A Comparative Study of the Effects of Four Choline Esters on the Secretion of Fluid and Glycoprotein from Rat Submandibular Glands

Yoshiki IWABUCHI, Chihiro AOKI and Taizo MASUHARA

Department of Dental Pharmacology, School of Dentistry at Niigata, The Nippon Dental University, Niigata 951, Japan

Accepted June 16, 1988

Abstract-The actions of four choline esters, acetylcholine (ACH), methacholine (MET), carbachol (CAR) and bethanechol (BET), on the secretion of saliva and the specific glycoprotein (GP) contained in the secretory cells of the submandibular gland (SMG) of the rat were compared under conditions with and without physo stigmine (PHY). The ED50 values with respect to salivation were 17 mg/kg for ACH, 1060 ag/kg for BET, 810 zig/kg for MET and 75 ug/kg for CAR, whereas after pretreatment with PHY, ED50 values were lowered to 7.5 mg/kg for ACH and 212 fig/kg for MET, but remained unchanged for CAR and BET. SDS-polyacrylamide gel electrophoresis demonstrated that the saliva from the SMG elicited by the four choline esters contained GP I (130 KDa) and GP IV (21.5 KDa), characteristic of the acinus, and a band of GP III (31 KDa), which originates from the granular tubules. The order of intensity of these bands was band l>>band lll=band IV. Among these bands, band I increased in intensity in a dose-dependent manner. These results suggest that the four choline esters act mainly on the acinar cells, but exert some effects on the granular tubules of the rat SMG.

