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The chronically reserpinized rat: Decreased glycolytic activity in the submandibular gland
BIOCHEMICAL
MEDICINE
33, 99-103
(1985)
The Chronically Reserpinized Rat: Decreased Glycolytic Activity in the Submandibular Gland ALICJA BARDO~.*‘$ R. MARGARETA MILLER,? OVE CEDER,* AND GODFRIED M. ROOMANS~ *Department of Pediatrics, University Hospital, S-901 85 UmeB, and f Department of Ultrastructure Research, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. and SDepartment of Biochemistry, Institute of Sport. Warsuw. Poland Received
October
17, 1983
The rat given reserpine chronically has frequently been used as an animal model in the study of cystic fibrosis (CF). Several functional abnormalities in the exocrine gland of these rats are found, resembling some of those found in CF patients (l-3): the electrolyte distribution is disturbed, increased mucus and glycoprotein contents are found, and the saliva of these rats contains a “CF factor.” In a series of publications (4-7) we have found evidence of a disturbed glycolytic metabolism in CF. This disturbance causes lactic acidosis and depletion of energy in the exocrine glands (7) and disturbs the secretory process. Since similar disturbances in electrolyte and mucus regulation of the salivary glands are found in CF and the reserpine-treated rat (l-3), we investigated some of the enzymes and metabolites of the glycolytic pathway in the submandibular glands of these rats. In this publication we report the findings of decreased glycolytic activity in the submandibular gland of reserpine-treated rats. MATERIAL
AND METHODS
Female Sprague-Dawley rats (200-300 g) were injected intraperitoneally with reserpine (0.5 mg/kg body wt) daily for 7 days (8). Female rats were used in previous studies of changes in elemental distribution in the submandibular gland (8) and in this respect the effects of chronic reserpine treatment are not sex specific (2,8). During this period the rats had access to food and water ad libitum. After being deprived of food overnight, the rats were deeply anesthetized with sodium pentobarbital and the submandibular glands were excised and immediately snap-frozen in liquid Freon 13 cooled by liquid nitrogen. The glands were then stored at -70°C until analysis was performed. For enzyme studies a 5% (w/v) homogenate was made with an all-glass homogenizer of the frozen gland in cold distilled water and the 15OOg supernatant was used for analysis. All of the above steps were carried out on ice, and all 99 0006-2944185
$3.00
Press. Inc. Copyright Q 1985 by Academic All rIghI? of reproduction in any form reserved.
100
BARDOh El-AI
analyses were done on freshly prepared homogenates. The following enzymes were analyzed: phosphofructokinase (PFK. ATI%-fructose 6-phosphate I-phosphotransferase, EC 2.7. I. 1I), glycerol-3-phosphate dehydrogenase (GDH, glycerol3-phosphate:NAD’ 2-oxidoreductase. EC I. 1. I .S), enolase (2-phospho-o-glycerate hydrolyase, EC 4.2.1.11), pyruvate kinase (PK. ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), and lactate dehydrogenase (LDH. r.