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The chronically pilocarpine-treated rat in the study of cystic fibrosis: Investigation on submandibular gland and pancreas
EXPERIMENTAL
AND
MOLECULAR
The Chronically Fibrosis:
Pilocarpine-Treated Rat in the Study of Cystic Investigations on Submandibular Gland and Pancreas
R. MARGARETA
MUELLER,] GEMMA
OVE CEDER,~ ‘Depurtment Stockholm.
of Ultrastructure Sweden,
Received
43, 97-106 (1985)
PATHOLOGY
and
November
A. J. KUIJPERS,*~~ ALICJA AND GODFRIED M. ROOMANS'
Reseurch.
Wenner-Gren
‘Department
of Pediutrics. UmeB. SH,eden
30.
1984.
and
Institllte, UmeB
in revisedform
BARDON,~
University
of Stockholm,
University
Hospital,
February
25.
S-10691 S-90185
1985
The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. The effect of chronic pilocarpine treatment on structure, composition. and function of the acinar cells of rat submandibular gland and pancreas was investigated by electron microscopy, X-ray microanalysis, and biochemical analysis. The morphological effects of chronic pilocarpine treatment were most pronounced in the pancreas. The number and size of the zymogen granules was increased, and the granules had a less electron-dense appearance. X-ray microanalysis at the cellular level showed in both the submandibular gland and the pancreas a significant increase in calcium and a decrease in sodium. The increase in cellular calcium concentrations can be explained by an increase in the relative volume of secretory material in the cell (assessed by morphometry) and an increase in the local calcium concentration in the secretory material (assessed by X-ray microanalysis at the subcellular level). Chronic pilocarpine treatment caused a disturbance of glycolysis and energy metabolism in the submandibular gland. but no significant effects in this respect were noted in the pancreas. On the other hand. a nearly twofold increase of the pancreatic amylase activity was noted. The pancreas appeared somewhat hyperreactive towards cholinergic stimulation. ill 19X5 Academic Press. Inc. INTRODUCTION
The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis (CF) (Sturgess and Reid, 1973). Chronic cholinergic stimulation induces changes in the structure and function of salivary glands and pancreas that, to a certain extent, resemble the changes observed in exocrine glands of patients with CF. The main points of agreement appear to be hypersecretion and hypertrophy of the glands (Sturgess and Reid, 1973). Recently, it has been shown that chronic pilocarpine treatment of rats induces hyperreactivity of the pancreas to muscarinic cholinergic stimulation indicated by increased amylase release after stimulation by carbachol in vitro (Kuijpers and Roomans, 1984). Other suggested animal models for cystic fibrosis are the chronically reserpinized rat and the chronically isoproterenol-treated rat (Sturgess and Reid, 1973; Martinez et al., 1975a, b; Wood and Martinez 1977; Mangos et al., 1981). In these models, the sympathetic nervous system and the adrenergic receptors are influenced (Bylund and Martinez, 1980; Roscher et al., 1981), whereas in the chronically pilocarpine-treated rat, the parasympathetic system is affected. In a series of recent studies, we have used X-ray microanalysis to study the elemental composition of the exocrine glands in the chronically reserpinized and the chronically ’ Permanent address: Department of Biochemistry, HB Nijmegen, The Netherlands.
University of Nijmegen, P.O. Box 9101, 6500
97 0014-4800/85 $3.00 Copyright “1 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
98
MtiLLER
ET AL.
isoproterenol-treated rat (Roomans et al., 1982a; Mtiller and Roomans 1984a, b). Furthermore, the effect of chronic treatment with reserpine on the activity of the enzymes and metabolites of the glycolytic pathway in the submandibular gland was investigated (Bardon et al., 1985b). In the present study, X-ray microanalysis was used to investigate the elemental composition of submandibular gland mucous acinar cells and pancreatic acinar cells in the chronically pilocarpine-treated rat. In addition, the activity of some enzymes of the glycolytic pathway in these organs was measured. MATERIALS
AND METHODS
Female Sprague-Dawley rats (200 g) were used in this study. Pilocarpine (50 mg/kg body wt) was administered daily for 7 days by intraperitoneal injection (Sturgess and Reid, 1973). Nontreated rats were used as controls. All rats were supplied with food and water ad libitum. After the last injection the animals were starved overnight prior to removal of the gland. The submandibular glands and pancreas were excised under heavy sodium pentobarbital anesthesia and cut into pieces of 1 mm3 or less. For X-ray and biochemical analysis, the specimens were shock-frozen in liquid Freon 13 subcooled by liquid nitrogen to about - 190°C (Barnard, 1982) and then transferred to liquid nitrogen. To investigate the effects of in vitro stimulation of the glands, fragments of freshly excised tissue were washed in Krebs-Ringer buffer, and preincubated for 30 min in this buffer at 37°C. The medium was continually gassed with 95% O,/ 5% CO,. After the period of equilibration, the incubation was started in fresh buffer; after 20 min the cholinergic agonist carbachol at a concentration giving maximal stimulation (10 l&Z) was added to the incubation medium (Kuijpers and Roomans, 1984). Tissue fragments to which only buffer was added served as control. After incubation for another 30 min. the tissue fragments were shockfrozen as described above. For X-ray microanalysis at the cellular level, 16 km cryosections were cut at - 20°C in a conventional cryostat, mounted on carbon specimen holders, freezedried overnight, and coated with a conductive carbon layer (Wroblewski et al., 1978; Roomans et al., 1982a). For analysis at the subcellular level, thin cryosections (100 nm) were cut at a temperature of about - 110°C. Sections were cut and collected dry on Formvar film-covered loo-mesh nickel grids, freeze-dried at -9O”C, brought to room temperature, and coated with a thin carbon layer (Roomans et al., 1982b). X-ray microanalysis was carried out with a Kevex energy-dispersive spectrometer in combination with a Kevex 7000 multichannel analyzer/computer system, in a JEOL 100 C electron microscope with ASID-4B scanning attachment. The thick cryosections were viewed in the scanning mode and analyzed at 20 kV (Wroblewski et al. 1978). The thin sections were analyzed in the scanning-transmission mode and analyzed at 80 kV. In both cases, a stationary spot was used for analysis. Quantitative analysis was performed as described elsewhere in detail (Roomans, 1980, 1981). Morphological and morphometrical studies were carried out on tissue fixed in glutaraldehyde, postfixed with osmium tetroxide, dehydrated in a graded ethanol series and propylene oxide, and embedded in Polarbed 812. Thin (50 nm) sections were stained with many1 acetate and lead citrate and studied in a JEOL 100 S transmission electron microscope. For the morpho-