In a study on five choline esters, acetyl granular convoluted tubules are secreted in choline, methacholine, carbachol, ethyl ether response to methoxamine at optimal doses of methacholine and carbachol, Molitor (1) (6, 9, 10) and dopamine at large doses (8). compared their toxicity, action on the circula The present study was designed to com tory system, miotic action, and effects on the pare the potencies of the effects on salivation gastrointestinal tract, isolated intestine and of four choline esters: acetylcholine, me heart. In addition, the effects of acetylcholine thacholine, bethanechol and carbachol, and carbachol in cats (2) and effects of which have been employed clinically, and methacholine and carbachol in rats, mice and to elucidate the site of action of these hamsters (3) have also been previously com compounds on the functional segments of pared. However, there have been no reports rat submandibular gland. comparing the potencies of the effects of these choline esters on salivation. Materials and Methods We reported previously that the various Collection of submandibular saliva: Male species of secretable glycoprotein contained Sprague-Dawley rats, eight weeks of age, in the rat submandibular gland differ markedly were fasted but allowed water ad libitum for between the acini and the granular convoluted the 24 hr prior to examination. Each rat was tubules (4) : the glycoproteins that are anesthetized with sodium pentobarbital (50 characteristic of the acini are secreted into mg/kg, i.p.) and then placed on a heating the saliva in response to pilocarpine (5), pad maintained at 37 °C. The excretory ducts isoproterenol (6), substance P (7) and of the sublingual gland were ligated after dopamine at small doses (8), whereas the they had been separated from the adherent glycoprotein species characteristic of the tissues around the submandibular gland. The trachea was cannulated with a polyethylene sample, containing 1 % (w/v) SDS, 5% tube (MRC, 2x2.7 mm). Submandibular 2-mercaptethanol and 20% glycerol, was saliva was then collected from the tip of the applied to the top of a 4-40% continuous ductal cannula with a capillary micropipette gradient polyacrylamide disc gel, in a 10 /cl (Drummond Microcaps, 10 and 20 ,ul) at capillary tube, as described by Rachel et al. periods of 5 min for 1 hr after the intraperi (12). Electrophoresis was carried out at toneal administration of the choline deriva 60 V for 60 min in 50 mM Tris-glycine buffer tives. The total volume of saliva elicited per (pH 8.4) in 0.1% SDS. The apparent 5 min was measured, and then the saliva was molecular weights of the glycoproteins promptly stored in small test tubes at -20°C detected on the densitometric scan was until the assays were conducted. At the end estimated from the relative positions of of each experiment, the submandibular glands myosin, a-galactosidase, phosphorylase b, were carefully removed, and flow rates were albumin, ova!bumin, carbonic anhydrase, calculated from the volume of fluid elicited trypsin inhibitor and a-lactalbumin, which per minute per milligram of wet weight of were used as molecular weight markers. each gland. Gels were stained either with 0.2% Coomas Preparation of functional segments: Paren sie Brilliant Blue R-250 for protein or with chymal components from the rat sub Periodic-Schiff's reagent (PAS) for glyco mandibular gland were isolated by the protein, and destained in 7% acetic acid. method of Masuhara and lwabuchi (4) . Rats Gels were scanned with a Joyce-Loebl 3CS were anesthetized with pentobarbital , and microdensitometer at a wavelength of 595 the submandibular gland was perfused via nm for gels stained with Coomassie Blue and the carotid artery with a solution of collage at 550 nm for those stained with PAS. nase which consisted of 0.1% collagenase Administration of drugs: Each of the four (Sigma, Type 11), 1.0 mM CaC12 and 0.1% choline derivatives, acetylcholine (5-30 mg/ bovine serum albumin in modified Hanks' kg), methacholine (150-2000 ug/kg), be medium (137 mM NaCl, 5 mM KCI, 0.8 mM thanechol (300-1500 fig/kg) and carbachol MgSO4, 0.33 mM NaH2PO4, 0 .44 mM (30-150 ,ug/kg), was injected intraperitoneal KH2PO4, 1 MM MgC12, 10 mM Tris-HCI , ly. Physostigmine was injected subcu pH 7.4). The submandibular gland was im taneously 10 min prior to the intraperitoneal mediately removed, sliced and then incubated injection of acetylcholine (20 mg/kg) at for 120 min at 37°C in a sample of the same doses of 10, 25, 50 and 75 /cg/kg. In addition, collagenase solution, in an atmosphere of 95% physostigmine at a dose of 50 ,ug/kg was 02 plus 5% CO2. The slices were rinsed with administered in the same manner prior to the ice-cold modified Hanks' solution to remove injections of acetylcholine (5-20 mg/kg), collagenase, and then under a stereo methacholine (150-700 fig/kg), carbachol microscope, each segment of the acini and (50 ,ig/kg) and bethanechol (700 ug/kg). granular convoluted tubules was dissected The volume of each injection in the present out with needles. study was 0.1 ml/100 g body weight. SDS-polyacrylamide micro-disc electro Drugs: Drugs used were acetylcholine phoresis: Each segment was dissolved in an chloride (Ovisot, Daiichi Seiyaku), me equal volume of 6% (w/v) sodium dodecyl thacholine chloride (Sigma), bethanechol sulphate (SDS) solution, which contained chloride (Sigma), carbachol (Sigma) and 10% 2-mercaptethanol, and was heated at physostigmine sulfate (Wako Pure Chem 90°C for 3 min. The protein content of each icals). of the samples of saliva and tissue was Statistical analysis: Data are presented as determined by the Lowry method (11) with the mean+S.E. of data from 6 rats. The bovine serum albumin as the standard. In statistical significance of differences was as the case of determination of protein content sessed by Student's t-test. in segments, standard solutions contain SDS and 2-mercaptethanol at the concentrations Results present in the samples. One ,,l of each Secretory response of submandibular saliva: The flow rates of saliva elicited from rat methacholine and during the period 5-10 min submandibular glands after intraperitoneal after injection for carbachol (30-150 iig/kg). injection of acetylcholine (10-30 mg/kg), In the case of bethanechol, the maximum methacholine (300-2000 ag/kg), carbachol flow rate was achieved during the period 5-10 (30-150 acg/kg) and bethanechol (300 min after injection of doses of 700, 1000 or 1500 cg/kg) increased in a dose-dependent 1 500 ,ug/kg and during the period 10-15 min manner during the observation period (Fig. after injection of doses of 300 or 500 ,ug/kg. 1); they reached a maximal level during the Disappearance of salivation produced by first 5 min in the case of acetylcholine and administration of either acetylcholine or

Fig. 1. Comparison of flow rates of the saliva elicited from at submandibular glands by i.p. adminis tration of acetylcholine (A), methacholine (B), carbachol (C) and bethanechcl (D). Each point re presents the mean±S.E. of results for six animals. Fig. 2. Dose-response curve of the volume of saliva elicited from rat submandibular glands by i.p. administration of acetylcholine, methacholine, carbachol and bethanechol. Each point represents the mean±S.E. of results for six animals.