-lactate:NAD oxidoreductase, EC 1.1.1.27). For all enzymes, methods described in (9) were used. One unit (U) of enzyme activity corresponds to the amount of enzyme that converts 1 pmole of substrate per minute (9). Free and total ribonuclease (RNase, ribonucleate 3-pyrimidinooligonucleotido hydrolase. EC 3. I .4.33) were estimated without and with paru-chloromercuribenzoic acid essentially as described earlier (10) but at pH 7.8. One unit (U) of RNase activity was defined as I mmole of acid-soluble products formed per hour (1 I ). For estimation of the concentration of metabolites a 20% (w/v) homogenate was made in cold 0.6 M perchloric acid with an all-glass homogenizer on ice. Neutralization was carefully done to pH 6.8-7.0 with 4 M KOH. The samples were centrifuged and the 15OOg supernatant was used for analysis. Glycogen. glucose, glycerate-2-phosphate, phosphoenolpyruvate, pyruvate, and lactate were analyzed by enzymatic reactions coupled to the oxidation or reduction of pyridine nucleotides (9). Protein was estimated both in the water homogenate and in the perchloric acid extract by the method of Lowry rt al. (12). with bovine serum albumin as standard. All results were calculated per gram, wet weight. of tissue and are presented as means (SEM). The statistical significance of the differences was evaluated with Student’s t test. RESULTS
A decreased activity was found for all of the measured glycolytic enzymes in the submandibular gland of the reserpine-treated rats (Table 1). Also the activity of glycerol-3-phosphate dehydrogenase showed a tendency to decrease (Table 1). On the contrary, the activity of ribonuclease, especially the free form. showed a tendency to increase in activity (Table I) in the glands of reserpine-treated rats. This could indicate a decreased glandular pH (13) in these animals. For metabolites increased concentrations were seen for glycogen and phosphoenolpyruvate whereas the concentrations of the end products of glycolysis, pyruvate and lactate, were decreased (Table 2). No effect of reserpine treatment was seen on the concentrations of glucose and glycerate-2-phosphate. No difference in total protein concentration was found between the two groups examined (Table 1). In contrast to this, the perchloric acid-soluble part of the proteins was clearly increased (Table 2) in the glands of reserpine-treated animals. DISCUSSION
This is, to our knowledge, the first investigation of glycolytic enzymes and metabolites in the submandibular gland of reserpine-treated rats. Good agreement between our values for control animals and values found in the literature is achieved (9,14,15). It is evident that reserpine treatment causes decreased
___
of Glycolytic
TABLE 2 and Protein in Perchloric Acid Extracts of Submandibular Gland from Reserprine-Treated and Control Rats __ ~____ ..~~ ___ .--~ .___ Glycerate-2PhosphophenolAcid-soluble pyruvate Glucose phosphate Pyruvate Lactate protein (pmole/g) l~moleig) Lumolekd (~moleig) (pmoleig) b-cidd ~~ ___..~~ ~..-~ ______. 0.033 (0.0054) 2.0 (0.09) 0.038 (0.0067) 0.087 (0.0085) 2.3 (0.12) 1 I .2 (0.44) 2.0 (0.12) 0.040 (0.0071) 0.084 (0.0065) 0.038 (0.0068) I .6 (0.065) 46.4 (4.1) 0.9 0.8 <0.0001 0.0005 0.0002
Metabolites
~~~-- __ ~ Glycogen (as glucose) @mole/g) __ ~~~ .~ Controls (n = 13) 9.2 (0.27)" Reserpine (n = IO) 12.7 (0.77) P 0.0002 _____ ’ Mean (SEM).
~~
Concentrations
TABLE I Enzyme Activities and Protein Concentration in Homogenates of Submandibular Glands from Reserpine-Treated and Control Rats ~_ .__.~ ~~ .___ ~~ -~ ___. .-. Phosphofructo Glycerol-3-phosphate Pyruvate Lactate Ribonuclease Total dehydrogenase Enolase kinase dehydrogenase kinase protein Free (U/g) Total (U/g) (U/g) NJk) Wig) (U/g) Wig) On&) ~___~.~.- ____ ___~. ~~. 28 (2.3) 48 (3.0) 106 (7.7) 2.1 (0.65) 18 (1.7) 118 (7.2) Controls (n = 10) 27 (2.6)” 4.6 (0.38) 20 (0.68) 38 (2.2) 59 (3.9) 4.3 (1.2) Reserpine (n = 6) 12 (1.7) 3.4 (0.28) 22 (4.6) 113 (8.0) P 0.002 0.06 0.03 0.05 0.009 0.1 0.4 0.7 ~~~~~.~-~~ .__ -~ ___~ _. ___ ~~ _-~ ___ ’ Mean (SEM).
