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Amino acid composition of salivary protein secreted by the parotid glands of rats in response to parasympathomimetic and sympathomimetic drugs
Archs oral Bid. Printed in Great
Vol. 29, No.
2, pp. 127-130,
1984
0003-9969184 $3.00 + 0.00
Pergamon Press Ltd
Britain
AMINO ACID COMPOSITION OF SALIVARY PROTEIN SECRETED BY THE PAROTID GLANDS OF RATS IN RESPONSE TO PARASYMPATHOMIMETIC AND SYMPATHOMIMETIC DRUGS K. ABE and H. NITTA* Department of Oral Biochemistry, and *Department of Oral Microbiology, Gifu College of Dentistry, Gifu, 501-02, Japan
Summary-Although the total protein concentration in the saliva varied markedly, depending on the nature of the stimulus, the proportions of the amino-acid residues were independent on the nature of the stimuli, where they were cholinergic, adrenergic or dopaminergic. However, the difference in protein comoosition of saliva nreviouslv shown in chronically isoprenaline-treated rats electrophoretically could be determined by amino-acid analysis.
INTRODUCTION
Several attempts have been made to determine whether the concentrations of the different proteins in various types of saliva always change concomitantly, or whether, the relative proportions change under different conditions of secretory stimulation. For the pancreas, some investigators (Tartakoff et al., 1974; Gilliland and Glazer, 1980; Kanagasuntheram and Lim, 1981) have claimed parallelism in the secretion of pancreatic enzymes, in contrast to other reports that the proportions of the different enzymes in pancreatic juice are dependent on diet (Howard and Yudkin, 1963; Ben, Abdelilli and Desnuelle, 1964; Dijkhof and Poort, 1978) and the nature of secretory stimulus (Rothman, 1977; Dagorn, 1978; Roberge and Beaudoin, 1982). In submandibular saliva from dogs (Dische et al., 1962) cats (Dische et al., 1970) and man (Caldwell and Pigman, 1966) the proportions of certain protein-bound carbohydrates were found to be dependent on stimulus conditions. Furthermore, we found clear differences in the types of protein in rat submandibular saliva depending on the nature of stimulus (Abe and Dawes, 1978; Abe et al., 1980; Abe and Dawes, 1982). Rat submandibular glands contain two major types of cells which secrete protein, the acinar cells and. the convoluted granular tubule cells. In contrast, l.he parotid glands contain only acinar cells and no difference in the types of protein has been found by electrophoretic analysis of proteins in rat parotid salivas elicited by cholinergic, /I- and cc-adrenergic stimuli (Abe and Dawes, 1978; Abe et al., 1980) or by a edopaminergic stimulation as described previously (Abe and Dawes, 1982). In human parotid saliva, the concentration of IgA and lysozyme was found to be much higher in unstimulated than in stimulated saliva (Mandel and Khurana, 1969; Brandzaeg, 1971) but for amylase the reverse is true. The present stud:y was designed mainly to determine whether the proportions of the amino-acid residues in rat parotid saliva elicited by parasym127
pathomimetic and sympathomimetic drugs, with and without different types of blockers, were constant or not under the different conditions of stimulation and to determine whether electrophoretic differences could be detected by amino-acid analysis. MATERIALS
Experimental
AND METHODS
animals
Adult male Sprague-Dawley rats, weighing 360-600 g, were distributed into experimental groups of about 10 animals. A 12-h light-dark cycle was maintained in the animal room by an automatic timing device with lights turned off at 18.00 h. The rats had ad libitum access to Oriental MF solid rat chow (Oriental Kobokogyo Co., Tokyo) and water; however, they were fasted from 09.00 to 17.00 h prior to saliva collection. For chronic administration of isoprenaiine, an intraperitoneal dose of 4 mg/kg was injected twice daily at 08.00 and 16.00 h for six consecutive days in rats in the experimental group (Abe and Dawes, 1980). Saliva collection
The rats were anaesthetized by intraperitoneal (ip.) injections of sodium pentobarbital (50 mg/kg), placed supine and tracheotomized. The two parotid ducts were cannulated at their openings and parotid saliva was collected into Pyrex tubes in ice for 15 min (Abe and Dawes, 1975). As secretory stimuli, carbachol (100 pg/kg), acetyl-p-methylcholine (methacholine, 5 mg/kg), pilocarpine (8 mg/kg), methoxamine (6 mg/kg), phenylephrine (6 mg/kg), adrenaline (2 mg/kg), noradrenaline (4 mg/kg), dopamine (100 mg/kg), and isoprenaline (30 mg/kg) were dissolved in isotonic saline and administered i.p. With dopamine (10, 20, 30, 40, 50 and 75 mg/kg), dose-response effects were also tested. On occasion, various autonomic blocking agents were injected i.p. 1.5-30 min prior to administration of dopamine (100 mg/kg) and these included a cholinergic blocker (atropine, I mg/kg), an
K. ABE and H. NITTA
128 Table tration,
1. The effects of parasympathomimetic the amounts
of protein
secreted different
Protein concentration (g/l00 cm))
Stimulants Methacholine Carbachol Pilocarpine Isoprenahne Methoxamine Phenylephrine Dopamine Noradrenaline Adrenaline
0.2 0.2 2.6 22.0 0.4 3.4 13.6 3.5 4.8
* + + + * * + f +
and sympathomimetic drugs on protein concenand amylase activity of the parotid saliva elicited by stimuli (means + SE)
Protein (mgih)
0.01 0.03 0.2 1.4 0.04 0.2 0.9 0.5 0.6
6.8 3.7 30.5 57.1 5.2 13.2 53.9 17.8 24.5
Saliva analysis A sample of saliva was analysed for protein by the method of Lowry et al. (1951) using casein as a standard. Amylase activity was determined by the Daiichi amylase test (Sasaki, Ohmizu and Tanemura, 1971). For amino-acid analysis, the saliva samples were dialysed against distilled water for 24 h using Cellulose Tubing (size; 24/32, UC Co., U.S.A.) which can retain substances of molecular weights over 10,000. Before and after dialysis, electrophoretic patterns did not show any change. Samples were then hydrolysed for 24, 48 and 72 h with 6M HCl to compensate for the effects of duration of hydrolysis, evaporated and re-solubilized with 0.001 M HCI.
