Effects of monensin and salinomycin on amylase release from the superfused parotid segments of sheep and rat

Research in Veterinary Science /986, 4/, 207-2/0

Effects of monensin and salinomycin on amylase release from the superfused parotid segments of sheep and rat K. KATOH, T. TSUDA, Department of Animal Physiology, Faculty of Agriculture, Tohoku University,

Tsutsumidori Amamiyamachi, Sendai 980, Miyagi, Japan

The effects of monensin and salinomycin (ionophorous antibiotics) on amylase release in the parotid glands were investigated in the superfused segments of sheep and rat. Monensin and salinomycin as well as acetylcholine and isoprenaline caused enhanced amylase release in a dose-dependent manner. Salinomycin was significantly more effective than monensin in both animals. The enhanced amylase release evoked by these ionophores and acetylcholine was significantly reduced in a calcium-free solution containing EGTA. From these results it is concluded that monensin and salinomycin transport calcium ions into the cytosol of parotid acinar cells from the extracellular fluid, resulting in amylase release. RECENTL Y, carboxylic ionophores such as monensin, salinomycin and lasalocid have been suggested as useful candidates as chemical additives which can efficiently promote the growth rate of beef cattle through the control of ruminal fermentation (Chalupa 1980, Goodrich et al 1984, Schelling 1984). These antibiotics, through their actions as ionophores, generally decrease the molar ratio of acetate to propionate, as well as methane production and amino acid degradation by the ruminal bacteria. However, the ruminal metabolic alteration is not sufficient to account for all the biological effects of these ionophores. More complex actions on the digestive and metabolic pathways might also be involved (Pressman 1976, Bergen and Bates 1984). It is likely that these ionophores administered orally are absorbed in part, metabolised and excreted in the bile (Donoho 1984). However, there are few reports on the physiological or pharmacological effects of the ionophores in the host animals. Only the effects on the cardiovascular system (Fahirn and Pressman 1981) and on mineral metabolism (Spears and Hervey 1982, Starnes et al 1984) are known. In the present study, the effects of monensin and salinomycin, as well as other autonomimetic drugs, on amylase release from tissue segments of parotid gland in sheep and rat, were investigated.

Materials and methods Five crossbred sheep (45 to 74 kg bodyweight) of both sexes and male Wistar-strain rats (230 to 310 g bodyweight) were used.

Superfusion experiments The test animals were killed by an overdose of sodium pentobarbitone. Then a part of the parotid gland in sheep or the whole organ in rats was isolated and cut into small segments with a pair of fine scissors in an oxygenated Tris-buffered superfusing solution, as previously described in detail (Katoh et al 1983). These segments (about 50 mg or 15 mg wet weight of tissue segments of sheep or rat, respectively) were transferred to a column-shaped tissue basket (about O' 5 ml), the bottom of which consisted of gauze, and incubated for 20 minutes at 37°C in 50 ml of oxygenated superfusing solution. In experiments to determine the time course of amylase release, the sampling procedure was similar to that described in the previous paper (Katoh and Tsuda 1984). Namely, after the preincubation, the tissue basket containing tissue segments was transferred into a 10 ml test-tube containing 2 ml of oxygenated superfusing solution (37°C) for one minute, then transferred into another test-tube containing 2 ml of oxygenated superfusing solution. The same procedure was repeated successively. The first to the fifth test-tube contained a control solution, while the sixth to the 20th (last) had the same solution containing secretagogues at a given concentration. After removal of the basket from the last test-tube, the tissue segments were weighed. In experiments to obtain the dose-response curves, the first and the second (basal) test-tubes contained a control solution, while the third to the seventh had a solution containing secretagogues, the concentration of which was increased IO-fold at each step. The incubation time for each test-tube was 10 minutes. In this case, amylase increment (per cent) was calculated according to the following formula:

207

K. Katoh, T. Tsuda

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amylase release (iu 10 min -I) stimulated by secretagogue - basal amylase release (iu 10 min -I)

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basal amylase release (iu 10 min -I) Any value less than the basal level was regarded as