z 2
g
z 2 T-
n z
0 2 8 s E 5
102
BARDOk’
ET Ai
activity of several of the enzymes connected to glycolysis. including the two main regulatory enzymes-phosphofructokinase and pyruvate kinase. Although. in the test tube, this decrease is only 20%~. it might. in the intact animal. be further aggravated by changes in the intracellular ion concentration. e.g.. the NaiK ratio (S), on which the activity of pyruvate kinase depends. This leads to accumulation of metabolites above these steps as i$ seen for phosphoenolpyruvatc and glycogen. The concentrations of pyruvate and lactate. the end products ot’ glycolysis, decrease, however. Measurement of the RNase activity can be used as an indicator of acidosis within the cells (13). Normally RNase is bound to its natural inhibitor but with decreasing pH the enzyme and the inhibitor are dissociated. Our finding of ;L tendency toward increased activity of free ribonuclease after rescrpine treatment. therefore, may indicate increased acidosis in the glands. In the submandibular gland of the reserpine-treated rat. decreased RNA content (16) and a decreased level of cell phosphorus (8) have been demonstrated. One possible cau~c l’or the acidosis in the gland is the increased accumulation of phosphoenolpyruvate. In addition to acidosis of the gland, disturbance in the production of energ) I\ expected. The total protein concentration of the hubmandibular gland did not change after reserpine treatment. which is in agreement with earlier reports (17). The perchloric acid-soluble part of the proteins (mainly glycoproteins) were increased fourfold, however, in both this and earlier (17) studies. Both body weight and gland weight were reduced after chronic reserpinc treatment but the ratio of gland weight to body weight was constant ( I ,2). The results of this study, thus, have shown clearly that the glycolytic pathw;l) is disturbed in the submandibular glands of chronically reserpine-treated rat\. Also in cystic fibrosis. a disease for which these rats ;tre used as an animal model. there is evidence for a disturbance of the glycolytic metabolism ~4-7). In cystic fibrosis this disturbance causes lactic acidosis and depletion of energy in the exocrine glands (4-7). In the reserpine-treated rats, a decreased ATP pool after cholinergic and a-adrenergic stimulation of the submandibular glands has been noted (IS). In these rats no evidence of lactic acidosis is found hut rather a decreased concentration of lactate and pyruvate. Instead. accumulation of phosphoenolpyruvate occurs (Table 21, leading to acidosis in the glands. A generally decreased activity of the glycolytic enzymes causes a disturbance of the energy metabolism in the glands and thereby mimics the situation (4-7) in the glands of patients with cystic fibrosis. SUMMARY Some of the enzymes and metabolites of the glycolytic pathway of an ammal model for cystic fibrosis (the chronically reserpine-treated rat) were investigated. The activities of the enzymes phosphofructokinase (P c 0.002). enolase (P -.: 0.03), pyruvate kinase (P < 0.005). and lactate dehydrogenase CP < 0.009) were decreased whereas the activity of glycerol-3-phosphate dehydrogenase was unaffected in the submandibular glands of the treated animals. For metabolitec. the reserpine treatment resulted in an increased concentration of glycogen (I’ .-
GLYCOLYSIS
IN RESERPINE-TREATED
103
RATS
0.0002) and phosphoenolpyruvate (P < 0.001) and a decreased concentration of pyruvate (P < 0.005) and lactate (P < 0.002) in the glands. The concentration of glucose and glycerate-2-phosphate was unaffected. The perchloric acid-soluble part of the proteins was also increased (P < 0.0001) in the submandibular glands of the reset-pine-treated animals, as was the activity of ribonuclease. These findings point to a disturbance in the metabolism of glucose and a possible acidosis in the submandibular glands of this animal model for cystic fibrosis. ACKNOWLEDGMENTS This study was financially supported by the Torsten and Ragnar Soderberg foundations, the Swedish National Association against Heart and Chest Diseases, and the Cystic Fibrosis Research Trust. The support is gratefully acknowledged.
REFERENCES I. Martinez, J. R.. Adelstein, E.. Quissel, D., and Barbero. G. J., Pediutr. Res. 9, 463 (1975). 2. Martinez, J. R.. Adshead, P. C.. Quissel. D. 0.. and Barbero, G. J., Pediutr. Res. 9, 470 (1975). 3. Roomans, G. M.. von Euler, A. M., and Mtiller. R. M.. Sunning Electron Microsc. 11, 697 (1983). 4. Bardon. A., Ceder, O., and Kollberg, H., C/in. Chim. Acru 133, 31 I (1983). 5. Ceder, 0.. Teien, D., Bardon, A., Hellsing. K.. and Kollberg, H., Actrr Poediatr. Sccmd. .Suppl. 309, 6.
