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Changes in rat parotid saliva protein composition following chronic reserpine treatment and their relation to inanition
Archs oral Bid. Vol. 33, No. 7, pp. 463&, Printed in Great Britain. All rights reserved
1988
0003-9969/88$3.00+ 0.00 Copyright 0 1988 Pergamon Press plc
CHANGES IN RAT PAROTID SALIVA PROTEIN COMPOSITION FOLLOWING CHRONIC RElSERPINE TREATMENT AND THEIR RELATION TO INANITION DORTHEA A. JOHNSON Department of Community Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. Summary-_(~hronic administration of the catecholamine-depleting agent, reserpine (0.5 mg/kg), resulted in a reduction in food intake after 3 days. To differentiate effects of the drug from those of reduced food intake a pair-fed group, whose daily caloric intake was restricted to the amount consumed by the reserpine-treated rats, was included. After 7 days, both the reserpine-treated and pair-fed control exhibited a marked retzluction in the volume of saliva collected in a 30 min interval following a secretory stimulus compared tcl untreated ad libitum-fed controls, and the proportion of salivary proteins attributable to acidic and basic proline-rich proteins and to minor 1b protein were decreased whereas deoxyribonuclease was increaseed. For two of the salivary proteins (fractions I and V) changes for the reserpine-treated and pair-fed groups were different. Fraction I was reduced in both groups, but exhibited a greater decrease in the pair&d than in the reserpine-treated, whereas fraction V was significantly increased only in the pair-fed group. Thus many of the salivary changes associated with reserpine treatment may have resulted from the change in feeding habits and not from reserpine treatment per se. The study demonstrates the importance of controlling for food intake under experimental circumstances which may lead to a marked change in daily feeding habits.
INTRODUCflON
MATERIALS AND METHODS
The weight of the rat parotid gland, as well as the synthesis of secretory proteins, is normally regulated in large measure by secretory activity induced during the feeding cycle. During feeding, neurally mediated reflex stimulation of the gland results in the output of fluid and protein; fluid output is primarily mediated by the parasympathetic branch and protein output primarily by the sympathetic branch @piers and Hodgson, 1976). When rats are fed a diet of liquid texture there is a :profound atrophy of the parotid gland within 7 days (Hall and Schneyer, 1964). A similar atrophy only occurs when gland stimulation via both the sympathetic and parasympathetic nerves is prevented (Hall and Schneyer, 1973). The dietinduced atrophy is accompanied by a change in the protein composition of parotid saliva such that the proline-rich proteins are reduced by over 50 per cent (Johnson, 1984). Conversely, the quantities of such proteins in rat parotid saliva can be increased by chronic treatment with isoproterenol, a drug which acts via the gland’s sympathetic beta-adrenergic receptors (Schneyer, 1969; Fernandez-Sorensen and Carlson, 1974; Muenzer et al., 1979; Johnson, 1984). This suggests that the proline-rich proteins are reduced in the parotlld saliva of rats fed a liquid diet because of decreased stimulation of the gland via the sympathetic pathway. I have now sought to evaluate the changes in parotid saliva protein composition resulting from decreased gland stimulation via the sympathetic pathway following administration of reserpine, a drug which depletes the neurotransmitter at the post-ganglionic sympathetic nerve endings (Benmiloud and vonEuler, 1963).
The daily food intake of adult male Sprague-Dawley rats was determined for 3 days. The rats were then distributed into 3 groups such that the daily food intake and initial body weights in each group were similar. The control group (n = 8) received daily injections of saline. The experimental group was given daily injections of reserpine (Serpasil, Ciba Pharmaceutical Company, Summit, NJ 07501, 0.5 mg/kg; n = 9) for 7 d,ys (Martinez et al., 1979). Each animal in the third group, a pair-fed control group (n = 9), was paired with an animal in the reserpine-treated group in such a manner that their initial body weight and mean daily food intake were the same. The food intake of each animal in this pair-fed control group was limited to that amount consumed on the previous night by the partner in the reserpine-treated group. One day after the last injection or restricted food intake, the parotid ducts were surgically cannulated under pentobarbital anaesthesia and secretion was stimulated by injection of both isoproterenol (5 mg/animal) and pilocarpine (5 mg/animal). Pilocarpine was used to increase salivary flow because saliva elicited by isoproterenol alone often gels at the end of the cannula owing to its high protein concentration and low flow rate. The saliva was collected for 30 min into a tared vial kept on ice and the weight collected was recorded. The protein concentration of the saliva was determined by the technique of Lowry et al. (1951), and by absorption at 215 nm (Arneberg, 1971) using bovine serum albumin as a standard. A sample of saliva containing 50 pg of protein by absorption at 215 nm was lyophilized, and then subjected to slab gel electrophoresis