CHRONICALLY
PILOCARPINE-TREATED
99
RAT
metrical studies, a HIPAD digitizer board (Houston Instruments, Houston, Tex.) in connection with an LSI-11 (Digital) computer, was used. For enzyme studies a 5% (w/v) homogenate was made of the frozen gland in cold distilled water with an all-glass homogenizer on ice. The 1500-g supernatant was stored frozen at - 70°C until analysis. The following enzymes were analyzed: hexokinase (HK, EC 2.7.1. l), phosphofructokinase (PFK, EC 2.7.1.1 l), pyruvate kinase (PK, EC 2.7.1.40), lactate dehydrogenase (LDH, EC 1.1.1.27). and creatine phosphokinase (CPK, EC 2.7.3.2.). For these enzymes methods described by Bergmeyer (1974) were used. One unit (U) of enzyme activity corresponds to the amount of enzyme that converts 1 pmole of substrate per minute. Amylase (EC 3.2.1.1) activity was determined by the Phadebas test (Pharmacia, Uppsala, Sweden). 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 1500-g supernatant was stored frozen until analysis. Lactate and creatine phosphate were analyzed by enzymatic reactions coupled to the oxidation or reduction of pyridine nucleotides as described by Bergmeyer (1974). Protein was estimated in both the water homogenate and in the perchloric acid extract with the method of Lowry et al. (1951). For statistical analysis of the data, the nonparametric Mann-Whitney U test (Siegel, 1956), or (in those cases where n > 8 and a normal distribution of the data could be assumed) Student’s t test was used. RESULTS Chronic treatment with pilocarpine slightly increased the relative volume of mucus in the mucous acinar cells of the submandibular gland. However, the cell size and the diameter of the mucous granules were not significantly different from the controls (Table I). The plications of the lateral cell membrane of the acinar cells were retained after chronic pilocarpine treatment (Figs. 1, 2). In the pilocarpine-treated rat, the zymogen granules were more densely packed and had a less electron-dense appearance than in the controls (Figs. 3, 4). The size of the pancreatic acinar cells was slightly decreased and the number of zymogen granules and their size were increased compared to the controls (Table I). X-ray microanalysis at the cellular level showed that pilocarpine treatment induced significantly lower Na and higher Ca levels in both submandibular gland and pancreas (Table II). It could be noted that in control animals the cellular calcium level of the pancreatic acinar cells was markedly lower than that of the mucous cells of the submandibular gland. Pancreatic acinar cells of pilocarpinetreated animals had a significantly higher sulfur concentration than the controls.
Morphometry
of Submandibular
Submandibular
TABLE 1 Gland and Pancreas of Chronically Pilocarpine-Treated
gland
Cell size Relative volume of mucus Diameter of mucous granules
Rats
Pancreas 98 t II 110 k 5 96 k 11
Cell size Number of zymogen granules per cell Diameter of zymogen granules
93 2 3 129 r I? 122 k 5
Nore. All data expressed in percentage of the control value. Mean and standard error of the mean for IO- 15 cells of each tissue are given.
100
MijLLER
ET AL.
For all other elements, no significant effects of pilocarpine treatment in any of the two glands investigated could be noted. X-ray microanalysis of rat submandibular gland at the subcellular level showed that, after pilocarpine treatment, the mucous granules contained increased con-
CHRONICALLY
PILOCARPINE-TREATED
RAT
101
G. 3. Acinar cell of the pancreas
centrations of Na and Ca compared to the controls. The concentrations of P, S, and K were significantly decreased (Table III). In the pancreas, the zymogen granules showed a significant decrease in Na and S concentrations, and a weakly significant increase in Ca concentration after chronic treatment with pilocarpine.
FIG. 4. Acinar cell of the pancreas in a chronically pilocarpine-treated rat, with relative large, densely packed, less electron-dense zymogen granules than in the control. x 8500.
102
MijLLER X-ray Microanalysis
TABLE II of Acinar Cells of Submandibular
Submandibular
122 72 506 190 134 497 43
t 2 ? 2 2 2 t
8 6 35 12 9 19 5
Gland and Pancreas
gland
Control Na Mg P S Cl K Ca
ET AL.
Pancreas
Pilocarpine 71 60 529 200 145 481 77
? 2 ” t f 5 f
5 (P < 0.01) 3 21 13 9 19 7 (P < 0.01)
Control 130 54 816 234 152 528 I1
2 2 2 2 ” + *
15 3 24 11 7 20 2
Pilocarpine 76 47 748 270 138 586 19
2 + k i2 2 2
6 (P < 0.01) 2 23 7 (P < 0.02) 6 24 4 (P < 0.05)
Note. Data in mmolikg dry weight, mean and standard error for 17 control animals and 10 pilocarpine-treated animals. The statistical significance of the difference was determined by Student’s f test.
Incubation of slices of submandibular gland or pancreas with 10 p&Z carbachol caused in both glands a decrease in K and an increase in Na concentration, leading to an increase in the cellular Na/K ratio, indicative of depolarization (Fig. 5). In the submandibular gland, also, a decrease in Ca and Mg levels was noted, indicative of the release of mucus. In pilocarpine-treated rats, the loss of Ca was significantly less than in control animals (P < 0.05). In the pancreas, however, the reverse was noted: a loss of Ca was found in the pilocarpine-treated rats but not in the control animals, and this difference was significant (P < 0.02). The influence of pilocarpine treatment on the activity of some enzymes and the concentration of protein and some metabolites in the acinar cells of the submandibular gland and pancreas is summarized in Table IV. In the submandibular gland, the activity of the enzymes hexokinase (HK), phosphofructokinase (PFK), and creatine phosphokinase (CPK) is decreased, whereas in the pancreas the activity of these enzymes appears unaffected. In contrast, amylase levels are markedly increased in the pancreas of chronically pilocarpine-treated rats. The concentration of creatine phosphate increased in the submandibular gland, but was unaffected in the pancreas of pilocarpine-treated rats. in vitro
DISCUSSION In the submandibular gland the cellular Ca levels are increased after chronic treatment with pilocarpine. Pilocarpine treatment results in an increase in the X-ray Microanalysis
TABLE III of Mucous Granules and Zymogen Granules
Mucous granules (submandibular Control (20) Na Mis P S Cl K Ca
72 70 544 166 134 421 78
2 2 k _’ 2 2 k
9 5 19 6 9 31 9
gland)
Pilocarpine (10) 107 69 429 138 109 271 108
” k + * f ? *
12 (P < 0.05) 8 8(P
Zymogen granules (pancreas) Control (35) 141 58 789 246 191 436 18
2 ” 2 2 2 2 2
20 3 25 9 26 23 2
Pilocarpine (I 3) 100 55 816 190 214 445 21
2 5 k ” 5 2 +
32 4 49 12 (P < 0.01) 12 21 2
Note. Data in mmol/kg dry weight, mean and standard error. The number of measurements is given in parentheses. The statistical significance of the difference was determined by Student’s r test.