2. Changes
of the amino-acid chronic isoprenaline Controls
Lysine Histidine NH: Arginine Aspartic acid Threonine Glutamic Serine
acid
35.6 19.7 201.8 35.7 109.9 42.7
& 1.6 + 0.6 + 7.2 & 2.7 * 2.2 + 2.3
(pg/mg
+ 0.8 * 0.7 i. 3.5 f 4.4 k 0.9 k 2.0 + 6.9 k 2.8 & 5.4
a-adrenergic blocker (phentolamine, 25 mg/kg) and a /I-adrenergic blocker (propranolol, 10 mg/kg). A specific dopaminergic blocker (haloperidol, 2 mg/kg) was simultaneously administered intravenously (i.v.) into the femoral vein with dopamine (100 mg/kg) in some experiments. In rats subjected to chronic administration of isoprenaline as well as control rats, methoxamine (6mg/kg) was also used as an acute secretory stimulus.
Table
secreted h)
Amylase activity (IUjl x 10-S)
No. of rats
6*3 2+1 87 + 2 1080 + 300 6*2 98 + 5 623 k 200 12+3 388 &-97
6 13 14 10 8 6 9 7 5
16.8 f. 2.0 9.1 * 1.4 79.9 + 10.5 190.8 f 28. I 12.3 + 1.6 37.8 &-6.4 151.6+29.1 69.7 k 8.0 68.8 k 16.4
Thereafter, amino-acid Tokyo).
amino-acid analysis was carried out in an analyser (JLC-6AH, Nihondenshi Co.,
RESULTS The protein concentration, the amount of protein secreted and level of amylase activity with different stimuli are shown in Table 1. The most effective stimulant for protein secretion was isoprenaline followed by dopamine, the cc-adrenergic and then the cholinergic stimuli. The amino-acid composition of parotid saliva proteins produced in response to three parasympathomimetic drugs (methacholine, carbachol and pilocarpine) a p-adrenergic drug, isoprenaline, five a-adrenergic drugs (methoxamine, phenylephrine, noradrenaline, adrenaline and dopamine) and also to different doses of dopamine (10-20, 30. 4&50 and 75 mg/kg) and dopamine in combination with atropine, propranolol, phentolamine or haloperidol, did not show major statistically significant differences although minor statistically significant differences were observed in a few amino-acid residues. The
composition of rat parotid saliva administration (means * SE) Isoprenaline (acute) 35.4 20.0 168.0 41.4 107.4 36.0
+ + k + + k
1.6 0.7 8.5 1.5 4.2 I .2
Experimentals (chronic) 25.5 14.3 190.8 34.8 47.7 7.6
+ 1.3 + 0.6 f 2.5 + 2.5 + 1.2 & 0.2
subjected
F-analysis t : NS NS :
81.0+2.6 53.6 + 2.6
77.8 & 3.3 46.8 & 2.8
186.3 8.0 18.4 f& 0.7
i
Glycine Proline Alanine
86.4 + k 2.6 2.3 86.4 44.9 * 1.0
82.0 *f 4.3 4.4 94.0 42.4 f 1.6
135.4 k + 4.3 258.8 3.1 17.0 f 1.3
: $
Methionine Valine Isoleucine Leucine
4.5 f+ 1.0 63.0 1.9 36.9+ 1.1 83.0 + 1.9
4.2 * f 0.8 60.6 3.4 31.6 f 1.3 79.0 f 2.9
1.6kO.3 7.9 + 0.9 4.9 + 0.4 15.7 f 0.5
i
Tyrosine Phenylalanine Times repeated
17.7 * 1.3 33.5 f 0.6 8
17.3 f 1.5 32.0 k 1.2 5
2.3 + 0.3 10.7 * 0.3 8
i 1:
*Denotes the F-analysis.
sum of glutamine and tp < 0.01. $p < 0.001.
asparagine
residues.