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Table 2. Effect of reserpine and restricted food intake on body weight, parotid saliva volume and protein concentration*
Table 1. Effect of reserpine on food intake* Food intake (g/day) Reserpine (n = 9)
Pair-fed (n = 9)
22.4 f 0.4 22.6 k 0.5 26.6 + 0.9 23.2 + 1.6 1.5 + 1.1 1.0 f 0.5 0.2kO.l 0.2 + 0. I
22.6 k 0.4 22.3 k 0.4 23.4 k 0.5 21.4 f 1.4 1.5 + 1.1 1.0 * 0.5 0.2+0.1 0.2 k 0.1
Control (n =8)
Pre-experimental Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
22.6 & 0.9 22.1 f 0.1 23.1 i 0.5 24.8 + 0.8 23.9 + 0.1 24.3 + 1.2 26.0 i 1.2 25.6 k 1.2
*Pre-experimental food intake refers to the mean intake per animal in each group for the 3 days prior to the start of the experiment. Day refers to experimental day as the pair-fed group started 1 day later than the other two groups because their food intake was restricted to that amount consumed the previous night by the partner in the reserpine-treated group. In some cases not all animals of the pair-fed group consumed their entire allotment and therefore the mean food intake in the first few
days was slightly less than that of the reserpine-treated animals. using the conditions described by Laemmli and Favre (1973). The gels were stained with Coomassie blue and then destained. Colour positive films taken of the gels were scanned with a microprocessor-controlled spectrophotometer (Gilford 2600, Gilford Systems, 132 Artino St. Oberlin, OH 44074) equipped with a scanning device and with output to an x-y plotter (Hewlett Packard, 7225B, Hewlett Packard, 16399 W. Bernard0 Drive, San Diego, CA 92127). The area subtended by each protein component was determined using software within the spectrophotometer. The individual areas were summed to give total area, and the per cent of total area attributable to each peak was calculated. The identity of the individual protein bands was known from previous studies (Keller et al., 1975; Johnson, 1984). The data were analysed by analysis of variance or, in the case of the gel scans, by analysis of covariance with gel number as the covariate (SPSS, Nie et al., 1975). RESULTS
The mean daily food intake per animal in the reset-pine-treated group was similar to that of the
Table 3. Protein composition
Control (n = 8)
Reserpine (n = 9)
Body weight (g)
Initial
209_+4 209 f 3 299 + 4’ 181 f 3’ Weight of saliva collected in 30min mg/gland pair 466 & 35’,2 345 * 211 Final
Protein concentration (mg/ml) Lowry 24.01 i 0.80
Absorption at 215 nm
Control (n = 8)
Basic PRP:peak A Amylase Basic PRP: SP- 1 Acidic PRPs Deoxyribonuclease Fraction V Basic PRP:SP-3 Fraction I Minor la Minor lb
1.91 & 0.16’,2 31.12 f. 1.26 1.69 + 0.35 1.16 k 0.78’,2 10.81 k 0.39’,* 15.13 kO.14’ 2.45 + 0.21’,2 20.19 z 0.16’ 0.91 f 0.06’ 1.91 * 0.11’,2
209 f 3 209k3’ 333 * 272
25.35 k 0.95’ 21.64 + 1.30’
29.26 i 1.66 29.11 + 1.08 26.63 + 1.58
*Means sharing a common superscript differ significantly (p d 0.05) by Duncan’s new multiple range test (Bruning and Kintz, 1911).
control group for the first 3 days (Table 1). On day 4, the food intake of the treated group was reduced by two-thirds and on days 5, 6 and 7, it was negligible. At the end of the study, the mean body weight of the reserpine-treated group was 28 g less than its starting body weight, while that for the pair-fed group, which started the experimental period 1 day after the reserpine-treated group, was the same as its starting weight (Table 2). The weight of saliva collected in the 30 min interval was reduced by about 25 per cent for both the reserpine-treated and pair-fed control animals (Table 2). By the Lowry assay the protein concentration for the pair-fed group was lower than for the reserpinetreated group but was not different from the control. There were no differences among the groups when protein concentration was measured by absorbance at 215 nm. Among the groups there were marked differences in protein composition as shown by changes in the proportional distribution of gel-scan area attributable to several of the protein components (Table 3). Many of these changes were the same for the reserpine-treated and pair-fed controls: both exhibited a decrease in acidic proline-rich proteins; in two of the three basic proline-rich proteins (peak A and SP-3); in fraction I and in minor lb; and an increase in deoxyribonuclease. For fraction V, the mean for the reserpine-treated group did not differ
of rat parotid saliva determined by gel scanning*
Protein component
Pair-fed (n = 9)
Reserpine (n = 9) 0.64 k 0.04’ 38.95 + 1.54 1.14~0.11 3.09 & 0.15’ 18.54 f 0.10’ 16.04 + 0.832 1.30 + 0.01’ 17.53 + 0.51’ 1.09+0.11 1.09 kO.13’
Pair-fed (n = 9) 0.68 f 0.092 36.01 k 1.39 2.06 + 0.23 3.39 f o.192 11.68 f 0.45* 20.56 k 0.95’,* 1.13 f0.112 15.35 kO.71’ 1.15 * 0.07’ 1.40*0.132
*Per cent of total area attributable to each component. Means sharing a common superscript differ significantly (p < 0.05) by Duncan’s new multiple range test (Bruning and Kintz, 1911).