Fig. 3. Effects of physostigmine on the flow rate (A) and volume (B) of saliva elicited from rat sub mandibular glands by acetylcholine at a dose of 20 mg/kg. Physostigmine at doses of 10, 25, 50 and 75 tg/kg was injected s.c. 10 min prior to i.p. administration of acetylcholine at a dose of 20 mg/kg. (A) Each point represents the mean±S.E. of results for six animals. 0------0 without physostigmine, 40 with physostigmine. (B) Each point represents a relative value expressed as a multiple of the volume of saliva secreted by acetylcholine which was taken as 1.00. methacholine proceeded rapidly after the salivation elicited by acetylcholine at a dose maximal flow rates were reached and ceased of 20 mg/kg was observed until 15-25 min, within about 10-35 min after administration, 20-25 min, 25-30 min and 20-30 min after whereas in the case of carbachol or betha administration of acetylcholine, respectively nechol, salivation decreased gradually until (Fig. 3A), and the total volume of saliva 60 min had elapsed. The total volumes of increased 1.1-, 1.6-, 2.3 and 2.4-fold, saliva elicited by administration of either respectively, compared to the total volumes in bethanechol or methacholine were similar in the absence of physostigmine (Fig. 3B). the dose range from 300 to 1000 ag/kg (Fig. Flow rates of saliva in rats under the influence 2). The total volume of saliva secreted during of physostigmine at a dose of 50 ag/kg are the 60 min after administration of the drugs shown in Fig. 4. In this case, administration increased in a dose-dependent manner, and of acetylcholine at a dose of 10 and 20 mg/kg the order of potency was carbachol>> prolonged the duration of salivation by about bethanechol>methacholine>>]acetylcholine. 10 and 15 min, respectively, and the total When rats were pretreated with physo volumes increased 6.6 and 2.3-fold, respec stigmine at doses of 10. 25, 50 and 75 gig/kg, tively, compared to the volume elicited in the

Fig. 4. Effects of physostigmine at a dose of 50 pg/kg on the flow rate of saliva elicited from rat sub mandibular glands by i.p. administration of acetylcholine (A), methacholine (B), carbachol (C) and bethanechol (D). Physostigmine at a dose of 50 ug/kg was injected s.c. 10 min prior to i.p. adminis tration of choline ester. 0------0 without physostigmine, 0-0 with physostigmine. Each point represents the mean±S.E. of results for six animals. absence of physostigmine. Acetylcholine at of physostigmine. By contrast, a single a dose of 5 mg/kg failed to elicit secretion administration of physostigmine at a dose of from the gland, but in combination with 50 ug/kg failed to elicit any saliva from the physostigmine, saliva was evoked for 10-15 submandibular gland (data not shown). min. In the case of methacholine at doses of The ED50 values for salivation during the 300, 500 or 700 ug/kg, salivation was 30 min after administration of the test com observed for 30 min, and total volumes pounds were 17 mg/kg for acetylcholine, increased 11.8-, 5.9 and 2.3-fold, respec 1060 ug/kg for bethanechol, 810 ug/kg for tively, compared to volumes elicited in the methacholine and 75 ug/kg for carbachol absence of physostigmine. Although me under conditions without physostigmine. thacholine alone at a dose of 150 ug/kg With rats under the influence of physostig failed to elicit salivation, saliva was evoked for mine, the values fell to 7.5 mg/kg for acetyl 20-25 min when methacholine was given in choline and 212 ag/kg for methacholine, but combination with physostigmine. Further were not altered for carbachol or bethanechol. more, in the case of carbachol at a dose of 50 Concentration of protein and total a ug/kg or bethanechol at a dose of 700 ug/kg, mounts of protein in saliva elicited from patterns of secretion and total volumes did submandibular glands: The concentrations not differ from those recorded in the absence and amounts of protein in saliva elicited from