NS:
Not
to
significant
by
Protein
secretion
control data in Table 2 is a representative pattern, in this instance elicited by methoxamine. The parotid saliva proteins were particularly rich in aspartic acid, glutamic acid, proline, glycine and leucine. No cystine was detected and methionine was present only in small amounts. The amino-acid composition of the parotid saliva proteins was independent of the nature of the stimulus, whether this was cholinergic, /?- or a-adrenergic or dopaminergic or dopaminergic in the presence of various blockers. However, the difl’erence in protein composition of parotid saliva in rats subjected to chronic administration of isoprenaline, as determined electrophoretically, and as shown previously, was detected by amino-acid analysis (Table 2). DISCUSSION
The results confirm previous findings (Byrt, 1966; Speirs et al., 1974; Abe and Dawes, 1975, 1978, 1982) that the protein concentration, the amounts of protein secreted and I:he level of amylase activity in parotid saliva are increased most markedly by p-adrenergic stimulation followed by primarily a-adrenergic stimuli, such as dopamine, adrenaline, phenylephrine and noradrenaline, and then cholinergic stimulation. The higher protein concentration in parotid saliva elicited by pilocarpine than in saliva elicited by some of l:he a-adrenergic stimuli is probably due to the fact. that pilocarpine stimulates not only cholinergic fibres but also post-ganglionic fibres in the superior cervical ganglion (Schneyer, 1965). In previous studies, it has been found that the protein concentration in parotid saliva varies with different stimuli, salivary flow rate, circadian rhythm, length of stimulation, time course and other factors (Abe and Dawes, 1975; Wallach, Kirshner and Schramm, 1975; Abe and Dawes, 1978; Abe et al., 1980; Johnson and Sreebny, 1982). However, the types of protein secreted, as judged by electrophoretic techniques, were not infuenced by the age of the rat, by circadian rhythms in the secretory content of the gland or by the nature of stimulus, whether electrical, cholinergic, a- or p-adrenergic (Abe and Dawes, 1975, 1978; Abe et al., 1980) or dopaminergic (Abe and Dawes, 1982). Propranolol inhibited flow rate by about 60 per cent of dopamine-induced secretion from the parotid gland and was particularly effective in reducing protein. secretion. Haloperidol reduced rate by about 70 per cent, but, like phentolamine, caused an increase of about 50 per cent in the protein concentration of the saliva secreted. However, the relative proportions of the different types of proteins secreted were not affected, as described previously (Abe and Dawes, 1982). That finding was confirmed by amino-acid analysis. Further studies are necessary to elucidate how the dopamine effects are mediated. It is well-known that the types of protein and amylase isozymes secreted by the parotid glands vary in rat glands enlarged by chronic administration of isoprenaline (Fernandez-Sorensen and Carlson, 1974; Robinovitch et al., 1977; Abe and Dawes, 1980) and in genetic polymorphisms in man (Azen and Oppenheim, 1973; Merritt et al., 1973) and of different blood groups (Siinju and Rolla, 1975). Amino-acid
by rat parotid
glands
129
analysis might confirm these observations (Abe and Dawes, 1975; Wallach et al., 1975; Abe and Dawes, 1978; Abe et al., 1980; Abe and Dawes, 1982; Johnson and Sreebny, 1982). There is little information on the general aminoacid composition of _ parotid saliva proteins (Armstrong, 1967; Armstrong, 1970; Levine and Ellison, 1973; Feller, Shannon and Wescott, 1974) although there are many reports,on specific proteins such as basic and acidic proline rich proteins (Mandel, Thompson and Ellison, 1965; Henkin et al., 1978) tyrosine rich proteins (Hay, 1973) and phosphoproteins (Wong, Hofmann and Bennick, 1979). The richness of human parotid saliva in proline, glutamic acid, glycine and aspartic acid is consistent with our results, although rat parotid saliva was richest in aspartic acid. Leucine, valine, serine and phenylalanine are also in moderately high concentration in human parotid saliva. Similar results were obtained in rat parotid saliva except that phenylalanine was not a major amino acid in rat parotid proteins. To the best of our knowledge, the present work is the first such study on rat parotid saliva. The aminoacid composition was largely independent of the nature and doses of the stimuli although minor differences in certain amino-acid residues were found. In man, the proportion of the different proteins varies considerably from one person to another and we assumed that the minor differences found in the amino-acid composition of rat parotid saliva might be due to individual variations between rats rather than to variations due to the nature of stimulus. Further in rats subjected to chronic administration of isoprenaline, the differences in protein composition of parotid saliva revealed electrophoretically can be detected by amino-acid analysis. Acknowledgements-We arc grateful to Professors C. Dawes, Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3, Y. Yokota, Department of Oral Biochemistry and I. Namikawa, Department of Oral Microbiology, Gifu College of Dentistry, Gifu, 501-02, Japan for advice.
REFERENCES
Abe K. and Dawes
C. (1975) Circadian variations in pilocarpine and isoprenaline-induced protein secretion by rat parotid glands. Archs oral Biol. 20, 543-546. Abe K. and Dawes C. (1978) The effects of electrical and pharmacological stimulation on the types of proteins secreted by rat parotid and submandibular glands. Archs oral Biol. 23, 367-372. Abe K. and Dawes C. (1980) The secretion of protein and of some, electrolytes in response to a- and /I-adrenergic agonists by rat parotid and submandibular salivary glands enlarged by chronic treatment with isoproterenol. J. dent. Res. 59, 1081-1089. Abe K, Yoneda K., Fujita R., Ydkota Y. and Dawes C. (1980) The effects of epinephrine, norepinephrine and phenylephrine on the types of proteins secreted by rat salivary glands, J. dent. Res. 59, 1627-1634. Abe K. and Dawes C. (1982). Dopamine-induced secretion of protein and of some electrolytes by rat submandibular and parotid glands. Archs oral Biol. 27, 635X43. Armstrong W. G. (1967) The composition of organic films formed on human teeth. Caries Res. 1, 89-103.
