dehydrogenase;32 lactate dehydrogenase (LDH), in a reaction mixture containing 48 mmol/l phosphate buffer pH 7.5, 0.6 mmol/l pyruvate and 0.18 mmol/l NADH;33 glucose-6-phosphate dehydrogenase (G6PD) in a reaction medium containing 50 mmol/l glycylglycine buffer pH 7.6, 2.3 mol/l MgCl2, 0.1 mmol/l NADP and 1.33 mmol/l glucose-6-phosphate.34 Protein was estimated in the supernatant fraction by the method of Lowry et al.35 with bovine serum albumin as standard. Statistical analysis: Data are presented as mean S:D. Statistical significance of experimental
Activities of hexokinase (HK) and phosphofructokinase-1 (PFK-1) in the submandibular and parotid glands of rats at different ages.
Age (days)
Submandibular
Parotid
HK
2 7 14 21 30 60
PFK 1
HK
PFK 1
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
U/mg protein, 102
U/g tissue
1.628 (12) a 2.478 (16) 3.431 (9) b 5.002 (6) 3.168 (21) b 1.010 (12) a
0.801 0.193 (12) a 1.251 0.318 (16) ab 1.290 0.176(7) ac 1.067 0.550 (7) ad 2.010 0.763 (17) 1.522 0.180 (18) bcd
0.580 (7) a 1.028 (10) b 1.311 (8) bcd 1.680 (6) c 1.705 (18) cd 0.755 (12) a
0.477 (7) 1.025 (10) a 1.290 (7) ab 2.238 (4) c 1.724 (19) bc 1.182 (12) ab
0.540 (18) a 0.945 (12) ab 1.387 (21) c 1.015 (16) b 0.709 (8) ab 1.430 (12) c
0.294 (18) a 0.443 (12) ab 0.718 (22) b 1.488 (17) 0.672 (12) b 0.814 (12) b
0.534 (18) a 0.135 (6) a 4.290 (16) 2.720 (12) 1.730 (9) 1.282 (10) a
0.268 (18) a 0.138 (6) a 4.508 (16) b 4.310 (12) b 2.173 (12) 0.753 (10)
0.451 0.899 0.619 0.670 0.503 0.407
0.223 0.144 0.337 0.361 0.550 0.264
0.029 0.257 0.428 0.434 0.405 0.330
0.160 0.477 0.274 0.343 0.278 0.568
0.058 0.141 0.151 0.771 0.259 0.205
0.176 0.031 1.185 0.905 0.444 0.420
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
0.089 0.033 0.981 1.120 0.539 0.253
Glycogen content and activities of enzymes involved in the carbohydrate metabolism
Table 1
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Table 2 Activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the submandibular and parotid glands of rats at different ages. Age (days)
Submandibular
Parotid
PK
2 7 14 21 30 60
LDH
LDH
PK
U/mg protein
U/g tissue
U/mg protein
U/g tissue
U/mg protein
U/g tissue
U/mg protein
U/g tissue
0.236 (10) a 0.211 (10) b 0.313 (10) a 0.330 (9) a 0.263 (7) a 0.315 (12) a
9.15 (10) a 11.85 (10) ab 14.26 (10) bc 19.87 (9) cd 18.78 (7) d 11.20 (12) ab
0.710 (10) 0.392 (9) a 0.365 (10) a 0.309 (10) a 0.195 (16) 0.069 (12)
18.12 (12) a 13.80 (16) a 19.11 (10) a 19.28 (9) a 17.65 (19) a 8.02 (9) b
0.231 (17) a 0.132 (12) b 0.203 (6) ab 0.217 (7) a 0.137 (11) b 0.310 (12)
11.53 (17) a 6.55 (12) b 10.74 (6) ac 13.90 (11) ac 8.20 (11) b 11.24 (12)
0.521 (18) 0.174 (12) a 0.352 (17) b 0.152 (10) a 0.348 (11) b 0.299 (12) b
25.8 8.86 (18) a 8.30 1.97 (12) 18.47 4.22 (17) b 14.82 4.04 (12) b 21.12 3.51 (11) ab 18.03 1.84 (12) b
0.016 0.014 0.178 0.024 0.047 0.110
1.22 2.24 2.40 1.59 3.40 3.33
0.12 0.08 0.08 0.10 0.06 0.025
8.96 8.10 2.90 4.42 3.86 1.18
0.05 0.03 0.05 0.04 0.01 0.11
1.73 0.66 2.87 4.32 0.60 2.79
0.188 0.072 0.083 0.024 0.069 0.086
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
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Glycogen content and activities of enzymes involved in the carbohydrate metabolism
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activity from 2 to 14 days although it was not statistically significant. The values found for rats aged 30 and 60 days were higher than in the preceding groups, although from 30 to 60 days there was a decrease in activity. In the parotid glands the specific activity of this enzyme also showed a tendency to increase from 2 to 14 days old, then decrease from 21 to 30 days old; whilst the value for the 60 days old group was statistically greater than in the other groups. Comparing the specific activity of hexokinase obtained in the two glands, the values for the submandibular were from 2.5 to 4.9 times higher than those found in the parotid gland for the 2 to 30 days old groups. However, in the 60 days old rats the mean value for the submandibular was lower than the mean value for the parotid glands. In terms of the activity per g of tissue, the mean value in the 21 days old group was higher than that in the other groups. The values for the submandibular glands were still higher than those in the parotid glands when compared as activity per gram of tissue, but the differences were smaller. Phosphofructokinase-1 showed a different
observations was determined by analysis of variance with the process of multiple comparison of Tukey. The level of significance was set at P < 0:05.