Saliva after reserpine or starvation statistically from that of the control group, but was significantly less than that for the pair-fed group. For fraction I, there were significant differences among all groups with the reduction for the pair-fed group being greater than that for the reserpine-treated group. DISCUSSION
Reserpine is a catecholamine-depleting drug often used to study stimulus-secretion coupling in rodent salivary glands. In parotid gland, the secretion of protein, electrolytes and fluids is impaired following 7 days of treatment with a dose of 0.5 mg/kg (Setser et al., 1979; Martinez et al., 1979; Watson et al., 1984). At the ultras,tructural level, Setser et al. (1979) reported that the acinar cells exhibit an accumulation of secretory material and a reduction in endoplasmic reticulum, changes suggestive of impaired discharge and synthesis of protein. They noted that the morphology of the acinar cells of the reserpine-treated animal is similar tcl that reported by Hand (1972) in acutely starved rats. The influence of reserpine treatment on the morphology of the parotid gland secretory granule has been reported by Muller and Roomans (1987) who suggest that the changes may occur in two stages. From days 1 to 3 there is an increase in electron-lucent granules; after day 3 the electronlucent granules rapidly disappear and are replaced by electron-dense granules, which are also common in acutely starved rats. As the synthesis and secretion of secretory proteins is regulated in large measure by gland stimulation via the sympathetic pathway, these morphological observations suggest that the transfer of a sympathetic stimulus is impaired by reserpine treatment. This is further supported by the study of Jirakulsomchok, Yu and Schneyer (1984) who found that 24 h after a single injection of reserpine, stimulation of the sympathetic nerve to the parotid gland does not evoke a secretory flow but that the flow elicited by parasympathetic nerve stimulation is not affected. In my study I have attempted to use reserpine as a means to prevent the transfer of a sympathetic stimulus to the rat parotid gland for a period of several days (leaving the parasympathetic transfer intact), thereby assessing the role of the sympathetic branch in the regulation of the synthesis of prolinerich proteins. As th’e mastication of food is a primary factor in regulating parotid function, a pair-fed control group was inchtded to differentiate the effects of reserpine from those that might result from an alteration in food intake. Many changes observed in my reserpine-treated animals were the same as in the pair-fed group, and therefore it cannot be concluded that reserpine per se had an effect on the salivary factors. The reduction in the proline-rich proteins in parotid saliva in the reserpine group was anticipated by the hypothesis that their synthesis is regulated, at least in part, by gland stimulation via the sympathetic pathway. Their reduction in the pair-fed group (where there was no mastication due to acute starvation) is in keeping with the concept that, in the absence of gland secretory activity, such proteins are reduced in parotid saliva. Although my study does not confirm that ;I decrease in these proteins in
465
parotid saliva is mediated by decreased gland stimulation via the sympathetic pathway, other studies indicate that this pathway is of major importance, For example, Asking et al. (1987) demonstrated that proline-rich proteins are reduced in rat parotid saliva 1 week following removal of the superior cervical ganglion, and Cortez and Johnson (1987) showed there was a reduction in these proteins after 1 week of treatment with the beta-adrenergic antagonists, metoprolol or propranolol. Mine is the first study as far as I am aware to include a pair-fed group when evaluating the effect of reserpine on salivary gland function, and it clearly shows that pair-fed controls must be included where experimental conditions result in marked alterations in daily food intake. The effect of alterations in feeding habits on parotid salivary proteins varies with the degree of food restriction and its duration. A limited reduction, i.e. 2 to 3 g per day for a period of 1 to 2 weeks, has no measurable effect on the composition of proteins in rat parotid saliva (Johnson et al., 1987). A severe reduction of 30 to 50 per cent for a period of 5 to 6 weeks results in a marked increase in their proportion in rat parotid saliva (Johnson and Alvares, 1984). In my study, the degree of food restriction was severe (almost 100 per cent), although the duration was minimal (3 days); under these conditions there was a decrease in proportion