130
K. ABE an Id H. NITTA
Armstrong W. G. (1970) Amino acid composition of human parotid salivary proteins selectively adsorbed by hydroxyapatite. Archs oral Biol. 15, 1001-1003. Azen E. A. and Oppenheim F. G. (1973) Genetic polymorphism of proline rich human salivary proteins. Science 180, 1067-1069. Ben Abdelilli A. and Desnuelle P. (1964) Sur l’adaptation des enzymes exocrines du pancreas a la composition du regime. Biochim. hiophys. Acta 81, 136149. Brandtzaeg P. (197 I) Human secretory immunoglobulinsVII. Concentrations of parotid IgA and other secretory proteins in relation to the rate of flow and duration of secretory stimulus. Archs oral Biol. 16, 1295-1310. Byrt T. (1966) Secretion and synthesis of amylase in the rat parotid gland after isoprenahne. Nature, Lond. 212, 1212-1215. Caldwell R. C. and Pigman W. (1966) Changes in protein and glycoprotein concentrations in human submaxillary saliva under various stimulatory conditions. Archs oral Biol. 11, 437-449. Dagorn J. C. (1978) Non-parallel enzyme secretion from rat pancreas: In viva studies. J. Physiol., Lond. 280, 435-448. Dijkhof J. and Poort C. (1978) Non-parallel secretion of newly synthesized rat pancreatic proteins after feeding a diet containing raw soybean flour. Biochim. biophys. Acta 543, 167-174. Dische Z., Paravicini C., Kawasaki H., Smirnow N., Cizek L. J. and Chien S. (1962) Influence of the nature of the secretory stimulus on the composition of the carbohydrate moiety of glycoproteins of the submaxillary saliva. Archs Biochem. Biophys. 91, 459464. Dische Z., Kahn N., Rothschild C., Danilchenko A., Liking J. and Wang S. C. (1970) Glycoproteins of submaxillary saliva of the cat: differences in composition produced by sympathetic and parasympathetic nerve stimulation. J. Neurochem. 11, 649-658. Feller R. P.. Shannon I. L. and Wescott W. B. (1974) Total amino acid levels of parotid saliva at different rates of gland function. J. dent. Res. 53, 772. Fernandez-Sorensen A. and Carlson D. (1974) Isolation of a proline-rich protein from rat parotid glands following isoproterenol treatment. Biochem. biophys. Res. Commun. 60, 249-256. Gilliland E. L. and Glazer G. (1980) Parallel secretion of enzymes by the rabbit pancreas. J. Physiol., Lond. 303, 33-41. Hay D. 1. (1973) The isolation from human parotid saliva of a tyrosine-rich acidic peptide which exhibits high affinity for hydroxyapatite surfaces. Archs oral Biol. 18, 1531-1541. Henkin R. I., Lippoldt R. E., Bilstad J., Wolf R. O., Lum C. K. L. and Edelhoch H. (1978) Fractionation of human parotid saliva proteins. ‘J. b;ol. Chem. 253, 755&7565. Howard F. and Yudkin J. (1963) Effect of dietary change upon the amylase and trypsin activities of the rat pancreas. Br. J. Nutr. 17, 281-294. Johnson D. A. and Sreebny L. M. (1982) Effect of increasing
the bulk content of the diet on the rat parotid gland and saliva. J. dent. Res. 61, 691-696. Kanagasuntheram P. and Lim S. H. (1981) Parallel secretion of secretory proteins and calcium by the rat parotid gland. J. Physiol., Land. 312, 445454. Levine M. J. and Ellison S. A. (1973) Immunoelectrophoretic and chemical analysis of human parotid saliva. Archs oral Biol. 18, 839-853. 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, 2655215. Mandel I. D., Thompson R. H. Jr and Ellison S. A. (1965) Studies on the mucoproteins of human parotid saliva. Archs oral Biol. 10, 499-507. Mandel I. D. and Khurana H. S. (1969) The relation of human salivary yA globulin and albumin to flow rate. Archs oral Biol. 14, 1433-1435. Merritt A. D., Rivas M. L., Bixler D. and Newell R. (1973) Salivary and pancreatic amylase: Electrophoretic characterizations and genetic studies. Am. J. hum. Genet. 25, 510-522. Roberge M. and Beaudoin A. R. (1982) Newly synthesized secretory proteins from pig pancreas are not released from a homogenous granule compartment. Biochim. biophys. Acfa 716, 331-336. Robinovitch M. R., Keller P. J., Johnson D. A., Iversen J. M. and Kauffman D. L. (1977) Changes in rat parotid salivary proteins induced by chronic isoproterenol administration J. dent. Res. 56, 290-303. Rothman S. S. (1977) The digestive enzymes of the pancreas: A mixture of inconstant proportions. A. Rev. Physiol. 39, 373-389. Sasaki T., Ohmizu Y. and Tanemura K. (1971) Serum amylase assay method by the “Daiichi” amylase test. The results of the basic properties tested. J. med. Tech. 15, 7 14-7 19 (Japanese). Schneyer C. A. (1965) Modification of the action of pilocarpine by adrenergic blocking agents. Proc. Sot. exp. Biol. Med. 120, 230-232. Sijnju T. and Rolla G. (1975) Further studies of the chemistry of a human salivary blood-group substance. Acta path. microbial. stand. C.83, 215-220. Speirs R. L., Herring J., Cooper W. D., Hardy C. C. and Hind C. R. K. (1974) The influence of sympathetic activity and isoprenaline on the secretion of amylase from human parotid gland. Archs oral Biol. 19, 745-752. Tartakoff A. M., Greene L. J., Jamieson J. D. and Parade G. E. (1974) Parallelism in the processing of pancreatic proteins. In: Advances in Cytopharmacology (Edited by Ceccarelh B., Clementi F. and Meldosi J.) Vol. 2. pp. 177-194. Raven Press, New York. Wallach D., Kirschner N. and Schramm M. (1975) Nonparallel transport of membrane proteins and content proteins during assembly of the secretory granule in rat parotid gland. Biochim. biophys. Acta 375, 87-105. Wong R. S. C., Hofmann T. and Bennick A. (1979) The complete primary structure of a proline-rich phosphoprotein from human saliva. J. biol. Chem. 254, 48OW808.