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Measurement of amylase concentration Amylase concentration in the 2 ml sample solution in each test-tube was determined by the blue starch method previously described in detail (Katoh et al 1984). Namely, one tablet of blue starch (Rinderknecht et al 1967) was added to each sample solution, or to a solution containing amylase at a defined concentration for calibration, under ice-cooling after dilution by the addition of 2 ml of distilled water. The mixture was warmed at 37°C for several minutes in a water bath. To stop the catalysis O' 5 ml of O' 5 N sodium hydroxide was then added to the mixture. This mixture was centrifuged at 12,000 rpm for five minutes and the absorbance of the supernatant was measured photometrically at 620 nm. From the amylase concentration thus determined (iu ml') and the tissue wet weight, amylase release (iu g-I min-I) was calculated. The composition of Tris-buffered superfusing solution used was similar to that previously described (Katoh et al 1983), except that the concentration of glucose was doubled (5'6 mM). The solution was gassed with 100 per cent oxygen. The pH of the solution was 7'4 at 37°e. In a calcium-free solution, calcium chloride was replaced by an equiosmolar amount of sodium chloride containing EGTA (ethylene glycol bis [(J-aminoethyl etherl-N,N,N / ,N /-tetraacetic acid, 10- 4 M). Drugs used were acetylcholine chloride, isoprenaline bitartrate, EGTA, atropine sulphate, propranolol HCl (Sigma), monensin (Eli Lilly), salinomycin (Kaken Pharm) phentolamine mesylate (Ciba) and A23187 (Calbiochem). All ionophores were dissolved in ethanol and stored at - 20°e. Amylase (Sigma, type VI-A) was used as a calibration standard for amylase activity.· Blue starch was purchased from Daiichi. Results are given as the mean ± SEM. For statistical analysis, Student's t test was employed.

Results Effects of monensin and salinomycin on amylase release in parotid segments ofsheep and rat As shown in Fig I, for sheep parotid salinomycin and monensin at 10- 3 M caused an increase in amylase release by 238'3 ± 58'7 or 58'5 ± 18'7 per

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cent, respectively. The amylase increment evoked by salinomycin was significantly larger than that evoked by monensin at the same dose. Although Patterson and Titchen (1979) reported that sheep parotid has an adrenergic innervation, the {J-adrenergic agonist isoprenaline (10- 6 M) caused only a small and unstable increase in amylase increment (about 10 per cent). No clear effect of acetylcholine (5' 5 X 10- 6 M) was seen. As shown in Fig 2, for rat parotid segments isoprenaline (10- 6 M), salinomycin (10- 4 M) and monensin (10- 4 M) each caused an increase in amylase release. However, the amylase release evoked by isoprenaline or monensin was sustained, whereas that evoked by salinomycin was transient. The amylase release evoked by both ionophores was persistent even in a solution containing atropine (I' 4 X 10- 6 M), phentolamine (10- 5 M) and propranolol (5 X 10- 6 M). Fig 3 shows dose-response curves in rat parotid segments for isoprenaline, acetylcholine, salino-

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FIG 2: The time course of amylase releaseevoked by stimulation with isoprenaline, salinomycin or monensin in the parotid segments of rat. The stimulation was carried out during the period indicated by the black strip. In the stimulation with salinomycin or monensin, the superfusing solution contained atropine 11·4x 10~6 Ml, phentolamine (10- 5 M) and propranolol 15x 10- 6 M). Results are given as the mean ± SEM of three different experiments

mycin, monensin and A23187, the ED 50 values of which were about 10- 6 , 2x 10- 8 , 2x 10- 5 , 2x 10- 4 and I· 5 x 10- 8 M, respectively. The potency or efficacy was largest in the stimulation with acetylcholine or salinomycin, respectively. On the other hand, other types of ionophores (gramicidine and valinomycin) also increased amylase release in a dosedependent manner, and these two ionophores showed similar potency and efficacy which were much less than that of salinomycin (not shown).