CHRONICALLY
PILOCARPINE-TREATED
t
NaiK
103
RAT Na/K
50
FIG. 5. Effect of stimulation with IO Q4 carbachol in r&o on the elemental composition of submandibular gland (a) and pancreas (b) in untreated and pilocapine-treated rats. All data are expressed relative to the unstimulated tissue slices (= 100). Open bars, untreated animals; filled bars, pilocarpinetreated animals. Mean and standard errors (denoted by thin bars) for four to six experiments with about 10 measurements each are given. Statistically significant differences between untreated and pilocarpine-treated animals (using the Mann-Whitney U test) were only demonstrated for the changes in calcium content in the submandibular gland (P < 0.05) and pancreas (P < 0.02).
relative volume of mucus in the acinar cells. In addition, analysis at the subcellular level shows this mucus to contain more calcium than in the controls. Together, these changes explain the increase in the cellular calcium concentration. The sodium levels in the submandibular acinar cells of pilocarpine-treated rats are significantly decreased. In previous X-ray microanalytical studies we have demonstrated decreased cellular Na levels in the rat submandibular gland after chronic treatment with isoproterenol (Mtiller and Roomans 1984a). after chronic TABLE IV Analysis of Enzymes and Metabolites in Submandibular Submandibular
HK PFK PK LDH CPK Amylase Lactate Creatine phosphate Total protein Acid-soluble protein
0.76 12.3 50.1 93.8 39.0
2 + 5 i5
0.06 0.9 I.4 3.3 1.7
1.8 2 0.1 0.38 2 0.02 104.8 * 3.1 7.2 k 0.6
gland Pilocarpine
Control 0.57 6.5 51.0 99.6 29.1
Gland and Pancreas
2 ” 2 IT k
0.04 (P < 0.02) 1.1 (P < 0.01) 1.4 6.9 1.4 (P < 0.01)
2.1 5 0.4 0.70 t- 0.06 (P < 0.01) 103.8 2 5.6 8.3 k 1.0
Pancreas Control 0.69 17.0 16.0 48.8 26.0 14.2 2.8
2 0.06 2 1.0 + 0.9 -+ 1.1 2 I.1 t 1.2 i 0.3
Pilocarpine 0.75 17.3 15.8 47.0 22.2 24.3 2.1
r 2 ‘k 2 k t
0.16 1.9 1.7 5.7 1.5 2.3 (P < 0.01) 0.2
0.10 k 0.04 155.1 + 8.2
0.11 2 0.03 161.0 k 8.7
2.3 ” 0.2
2.0 2 0.2
Note. Data in U/g tissue wet weight (for enzymes), mmolig wet weight (metabolites), or mgig wet weight (protein): mean and standard error of the glands of six animals. The Mann-Whitney U test was used for statistical analysis of the data.
104
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ET AL.
metabolic acidosis (Roomans and Bardon, 1984) and after a single dose of reserpine or isoproterenol (Mtiller et al., 1985). In all these cases, the cellular Na/K level is significantly decreased. The significance of this common finding in a variety of different experimental conditions remains to be clarified. The ultrastructure of the pancreatic acinar cells is markedly affected by chronic pilocarpine treatment. The number and size of the zymogen granules is slightly increased, as well as their calcium concentration. These three factors together result in increased cellular calcium levels. The increase in number and possibly also in size of the amylase-containing zymogen granules induced by pilocarpine treatment could explain both the increased amylase activity in the pancreas noted in the present study and the increased basal amylase release noted in a previous study (Kuijpers and Roomans, 1984). Zymogen granules in rat pancreas are rich in sulfur (Roomans and Wei, 1983, due to the presence of a sulfated polyanion responsible for the condensation process in the granules (Reggio and Palade, 1978). The less condensed appearance of the granules in pilocarpine-treated rats may correlate with the decrease in sulfur content. At the cellular level, the increase in number and size of the zymogen granules apparently compensates for the decreased sulfur levels in the individual granules, and results in a slight increase of the cellular sulfur level. The effect of chronic pilocarpine treatment on glycolysis and energy metabolism appears to be different for the two glands investigated. In the pancreas, the glycolytic enzymes and metabolites are not affected at all, whereas in the submandibular gland, several enzymes show a decreased activity, in accordance with findings in the reserpinized rat (Bardon et al., 1985a, b). This would result in accumulation of acidic intermediates of the glycolysis and possible acidosis of the gland. A disturbance of the energy production is also reflected in accumulation of creatine phosphate in the gland. Similar differences in response to chronic pilocarpine treatment are evident in the experiments in which the effects of in vitro stimulation of the glands were investigated. Loss of cellular calcium after stimulation represents loss of secretory material, which in both cell types is the structure containing most calcium (Roomans and Barnard, 1982). In the pancreas, loss of secretory material after maximal stimulation is most pronounced in the pilocarpine-treated rats, which fits in with the hyperreactivity towards muscarinic cholinergic stimulation noted in a previous study (Kuijpers and Roomans, 1984). The hyperreactivity at maximal cholinergic stimulation may be the result of the increased accumulation of secretory material in the cells, rather than to hypersensitivity. In the submandibular gland, the situation is different, and this would be in agreement with the findings of Ohlin (1966) who demonstrated that chronic pilocarpine treatment abolished the hypersensitivity in denervated submandibular glands of the cat. The changes observed in the rat submandibular gland after chronic treatment with pilocarpine resemble to some extent those seen in other animal models for CF, but differences can also be observed. A marked increase in the relative volume of mucus is also caused by chronic treatment with isoproterenol (Mtiller and Roomans, 1984a). On the other hand, the increase in cell size noted after chronic isoproterenol treatment was not seen with pilocarpine. The plications of the lateral cell membrane remain after chronic pilocarpine treatment, but disappear after chronic treatment with isoproterenol or reset-pine (Mtiller and Roomans, 1984a) or after chronic metabolic acidosis (Roomans and Bardon, 1984).