of total protein in parotid saliva attributable to proline-rich proteins. The need to control for alterations in feeding habits extends to other organ tissues. Hazlett, Korc and Brannon (1986) reported that many of the changes in pancreatic exocrine function in chronically reserpinized rats (0.5 mg/day for 7days) were also found in pair-fed controls, and they concluded that such controls were needed to differentiate effects of reserpine from those related to the associated state of malnutrition. Acknowledgements-The expert technical support of Mr Joseph E. Cortez, MS Lucinda Girard-Aparicio and MS Greta Vance are gratefully acknowledged. This research was supported by USPHS Grant ROI-DE-06000 from the National Institutes of Dental Research, National Institutes of Health, Bethesda, MD, 20892, U.S.A. REFERENCES Arneberg P. (1971) Quantitative determination of protein in saliva. A comparison of analytical methods. Stand. J. dent. Res. 19, 60-64. Asking B., Clarke G., Garrett J. R. and Proctor G. B. (1987) Effects of sympathectomy on the protein content of parasympathetic saliva from parotid glands of rats. J. Physiol. 390, 171P. Benmiloud M. and vonEuler U. S. (1963) Effects of bretyhum, reserpine, guanethidine and sympathetic denervation on the noradrenaline content of the rat submaxillary gland. Acta physiol. stand. 59, 3442. Bruning J. L. and Kintz B. L. (1977) Computational Handbook of Statistics, 2nd edn, pp. 116119, Scott, Foresman, Glenview, Ill. Cortez J. E. and Johnson D. A. (1987) Reduction in salivary proline-rich proteins following beta receptor blockade. J. dent Res. 66, 306 (abstr. 1597). Fernandez-Sorensen A. and Carlson D. M. (1974) Isolation of a “proline-rich” protein from rat parotid glands following isoproterenol treatment. Biochem. biophys. Res. Commun. 60, 249-256.
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DORTHEA A. JOHNKIN
Hall H. D. and Schneyer C. A. (1964) Salivary gland atrophy in rat induced by liquid diet. Proc. Sot. exp. Biol. Med. 117, 789-793. Hall H. D. and Schneyer C. A. (1973) Role of autonomic pathways in disuse atrophy of rat parotid. Proc. Sot. exp. Biol. Med. 143, 19-22.
Hand A. R. (1972) The effects of acute starvation on parotid acinar cells. Ultrastructural and cytochemical observations on ad libitum-fed and starved rats. Am J. Anaf. 135, 71-92. Hazlett D., Korc M. and Brannon P. M. (1986) Effects of malnutrition and chronic reserpine treatment on pancreatic exocrine function. Pediat. Res. 20, 12361239. Jirakulsomchok D., Yu J.-H. and Schneyer C. A. (1984) Secretory responses to autonomic stimulation of rat salivary glands following reserpine treatment. Archs oral Biol. 29, 394.
Johnson D. A. (1984) Changes in rat parotid salivary proteins associated with liquid diet-induced gland atrophy and isoproterenol-induced gland enlargement. Archs oral Biol. 29, 215-221. Johnson D. A. and Alvares 0. F. (1984) Zinc deficiency-induced changes in rat parotid salivary proteins. J. Nufr. 114, 19551964. Johnson D. A., Etzel K. R., Alvares 0. F. and Cortez J. E. (1987) Regulation of parotid salivary proteins by glucocorticoids. J. dent. Res. 66, 1563-1568. Keller P. J., Robinovitch M., Iversen J. and Kauffman D. L. (1975) The protein composition of rat parotid saliva and secretory granules. Biochim. biophys. Acra 379, 562-570. Laemmli U. K. and Favre M. (1973) Maturation ofthe head of bacteriophage T4. J. molec. Biol. 80, 575-599.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin nhenol reagent. J. ‘biol. Chem. 193, 265-275. Martinez J. R., Braddock M. E., Martinez A. M. and Cooper C. (1979) Effect of chronic reserpine administration on K+ and amylase release from the rat parotid gland. Pediut. Res. 13, 1024-1029. Muenzer J., Bildstein C., Gleason M. and Carlson D. M. (1979) Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats. J. biol. Chem. 254, 5623-5628.
Muller R. M. and Roomans G. M. (1987) Effects of reserpine treatment on the ultrastructure of the rat parotid and submandibular gland. J. Submicrosc. Cytol. 19, 283-289.
Nie N. H., Hull C. H., Jenkins J. G., Steinbrenner K. and Bent D. H. (1975) SPSS: Statistical Package for the Social Sciences, 2nd edn, Chap. 22, pp. 398422.-McGraw-Hill, New York. Schneyer C. A. (1969) /I-Adrenergic effects by autonomic agents on mitosis and hypertrophy in rat parotid. Proc. Sot. exp. Biol. Med. 131, 71-75.
Setser M. E., Spicer S. S., Simson J. A. V. and Martinez J. R. (1979) Altered granule discharge and amylase secretion of parotid glands in reserpine-treated rats. Lab. Invest. 41, 256264. Spiers R. L. and Hodgson C. (1976) Control of amylase secretion in the parotid gland during feeding. Archs oral Biol. 21, 539-544.