Vol. 29, No.
2, pp. 127-130,
1984
0003-9969184 $3.00 + 0.00
Pergamon Press Ltd
Britain
AMINO ACID COMPOSITION OF SALIVARY PROTEIN SECRETED BY THE PAROTID GLANDS OF RATS IN RESPONSE TO PARASYMPATHOMIMETIC AND SYMPATHOMIMETIC DRUGS K. ABE and H. NITTA* Department of Oral Biochemistry, and *Department of Oral Microbiology, Gifu College of Dentistry, Gifu, 501-02, Japan
Summary-Although the total protein concentration in the saliva varied markedly, depending on the nature of the stimulus, the proportions of the amino-acid residues were independent on the nature of the stimuli, where they were cholinergic, adrenergic or dopaminergic. However, the difference in protein comoosition of saliva nreviouslv shown in chronically isoprenaline-treated rats electrophoretically could be determined by amino-acid analysis.
INTRODUCTION
Several attempts have been made to determine whether the concentrations of the different proteins in various types of saliva always change concomitantly, or whether, the relative proportions change under different conditions of secretory stimulation. For the pancreas, some investigators (Tartakoff et al., 1974; Gilliland and Glazer, 1980; Kanagasuntheram and Lim, 1981) have claimed parallelism in the secretion of pancreatic enzymes, in contrast to other reports that the proportions of the different enzymes in pancreatic juice are dependent on diet (Howard and Yudkin, 1963; Ben, Abdelilli and Desnuelle, 1964; Dijkhof and Poort, 1978) and the nature of secretory stimulus (Rothman, 1977; Dagorn, 1978; Roberge and Beaudoin, 1982). In submandibular saliva from dogs (Dische et al., 1962) cats (Dische et al., 1970) and man (Caldwell and Pigman, 1966) the proportions of certain protein-bound carbohydrates were found to be dependent on stimulus conditions. Furthermore, we found clear differences in the types of protein in rat submandibular saliva depending on the nature of stimulus (Abe and Dawes, 1978; Abe et al., 1980; Abe and Dawes, 1982). Rat submandibular glands contain two major types of cells which secrete protein, the acinar cells and. the convoluted granular tubule cells. In contrast, l.he parotid glands contain only acinar cells and no difference in the types of protein has been found by electrophoretic analysis of proteins in rat parotid salivas elicited by cholinergic, /I- and cc-adrenergic stimuli (Abe and Dawes, 1978; Abe et al., 1980) or by a edopaminergic stimulation as described previously (Abe and Dawes, 1982). In human parotid saliva, the concentration of IgA and lysozyme was found to be much higher in unstimulated than in stimulated saliva (Mandel and Khurana, 1969; Brandzaeg, 1971) but for amylase the reverse is true. The present stud:y was designed mainly to determine whether the proportions of the amino-acid residues in rat parotid saliva elicited by parasym127
pathomimetic and sympathomimetic drugs, with and without different types of blockers, were constant or not under the different conditions of stimulation and to determine whether electrophoretic differences could be detected by amino-acid analysis. MATERIALS
Experimental
AND METHODS
animals
Adult male Sprague-Dawley rats, weighing 360-600 g, were distributed into experimental groups of about 10 animals. A 12-h light-dark cycle was maintained in the animal room by an automatic timing device with lights turned off at 18.00 h. The rats had ad libitum access to Oriental MF solid rat chow (Oriental Kobokogyo Co., Tokyo) and water; however, they were fasted from 09.00 to 17.00 h prior to saliva collection. For chronic administration of isoprenaiine, an intraperitoneal dose of 4 mg/kg was injected twice daily at 08.00 and 16.00 h for six consecutive days in rats in the experimental group (Abe and Dawes, 1980). Saliva collection
The rats were anaesthetized by intraperitoneal (ip.) injections of sodium pentobarbital (50 mg/kg), placed supine and tracheotomized. The two parotid ducts were cannulated at their openings and parotid saliva was collected into Pyrex tubes in ice for 15 min (Abe and Dawes, 1975). As secretory stimuli, carbachol (100 pg/kg), acetyl-p-methylcholine (methacholine, 5 mg/kg), pilocarpine (8 mg/kg), methoxamine (6 mg/kg), phenylephrine (6 mg/kg), adrenaline (2 mg/kg), noradrenaline (4 mg/kg), dopamine (100 mg/kg), and isoprenaline (30 mg/kg) were dissolved in isotonic saline and administered i.p. With dopamine (10, 20, 30, 40, 50 and 75 mg/kg), dose-response effects were also tested. On occasion, various autonomic blocking agents were injected i.p. 1.5-30 min prior to administration of dopamine (100 mg/kg) and these included a cholinergic blocker (atropine, I mg/kg), an
K. ABE and H. NITTA
128 Table tration,
1. The effects of parasympathomimetic the amounts
of protein
secreted different
Protein concentration (g/l00 cm))
Stimulants Methacholine Carbachol Pilocarpine Isoprenahne Methoxamine Phenylephrine Dopamine Noradrenaline Adrenaline
0.2 0.2 2.6 22.0 0.4 3.4 13.6 3.5 4.8
* + + + * * + f +
and sympathomimetic drugs on protein concenand amylase activity of the parotid saliva elicited by stimuli (means + SE)
Protein (mgih)
0.01 0.03 0.2 1.4 0.04 0.2 0.9 0.5 0.6
6.8 3.7 30.5 57.1 5.2 13.2 53.9 17.8 24.5
Saliva analysis A sample of saliva was analysed for protein by the method of Lowry et al. (1951) using casein as a standard. Amylase activity was determined by the Daiichi amylase test (Sasaki, Ohmizu and Tanemura, 1971). For amino-acid analysis, the saliva samples were dialysed against distilled water for 24 h using Cellulose Tubing (size; 24/32, UC Co., U.S.A.) which can retain substances of molecular weights over 10,000. Before and after dialysis, electrophoretic patterns did not show any change. Samples were then hydrolysed for 24, 48 and 72 h with 6M HCl to compensate for the effects of duration of hydrolysis, evaporated and re-solubilized with 0.001 M HCI.