CHRONICALLY
PILOCARPINE-TREATED
RAT
105
The way glycolysis is affected in the submandibular glands of pilocarpine-treated rats is similar to that seen after chronic reset-pine treatment but different from that seen after chronic metabolic acidosis (Bardon et al., 1985a, b). A common feature of all animal models for CF investigated by X-ray microanalysis is the increase in cellular calcium levels in the acinar cells of the submandibular gland. The decrease in Na levels, mentioned above, occurs in most animal models, except in the chronically reserpinized rat (Roomans et al., 1982a; Miiller and Roomans, 1984a). The findings on the changes in elemental composition of the pancreas after chronic pilocarpine treatment cannot be compared directly to the other animal models, since in the pancreas adrenergic receptors, although probably present (Pearson et al., 1984), do not appear to be quantitatively significant. Davis et al. (1980) have shown that in CF patients the reactivity towards cholinergic stimulation is disturbed. The present paper shows that such a disturbance can be associated with changes in structure and function of the exocrine glands that are directly relevant to CF. The increased Ca content of the mucous granules will result in increased Ca levels of the secreted mucus. Exocrine secretions in CF patients have abnormally high calcium levels, which may be responsible for at least some of the clinical symptoms. The decrease in cellular Na content may point to a disturbed transport of this ion: abnormal Na levels in exocrine secretions (e.g., sweat) are a classical feature of CF. In this respect, it is also of interest that the elemental changes observed in the exocrine glands of the chronically pilocarpine-treated rat are also found in cultured tibroblasts from CF patients (Roomans et al., 1984) and after chronic treatment of rats with conditioned culture medium from CF fibroblasts (von Euler et al., 1985). Although the chronically pilocarpine-treated rat certainly has features of relevance in the study of CF, this does not necessarily imply that the same mechanism is responsible for the malfunctioning of the exocrine glands. The calcium content of the secretory material of exocrine glands can also be increased by other mechanisms, some of which are more effective than chronic pilocarpine treatment. ACKNOWLEDGMENTS The excellent technical assistance of Ms. Eva Bjorkner and Ms. Carina Starkerud is gratefully acknowledged. This study was financially supported by grants from the Cystic Fibrosis Research Trust (R.M.M., G.M.R.), the Swedish National Association against Heart and Chest Diseases (O.C.. G.M.R.), the Swedish Medical Research Council (Grant 12X-07125, G.M.R.), the Torsten and Ragnar Soderberg Foundation (A.B., O.C.), and the Netherlands Foundation for Biophysics (G.A.J.K.).
REFERENCES BARDON. A., MILLER, R. M., CEDER, 0.. and ROOMANS, G. M. (1985a). The chronically reserpinized rat: Decreased glycolytic activity in the submandibular gland. Biochem. Med., in press. BARDON, A.. MOLLER, R. M., CEDER. 0.. ROOMANS, G. M.. and KOLLBERG, H. (1985b). Glycolytic enzymes in the submandibular gland of reserpine treated rats. In “Animal Models for Cystic Fibrosis: The Reserpine Treated Rat” (J. R. Martinez and G. J. Barbero, ed.). San Francisco Press, San Francisco, in press. BARNARD, T. (1982). Thin frozen-dried cryosections and biological X-ray microanalysis. J. Microsc. 126, 317-332. BERGMEYER, H. U. (1974). “Methods of Enzymatic Analysis.” Academic Press, New York. BYLUND, D. B., and MARTINEZ, J. R. (1980). az-Adrenergic receptors appear in rat salivary glands after reserpine treatment. Nurure (London) 285, 229-230. DAVIS, P. B., SHELHAMER, J. R., and KALINER M. (1980). Abnormal adrenergic and chohnergic sensitivity in cystic fibrosis. N. Engl. J. Med. 302, 1453- 1456.
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KUUPERS, G. A. J., and ROOMANS, G. M. (1984). Pancreatic amylase release in chronically pilocarpine-treated rats. Acra Physiol. Stand. 121, 85-87. LOWRY, 0. H., ROSEBROUGH.N. J., FARR, A. L.. and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MANGOS, J. A., BOYD. R. L., BOUCHLAS, D., and FARNHAM. W. (1981). Isolated parotid acinar cells from oL-isoproterenol-treated rats: A cellular model for cystic fibrosis. J. Dent. Res. 60, 80-85. MARTINEZ, J. R.. ADELSTEIN, E., QUISSELL, D., and BARBERO, G. J. (1975a). The chronically reserpinized rat as a possible model for cystic fibrosis. I. Submaxillary gland morphology and ultrastructure. Pediatr. Res. 9, 463-469. MARTINEZ, J. R., ADSHEAD, P. C.. QUISSELL. D. O., and BARBERO, G. J. (1975b). The chronically reserpinized rat as a possible model for cystic fibrosis. II. Composition and cilioinhibitory effects of submaxillary saliva. Pediatr. Res. 9, 470-475. MUELLER, R. M., GRUNDIN. T. G., and ROOMANS, G. M. (1985). Effects of reserpine and isoproterenol on elemental distribution in submandibular gland of rat. Acta Physiol. Stand., 123, 383-391. MILLER, R. M., and ROOMANS. G. M. (1984a). The chronically isoproterenol-treated rat in the study of cystic fibrosis: X-ray microanalysis of the submandibular gland. Exp. Mol. P&o/. 40, 391-400. MOLLER, R. M., and ROOMANS, G. M. (1984b). X-ray microanalysis of the rat parotid gland after chronic sympathetomimetic stimulation. Exp. Mol. Purhol. 41, 363-370. OHLIN. P. (1966). Effects of treatments with isoprenaline or pilocarpine on the secretory responses of the parasympathetically denervated submaxillary gland of the rat. /. Oral Ther. Phartnaco/. 2, 376-379. PEARSON, G. T., SINGH. J., and PETERSON, 0. H. (1984). Adrenergic nervous control of CAMP-mediated amylase secretion in the rat pancreas. Am. J. Physiol. 