Watson E., Dowd F., Jacobson K. and Horwitz H. (1984) Reserpinization: effects on parotid gland function. J. dent. Res. 63, 82-86.
1988
0003-9969/88$3.00+ 0.00 Copyright 0 1988 Pergamon Press plc
CHANGES IN RAT PAROTID SALIVA PROTEIN COMPOSITION FOLLOWING CHRONIC RElSERPINE TREATMENT AND THEIR RELATION TO INANITION DORTHEA A. JOHNSON Department of Community Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. Summary-_(~hronic administration of the catecholamine-depleting agent, reserpine (0.5 mg/kg), resulted in a reduction in food intake after 3 days. To differentiate effects of the drug from those of reduced food intake a pair-fed group, whose daily caloric intake was restricted to the amount consumed by the reserpine-treated rats, was included. After 7 days, both the reserpine-treated and pair-fed control exhibited a marked retzluction in the volume of saliva collected in a 30 min interval following a secretory stimulus compared tcl untreated ad libitum-fed controls, and the proportion of salivary proteins attributable to acidic and basic proline-rich proteins and to minor 1b protein were decreased whereas deoxyribonuclease was increaseed. For two of the salivary proteins (fractions I and V) changes for the reserpine-treated and pair-fed groups were different. Fraction I was reduced in both groups, but exhibited a greater decrease in the pair&d than in the reserpine-treated, whereas fraction V was significantly increased only in the pair-fed group. Thus many of the salivary changes associated with reserpine treatment may have resulted from the change in feeding habits and not from reserpine treatment per se. The study demonstrates the importance of controlling for food intake under experimental circumstances which may lead to a marked change in daily feeding habits.
INTRODUCflON
MATERIALS AND METHODS
The weight of the rat parotid gland, as well as the synthesis of secretory proteins, is normally regulated in large measure by secretory activity induced during the feeding cycle. During feeding, neurally mediated reflex stimulation of the gland results in the output of fluid and protein; fluid output is primarily mediated by the parasympathetic branch and protein output primarily by the sympathetic branch @piers and Hodgson, 1976). When rats are fed a diet of liquid texture there is a :profound atrophy of the parotid gland within 7 days (Hall and Schneyer, 1964). A similar atrophy only occurs when gland stimulation via both the sympathetic and parasympathetic nerves is prevented (Hall and Schneyer, 1973). The dietinduced atrophy is accompanied by a change in the protein composition of parotid saliva such that the proline-rich proteins are reduced by over 50 per cent (Johnson, 1984). Conversely, the quantities of such proteins in rat parotid saliva can be increased by chronic treatment with isoproterenol, a drug which acts via the gland’s sympathetic beta-adrenergic receptors (Schneyer, 1969; Fernandez-Sorensen and Carlson, 1974; Muenzer et al., 1979; Johnson, 1984). This suggests that the proline-rich proteins are reduced in the parotlld saliva of rats fed a liquid diet because of decreased stimulation of the gland via the sympathetic pathway. I have now sought to evaluate the changes in parotid saliva protein composition resulting from decreased gland stimulation via the sympathetic pathway following administration of reserpine, a drug which depletes the neurotransmitter at the post-ganglionic sympathetic nerve endings (Benmiloud and vonEuler, 1963).
The daily food intake of adult male Sprague-Dawley rats was determined for 3 days. The rats were then distributed into 3 groups such that the daily food intake and initial body weights in each group were similar. The control group (n = 8) received daily injections of saline. The experimental group was given daily injections of reserpine (Serpasil, Ciba Pharmaceutical Company, Summit, NJ 07501, 0.5 mg/kg; n = 9) for 7 d,ys (Martinez et al., 1979). Each animal in the third group, a pair-fed control group (n = 9), was paired with an animal in the reserpine-treated group in such a manner that their initial body weight and mean daily food intake were the same. The food intake of each animal in this pair-fed control group was limited to that amount consumed on the previous night by the partner in the reserpine-treated group. One day after the last injection or restricted food intake, the parotid ducts were surgically cannulated under pentobarbital anaesthesia and secretion was stimulated by injection of both isoproterenol (5 mg/animal) and pilocarpine (5 mg/animal). Pilocarpine was used to increase salivary flow because saliva elicited by isoproterenol alone often gels at the end of the cannula owing to its high protein concentration and low flow rate. The saliva was collected for 30 min into a tared vial kept on ice and the weight collected was recorded. The protein concentration of the saliva was determined by the technique of Lowry et al. (1951), and by absorption at 215 nm (Arneberg, 1971) using bovine serum albumin as a standard. A sample of saliva containing 50 pg of protein by absorption at 215 nm was lyophilized, and then subjected to slab gel electrophoresis