Table 1. Concentration and amounts of protein in saliva secreted from rat submandibular glands after administration of acetylcholine, methacholine, carbachol and bethanechol, with or without physostigmine administration the submandibular glands during the first 5 creased when the dose was 10 mg/kg and min after administration of the four choline increased when the dose was 20 mg/kg. By esters, with and without physostigmine at a contrast, the concentrations of protein in dose of 50 ,ug/kg, are shown in Table 1. saliva elicited by methacholine at doses of When the rats were administered the 300, 500 and 700 ug/kg decreased 0.20-, choline esters alone, the concentration of 0.33 and 0.79 fold, respectively, compared protein in the saliva decreased in a dose to protein concentrations in the absence of dependent manner, while the total amounts physostigmine. Furthermore, the concen of protein increased conversly. trations of protein in saliva elicited by car When the rats were given physostigmine at bachol at a dose of 50 ug/kg and bethanechol a dose of 10, 25, 50 or 75 ag/kg prior to the at a dose of 700 ug/kg increased 1.7 and injection of acetylcholine at a dose of 20 mg/ 1.4-fold, respectively, compared to values in kg, the protein concentration in the saliva did the absence of physostigmine. By contrast, not change significantly, whereas the amounts total amounts of protein produced after in of protein secreted into the saliva increased jection of acetylcholine at doses of 10 and 20 1.2-, 1.5-, 2.1 and 1.4-fold, respectively, mg/kg in combination with physostigmine compared to the amounts in the absence of increased 2.8 and 2.1-fold when compared physostigmine (data not shown). When rats to the amounts elicited without physostig were under the influence of physostigmine at mine; and, in the same manner, the total a dose of 50 fig/kg, the concentration of amounts of protein increased 1.3-, 1.5 and protein in acetylcholine-evoked saliva de 1.2-fold for methacholine at doses of 300,

Fig. 5. Typical densitometric scans of glycoproteins from saliva elicited from rat submandibular glands by administration of acetylcholine (A), methacholine (B), carbachol (C) and bethanechol (D). Each sample (0.375 ug protein) of saliva elicited during the period 0-5 min after i.p. administration of acetyl choline at a dose of 10 mg/kg, methacholine or carbachol at a dose of 1 mg/kg, and bethanechol at a dose of 0.1 mg/kg was applied to the gel, and electrophoresis was performed as described in Materials and Methods. 500 and 700 jcg/kg respectively; 2.2-fold for choline esters administered intravenously to carbachol at a dose of 50 ug/kg; and 2.0 rats, their miotic action in rabbits, and their fold for bethanechol at a dose of 700 ug/kg. cathartic action in dogs after the subcu Electrophoretic profiles of the glycopro taneous injection of these drugs (1). teins in saliva and segments: The typical It is well-known that some choline esters, electrophoretic patterns of glycoproteins con in particular, acetylcholine, are hydrolyzed to tained in the submandibular saliva elicited a greater or lesser extent by both acetyl during the first 5 min after intraperitoneal cholinesterase and pseudocholinesterase. administration of acetylcholine at a dose of The duration of the salivation induced by 10 mg/kg, methacholine or bethanechol at a acetylcholine is considerably shorter than that dose of 1 mg/kg, and carbachol at a dose of of salivation induced by methacholine, as 0.1 mg/kg, are shown in Fig. 5. The number shown in Fig. 1. Thus, for the sake of com of bands of glycoprotein did not differ sig parisons, this inconvenient difference was nificantly between acetylcholine-, methacho elimated by performing separate experiments line-, bethanechol and carbachol-evoked on choline esters in the presence of physo saliva. The profiles showed a heavily stained stigmine, an anticholinesterase. When physo band I, and two slightly stained bands, III stigmine at different doses was administered and IV. However, the intensities of band I of prior to acetylcholine at a dose of 20 mg/kg, the glycoproteins from either methacholine prolongation of salivation (Fig. 3A) and an or carbachol -evoked saliva were stronger increase in salivary volume (Fig. 3B) occurred than those of band I from acetylcholine and in a dose-dependent manner. Physostigmine bethanechol-evoked saliva. Also, the intensity at a dose of 50 jig/kg, which produces a of this band in saliva evoked by administration maximal response, did not by itself elicit of the four choline esters was greater at high saliva from the submandibular glands (data doses than at low doses of these drugs. The not shown). Maayani et al. (13) have patterns of glycoproteins from the secretory reported that the ED50 value for salivation in segments isolated from the submandibular mice induced by a single subcutaneous in gland of the normal rat showed that the acinar jection of physostigmine was 0.15 mg/kg segments contain one major band (I) and and that, at a dose of 50 jig/kg, salivation three minor bands (II, III and IV), whereas was not observed. Thus, the potencies of the granular convoluted tubules contain one the effects on salivation of the four choline major band (III) and two minor bands (I esters was compared in rats under the in and II), as reported previously (4). When fluence of physostigmine at a dose of 50 ug/ samples of saliva elicited by the four choline kg. In this case, the dose-response curve for esters were subjected to electrophoresis with acetylcholine shifted to the left on the abscissa solubilized segments of the acinus or of the (data not shown). A similar finding has been granular tubules, the main bands I and IV in recorded in cats (14). Furthermore, the dose saliva were electrophoretically identical with response curve for methacholine also shifted the main bands I and IV in the acinar seg to the left, but the extent of the shift was ment, whereas band III in saliva was electro considerably smaller than that of the dose phoretically identical to the major band (III) response curve for acetylcholine (Fig. not contained in granular tubules (data not shown). In contrast, the effects of both shown). carbachol and bethanechol were not affected by physostigmine, as generally recognized. Discussion In the present study, we found that the order In the present study, the relative potencies of potency among the four choline esters in of their effects on salivation from rat sub rats under the influence of physostigmine was mandibular glands of the four choline esters carbachol>methacholine>bethanechol> were carbachol>>bethanechol>methacholine acetylcholine. >>acetylcholine (Fig. 2). The same order of The protein concentrations in the saliva potency has also been observed for the four elicited from the submandibular glands choline esters with regards to the toxicity of during the first 5 min after administration of

