Changes of salivary amylase in serum and parotid glandduring pharmacological and physiological stimulation

Akos Nagya, Adrienn Bartaa,b, Gabor Vargab, Tivadar Zellesa,*aDepartment of Oral Biology, Nagyvarad ter 4, 1084 Budapest, Hungary

bInstitute of Experimental Medicine, Hungarian Academy of Sciences, Hungary

Abstract

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information isavailable regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to

investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to variousstimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1

h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fedanimals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch asa substrate. Pancreatic and parotid isoamylase levels in serum were separated by gelelectrophoresis utilizing differences in ionic

properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation.Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats duringtheir peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, aswell as a mild stimulation of parotid function by spontaneuous food intake during nonfasted state results in a decrease in parotid

tissue amylase activity and a proportional increase in serum levels of parotid isoamylase. # 2001 Published by Elsevier Science Ltd.

Keywords: Parotid gland; Isoenzymes; Parotid amylase; Serum; Pilocarpine; Food intake

1. Introduction

According to the classical view there are two distincttypes of secretory glands, exocrine and endocrine. Exo-crine glands secrete the products they manufacture intothe external environment, such as the lumen of the gas-trointestinal tract, via the duct system of the gland. Onthe other hand, endocrine glands, release their productsinternally, initially into interstitial fluid and then intothe bloodstream. It has been long known, however, thatdigestive enzymes such as trypsin, lipase and amylase,can be detected in blood serum [10]. Their appearance inblood is generally believed to be the result of patholo-gical events or accidental byproducts of exocrine secre-tion. Consistent with this view, elevated concentrationsof digestive enzymes in serum are common symptomsand important diagnostic criteria for inflammatory dis-eases such as acute pancreatitis or mumps [16,17,31].But digestive enzymes are normal constituents of blood

under physiological conditions as well, and a number ofstudies indicate that their levels in serum are regulatedby physiological mechanisms [10].The two major sources of a-amylase in rat and man

are the parotid gland and the pancreas [11]. Thesesources were shown to contribute to serum amylasesecretion equally in man [22] whereas in rat, the pan-creas seems to secrete much less amylase into the circu-lation than the parotid gland under physiologicalconditions [24]. It has been also shown that pharmaco-logical stimulation of parotid function by pilocarpine[8,9] as well as parasympathetic nerve stimulation [25]leads to an increase in serum amylase activity. In fastedrats food intake was also shown to elevate the level ofthe parotid isoform of the enzyme [23]. It is not known,however, how serum isoamylase level is affected byspontaneous feeding in nonfasted animals. The purposeof the present study was twofold. First we attempted toclarify whether normal feeding affects parotid iso-amylase level in serum. Second we compared the rate ofdisappearance of amylase from parotid tissue and itsappearance in serum in response to different pharma-cological and physiological stimulations.

0928-4257/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.

PI I : S0928-4257(01 )00018-3

Journal of Physiology - Paris 95 (2001) 141–145

www.elsevier.com/locate/jphysparis

* Corresponding author. Tel.: +36-1-210-4415; fax:+36-1-210-4421.

E-mail address: zeltiv@net.sote.hu (T. Zelles).

2. Methods

2.1. Animals

Female 250–320 g Wistar rats (Charles River,Hungary) were housed under conditions of constanttemperature (24 �C) and a 12–12 h light cycle (lights onfrom 07:00 h until 19:00 h). They were adapted for atleast 14 days to this cycle before starting experiments.Animals received standard rat chow and water adlibitum.

2.2. Experimental design

In the first experiments, after 16 h fasting rats receivedeither 5 mg/kg pilocarpine-HCl or saline (n=10–20).After 1 h, blood and parotid glands were collected. Inthe second experiment 18 rats were used. After 16 hfasting half of the animals were fed with standardlaboratory rodent chow for 1 h, the other half receivedno food. After 1 h blood and parotid glands were col-lected. In the third experiment 16 rats were allocatedinto two groups. All of the animals were fed ad libitumup to the start of the experiment. Then in the first groupjust before the light was turned off at 19:00 h, food waswithdrawn and animals were sacrificed 2 h later. Foodwas not withdrawn in the other group of rats; these ratswere fed ad libitum and were also sacrificed at 21:00 h.