0.a
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464
Table 2. Effect of reserpine and restricted food intake on body weight, parotid saliva volume and protein concentration*
Table 1. Effect of reserpine on food intake* Food intake (g/day) Reserpine (n = 9)
Pair-fed (n = 9)
22.4 f 0.4 22.6 k 0.5 26.6 + 0.9 23.2 + 1.6 1.5 + 1.1 1.0 f 0.5 0.2kO.l 0.2 + 0. I
22.6 k 0.4 22.3 k 0.4 23.4 k 0.5 21.4 f 1.4 1.5 + 1.1 1.0 * 0.5 0.2+0.1 0.2 k 0.1
Control (n =8)
Pre-experimental Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
22.6 & 0.9 22.1 f 0.1 23.1 i 0.5 24.8 + 0.8 23.9 + 0.1 24.3 + 1.2 26.0 i 1.2 25.6 k 1.2
*Pre-experimental food intake refers to the mean intake per animal in each group for the 3 days prior to the start of the experiment. Day refers to experimental day as the pair-fed group started 1 day later than the other two groups because their food intake was restricted to that amount consumed the previous night by the partner in the reserpine-treated group. In some cases not all animals of the pair-fed group consumed their entire allotment and therefore the mean food intake in the first few
days was slightly less than that of the reserpine-treated animals. using the conditions described by Laemmli and Favre (1973). The gels were stained with Coomassie blue and then destained. Colour positive films taken of the gels were scanned with a microprocessor-controlled spectrophotometer (Gilford 2600, Gilford Systems, 132 Artino St. Oberlin, OH 44074) equipped with a scanning device and with output to an x-y plotter (Hewlett Packard, 7225B, Hewlett Packard, 16399 W. Bernard0 Drive, San Diego, CA 92127). The area subtended by each protein component was determined using software within the spectrophotometer. The individual areas were summed to give total area, and the per cent of total area attributable to each peak was calculated. The identity of the individual protein bands was known from previous studies (Keller et al., 1975; Johnson, 1984). The data were analysed by analysis of variance or, in the case of the gel scans, by analysis of covariance with gel number as the covariate (SPSS, Nie et al., 1975). RESULTS
The mean daily food intake per animal in the reset-pine-treated group was similar to that of the
Table 3. Protein composition
Control (n = 8)
Reserpine (n = 9)
Body weight (g)
Initial
209_+4 209 f 3 299 + 4’ 181 f 3’ Weight of saliva collected in 30min mg/gland pair 466 & 35’,2 345 * 211 Final
Protein concentration (mg/ml) Lowry 24.01 i 0.80
Absorption at 215 nm
Control (n = 8)
Basic PRP:peak A Amylase Basic PRP: SP- 1 Acidic PRPs Deoxyribonuclease Fraction V Basic PRP:SP-3 Fraction I Minor la Minor lb
1.91 & 0.16’,2 31.12 f. 1.26 1.69 + 0.35 1.16 k 0.78’,2 10.81 k 0.39’,* 15.13 kO.14’ 2.45 + 0.21’,2 20.19 z 0.16’ 0.91 f 0.06’ 1.91 * 0.11’,2
209 f 3 209k3’ 333 * 272
25.35 k 0.95’ 21.64 + 1.30’
29.26 i 1.66 29.11 + 1.08 26.63 + 1.58
*Means sharing a common superscript differ significantly (p d 0.05) by Duncan’s new multiple range test (Bruning and Kintz, 1911).
control group for the first 3 days (Table 1). On day 4, the food intake of the treated group was reduced by two-thirds and on days 5, 6 and 7, it was negligible. At the end of the study, the mean body weight of the reserpine-treated group was 28 g less than its starting body weight, while that for the pair-fed group, which started the experimental period 1 day after the reserpine-treated group, was the same as its starting weight (Table 2). The weight of saliva collected in the 30 min interval was reduced by about 25 per cent for both the reserpine-treated and pair-fed control animals (Table 2). By the Lowry assay the protein concentration for the pair-fed group was lower than for the reserpinetreated group but was not different from the control. There were no differences among the groups when protein concentration was measured by absorbance at 215 nm. Among the groups there were marked differences in protein composition as shown by changes in the proportional distribution of gel-scan area attributable to several of the protein components (Table 3). Many of these changes were the same for the reserpine-treated and pair-fed controls: both exhibited a decrease in acidic proline-rich proteins; in two of the three basic proline-rich proteins (peak A and SP-3); in fraction I and in minor lb; and an increase in deoxyribonuclease. For fraction V, the mean for the reserpine-treated group did not differ
of rat parotid saliva determined by gel scanning*
Protein component
Pair-fed (n = 9)
Reserpine (n = 9) 0.64 k 0.04’ 38.95 + 1.54 1.14~0.11 3.09 & 0.15’ 18.54 f 0.10’ 16.04 + 0.832 1.30 + 0.01’ 17.53 + 0.51’ 1.09+0.11 1.09 kO.13’
Pair-fed (n = 9) 0.68 f 0.092 36.01 k 1.39 2.06 + 0.23 3.39 f o.192 11.68 f 0.45* 20.56 k 0.95’,* 1.13 f0.112 15.35 kO.71’ 1.15 * 0.07’ 1.40*0.132
*Per cent of total area attributable to each component. Means sharing a common superscript differ significantly (p < 0.05) by Duncan’s new multiple range test (Bruning and Kintz, 1911).