25 (1983).
Ceder, 0.. Bardon. A., Hellsing, K., and Kollberg.
H.. Acru Paediarr.
Scund.
Suppl.
309, 33
(1983).
13. 14. IS. 16. 17.
Kollberg, H., Bardon, A., and Ceder. O., Acta Paediatr. Stand. Suppl. 309, 41 (1983). Roomans. G. M.. Wei. X., Ceder, 0.. and Kollberg. H., Uhrastrucf. Pathal. 3, 285 (1982). Bergmeyer, H. U. In “Methods of Enzymology.” Academic Press, New York, 1974. Bardon, A., and Shugar, D., C/in. Chim. Acru 101, I7 (1980). Beard. J. R., and Razzel, W. E.. J. Biol. Chem. 239, 4186 (1964). Lowry, 0. H.. Rosebrough. N. J.. Farr, A. L., and Randall. R. J.. J. Biol. C/rem. 193, 265 (1951). Szczesna-Kaczmarek. A., Experienrin 32, 1499 (1976). Mikhaylov, V. V., and Rusanova, A. G.. Vopr. Med. Kf7h7. 26, 99 (1980). Nicolau. J., and Sassaki. K. T.. Gen. Pharmocol. 13, I53 (1982). Simson. J. A. V., Spicer. S. S.. Setser, M. E.. and Martinez, J. R., Lab. InveSt. 39, I57 (1978). Taylor, P. W.. Richardsson. K. C.. Roddy, P. M.. and Titus, E., J. Pharmncol. Exp. Ther. 156,
18.
Quissel, D. 0.. Martinez,
7. 8.
9. IO. I I. 12.
483 (1967).
J. R.. and Giles. D.. Arch.
Oral
Biol.
24, 638
(1979).
MEDICINE
33, 99-103
(1985)
The Chronically Reserpinized Rat: Decreased Glycolytic Activity in the Submandibular Gland ALICJA BARDO~.*‘$ R. MARGARETA MILLER,? OVE CEDER,* AND GODFRIED M. ROOMANS~ *Department of Pediatrics, University Hospital, S-901 85 UmeB, and f Department of Ultrastructure Research, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden. and SDepartment of Biochemistry, Institute of Sport. Warsuw. Poland Received
October
17, 1983
The rat given reserpine chronically has frequently been used as an animal model in the study of cystic fibrosis (CF). Several functional abnormalities in the exocrine gland of these rats are found, resembling some of those found in CF patients (l-3): the electrolyte distribution is disturbed, increased mucus and glycoprotein contents are found, and the saliva of these rats contains a “CF factor.” In a series of publications (4-7) we have found evidence of a disturbed glycolytic metabolism in CF. This disturbance causes lactic acidosis and depletion of energy in the exocrine glands (7) and disturbs the secretory process. Since similar disturbances in electrolyte and mucus regulation of the salivary glands are found in CF and the reserpine-treated rat (l-3), we investigated some of the enzymes and metabolites of the glycolytic pathway in the submandibular glands of these rats. In this publication we report the findings of decreased glycolytic activity in the submandibular gland of reserpine-treated rats. MATERIAL
AND METHODS
Female Sprague-Dawley rats (200-300 g) were injected intraperitoneally with reserpine (0.5 mg/kg body wt) daily for 7 days (8). Female rats were used in previous studies of changes in elemental distribution in the submandibular gland (8) and in this respect the effects of chronic reserpine treatment are not sex specific (2,8). During this period the rats had access to food and water ad libitum. After being deprived of food overnight, the rats were deeply anesthetized with sodium pentobarbital and the submandibular glands were excised and immediately snap-frozen in liquid Freon 13 cooled by liquid nitrogen. The glands were then stored at -70°C until analysis was performed. For enzyme studies a 5% (w/v) homogenate was made with an all-glass homogenizer of the frozen gland in cold distilled water and the 15OOg supernatant was used for analysis. All of the above steps were carried out on ice, and all 99 0006-2944185
$3.00
Press. Inc. Copyright Q 1985 by Academic All rIghI? of reproduction in any form reserved.