246G, 563-573. REGGIO. H. A., and PALADE. G. E. (1978). Sulfated compounds in the zymogen granules of the guinea pig pancreas. J. Cell Biol. 77, 288-314. ROOMANS, G. M. (1980). Quantitative X-ray microanalysis of thin sections. In “X-ray Microanalysis in Biology” (M. A. Hayat. ed.), pp. 401-453. Univ. Park Press, Baltimore. ROOMANS, G. M. (1981). Quantitative electron probe X-ray microanalysis of biological bulk specimens. Scanning Electron Microsc. 2, 345-356. ROOMANS, G. M.. and BARDON, A. (1984). Effects of metabolic acidosis on acinar cells of rat submandibular gland. Res. Comtnun. Chetn. P&o/. Pharmacol. 46, 155-158. ROOMANS, G. M., and BARNARD, T. (1982). Calcium and magnesium in exocrine secretion: An X-ray microanalytical study. Scanning Elecrron Microsc. 1, 229-242. ROOMANS, G. M.. VON EULER, A. M.. and CEDER, 0. (1984). Microprobe analysis in studies and diagnosis of cystic fibrosis. Ann. N.Y. Acad. Sci. 428, 121-132. ROOMANS, G. M., and WEI, X. (1985). X-ray microanalysis of resting and stimulated rat pancreas. Acra Physiol. Stand.. in press. ROOMANS, G. M.. WEI, X.. CEDER. 0.. and KOLLBERG, H. (1982a). The reserpinized rat in the study of cystic fibrosis-X-ray microanalysis of submandibular gland and pancreas. Ulfrastruc~. Pathol. 3, 285-293. ROOMANS, G. M., WEI, X., and SEVEUS, L. (1982b). Cryoultramicrotomy as a preparative method for X-ray microanalysis in pathology. Ul~rasfrucf. Parho/. 3, 65-84. ROSCHER, A. A., WIESMANN. U. N., and HONEGGER, U. E. (1981). Changes in beta-adrenergic receptors in submaxillary glands of chronically reserpine- or isoproterenol-treated rats. J. Pharmacol. Exp.
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SIEGEL, S. (1956). “Non-parametric Statistics.” McGraw-Hill, New York. STURGESS,J., and REID, L. (1973). The effect of isoprenaline and pilocarpine on (a) bronchial mucussecreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen. Brir. J. Exp. Parhol.
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VON EULER, A. M.. CEDER, O., KOLLBERG, H.. and ROOMANS, G. M. (1985). Effects of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells. Exp. Mol. Parhol.
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WOOD, D. L.. and MARTINEZ. J. R. (1977). The chronically reserpinized rat as a possible model for cystic fibrosis. VI. Synergistic effects of isoproterenol on Ca+ + and protein in the submaxillary gland. Pediarr. Res. 11, 827-832. WR~BLEWSKI, R., ROOMANS, G. M., JANSSON. E., and EDSTR~M, L. (1978). Electron probe X-ray microanalysis of human muscle biopsies. Histochemistry 55, 281-292.
AND
MOLECULAR
The Chronically Fibrosis:
Pilocarpine-Treated Rat in the Study of Cystic Investigations on Submandibular Gland and Pancreas
R. MARGARETA
MUELLER,] GEMMA
OVE CEDER,~ ‘Depurtment Stockholm.
of Ultrastructure Sweden,
Received
43, 97-106 (1985)
PATHOLOGY
and
November
A. J. KUIJPERS,*~~ ALICJA AND GODFRIED M. ROOMANS'
Reseurch.
Wenner-Gren
‘Department
of Pediutrics. UmeB. SH,eden
30.
1984.
and
Institllte, UmeB
in revisedform
BARDON,~
University
of Stockholm,
University
Hospital,
February
25.
S-10691 S-90185
1985
The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. The effect of chronic pilocarpine treatment on structure, composition. and function of the acinar cells of rat submandibular gland and pancreas was investigated by electron microscopy, X-ray microanalysis, and biochemical analysis. The morphological effects of chronic pilocarpine treatment were most pronounced in the pancreas. The number and size of the zymogen granules was increased, and the granules had a less electron-dense appearance. X-ray microanalysis at the cellular level showed in both the submandibular gland and the pancreas a significant increase in calcium and a decrease in sodium. The increase in cellular calcium concentrations can be explained by an increase in the relative volume of secretory material in the cell (assessed by morphometry) and an increase in the local calcium concentration in the secretory material (assessed by X-ray microanalysis at the subcellular level). Chronic pilocarpine treatment caused a disturbance of glycolysis and energy metabolism in the submandibular gland. but no significant effects in this respect were noted in the pancreas. On the other hand. a nearly twofold increase of the pancreatic amylase activity was noted. The pancreas appeared somewhat hyperreactive towards cholinergic stimulation. ill 19X5 Academic Press. Inc. INTRODUCTION
The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis (CF) (Sturgess and Reid, 1973). Chronic cholinergic stimulation induces changes in the structure and function of salivary glands and pancreas that, to a certain extent, resemble the changes observed in exocrine glands of patients with CF. The main points of agreement appear to be hypersecretion and hypertrophy of the glands (Sturgess and Reid, 1973). Recently, it has been shown that chronic pilocarpine treatment of rats induces hyperreactivity of the pancreas to muscarinic cholinergic stimulation indicated by increased amylase release after stimulation by carbachol in vitro (Kuijpers and Roomans, 1984). Other suggested animal models for cystic fibrosis are the chronically reserpinized rat and the chronically isoproterenol-treated rat (Sturgess and Reid, 1973; Martinez et al., 1975a, b; Wood and Martinez 1977; Mangos et al., 1981). In these models, the sympathetic nervous system and the adrenergic receptors are influenced (Bylund and Martinez, 1980; Roscher et al., 1981), whereas in the chronically pilocarpine-treated rat, the parasympathetic system is affected. In a series of recent studies, we have used X-ray microanalysis to study the elemental composition of the exocrine glands in the chronically reserpinized and the chronically ’ Permanent address: Department of Biochemistry, HB Nijmegen, The Netherlands.