In summary, our data indicate that the order of potency of their effects on salivation is carbachol>>bethanechol>methacholine> acetylcholine; while in combination with physostigmine, the order is altered to carba chol > methacholine > bethanechol > acetyl choline. Furthermore, the four choline esters tested appear to act mainly on the acinar cells of rat submandibular glands, but have some effect on the granular tubules through an indirect action of the choline esters.

the four choline esters were reduced in a proportions of epinephrine and norepineph dose-dependent manner (Table 1). In con rine in cats. If this indirect action of choline trast, the amounts of protein secreted in esters is applicable to the granular tubules of these samples of saliva were increased in a the salivary gland, the fact that the intensity dose-dependent manner (Table 1). In rats of band III is equal to that of band IV from under the influence of physostigmine at a the acinus could be explained as being due to dose of 50 ,ug/kg, the concentrations of pro the action of epinephrine released from the tein in both acetylcholine and methacholine adrenal gland in response to choline esters. evoked saliva were reduced, whereas the concentrations in carbachol and be thanechol-evoked saliva were increased sig nificantly (Table 1). However, the amounts of protein secreted into saliva increased in the case of all choline esters tested (Table 1). The increase in the amounts of protein can be attributed to the prevention of hydrolysis of acetylcholine by acetylcholinesterase at the sites of cholinergic transmission. On the other hand, the changes in concentration of protein induced by pretreatment with physostigmine may be attributed to the secretion of fluid Acknowledgment: This study was supported in rather than protein, since secretion of fluid part by a Grant-in-Aid for Scientific Research (No. increased after acetylcholine and metha 60480404) from the Ministry of Education, Science choline, and it did not change after carbachol and Culture of Japan. and bethanechol. The administration of the choline esters References elicited the secretion of glycoproteins that 1 Molitor, H.: A comparative study of the effects migrated as the three bands designated as I, of five choline compounds used in therapeutics: III and IV on gels (Fig. 5). The intensity of acetylcholine chloride, acetyl beta-methylcholine band was considerably higher than those of chloride, carbaminoyl choline, ethyl ether methylcholine chloride, carbaminoyl beta bands III and IV. In our previous (4) and methylcholine chloride. J. Pharmacol. Exp. present studies, glycoprotein bands I and Ther. 58, 337-360 (1936) IV were characteristically found in the acinus 2 Emmelin, N.: On the innervation of the sub and were secreted in response to adminis maxillary gland cells in cats. Acta Physiol. tration of the choline esters. Junqueira et al. Scand. 34, 11-21 (1955) (15) have indicated, from histological studies, 3 Abe, K. and Dawes, C.: Secretion of protein by that carbachol acts mainly on the acinar the submandibular glands of the rat, mouse, and cells of the submandibular gland of rats and hamster in response to various parasympatho mice. Their findings indirectly support our and sympathomirnetic drugs. J. Dent. Res. 61, data. In the present study, we also found that 1454-1457 (1982) the intensity of band III originating in the 4 Masuhara, T. and Iwabuchi, Y.: Micro-discelec granular tubules was almost equal to that of trophoretic study on glycoproteins of parenchy band IV from the acinus. The glycoprotein mal components dissociated from rat sub which migrates as band III is secreted selec mandibular gland. Japan. J. Oral Biol. 25, 1177 tively into the saliva after administration of 1180 (1983) a1-adrenergic agonists such as methoxamine 5 Masuhara, T. and Iwabuchi, Y.: Characterization of glycoproteins in submandibular acinus and (6, 9, 10). Therefore, it is suggested that, to saliva of rats elicited by pilocarpine. Japan. J. some extent, choline esters may act as a, Oral Biol. 26, 280-283 (1984) adrenergic receptors in granular tubules. 6 Iwabuchi, Y., Aoki, C. and Masuhara, T,: Effects According to Douglas and Poisner (16), of adrenergic agents on glycoprotein secretion stimulation of adrenal perfusion by acetyl from acini and granular convoluted tubules of choline caused the release of almost equal the rat submandibular gland. Japan. J. Oral Biol.