100
BARDOh El-AI
analyses were done on freshly prepared homogenates. The following enzymes were analyzed: phosphofructokinase (PFK. ATI%-fructose 6-phosphate I-phosphotransferase, EC 2.7. I. 1I), glycerol-3-phosphate dehydrogenase (GDH, glycerol3-phosphate:NAD’ 2-oxidoreductase. EC I. 1. I .S), enolase (2-phospho-o-glycerate hydrolyase, EC 4.2.1.11), pyruvate kinase (PK. ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), and lactate dehydrogenase (LDH. r.-lactate:NAD oxidoreductase, EC 1.1.1.27). For all enzymes, methods described in (9) were used. One unit (U) of enzyme activity corresponds to the amount of enzyme that converts 1 pmole of substrate per minute (9). Free and total ribonuclease (RNase, ribonucleate 3-pyrimidinooligonucleotido hydrolase. EC 3. I .4.33) were estimated without and with paru-chloromercuribenzoic acid essentially as described earlier (10) but at pH 7.8. One unit (U) of RNase activity was defined as I mmole of acid-soluble products formed per hour (1 I ). For estimation of the concentration of metabolites a 20% (w/v) homogenate was made in cold 0.6 M perchloric acid with an all-glass homogenizer on ice. Neutralization was carefully done to pH 6.8-7.0 with 4 M KOH. The samples were centrifuged and the 15OOg supernatant was used for analysis. Glycogen. glucose, glycerate-2-phosphate, phosphoenolpyruvate, pyruvate, and lactate were analyzed by enzymatic reactions coupled to the oxidation or reduction of pyridine nucleotides (9). Protein was estimated both in the water homogenate and in the perchloric acid extract by the method of Lowry rt al. (12). with bovine serum albumin as standard. All results were calculated per gram, wet weight. of tissue and are presented as means (SEM). The statistical significance of the differences was evaluated with Student’s t test. RESULTS
A decreased activity was found for all of the measured glycolytic enzymes in the submandibular gland of the reserpine-treated rats (Table 1). Also the activity of glycerol-3-phosphate dehydrogenase showed a tendency to decrease (Table 1). On the contrary, the activity of ribonuclease, especially the free form. showed a tendency to increase in activity (Table I) in the glands of reserpine-treated rats. This could indicate a decreased glandular pH (13) in these animals. For metabolites increased concentrations were seen for glycogen and phosphoenolpyruvate whereas the concentrations of the end products of glycolysis, pyruvate and lactate, were decreased (Table 2). No effect of reserpine treatment was seen on the concentrations of glucose and glycerate-2-phosphate. No difference in total protein concentration was found between the two groups examined (Table 1). In contrast to this, the perchloric acid-soluble part of the proteins was clearly increased (Table 2) in the glands of reserpine-treated animals. DISCUSSION
This is, to our knowledge, the first investigation of glycolytic enzymes and metabolites in the submandibular gland of reserpine-treated rats. Good agreement between our values for control animals and values found in the literature is achieved (9,14,15). It is evident that reserpine treatment causes decreased
___
of Glycolytic
TABLE 2 and Protein in Perchloric Acid Extracts of Submandibular Gland from Reserprine-Treated and Control Rats __ ~____ ..~~ ___ .--~ .___ Glycerate-2PhosphophenolAcid-soluble pyruvate Glucose phosphate Pyruvate Lactate protein (pmole/g) l~moleig) Lumolekd (~moleig) (pmoleig) b-cidd ~~ ___..~~ ~..-~ ______. 0.033 (0.0054) 2.0 (0.09) 0.038 (0.0067) 0.087 (0.0085) 2.3 (0.12) 1 I .2 (0.44) 2.0 (0.12) 0.040 (0.0071) 0.084 (0.0065) 0.038 (0.0068) I .6 (0.065) 46.4 (4.1) 0.9 0.8 <0.0001 0.0005 0.0002
Metabolites
~~~-- __ ~ Glycogen (as glucose) @mole/g) __ ~~~ .~ Controls (n = 13) 9.2 (0.27)" Reserpine (n = IO) 12.7 (0.77) P 0.0002 _____ ’ Mean (SEM).