2. Changes
of the amino-acid chronic isoprenaline Controls
Lysine Histidine NH: Arginine Aspartic acid Threonine Glutamic Serine
acid
35.6 19.7 201.8 35.7 109.9 42.7
& 1.6 + 0.6 + 7.2 & 2.7 * 2.2 + 2.3
(pg/mg
+ 0.8 * 0.7 i. 3.5 f 4.4 k 0.9 k 2.0 + 6.9 k 2.8 & 5.4
a-adrenergic blocker (phentolamine, 25 mg/kg) and a /I-adrenergic blocker (propranolol, 10 mg/kg). A specific dopaminergic blocker (haloperidol, 2 mg/kg) was simultaneously administered intravenously (i.v.) into the femoral vein with dopamine (100 mg/kg) in some experiments. In rats subjected to chronic administration of isoprenaline as well as control rats, methoxamine (6mg/kg) was also used as an acute secretory stimulus.
Table
secreted h)
Amylase activity (IUjl x 10-S)
No. of rats
6*3 2+1 87 + 2 1080 + 300 6*2 98 + 5 623 k 200 12+3 388 &-97
6 13 14 10 8 6 9 7 5
16.8 f. 2.0 9.1 * 1.4 79.9 + 10.5 190.8 f 28. I 12.3 + 1.6 37.8 &-6.4 151.6+29.1 69.7 k 8.0 68.8 k 16.4
Thereafter, amino-acid Tokyo).
amino-acid analysis was carried out in an analyser (JLC-6AH, Nihondenshi Co.,
RESULTS The protein concentration, the amount of protein secreted and level of amylase activity with different stimuli are shown in Table 1. The most effective stimulant for protein secretion was isoprenaline followed by dopamine, the cc-adrenergic and then the cholinergic stimuli. The amino-acid composition of parotid saliva proteins produced in response to three parasympathomimetic drugs (methacholine, carbachol and pilocarpine) a p-adrenergic drug, isoprenaline, five a-adrenergic drugs (methoxamine, phenylephrine, noradrenaline, adrenaline and dopamine) and also to different doses of dopamine (10-20, 30. 4&50 and 75 mg/kg) and dopamine in combination with atropine, propranolol, phentolamine or haloperidol, did not show major statistically significant differences although minor statistically significant differences were observed in a few amino-acid residues. The
composition of rat parotid saliva administration (means * SE) Isoprenaline (acute) 35.4 20.0 168.0 41.4 107.4 36.0
+ + k + + k
1.6 0.7 8.5 1.5 4.2 I .2
Experimentals (chronic) 25.5 14.3 190.8 34.8 47.7 7.6
+ 1.3 + 0.6 f 2.5 + 2.5 + 1.2 & 0.2
subjected
F-analysis t : NS NS :
81.0+2.6 53.6 + 2.6
77.8 & 3.3 46.8 & 2.8
186.3 8.0 18.4 f& 0.7
i
Glycine Proline Alanine
86.4 + k 2.6 2.3 86.4 44.9 * 1.0
82.0 *f 4.3 4.4 94.0 42.4 f 1.6
135.4 k + 4.3 258.8 3.1 17.0 f 1.3
: $
Methionine Valine Isoleucine Leucine
4.5 f+ 1.0 63.0 1.9 36.9+ 1.1 83.0 + 1.9
4.2 * f 0.8 60.6 3.4 31.6 f 1.3 79.0 f 2.9
1.6kO.3 7.9 + 0.9 4.9 + 0.4 15.7 f 0.5
i
Tyrosine Phenylalanine Times repeated
17.7 * 1.3 33.5 f 0.6 8
17.3 f 1.5 32.0 k 1.2 5
2.3 + 0.3 10.7 * 0.3 8
i 1:
*Denotes the F-analysis.
sum of glutamine and tp < 0.01. $p < 0.001.
asparagine
residues.