impulse and of its chemical transmission. Q. J. Exp. Physiol. 26, 265-283 (1937)

in terms of the present knowledge of the nervous

27, 741-745 (1985) ten in Kapillaren; ihre Anwendung zur Protein 7 Iwabuchi, Y., Aoki, C. and Masuhara, T.: Effects fraktionierung and Molgewichts-bestimmung. of substance P on glycoprotein secretion from Hoppe Seylers Z. Physiol. Chem. 354, 1351 acinar cells of the rat submandibular gland. 1368 (1973) Japan. J. Pharmacol. 42, 453-456 (1986) 13 Maayani, S., Egozi, Y., Pinchasi, I. and Sokolo 8 Iwabuchi, Y., Aoki, C. and Masuhara, T.: Effects vsky, M.: On the interaction of drugs with the of dopamine on the secretion of glycoproteins cholinergic nervous system. V. Characterizaticn from the functional segments of the rat sub of some effects induced by physostigmine in mandibular gland. Japan. J. Pharmacol. 45, 149 mice: in vivo and in vitro studies. Biochem. 158 (1987) Pharmacol. 27, 203-211 (1978) 9 Iwabuchi, Y., Aoki, C. and Masuhara, T.: Secre 14 Beznak, M. and Farkas, E.: The interpretation of tory pattern of characteristic glycoprotein species some phenomena of salivary secretion caused by from segments of the rat submandibular gland direct electrical stimulation of the effector nerve, following methoxamine stimulation. Japan. J. Oral Biol. 27, 1267-1271 (1985) 10 Iwabuchi, Y., Aoki, C. and Masuhara, T.: Effects of adrenergic antagonists on glycoprotein 15 Junquira, L.C.U., Toledo, A.M.S. and Saad, A.: secretion from granular convoluted tubules of Studies on the physiology of rat and mouse the rat submandibular gland induced by me submaxillary glands. I. Amylase and protease thoxamine. Japan. J. Oral Biol. 28, 761-765 activities in serum, submaxillary gland, and (1986) submaxillary saliva of rat and mouse. In Salivary 11 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Glands and Their Secretions, Edited by Sreebny, Randall, R.J.: Protein measurement with the L.M. and Meyer, J., p. 105-118, Pergamon Folin phenol reagent. J. Biol. Chem. 193, 265 Press, London (1964) 275 (1951) 16 Douglas, W.W. and Poisner, A.M.: Preferential 12 Rachel, R., Mesecke, S., Wolfrum, D.I. and release of adrenaline from the adrenal medulla by Neuhoff, V.: Mikro-elektrophorese an Kon muscarine and pilocarpine. Nature 208, 1102 tinuierlichen Polyacrylamide Gradiented Gelen. 1103 (1965) I. Herstellung and Eigenschaften von Gelgradien