~~
Concentrations
TABLE I Enzyme Activities and Protein Concentration in Homogenates of Submandibular Glands from Reserpine-Treated and Control Rats ~_ .__.~ ~~ .___ ~~ -~ ___. .-. Phosphofructo Glycerol-3-phosphate Pyruvate Lactate Ribonuclease Total dehydrogenase Enolase kinase dehydrogenase kinase protein Free (U/g) Total (U/g) (U/g) NJk) Wig) (U/g) Wig) On&) ~___~.~.- ____ ___~. ~~. 28 (2.3) 48 (3.0) 106 (7.7) 2.1 (0.65) 18 (1.7) 118 (7.2) Controls (n = 10) 27 (2.6)” 4.6 (0.38) 20 (0.68) 38 (2.2) 59 (3.9) 4.3 (1.2) Reserpine (n = 6) 12 (1.7) 3.4 (0.28) 22 (4.6) 113 (8.0) P 0.002 0.06 0.03 0.05 0.009 0.1 0.4 0.7 ~~~~~.~-~~ .__ -~ ___~ _. ___ ~~ _-~ ___ ’ Mean (SEM).
z 2
g
z 2 T-
n z
0 2 8 s E 5
102
BARDOk’
ET Ai
activity of several of the enzymes connected to glycolysis. including the two main regulatory enzymes-phosphofructokinase and pyruvate kinase. Although. in the test tube, this decrease is only 20%~. it might. in the intact animal. be further aggravated by changes in the intracellular ion concentration. e.g.. the NaiK ratio (S), on which the activity of pyruvate kinase depends. This leads to accumulation of metabolites above these steps as i$ seen for phosphoenolpyruvatc and glycogen. The concentrations of pyruvate and lactate. the end products ot’ glycolysis, decrease, however. Measurement of the RNase activity can be used as an indicator of acidosis within the cells (13). Normally RNase is bound to its natural inhibitor but with decreasing pH the enzyme and the inhibitor are dissociated. Our finding of ;L tendency toward increased activity of free ribonuclease after rescrpine treatment. therefore, may indicate increased acidosis in the glands. In the submandibular gland of the reserpine-treated rat. decreased RNA content (16) and a decreased level of cell phosphorus (8) have been demonstrated. One possible cau~c l’or the acidosis in the gland is the increased accumulation of phosphoenolpyruvate. In addition to acidosis of the gland, disturbance in the production of energ) I\ expected. The total protein concentration of the hubmandibular gland did not change after reserpine treatment. which is in agreement with earlier reports (17). The perchloric acid-soluble part of the proteins (mainly glycoproteins) were increased fourfold, however, in both this and earlier (17) studies. Both body weight and gland weight were reduced after chronic reserpinc treatment but the ratio of gland weight to body weight was constant ( I ,2). The results of this study, thus, have shown clearly that the glycolytic pathw;l) is disturbed in the submandibular glands of chronically reserpine-treated rat\. Also in cystic fibrosis. a disease for which these rats ;tre used as an animal model. there is evidence for a disturbance of the glycolytic metabolism ~4-7). In cystic fibrosis this disturbance causes lactic acidosis and depletion of energy in the exocrine glands (4-7). In the reserpine-treated rats, a decreased ATP pool after cholinergic and a-adrenergic stimulation of the submandibular glands has been noted (IS). In these rats no evidence of lactic acidosis is found hut rather a decreased concentration of lactate and pyruvate. Instead. accumulation of phosphoenolpyruvate occurs (Table 21, leading