University of Nijmegen, P.O. Box 9101, 6500
97 0014-4800/85 $3.00 Copyright “1 1985 by Academic Press, Inc. All rights of reproduction in any form reserved
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isoproterenol-treated rat (Roomans et al., 1982a; Mtiller and Roomans 1984a, b). Furthermore, the effect of chronic treatment with reserpine on the activity of the enzymes and metabolites of the glycolytic pathway in the submandibular gland was investigated (Bardon et al., 1985b). In the present study, X-ray microanalysis was used to investigate the elemental composition of submandibular gland mucous acinar cells and pancreatic acinar cells in the chronically pilocarpine-treated rat. In addition, the activity of some enzymes of the glycolytic pathway in these organs was measured. MATERIALS
AND METHODS
Female Sprague-Dawley rats (200 g) were used in this study. Pilocarpine (50 mg/kg body wt) was administered daily for 7 days by intraperitoneal injection (Sturgess and Reid, 1973). Nontreated rats were used as controls. All rats were supplied with food and water ad libitum. After the last injection the animals were starved overnight prior to removal of the gland. The submandibular glands and pancreas were excised under heavy sodium pentobarbital anesthesia and cut into pieces of 1 mm3 or less. For X-ray and biochemical analysis, the specimens were shock-frozen in liquid Freon 13 subcooled by liquid nitrogen to about - 190°C (Barnard, 1982) and then transferred to liquid nitrogen. To investigate the effects of in vitro stimulation of the glands, fragments of freshly excised tissue were washed in Krebs-Ringer buffer, and preincubated for 30 min in this buffer at 37°C. The medium was continually gassed with 95% O,/ 5% CO,. After the period of equilibration, the incubation was started in fresh buffer; after 20 min the cholinergic agonist carbachol at a concentration giving maximal stimulation (10 l&Z) was added to the incubation medium (Kuijpers and Roomans, 1984). Tissue fragments to which only buffer was added served as control. After incubation for another 30 min. the tissue fragments were shockfrozen as described above. For X-ray microanalysis at the cellular level, 16 km cryosections were cut at - 20°C in a conventional cryostat, mounted on carbon specimen holders, freezedried overnight, and coated with a conductive carbon layer (Wroblewski et al., 1978; Roomans et al., 1982a). For analysis at the subcellular level, thin cryosections (100 nm) were cut at a temperature of about - 110°C. Sections were cut and collected dry on Formvar film-covered loo-mesh nickel grids, freeze-dried at -9O”C, brought to room temperature, and coated with a thin carbon layer (Roomans et al., 1982b). X-ray microanalysis was carried out with a Kevex energy-dispersive spectrometer in combination with a Kevex 7000 multichannel analyzer/computer system, in a JEOL 100 C electron microscope with ASID-4B scanning attachment. The thick cryosections were viewed in the scanning mode and analyzed at 20 kV (Wroblewski et al. 1978). The thin sections were analyzed in the scanning-transmission mode and analyzed at 80 kV. In both cases, a stationary spot was used for analysis. Quantitative analysis was performed as described elsewhere in detail (Roomans, 1980, 1981). Morphological and morphometrical studies were carried out on tissue fixed in glutaraldehyde, postfixed with osmium tetroxide, dehydrated in a graded ethanol series and propylene oxide, and embedded in Polarbed 812. Thin (50 nm) sections were stained with many1 acetate and lead citrate and studied in a JEOL 100 S transmission electron microscope. For the morpho-
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metrical studies, a HIPAD digitizer board (Houston Instruments, Houston, Tex.) in connection with an LSI-11 (Digital) computer, was used. For enzyme studies a 5% (w/v) homogenate was made of the frozen gland in cold distilled water with an all-glass homogenizer on ice. The 1500-g supernatant was stored frozen at - 70°C until analysis. The following enzymes were analyzed: hexokinase (HK, EC 2.7.1. l), phosphofructokinase (PFK, EC 2.7.1.1 l), pyruvate kinase (PK, EC 2.7.1.40), lactate dehydrogenase (LDH, EC 1.1.1.27). and creatine phosphokinase (CPK, EC 2.7.3.2.). For these enzymes methods described by Bergmeyer (1974) were used. One unit (U) of enzyme activity corresponds to the amount of enzyme that converts 1 pmole of substrate per minute. Amylase (EC 3.2.1.1) activity was determined by the Phadebas test (Pharmacia, Uppsala, Sweden). 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 1500-g supernatant was stored frozen until analysis. Lactate and creatine phosphate were analyzed by enzymatic reactions coupled to the oxidation or reduction of pyridine nucleotides as described by Bergmeyer (1974). Protein was estimated in both the water homogenate and in the perchloric acid extract with the method of Lowry et al. (1951). For statistical analysis of the data, the nonparametric Mann-Whitney U test (Siegel, 1956), or (in those cases where n > 8 and a normal distribution of the data could be assumed) Student’s t test was used. RESULTS Chronic treatment with pilocarpine slightly increased the relative volume of mucus in the mucous acinar cells of the submandibular gland. However, the cell size and the diameter of the mucous granules were not significantly different from the controls (Table I). The plications of the lateral cell membrane of the acinar cells were retained after chronic pilocarpine treatment (Figs. 1, 2). In the pilocarpine-treated rat, the zymogen granules were more densely packed and had a less electron-dense appearance than in the controls (Figs. 3, 4). The size of the pancreatic acinar cells was slightly decreased and the number of zymogen granules and their size were increased compared to the controls (Table I). X-ray microanalysis at the cellular level showed that pilocarpine treatment induced significantly lower Na and higher Ca levels in both submandibular gland and pancreas (Table II). It could be noted that in control animals the cellular calcium level of the pancreatic acinar cells was markedly lower than that of the mucous cells of the submandibular gland. Pancreatic acinar cells of pilocarpinetreated animals had a significantly higher sulfur concentration than the controls.
Morphometry
of Submandibular
Submandibular
TABLE 1 Gland and Pancreas of Chronically Pilocarpine-Treated
gland
Cell size Relative volume of mucus Diameter of mucous granules
Rats
Pancreas 98 t II 110 k 5 96 k 11
Cell size Number of zymogen granules per cell Diameter of zymogen granules
93 2 3 129 r I? 122 k 5
Nore. All data expressed in percentage of the control value. Mean and standard error of the mean for IO- 15 cells of each tissue are given.