NS:
Not
to
significant
by
Protein
secretion
control data in Table 2 is a representative pattern, in this instance elicited by methoxamine. The parotid saliva proteins were particularly rich in aspartic acid, glutamic acid, proline, glycine and leucine. No cystine was detected and methionine was present only in small amounts. The amino-acid composition of the parotid saliva proteins was independent of the nature of the stimulus, whether this was cholinergic, /?- or a-adrenergic or dopaminergic or dopaminergic in the presence of various blockers. However, the difl’erence in protein composition of parotid saliva in rats subjected to chronic administration of isoprenaline, as determined electrophoretically, and as shown previously, was detected by amino-acid analysis (Table 2). DISCUSSION
The results confirm previous findings (Byrt, 1966; Speirs et al., 1974; Abe and Dawes, 1975, 1978, 1982) that the protein concentration, the amounts of protein secreted and I:he level of amylase activity in parotid saliva are increased most markedly by p-adrenergic stimulation followed by primarily a-adrenergic stimuli, such as dopamine, adrenaline, phenylephrine and noradrenaline, and then cholinergic stimulation. The higher protein concentration in parotid saliva elicited by pilocarpine than in saliva elicited by some of l:he a-adrenergic stimuli is probably due to the fact. that pilocarpine stimulates not only cholinergic fibres but also post-ganglionic fibres in the superior cervical ganglion (Schneyer, 1965). In previous studies, it has been found that the protein concentration in parotid saliva varies with different stimuli, salivary flow rate, circadian rhythm, length of stimulation, time course and other factors (Abe and Dawes, 1975; Wallach, Kirshner and Schramm, 1975; Abe and Dawes, 1978; Abe et al., 1980; Johnson and Sreebny, 1982). However, the types of protein secreted, as judged by electrophoretic techniques, were not infuenced by the age of the rat, by circadian rhythms in the secretory content of the gland or by the nature of stimulus, whether electrical, cholinergic, a- or p-adrenergic (Abe and Dawes, 1975, 1978; Abe et al., 1980) or dopaminergic (Abe and Dawes, 1982). Propranolol inhibited flow rate by about 60 per cent of dopamine-induced secretion from the parotid gland and was particularly effective in reducing protein. secretion. Haloperidol reduced rate by about 70 per cent, but, like phentolamine, caused an increase of about 50 per cent in the protein concentration of the saliva secreted. However, the relative proportions of the different types of proteins secreted were not affected, as described previously (Abe and Dawes, 1982). That finding was confirmed by amino-acid analysis. Further studies are necessary to elucidate how the dopamine effects are mediated. It is well-known that the types of protein and amylase isozymes secreted by the parotid glands vary in rat glands enlarged by chronic administration of isoprenaline (Fernandez-Sorensen and Carlson, 1974; Robinovitch et al., 1977; Abe and Dawes, 1980) and in genetic polymorphisms in man (Azen and Oppenheim, 1973; Merritt et al., 1973) and of different blood groups (Siinju and Rolla, 1975). Amino-acid
by rat parotid
glands
129
analysis might confirm these observations (Abe and Dawes, 1975; Wallach et al., 1975; Abe and Dawes, 1978; Abe et al., 1980; Abe and Dawes, 1982; Johnson and Sreebny, 1982). There is little information on the general aminoacid composition of _ parotid saliva proteins (Armstrong, 1967; Armstrong, 1970; Levine and Ellison, 1973; Feller, Shannon and Wescott, 1974) although there are many reports,on specific proteins such as basic and acidic proline rich proteins (Mandel, Thompson and Ellison, 1965; Henkin et al., 1978) tyrosine rich proteins (Hay, 1973) and phosphoproteins (Wong, Hofmann and Bennick, 1979). The richness of human parotid saliva in proline, glutamic acid, glycine and aspartic acid is consistent with our results, although rat parotid saliva was richest in aspartic acid. Leucine, valine, serine and phenylalanine are also in moderately high concentration in human parotid saliva. Similar results were obtained in rat parotid saliva except that phenylalanine was not a major amino acid in rat parotid proteins. To the best of our knowledge, the present work is the first such study on rat parotid saliva. The aminoacid composition was largely independent of the nature and doses of the stimuli although minor differences in certain amino-acid residues were found. In man, the proportion of the different proteins varies considerably from one person to another and we assumed that the minor differences found in the amino-acid composition of rat parotid saliva might be due to individual variations between rats rather than to variations due to the nature of stimulus. Further in rats subjected to chronic administration of isoprenaline, the differences in protein composition of parotid saliva revealed electrophoretically can be detected by amino-acid analysis. Acknowledgements-We arc grateful to Professors C. Dawes, Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3, Y. Yokota, Department of Oral Biochemistry and I. Namikawa, Department of Oral Microbiology, Gifu College of Dentistry, Gifu, 501-02, Japan for advice.
REFERENCES
Abe K. and Dawes
C. (1975) Circadian variations in pilocarpine and isoprenaline-induced protein secretion by rat parotid glands. Archs oral Biol. 20, 543-546. Abe K. and Dawes C. (1978) The effects of electrical and pharmacological stimulation on the types of proteins secreted by rat parotid and submandibular glands. Archs oral Biol. 23, 367-372. Abe K. and Dawes C. (1980) The secretion of protein and of some, electrolytes in response to a- and /I-adrenergic agonists by rat parotid and submandibular salivary glands enlarged by chronic treatment with isoproterenol. J. dent. Res. 59, 1081-1089. Abe K, Yoneda K., Fujita R., Ydkota Y. and Dawes C. (1980) The effects of epinephrine, norepinephrine and phenylephrine on the types of proteins secreted by rat salivary glands, J. dent. Res. 59, 1627-1634. Abe K. and Dawes C. (1982). Dopamine-induced secretion of protein and of some electrolytes by rat submandibular and parotid glands. Archs oral Biol. 27, 635X43. Armstrong W. G. (1967) The composition of organic films formed on human teeth. Caries Res. 1, 89-103.