Saliva after reserpine or starvation statistically from that of the control group, but was significantly less than that for the pair-fed group. For fraction I, there were significant differences among all groups with the reduction for the pair-fed group being greater than that for the reserpine-treated group. DISCUSSION
Reserpine is a catecholamine-depleting drug often used to study stimulus-secretion coupling in rodent salivary glands. In parotid gland, the secretion of protein, electrolytes and fluids is impaired following 7 days of treatment with a dose of 0.5 mg/kg (Setser et al., 1979; Martinez et al., 1979; Watson et al., 1984). At the ultras,tructural level, Setser et al. (1979) reported that the acinar cells exhibit an accumulation of secretory material and a reduction in endoplasmic reticulum, changes suggestive of impaired discharge and synthesis of protein. They noted that the morphology of the acinar cells of the reserpine-treated animal is similar tcl that reported by Hand (1972) in acutely starved rats. The influence of reserpine treatment on the morphology of the parotid gland secretory granule has been reported by Muller and Roomans (1987) who suggest that the changes may occur in two stages. From days 1 to 3 there is an increase in electron-lucent granules; after day 3 the electronlucent granules rapidly disappear and are replaced by electron-dense granules, which are also common in acutely starved rats. As the synthesis and secretion of secretory proteins is regulated in large measure by gland stimulation via the sympathetic pathway, these morphological observations suggest that the transfer of a sympathetic stimulus is impaired by reserpine treatment. This is further supported by the study of Jirakulsomchok, Yu and Schneyer (1984) who found that 24 h after a single injection of reserpine, stimulation of the sympathetic nerve to the parotid gland does not evoke a secretory flow but that the flow elicited by parasympathetic nerve stimulation is not affected. In my study I have attempted to use reserpine as a means to prevent the transfer of a sympathetic stimulus to the rat parotid gland for a period of several days (leaving the parasympathetic transfer intact), thereby assessing the role of the sympathetic branch in the regulation of the synthesis of prolinerich proteins. As th’e mastication of food is a primary factor in regulating parotid function, a pair-fed control group was inchtded to differentiate the effects of reserpine from those that might result from an alteration in food intake. Many changes observed in my reserpine-treated animals were the same as in the pair-fed group, and therefore it cannot be concluded that reserpine per se had an effect on the salivary factors. The reduction in the proline-rich proteins in parotid saliva in the reserpine group was anticipated by the hypothesis that their synthesis is regulated, at least in part, by gland stimulation via the sympathetic pathway. Their reduction in the pair-fed group (where there was no mastication due to acute starvation) is in keeping with the concept that, in the absence of gland secretory activity, such proteins are reduced in parotid saliva. Although my study does not confirm that ;I decrease in these proteins in
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parotid saliva is mediated by decreased gland stimulation via the sympathetic pathway, other studies indicate that this pathway is of major importance, For example, Asking et al. (1987) demonstrated that proline-rich proteins are reduced in rat parotid saliva 1 week following removal of the superior cervical ganglion, and Cortez and Johnson (1987) showed there was a reduction in these proteins after 1 week of treatment with the beta-adrenergic antagonists, metoprolol or propranolol. Mine is the first study as far as I am aware to include a pair-fed group when evaluating the effect of reserpine on salivary gland function, and it clearly shows that pair-fed controls must be included where experimental conditions result in marked alterations in daily food intake. The effect of alterations in feeding habits on parotid salivary proteins varies with the degree of food restriction and its duration. A limited reduction, i.e. 2 to 3 g per day for a period of 1 to 2 weeks, has no measurable effect on the composition of proteins in rat parotid saliva (Johnson et al., 1987). A severe reduction of 30 to 50 per cent for a period of 5 to 6 weeks results in a marked increase in their proportion in rat parotid saliva (Johnson and Alvares, 1984). In my study, the degree of food restriction was severe (almost 100 per cent), although the duration was minimal (3 days); under these conditions there was a decrease in proportion of total protein in parotid saliva attributable to proline-rich proteins. The need to control for alterations in feeding habits extends to other organ tissues. Hazlett, Korc and Brannon (1986) reported that many of the changes in pancreatic exocrine function in chronically reserpinized rats (0.5 mg/day for 7days) were also found in pair-fed controls, and they concluded that such controls were needed to differentiate effects of reserpine from those related to the associated state of malnutrition. Acknowledgements-The expert technical support of Mr Joseph E. Cortez, MS Lucinda Girard-Aparicio and MS Greta Vance are gratefully acknowledged. This research was supported by USPHS Grant ROI-DE-06000 from the National Institutes of Dental Research, National Institutes of Health, Bethesda, MD, 20892, U.S.A. REFERENCES Arneberg P. (1971) Quantitative determination of protein in saliva. A comparison of analytical methods. Stand. J. dent. Res. 19, 60-64. Asking B., Clarke G., Garrett J. R. and Proctor G. B. (1987) Effects of sympathectomy on the protein content of parasympathetic saliva from parotid glands of rats. J. Physiol. 390, 171P. Benmiloud M. and vonEuler U. S. (1963) Effects of bretyhum, reserpine, guanethidine and sympathetic denervation on the noradrenaline content of the rat submaxillary gland. Acta physiol. stand. 59, 3442. Bruning J. L. and Kintz B. L. (1977) Computational Handbook of Statistics, 2nd edn, pp. 116119, Scott, Foresman, Glenview, Ill. Cortez J. E. and Johnson D. A. (1987) Reduction in salivary proline-rich proteins following beta receptor blockade. J. dent Res. 66, 306 (abstr. 1597). Fernandez-Sorensen A. and Carlson D. M. (1974) Isolation of a “proline-rich” protein from rat parotid glands following isoproterenol treatment. Biochem. biophys. Res. Commun. 60, 249-256.