2.3. Evaluation of amylase activity

In all studies, upon completion of experiments, ani-mals were sacrificed by thiopental-Na anesthesia (50mg/kg) followed by exsanguination from the abdominalvein. After collection, blood was centrifuged at 2500 gat 4 �C, and the serum was stored at �20 �C untilassayed. Electrophoresis of serum samples was per-formed in 0.9% agarose gels at pH 8.6 in Veronal bufferat 80V. Parotid and pancreatic amylase isoforms wereseparated utilizing their opposite charges at pH 8.6.Amylase activity was determined from isoenzyme bandsof the gel.For tissue analysis, following careful dissection, the

parotid glands were trimmed out of fat, weighed andhomogenized in Tris-buffer (pH 7.4). The homo-genates were centrifuged at 4 �C at 2500 g for 10 min.The supernatant was used for evaluation of amylaseactivity.Amylase activity in tissue and serum was determined

by starch-iodine color reaction as described previouslyin details [34]. Briefly, the substrate solution consisted ofstarch (4 g/l), Tris–HCl (60 mM), NaCl (50 mM), CaCl2(0.001 mM) at pH 7.4. The iodine solution included I2(0.2 mM), KI (3 mM), HCl (30 mM). Samples weregiven to substrate solution for 5–10 min at 37 �C. Thereaction was stopped by pipetting equal volume of the

incubation mixture into the iodine solution with simul-taneous stirring. The optical density of the reactionmixture was read at 620 nm. Dilution series of starchsolution served as internal calibration points. Underthese conditions there was a linear correlation betweenthe change in optical density of the reaction mixture andthe amount of digested starch. The value of amylaseactivity is given as unit (U) that equals 1 g starch diges-ted in 1 min.

2.4. Statistical analysis

Values were given as mean�S.E.M. Experimentswere performed using at least eight parallel samples.Percent of inhibition was calculated as percentage ofcontrol values (i.e. in vehicle treated rats, or in ratsreceiving no food). Comparison among the groups wasperformed by analysis of variance (ANOVA).

3. Results

Serum parotid isoamylase level in nonfasted rats was5.78�0.79 U/l. In rats fasted for 16 h we found that thisvalue was slightly decreased and reached the level of4.10�0.68 U/l. We observed that under our experi-mental conditions parotid tissue amylase activity innonfasted animals was 5.00�0.94 U/100 mg. Following16 h food withdrawal this value was found to be7.69�1.52 U/100 mg. We also observed that the elec-trophoretic mobilities of parotid amylase and serumparotid isoamylase were very similar, while the pan-creatic amylase in serum samples moved to the oppositedirection during electrophoresis.In the first experiment we studied the effect of pilo-

carpine, a potent parasympathetic agent on changes ofserum and parotid tissue levels of the isoenzyme. Wefound a 39�9% (P<0.01) decrease in tissue activity,and a 101�39% (P<0.01) increase in serum activityafter one hour of pilocarpine administration (Fig. 1).In the next experiment, in fasted animals the effect of

re-feeding was investigated. In rats fasted for 16 h, 1-hfeeding resulted in a 35�15% (P<0.01) decrease in tis-sue activity, and a 41�17% (P<0.05) increase in serumactivity (Fig. 2).Finally, the effect of food intake on parotid and

serum amylase was studied in nonfasted animals duringthe first 2 h of the dark period, that is, during the phasewhen rats consume a large amount of food followingthe light period when very little food is taken. Underthese experimental conditions, during the first 2 h ofspontaneous food intake, tissue amylase level decreasedby 27�7% (P<0.01). In parallel, serum amylase levelincreased by 16�1% (P<0.05) compared to valuesobtained in animals from which food was withdrawnjust before the light was turned off (Fig. 3).

142 A. Nagy et al. / Journal of Physiology - Paris 95 (2001) 141–145

Fig. 1. Percent changes of parotid tissue (U/100 mg) and serum parotid amylase (U/l) concentration in rats for 1 h after pilocarpine (piloc) adminis-

tration following 16 h fasting, relative to vehicle (veh) treated rats (100%). Data are means�S.E.M. of 10–20 experiments; **P<0.01 vs. control.