130
K. ABE an Id H. NITTA
Armstrong W. G. (1970) Amino acid composition of human parotid salivary proteins selectively adsorbed by hydroxyapatite. Archs oral Biol. 15, 1001-1003. Azen E. A. and Oppenheim F. G. (1973) Genetic polymorphism of proline rich human salivary proteins. Science 180, 1067-1069. Ben Abdelilli A. and Desnuelle P. (1964) Sur l’adaptation des enzymes exocrines du pancreas a la composition du regime. Biochim. hiophys. Acta 81, 136149. Brandtzaeg P. (197 I) Human secretory immunoglobulinsVII. Concentrations of parotid IgA and other secretory proteins in relation to the rate of flow and duration of secretory stimulus. Archs oral Biol. 16, 1295-1310. Byrt T. (1966) Secretion and synthesis of amylase in the rat parotid gland after isoprenahne. Nature, Lond. 212, 1212-1215. Caldwell R. C. and Pigman W. (1966) Changes in protein and glycoprotein concentrations in human submaxillary saliva under various stimulatory conditions. Archs oral Biol. 11, 437-449. Dagorn J. C. (1978) Non-parallel enzyme secretion from rat pancreas: In viva studies. J. Physiol., Lond. 280, 435-448. Dijkhof J. and Poort C. (1978) Non-parallel secretion of newly synthesized rat pancreatic proteins after feeding a diet containing raw soybean flour. Biochim. biophys. Acta 543, 167-174. Dische Z., Paravicini C., Kawasaki H., Smirnow N., Cizek L. J. and Chien S. (1962) Influence of the nature of the secretory stimulus on the composition of the carbohydrate moiety of glycoproteins of the submaxillary saliva. Archs Biochem. Biophys. 91, 459464. Dische Z., Kahn N., Rothschild C., Danilchenko A., Liking J. and Wang S. C. (1970) Glycoproteins of submaxillary saliva of the cat: differences in composition produced by sympathetic and parasympathetic nerve stimulation. J. Neurochem. 11, 649-658. Feller R. P.. Shannon I. L. and Wescott W. B. (1974) Total amino acid levels of parotid saliva at different rates of gland function. J. dent. Res. 53, 772. Fernandez-Sorensen A. and Carlson D. (1974) Isolation of a proline-rich protein from rat parotid glands following isoproterenol treatment. Biochem. biophys. Res. Commun. 60, 249-256. Gilliland E. L. and Glazer G. (1980) Parallel secretion of enzymes by the rabbit pancreas. J. Physiol., Lond. 303, 33-41. Hay D. 1. (1973) The isolation from human parotid saliva of a tyrosine-rich acidic peptide which exhibits high affinity for hydroxyapatite surfaces. Archs oral Biol. 18, 1531-1541. Henkin R. I., Lippoldt R. E., Bilstad J., Wolf R. O., Lum C. K. L. and Edelhoch H. (1978) Fractionation of human parotid saliva proteins. ‘J. b;ol. Chem. 253, 755&7565. Howard F. and Yudkin J. (1963) Effect of dietary change upon the amylase and trypsin activities of the rat pancreas. Br. J. Nutr. 17, 281-294. Johnson D. A. and Sreebny L. M. (1982) Effect of increasing
the bulk content of the diet on the rat parotid gland and saliva. J. dent. Res. 61, 691-696. Kanagasuntheram P. and Lim S. H. (1981) Parallel secretion of secretory proteins and calcium by the rat parotid gland. J. Physiol., Land. 312, 445454. Levine M. J. and Ellison S. A. (1973) Immunoelectrophoretic and chemical analysis of human parotid saliva. Archs oral Biol. 18, 839-853. 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, 2655215. Mandel I. D., Thompson R. H. Jr and Ellison S. A. (1965) Studies on the mucoproteins of human parotid saliva. Archs oral Biol. 10, 499-507. Mandel I. D. and Khurana H. S. (1969) The relation of human salivary yA globulin and albumin to flow rate. Archs oral Biol. 14, 1433-1435. Merritt A. D., Rivas M. L., Bixler D. and Newell R. (1973) Salivary and pancreatic amylase: Electrophoretic characterizations and genetic studies. Am. J. hum. Genet. 25, 510-522. Roberge M. and Beaudoin A. R. (1982) Newly synthesized secretory proteins from pig pancreas are not released from a homogenous granule compartment. Biochim. biophys. Acfa 716, 331-336. Robinovitch M. R., Keller P. J., Johnson D. A., Iversen J. M. and Kauffman D. L. (1977) Changes in rat parotid salivary proteins induced by chronic isoproterenol administration J. dent. Res. 56, 290-303. Rothman S. S. (1977) The digestive enzymes of the pancreas: A mixture of inconstant proportions. A. Rev. Physiol. 39, 373-389. Sasaki T., Ohmizu Y. and Tanemura K. (1971) Serum amylase assay method by the “Daiichi” amylase test. The results of the basic properties tested. J. med. Tech. 15, 7 14-7 19 (Japanese). Schneyer C. A. (1965) Modification of the action of pilocarpine by adrenergic blocking agents. Proc. Sot. exp. Biol. Med. 120, 230-232. Sijnju T. and Rolla G. (1975) Further studies of the chemistry of a human salivary blood-group substance. Acta path. microbial. stand. C.83, 215-220. Speirs R. L., Herring J., Cooper W. D., Hardy C. C. and Hind C. R. K. (1974) The influence of sympathetic activity and isoprenaline on the secretion of amylase from human parotid gland. Archs oral Biol. 19, 745-752. Tartakoff A. M., Greene L. J., Jamieson J. D. and Parade G. E. (1974) Parallelism in the processing of pancreatic proteins. In: Advances in Cytopharmacology (Edited by Ceccarelh B., Clementi F. and Meldosi J.) Vol. 2. pp. 177-194. Raven Press, New York. Wallach D., Kirschner N. and Schramm M. (1975) Nonparallel transport of membrane proteins and content proteins during assembly of the secretory granule in rat parotid gland. Biochim. biophys. Acta 375, 87-105. Wong R. S. C., Hofmann T. and Bennick A. (1979) The complete primary structure of a proline-rich phosphoprotein from human saliva. J. biol. Chem. 254, 48OW808.