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For all other elements, no significant effects of pilocarpine treatment in any of the two glands investigated could be noted. X-ray microanalysis of rat submandibular gland at the subcellular level showed that, after pilocarpine treatment, the mucous granules contained increased con-
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G. 3. Acinar cell of the pancreas
centrations of Na and Ca compared to the controls. The concentrations of P, S, and K were significantly decreased (Table III). In the pancreas, the zymogen granules showed a significant decrease in Na and S concentrations, and a weakly significant increase in Ca concentration after chronic treatment with pilocarpine.
FIG. 4. Acinar cell of the pancreas in a chronically pilocarpine-treated rat, with relative large, densely packed, less electron-dense zymogen granules than in the control. x 8500.
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TABLE II of Acinar Cells of Submandibular
Submandibular
122 72 506 190 134 497 43
t 2 ? 2 2 2 t
8 6 35 12 9 19 5
Gland and Pancreas
gland
Control Na Mg P S Cl K Ca
ET AL.
Pancreas
Pilocarpine 71 60 529 200 145 481 77
? 2 ” t f 5 f
5 (P < 0.01) 3 21 13 9 19 7 (P < 0.01)
Control 130 54 816 234 152 528 I1
2 2 2 2 ” + *
15 3 24 11 7 20 2
Pilocarpine 76 47 748 270 138 586 19
2 + k i2 2 2
6 (P < 0.01) 2 23 7 (P < 0.02) 6 24 4 (P < 0.05)
Note. Data in mmolikg dry weight, mean and standard error for 17 control animals and 10 pilocarpine-treated animals. The statistical significance of the difference was determined by Student’s f test.
Incubation of slices of submandibular gland or pancreas with 10 p&Z carbachol caused in both glands a decrease in K and an increase in Na concentration, leading to an increase in the cellular Na/K ratio, indicative of depolarization (Fig. 5). In the submandibular gland, also, a decrease in Ca and Mg levels was noted, indicative of the release of mucus. In pilocarpine-treated rats, the loss of Ca was significantly less than in control animals (P < 0.05). In the pancreas, however, the reverse was noted: a loss of Ca was found in the pilocarpine-treated rats but not in the control animals, and this difference was significant (P < 0.02). The influence of pilocarpine treatment on the activity of some enzymes and the concentration of protein and some metabolites in the acinar cells of the submandibular gland and pancreas is summarized in Table IV. In the submandibular gland, the activity of the enzymes hexokinase (HK), phosphofructokinase (PFK), and creatine phosphokinase (CPK) is decreased, whereas in the pancreas the activity of these enzymes appears unaffected. In contrast, amylase levels are markedly increased in the pancreas of chronically pilocarpine-treated rats. The concentration of creatine phosphate increased in the submandibular gland, but was unaffected in the pancreas of pilocarpine-treated rats. in vitro
DISCUSSION In the submandibular gland the cellular Ca levels are increased after chronic treatment with pilocarpine. Pilocarpine treatment results in an increase in the X-ray Microanalysis
TABLE III of Mucous Granules and Zymogen Granules
Mucous granules (submandibular Control (20) Na Mis P S Cl K Ca
72 70 544 166 134 421 78
2 2 k _’ 2 2 k
9 5 19 6 9 31 9
gland)
Pilocarpine (10) 107 69 429 138 109 271 108
” k + * f ? *
12 (P < 0.05) 8 8(P
Zymogen granules (pancreas) Control (35) 141 58 789 246 191 436 18
2 ” 2 2 2 2 2
20 3 25 9 26 23 2
Pilocarpine (I 3) 100 55 816 190 214 445 21
2 5 k ” 5 2 +
32 4 49 12 (P < 0.01) 12 21 2
Note. Data in mmol/kg dry weight, mean and standard error. The number of measurements is given in parentheses. The statistical significance of the difference was determined by Student’s r test.
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NaiK
103
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50
FIG. 5. Effect of stimulation with IO Q4 carbachol in r&o on the elemental composition of submandibular gland (a) and pancreas (b) in untreated and pilocapine-treated rats. All data are expressed relative to the unstimulated tissue slices (= 100). Open bars, untreated animals; filled bars, pilocarpinetreated animals. Mean and standard errors (denoted by thin bars) for four to six experiments with about 10 measurements each are given. Statistically significant differences between untreated and pilocarpine-treated animals (using the Mann-Whitney U test) were only demonstrated for the changes in calcium content in the submandibular gland (P < 0.05) and pancreas (P < 0.02).
relative volume of mucus in the acinar cells. In addition, analysis at the subcellular level shows this mucus to contain more calcium than in the controls. Together, these changes explain the increase in the cellular calcium concentration. The sodium levels in the submandibular acinar cells of pilocarpine-treated rats are significantly decreased. In previous X-ray microanalytical studies we have demonstrated decreased cellular Na levels in the rat submandibular gland after chronic treatment with isoproterenol (Mtiller and Roomans 1984a). after chronic TABLE IV Analysis of Enzymes and Metabolites in Submandibular Submandibular
HK PFK PK LDH CPK Amylase Lactate Creatine phosphate Total protein Acid-soluble protein
0.76 12.3 50.1 93.8 39.0
2 + 5 i5
0.06 0.9 I.4 3.3 1.7
1.8 2 0.1 0.38 2 0.02 104.8 * 3.1 7.2 k 0.6
gland Pilocarpine
Control 0.57 6.5 51.0 99.6 29.1
Gland and Pancreas
2 ” 2 IT k
0.04 (P < 0.02) 1.1 (P < 0.01) 1.4 6.9 1.4 (P < 0.01)
2.1 5 0.4 0.70 t- 0.06 (P < 0.01) 103.8 2 5.6 8.3 k 1.0
Pancreas Control 0.69 17.0 16.0 48.8 26.0 14.2 2.8
2 0.06 2 1.0 + 0.9 -+ 1.1 2 I.1 t 1.2 i 0.3
Pilocarpine 0.75 17.3 15.8 47.0 22.2 24.3 2.1
r 2 ‘k 2 k t
0.16 1.9 1.7 5.7 1.5 2.3 (P < 0.01) 0.2
0.10 k 0.04 155.1 + 8.2
0.11 2 0.03 161.0 k 8.7
2.3 ” 0.2
2.0 2 0.2
Note. Data in U/g tissue wet weight (for enzymes), mmolig wet weight (metabolites), or mgig wet weight (protein): mean and standard error of the glands of six animals. The Mann-Whitney U test was used for statistical analysis of the data.