Results In the present work we did not separate results from male from female animals. It has been reported that no differences in the development of the salivary glands were observed until day 60 after birth,36 and data on cell cycle, labelling and mitotic indexes show no significant differences between sexes.37 The results of our investigation are expressed in Tables 1—5. Table 1 shows the data for hexokinase and phosphofructokinase-1 in the submandibular and parotid glands of rats aged 2—60 days. The specific activity of hexokinase in submandibular glands increased from days 2 to 21 then decreased up to 60 days; there was no difference between the values at this age and those in 2 days old rats. Concerning the results expressed as activity per g of tissue, there is a tendency for an increase in
Table 3 Activity of glucose-6-phosphate dehydrogenase (G6PD) in submandibular and parotid glands of rats at different ages. Age (days)
G6PD Submandibular
Parotid 2
U/mg protein, 10 2 7 14 21 30 60
1.757 2.497 2.933 2.892 2.616 2.130
0.316 0.672 0.288 0.630 0.245 0.768
(9) a (10) b (13) b (13) b (16) b (12) ab
U/mg protein, 102
U/g tissue 0.666 1.778 1.244 1.190 2.021 0.56
0.106 0.129 0.261 0.300 0.335 0.158
(9) (10) a (10) a (13) a (21) (12)
0.957 1.178 2.511 2.190 0.828 1.000
0.271 0.559 0.805 0.705 0.230 0.370
(18) a (12) ab (8) c (16) bc (12) a (12) a
U/g tissue 0.478 0.560 1.336 1.429 0.592 0.596
0.108 0.158 0.389 0.317 0.114 0.152
(18) a (12) a (8) b (23) b (12) a (12) a
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05). Table 4 Data of the activities of phosphofructokinase-1 (PFK-1) and glucose-6-phosphate dehydrogenase (G6PD) to show the ratio between the two enzymes which give the potential capacity of the pentose-phosphate pathway and glycolysis. Age (days)
2 7 14 21 30 60
Submandibular
Parotid
PFK1
G6PD
U/mg protein, 102
U/mg protein, 102
0.58 1.03 1.31 1.68 1.70 0.75
1.76 2.50 2.93 2.90 2.62 2.13
PFK1
G6PD
G6PD/PFK-1
U/mg protein, 102
U/mg protein, 102
G6PD/PFK-1
3.0 2.4 2.2 1.7 1.5 2.8
0.53 0.13 4.29 2.72 1.73 1.28
0.96 1.17 2.51 2.19 0.83 1.00
1.8 9.0 0.6 0.8 0.5 0.8
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Table 5 The content of glycogen in the submandibular and parotid glands of rats at different ages. Age (days)
Submandibular (mg glycogen/mg tissue)
2 7 14 21 30 60
(10) (43) (21) (22) (20) (15)
2.747 2.369 1.649 1.020 0.772 0.349
0.761 0.645 0.451 0.360 0.202 0.051
a a
Parotid (mg glycogen/mg tissue) (12) (12) (10) (12) (14) (9)
b bc c
0.974 0.964 0.459 0.470 0.397 0.361
0.251 0.196 0.071 0.154 0.107 0.140
a a b b b b
Mean S:D: In parenthesis is the number of samples. The same letter indicate not statistically significant between the groups. (P < 0:05).