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FIG 4: Dose-response curves of amylase release increment in the calcium-free solution containing EGTA 110- 4 M) for stimulation with isoprenaline, salinornvcin, rnonensin, acetylcholine or A23187 in the parotid segments of rat. Results are given as the mean ± SEM of three different experiments

Effects of extracellular calcium removal on amylase release In a calcium-free solution containing EGTA (10- 4 M), the amylase increment evoked by 10- 3 M salinomycin was significantly reduced (P<0'05) to 52'8 ± 39'1 per cent in sheep parotid segments (Fig I). In rat parotid segments, calcium removal also reduced the amylase increment evoked by stimulation with acetylcholine, salinornycin, monensin or A23187, but had less effect on that evoked by stimulation with isoprenaline, as shown in Fig 4. The ED 50 values of isoprenaline, acetylcholine, salinornycin, monensin and A23187 were about 2 x 10-7, 5 x 10-7, 2x 10-4, 10- 5 and 10- 8 M, respectively. The ED 50 values of isoprenaline, salinomycin and monensin were increased about 10 to 20 times in the calciumfree solution, while that of acetylcholine was decreased about 25 times compared with those in the control solution. Discussion

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FIG 3: Dose-response curves for amylase release increment evoked by stimulation with isoprenaline, acetylcholine. salinornvcin, monensin or A23187 in the parotid segments of rat. The scale for A23187 is shown on the right-hand side. Results are given as the mean ± SEM of three to five different experiments

Acinar cells of parotid and submaxillary glands of rat and mouse are known to possess two distinct cellular pathways through which amylase and fluid secretion are controlled (Butcher and Putney 1980, Gallacher and Petersen 1983). One pathway is through a calcium-mediated process which is shared by the stimulation of cholinergic and a-adrenergic receptors and substance P receptor. The other is through a cyclic adenosine monophosphate (cAMP)mediated process which is activated by the stimulation of fJ-adrenergic receptor. The secretory mechanisms of the parotid glands of sheep should be in principle similar to the model established in these rodents, since it is known that fluid secretion of sheep parotid is

210

K. Katoh, T. Tsuda

increased by electrical stimulation of cholinergic or adrenergic nerves or by the injection of the parasympathetic or sympathomimetic drugs (Kay 1958, Patterson and Titchen 1979, Heal 1979). However, the importance of salivary amylase is unclear in sheep because bacteria in the reticulo-rumen might metabolise all starch into short chain fatty acids. Ionophores are generally classified into three types (carboxylic, neutral and channel-forming quasi-ionoph ores) as described by Pressman (1976). Monensin is the only ionophore permitted as an additive in cattle feed in the USA and belongs to the class of carboxylic ionophores, like salinomycin and A23187. On the other hand, gramicidin belongs to the class of channel-forming, quasi-ionophores, while valinomycin is one of the neutral ionophores. In the present study, all types of ionophores used stimulated amylase release and salinomycin was the most effective. The fact that monensin and salinomycin were effective in sheep, rat and pig (K. Katoh, unpublished data) suggests that these ionophores have no species-specificity. The amylase release evoked by acetylcholine or ionophores should be caused mainly by an increase in cellular calcium concentration, which is due to an increase in calcium influx from extracellular fluid into cytosol, because the amylase increment evoked by these stimulants was drastically reduced by the removal of extracellular calcium (Figs I and 4). It has not been reported that salinomycin and monensin have such a high affinity for calcium resulting in a large increment in amylase release, although Pressman (1976) reported that monensin mediates mainly' sodium-hydrogen ion exchange while salinomycin and lasalocid display a higher affinity for potassium. The result for acetylcholine on the calcium-dependency agrees with the previous findings that amylase release from rat and mouse parotid segments evoked by acetylcholine was markedly reduced by the removal of extracellular calcium (Petersen et al 1977, Watson et al 1979). The residual amylase release evoked by both ionophores in a calcium-free solution containing EGTA, as shown in Fig 4, might be caused by the toxity of ionophores (Todd et al 1984) or by an increase in cellular calcium concentration liberated from the internal calcium stores (Watson et aI1981). In conclusion, ionophorous antibiotics such as salinomycin and monensin were shown to transport divalent cations. The activity of salinomycin for calcium transport was larger than that of monensin. This result seems to be consistent with the general

observation that salinomycin is more effective in controlling ruminal fermentation at a given dosage.

Acknowledgements This work was supported in part by financial aid from Kaken Pharm Co, Japan.

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Accepted October 25, 1985