to acidosis in the glands. A generally decreased activity of the glycolytic enzymes causes a disturbance of the energy metabolism in the glands and thereby mimics the situation (4-7) in the glands of patients with cystic fibrosis. SUMMARY Some of the enzymes and metabolites of the glycolytic pathway of an ammal model for cystic fibrosis (the chronically reserpine-treated rat) were investigated. The activities of the enzymes phosphofructokinase (P c 0.002). enolase (P -.: 0.03), pyruvate kinase (P < 0.005). and lactate dehydrogenase CP < 0.009) were decreased whereas the activity of glycerol-3-phosphate dehydrogenase was unaffected in the submandibular glands of the treated animals. For metabolitec. the reserpine treatment resulted in an increased concentration of glycogen (I’ .-
GLYCOLYSIS
IN RESERPINE-TREATED
103
RATS
0.0002) and phosphoenolpyruvate (P < 0.001) and a decreased concentration of pyruvate (P < 0.005) and lactate (P < 0.002) in the glands. The concentration of glucose and glycerate-2-phosphate was unaffected. The perchloric acid-soluble part of the proteins was also increased (P < 0.0001) in the submandibular glands of the reset-pine-treated animals, as was the activity of ribonuclease. These findings point to a disturbance in the metabolism of glucose and a possible acidosis in the submandibular glands of this animal model for cystic fibrosis. ACKNOWLEDGMENTS This study was financially supported by the Torsten and Ragnar Soderberg foundations, the Swedish National Association against Heart and Chest Diseases, and the Cystic Fibrosis Research Trust. The support is gratefully acknowledged.
REFERENCES I. Martinez, J. R.. Adelstein, E.. Quissel, D., and Barbero. G. J., Pediutr. Res. 9, 463 (1975). 2. Martinez, J. R.. Adshead, P. C.. Quissel. D. 0.. and Barbero, G. J., Pediutr. Res. 9, 470 (1975). 3. Roomans, G. M.. von Euler, A. M., and Mtiller. R. M.. Sunning Electron Microsc. 11, 697 (1983). 4. Bardon. A., Ceder, O., and Kollberg, H., C/in. Chim. Acru 133, 31 I (1983). 5. Ceder, 0.. Teien, D., Bardon, A., Hellsing. K.. and Kollberg, H., Actrr Poediatr. Sccmd. .Suppl. 309, 6.
25 (1983).
Ceder, 0.. Bardon. A., Hellsing, K., and Kollberg.
H.. Acru Paediarr.
Scund.
Suppl.
309, 33
(1983).
13. 14. IS. 16. 17.
Kollberg, H., Bardon, A., and Ceder. O., Acta Paediatr. Stand. Suppl. 309, 41 (1983). Roomans. G. M.. Wei. X., Ceder, 0.. and Kollberg. H., Uhrastrucf. Pathal. 3, 285 (1982). Bergmeyer, H. U. In “Methods of Enzymology.” Academic Press, New York, 1974. Bardon, A., and Shugar, D., C/in. Chim. Acru 101, I7 (1980). Beard. J. R., and Razzel, W. E.. J. Biol. Chem. 239, 4186 (1964). Lowry, 0. H.. Rosebrough. N. J.. Farr, A. L., and Randall. R. J.. J. Biol. C/rem. 193, 265 (1951). Szczesna-Kaczmarek. A., Experienrin 32, 1499 (1976). Mikhaylov, V. V., and Rusanova, A. G.. Vopr. Med. Kf7h7. 26, 99 (1980). Nicolau. J., and Sassaki. K. T.. Gen. Pharmocol. 13, I53 (1982). Simson. J. A. V., Spicer. S. S.. Setser, M. E.. and Martinez, J. R., Lab. InveSt. 39, I57 (1978). Taylor, P. W.. Richardsson. K. C.. Roddy, P. M.. and Titus, E., J. Pharmncol. Exp. Ther. 156,
18.
Quissel, D. 0.. Martinez,
7. 8.
9. IO. I I. 12.
483 (1967).
J. R.. and Giles. D.. Arch.
Oral
Biol.
24, 638
(1979).