pattern of variation in the two glands. In submandibular glands the specific activity increased from 2nd to 30th day after birth. Although the value of the 60 days group was higher than that of 2 days, the difference was not statistically significant. In the parotid gland the specific activity of phosphofructokinase-1 decreased between 2 and 7 days of age, increased in the 14 days old group and then decreased until 60 days of age. The specific activities and activities per gram of tissue of pyruvate kinase and lactate dehydrogenase from the submandibular and parotid glands are shown in Table 2. In the submandibular glands, except in the 7 days old rats, the specific activity of pyruvate kinase was similar in all the age groups. The specific activity of lactate dehydrogenase in submandibular glands increased from 2 to 7 days old rats and decreased again in the 30 and 60 days old groups. In the parotid glands the specific activity of pyruvate kinase remained similar in the 2, 14 and 21 days old groups. The values in the 60 days old group were higher than those in younger groups. There was a biphasic pattern of lactate dehydrogenase expression with higher values at 2 days of age, and then decrease to a subsequent plateau up to 60 days of age. Table 3 shows the specific activity and the activity per gram of tissue of glucose-6-phosphate dehydrogenase. In the submandibular glands the specific activity of this enzyme showed a significant increase from the 2nd to the 14th day but thereafter showed no significant differences between ages. In the parotid glands no difference in glucose-6-phosphate dehydrogenase activities were observed at 2, 7, 30 and 60 days of age. The potential capacity of the pentose phosphate and glycolytic pathways as indicated by the ratio between the activities of glucose-6-phosphate dehydrogenase and phosphofructokinase-1 is shown in Table 4. In the submandibular glands the highest mean values were found in glands from 2 days old rats. After this age there was a decrease in the ratio until 30 days of age. There was, however,
an increase in the 60 days age group. In the parotid glands there was a peak in the ratio at 7 days suggesting a predominance of the pentose phosphate pathway over glycolysis. The glycogen content of both glands is shown in Table 5. The pattern was similar for both glands, the content being higher in the 2 days old rats and then decreasing up to 60 days of age.
Discussion The data from this study must be considered in the context of the changes in metabolism that occurs between the perinatal and postnatal period. The rat foetus has a plentiful supply of glucose from the maternal blood via the placenta. Hence, in the rat foetus the main source of energy is carbohydrate. For at least the first weeks after birth, the rat appears to require a liquid diet rich in fat and protein and relatively poor in carbohydrate.38 In fact, the reported milk composition gives a concentration for lactose that varies from 2.5 to 3.8%39,40 and for lipids a concentration between 16.8 and 20.7%.40 Milk consumption by the pups increases progressively to 18 days, then declines until weaning.41 In our experiments, weaning occurred at 21 days of age, so that there were four suckling groups (2, 7, 14 and 21 days after birth) and two groups on solid food (30 and 60 days after birth). According to Thomas and Fell,42 for a change in activity of any enzyme to cause a change in metabolic flux, there has to be a consequent change in concentration of one or more metabolites in the pathways and any enzyme will become rate-limiting if it is inhibited sufficiently. According to these authors, this is a mechanism by which the signal (change in enzyme activity), is propagated to cause a change in flux. The specific activity of hexokinase was higher in the submandibular glands than that in parotid glands. In muscle, according to Fell43 this enzyme is not considered as part of the glycolytic pathway,
Glycogen content and activities of enzymes involved in the carbohydrate metabolism
because the substrate of muscle glycolysis is glycogen, and hexokinase activity is related to glycogen synthesis. By analogy, if we consider this in the salivary glands, it is possible that the specific activity of hexokinase in the submandibular glands being higher than in the parotid glands was related to the higher glycogen content in the submandibular glands. The specific activity of phosphofructokinase-1 showed some interesting variations. Although the submandibular and parotid glands of 2 days old rats are in different stages of development no difference was observed in the specific activity of this enzyme. In the submandibular glands at this age the majority of cells belong to two categories, type III and type I whilst in the parotid glands, the terminal cluster form typical acini and their cells undergo cytodifferentiation.10 However, the mean values found for the parotid glands in 7 days old were approximately eight times lower than the mean values found for the submandibular glands, and at this age the glands are still in the neonatal period.10 Despite this difference for the 7 days old animals, from 14 days old onwards the mean values are higher for the parotid glands. The differences observed between the submandibular and parotid glands are understandable, because they are in different stages of maturation. Further, whilst in submandibular glands anaerobic metabolism is predominant, in the parotid gland aerobic metabolism predominates.44 Although the maturation of the acinar cells of the submandibular gland precedes that in the parotid, no measurable secretory response through a ductal cannula was observed earlier than 2 weeks of age with pilocarpine or 3 weeks of age with isoproterenol.18 