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metabolic acidosis (Roomans and Bardon, 1984) and after a single dose of reserpine or isoproterenol (Mtiller et al., 1985). In all these cases, the cellular Na/K level is significantly decreased. The significance of this common finding in a variety of different experimental conditions remains to be clarified. The ultrastructure of the pancreatic acinar cells is markedly affected by chronic pilocarpine treatment. The number and size of the zymogen granules is slightly increased, as well as their calcium concentration. These three factors together result in increased cellular calcium levels. The increase in number and possibly also in size of the amylase-containing zymogen granules induced by pilocarpine treatment could explain both the increased amylase activity in the pancreas noted in the present study and the increased basal amylase release noted in a previous study (Kuijpers and Roomans, 1984). Zymogen granules in rat pancreas are rich in sulfur (Roomans and Wei, 1983, due to the presence of a sulfated polyanion responsible for the condensation process in the granules (Reggio and Palade, 1978). The less condensed appearance of the granules in pilocarpine-treated rats may correlate with the decrease in sulfur content. At the cellular level, the increase in number and size of the zymogen granules apparently compensates for the decreased sulfur levels in the individual granules, and results in a slight increase of the cellular sulfur level. The effect of chronic pilocarpine treatment on glycolysis and energy metabolism appears to be different for the two glands investigated. In the pancreas, the glycolytic enzymes and metabolites are not affected at all, whereas in the submandibular gland, several enzymes show a decreased activity, in accordance with findings in the reserpinized rat (Bardon et al., 1985a, b). This would result in accumulation of acidic intermediates of the glycolysis and possible acidosis of the gland. A disturbance of the energy production is also reflected in accumulation of creatine phosphate in the gland. Similar differences in response to chronic pilocarpine treatment are evident in the experiments in which the effects of in vitro stimulation of the glands were investigated. Loss of cellular calcium after stimulation represents loss of secretory material, which in both cell types is the structure containing most calcium (Roomans and Barnard, 1982). In the pancreas, loss of secretory material after maximal stimulation is most pronounced in the pilocarpine-treated rats, which fits in with the hyperreactivity towards muscarinic cholinergic stimulation noted in a previous study (Kuijpers and Roomans, 1984). The hyperreactivity at maximal cholinergic stimulation may be the result of the increased accumulation of secretory material in the cells, rather than to hypersensitivity. In the submandibular gland, the situation is different, and this would be in agreement with the findings of Ohlin (1966) who demonstrated that chronic pilocarpine treatment abolished the hypersensitivity in denervated submandibular glands of the cat. The changes observed in the rat submandibular gland after chronic treatment with pilocarpine resemble to some extent those seen in other animal models for CF, but differences can also be observed. A marked increase in the relative volume of mucus is also caused by chronic treatment with isoproterenol (Mtiller and Roomans, 1984a). On the other hand, the increase in cell size noted after chronic isoproterenol treatment was not seen with pilocarpine. The plications of the lateral cell membrane remain after chronic pilocarpine treatment, but disappear after chronic treatment with isoproterenol or reset-pine (Mtiller and Roomans, 1984a) or after chronic metabolic acidosis (Roomans and Bardon, 1984).
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The way glycolysis is affected in the submandibular glands of pilocarpine-treated rats is similar to that seen after chronic reset-pine treatment but different from that seen after chronic metabolic acidosis (Bardon et al., 1985a, b). A common feature of all animal models for CF investigated by X-ray microanalysis is the increase in cellular calcium levels in the acinar cells of the submandibular gland. The decrease in Na levels, mentioned above, occurs in most animal models, except in the chronically reserpinized rat (Roomans et al., 1982a; Miiller and Roomans, 1984a). The findings on the changes in elemental composition of the pancreas after chronic pilocarpine treatment cannot be compared directly to the other animal models, since in the pancreas adrenergic receptors, although probably present (Pearson et al., 1984), do not appear to be quantitatively significant. Davis et al. (1980) have shown that in CF patients the reactivity towards cholinergic stimulation is disturbed. The present paper shows that such a disturbance can be associated with changes in structure and function of the exocrine glands that are directly relevant to CF. The increased Ca content of the mucous granules will result in increased Ca levels of the secreted mucus. Exocrine secretions in CF patients have abnormally high calcium levels, which may be responsible for at least some of the clinical symptoms. The decrease in cellular Na content may point to a disturbed transport of this ion: abnormal Na levels in exocrine secretions (e.g., sweat) are a classical feature of CF. In this respect, it is also of interest that the elemental changes observed in the exocrine glands of the chronically pilocarpine-treated rat are also found in cultured tibroblasts from CF patients (Roomans et al., 1984) and after chronic treatment of rats with conditioned culture medium from CF fibroblasts (von Euler et al., 1985). Although the chronically pilocarpine-treated rat certainly has features of relevance in the study of CF, this does not necessarily imply that the same mechanism is responsible for the malfunctioning of the exocrine glands. The calcium content of the secretory material of exocrine glands can also be increased by other mechanisms, some of which are more effective than chronic pilocarpine treatment. ACKNOWLEDGMENTS The excellent technical assistance of Ms. Eva Bjorkner and Ms. Carina Starkerud is gratefully acknowledged. This study was financially supported by grants from the Cystic Fibrosis Research Trust (R.M.M., G.M.R.), the Swedish National Association against Heart and Chest Diseases (O.C.. G.M.R.), the Swedish Medical Research Council (Grant 12X-07125, G.M.R.), the Torsten and Ragnar Soderberg Foundation (A.B., O.C.), and the Netherlands Foundation for Biophysics (G.A.J.K.).
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WOOD, D. L.. and MARTINEZ. J. R. (1977). The chronically reserpinized rat as a possible model for cystic fibrosis. VI. Synergistic effects of isoproterenol on Ca+ + and protein in the submaxillary gland. Pediarr. Res. 11, 827-832. WR~BLEWSKI, R., ROOMANS, G. M., JANSSON. E., and EDSTR~M, L. (1978). Electron probe X-ray microanalysis of human muscle biopsies. Histochemistry 55, 281-292.