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Hall H. D. and Schneyer C. A. (1964) Salivary gland atrophy in rat induced by liquid diet. Proc. Sot. exp. Biol. Med. 117, 789-793. Hall H. D. and Schneyer C. A. (1973) Role of autonomic pathways in disuse atrophy of rat parotid. Proc. Sot. exp. Biol. Med. 143, 19-22.
Hand A. R. (1972) The effects of acute starvation on parotid acinar cells. Ultrastructural and cytochemical observations on ad libitum-fed and starved rats. Am J. Anaf. 135, 71-92. Hazlett D., Korc M. and Brannon P. M. (1986) Effects of malnutrition and chronic reserpine treatment on pancreatic exocrine function. Pediat. Res. 20, 12361239. Jirakulsomchok D., Yu J.-H. and Schneyer C. A. (1984) Secretory responses to autonomic stimulation of rat salivary glands following reserpine treatment. Archs oral Biol. 29, 394.
Johnson D. A. (1984) Changes in rat parotid salivary proteins associated with liquid diet-induced gland atrophy and isoproterenol-induced gland enlargement. Archs oral Biol. 29, 215-221. Johnson D. A. and Alvares 0. F. (1984) Zinc deficiency-induced changes in rat parotid salivary proteins. J. Nufr. 114, 19551964. Johnson D. A., Etzel K. R., Alvares 0. F. and Cortez J. E. (1987) Regulation of parotid salivary proteins by glucocorticoids. J. dent. Res. 66, 1563-1568. Keller P. J., Robinovitch M., Iversen J. and Kauffman D. L. (1975) The protein composition of rat parotid saliva and secretory granules. Biochim. biophys. Acra 379, 562-570. Laemmli U. K. and Favre M. (1973) Maturation ofthe head of bacteriophage T4. J. molec. Biol. 80, 575-599.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin nhenol reagent. J. ‘biol. Chem. 193, 265-275. Martinez J. R., Braddock M. E., Martinez A. M. and Cooper C. (1979) Effect of chronic reserpine administration on K+ and amylase release from the rat parotid gland. Pediut. Res. 13, 1024-1029. Muenzer J., Bildstein C., Gleason M. and Carlson D. M. (1979) Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats. J. biol. Chem. 254, 5623-5628.
Muller R. M. and Roomans G. M. (1987) Effects of reserpine treatment on the ultrastructure of the rat parotid and submandibular gland. J. Submicrosc. Cytol. 19, 283-289.
Nie N. H., Hull C. H., Jenkins J. G., Steinbrenner K. and Bent D. H. (1975) SPSS: Statistical Package for the Social Sciences, 2nd edn, Chap. 22, pp. 398422.-McGraw-Hill, New York. Schneyer C. A. (1969) /I-Adrenergic effects by autonomic agents on mitosis and hypertrophy in rat parotid. Proc. Sot. exp. Biol. Med. 131, 71-75.
Setser M. E., Spicer S. S., Simson J. A. V. and Martinez J. R. (1979) Altered granule discharge and amylase secretion of parotid glands in reserpine-treated rats. Lab. Invest. 41, 256264. Spiers R. L. and Hodgson C. (1976) Control of amylase secretion in the parotid gland during feeding. Archs oral Biol. 21, 539-544.
Watson E., Dowd F., Jacobson K. and Horwitz H. (1984) Reserpinization: effects on parotid gland function. J. dent. Res. 63, 82-86.