Looking at the several enzymes involved in a pathway, mainly in the 7 and 14 days old rats, we observed that, despite the increase in the specific activities of hexokinase and phosphofructokinase-1 in the submandibular glands of the 7 days old rats, the specific activities of phosphofructokinase and lactate dehydrogenase decreased. In 14 days old rats changes in pyruvate kinase activity were similar to those in hexokinase and phosphofructokinase-1 activity. On the other hand, although the specific activity of hexokinase had increased in the parotid glands of 7 days old rats, the specific activity of phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase decreased. In these glands, all the enzymes studied showed increased specific activities in 14 days old animals. However, in interpreting the results of our work we must consider that at birth the suckling rat turns to a diet of milk. During this early postnatal period the specific activities of hexokinase and phosphofructokinase-1 in the sub-
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mandibular glands increased up to 21 days of age. At this age the values were higher than those preceding, and this coincides with the end of lactation and the switch to solid food. It is interesting that in the parotid glands the higher values for the specific activities of hexokinase and phosphofructokinase1 appeared at 14 days of age, during suckling period. However, at weaning (21 days of age) the specific activity of hexokinase was higher than at 2 days but lower than at 14 days. On the other hand, an eightfold increase in the activity of phosphofructokinase1 was noted in the parotid glands of 14 days old group, and five-fold increase in the 21 days old group as compared with the group 2 days after birth. Between 8 and 15 days of age the prolongation of the rat parotid acinar cell cycle is reported to be primarily due to an increase in the duration of the G-1 phase.45 During G-1 phase there is considerable synthesis of enzymes involved in nucleotide metabolism46 which may explain our findings, as synthetic processes are energy-dependent. The product of the action of hexokinase, glucose6-phosphate, is an intermediate in three pathways– —namely glycolysis, synthesis of glycogen and the pentose phosphate pathway. According to Fell43 hexokinase does not change in concert with the glycolytic enzymes, and in muscle it changes activity in the opposite direction to the other enzymes.47 In the present work, hexokinase activity changed in the same direction as phosphofructokinase-1 and glucose-6-phosphate dehydrogenase in the submandibular glands, but in the parotid glands this occurred only with glucose-6-phosphate dehydrogenase and not with phosphofructokinase-1. The treatment of Shonk and Boxer48 for the potential capacity of the pentose phosphate and glycolytic pathways, demonstrated by the ratio of glucose-6-phosphate dehydrogenase and phosphfructokinase-1 activities, give us interesting information. While phosphofructokinase-1 is the key enzyme in glycolysis, glucose-6-phosphate dehydrogenase is the first enzyme in the pentosephosphate-pathway. This enzyme supplies NADPH, which is important in the reducing processes of biosynthesis of ribose-5-phosphate constituent of nucleotides. For the submandibular gland at 2 days old, the ratio (3.0) favoured the pentose phosphate pathway, but as the age of the animals increased this ratio became smaller, indicating increased glycolytic activity. In the parotid glands, it is noticeable that at 7 days of age the ratio (9.0) is markedly in favour of the pentose-phosphate pathway. However, from 14 days on the ratio favours glycolysis. It should be point out that the glands are at different stages of development at the time when the animals were killed, and that no secretion of saliva has been
108 observed before day 14.18,24 Considering that all functions of a cell are energy dependent, we may speculate, mainly in the relation to the parotid gland, that up to 7 days after birth the metabolism of the cells are particularly directed towards biosynthetic processes. The glycogen content in the submandibular glands was greater than that in the parotid glands, and in both glands it decreased with increasing age. This decrease in animals from 2 to 60 days old seems to agree with the reported for the liver.49 They showed that at birth the foetal rat liver accumulates large amounts of glycogen. It is suggested that the accumulated polysaccharides are utilised after birth to sustain the animal until feeding is established.50,51 This suggestion is understandable, as the liver is an homeostatic organ. Our results on glycogen concentration agree with those reported for dog salivary glands.25 It is possible that the glycogen concentration in the glands might vary during the 2 h period over which different animals were killed. However, preliminary results from our laboratory showed no differences in glandular polysaccharide concentration between rats killed at 9:00 a.m. and those killed at 11:00 a.m. We believe that the high content of glycogen in the glands during the first 2 weeks after birth provides a reserve to enable the glands to mature fully. In conclusion, this study shows differences in the specific activity of some enzymes involved in carbohydrate metabolism and in glycogen concentrations in developing salivary glands. It is possible that these differences may have some influence on the process of salivary secretion in baby rats.
Acknowledgements This work was supported by a grant from Fundac¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP). JN is a recipient of a fellowship from Conselho Nacional de Cie ˆncia e Tecnologia (CNPq).
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