Fig. 2. Percent changes of parotid tissue (U/100 mg) and serum parotid amylase (U/l) concentrations in rats fed for 1 h following 16 h fasting,

relative to the fasted rats (100%). Data are means�S.E.M. of nine experiments; **P<0.01, *P<0.05 vs. control.

A. Nagy et al. / Journal of Physiology - Paris 95 (2001) 141–145 143

4. Discussion

In the present study we found that pilocarpineadministration-induced tissue amylase discharge wasaccompanied by a considerable increase in serum par-otid isoamylase level. We observed a similar decline inparotid tissue and rise in serum levels of the enzymewhen fasted rats received were re-fed. Finally in non-fasted, ad libitum fed rats, food intake in the early darkperiod also induced a decrease in parotid tissue amylase,and an increase in serum isoamylase levels.Our observation that the electrophoretic mobilities of

parotid amylase and serum parotid isoamylase are verysimilar, while the pancreatic amylase in serum samplesmoves to the opposite direction during electrophoresis,confirms the data from earlier investigations[5,30,32,33]. Those studies showed that parotid iso-amylase is the primary component of circulating amy-lase, although at a lower degree, amylases originatingfrom the pancreas [6,23,33] and the liver [24] also con-tribute to the sum of serum enzyme activity.Our findings regarding the effect of pilocarpine on

serum and parotid amylase activity are also in line withprevious reports. Proctor and his coworkers [25] foundthat parasympathetic nerve stimulation produced salivawith relatively low amylase level but yields a substantialincrease in serum amylase concentration. On the contrary,

sympathetic nerve stimulation resulted in a high amylaseconcentration of saliva but little or no change in serumamylase activity [25]. That work along with other studiesusing marker proteins instilled into the duct system[3,7,10,18] suggested that amylase moves to the serum viaparacellular passage.When fasted rats were fed with standard rodent chow,

parotid isoamylase level also decreased in parotid tissueand proportionally increased in serum. This effect hasbeen attributed to a synergistic action of both sympa-thetic and parasympathetic nerves since the increase inserum was largely inhibited by parasympathectomy orsympathetic denervation of the parotid gland, and alsoby b-adrenergic blockade with propranolol [23].Our study is the first, however, to show that both

parotid and serum amylase level is controlled by spon-taneous food intake in rats. These data show that, in adlibitum fed rats, serum amylase level increases and par-otid amylase concentration decreases during the firsthours of dark, the period when rats eat a major portionof daily food consumption. These antiparallel changescan be attributed to food-stimulated activation of neu-ronal and hormonal pathways leading to the dischargeof parotid amylase into saliva, and also to the elevationof the enzyme activity in serum. Rats eat much more atnight than during the day. About 80–90% of the food isconsumed during the dark period when rats are kept at

Fig. 3. Percent changes of parotid tissue (U/100 mg) and serum parotid amylase (U/l) concentrations in ad libitum fed rats 2 h following the

beginning of the dark period (i.e. at 21:00 h). The food was withdrawn in the ‘‘no-food’’ group (100%) just before the light was turned off. Data are

means�S.E.M. of eight experiments; **P<0.01, *P<0.05 vs. control.

144 A. Nagy et al. / Journal of Physiology - Paris 95 (2001) 141–145

a 12–12 h light-dark cycle [4,13,14]. Diurnal patterns arecommon in nature [12]. In rats there is a diurnal varia-tion in food intake that is associated with positiveenergy balance during the night, when most food iseaten, and negative energy balance during the day, whenlittle food is eaten [15]. This diurnal pattern of feeding isentrained by light. Either continuous light or con-tinuous darkness will attenuate the diurnal feeding pat-tern [2]. The entraining of food intake to the light cycleprobably involves an oscillator in the suprachiasmaticnucleus [19,21,28,29]. Diurnal feeding patterns can alsobe abolished by destructive lesions in the suprachias-matic nucleus [19]. Food intake is also controlled by alarge number of hormones and neurotransmitters suchas CCK [26,27], neuropeptide Y [1] and leptin [20].

Acknowledgements

This work was supported by grants from the Hun-garian Ministry of Health and the Hungarian Ministryof Education.

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