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J Osaka Dent Univ 2009 (April) ;43 (1)[119-127.

Effectsof premedication on salivary secretionin the rat submandibular gland

Satoko Taniwa, Kenii Uchihashi*, Junichiro Kotani** and Yasuo Nishikawa*

' Graduate Schoo/ of Dentistr)t e4nesthesioJog;c), **Department ofAnesthesiologJt and Departrnent of Physioiog}c Osaka Dentat

Univetsity, 8-1 Kuiuhahanazono-cho, Hirakata-shi, Osaka 57]-1121, .lapan.

During anesthesia, muscarinic parasympathetic antagonists can be used as a preopera- tive medication on order to reduce salivary tlew, tracheobronchial secretions, and pha- ryngeal secretions, as well as to decrease the acidity ot gastric secretions. We did an in vivo study to examine whether muscarinic receptor subtypes play a role in the exocrine function of the rat submandibular gland. The effects ot muscarinic antagonists (piren- zepine, methoctramine, 4-diphenylacetoxy-N-dimethylpiperidinlum (4-DAMP) and gly- copyrroLate) were examined on secretion from the submandibular gland evoked by ace- tylcholine in pentobarbital-anesthetized rats. Glycopyrrolate caused less elevation in heart rate than the other anticholinergics. 4-DAMP and glycopyrrolate markedly inhibited the acety)choline-evoked fluid responses. Pirenzepine showed signiticantly ]ower inhibi- tory potency than 4-DAMP or glycopyrrolate, while methoctramine had even less of an in-

hibitory effect.

Pirenzepine, 4-DAMP and glycopyrrolate significantly inhibited both protein concentra- tion and amylase activity in the acetylcholine-evoked submandibular saliva, while

methoctramine did not affect the responses. The reduction ot the protein concentration and amylase activity in submandibular saliva caused by pirenzepine, 4-DAMP anci gly- copyrrolate were inhibited by NG-nitro-L-arginine methyl ester (L-NAME). Thus, it might be etfective to administer anti-cholinergic drugs together with L-NAME as premedication. These results suggest that glycopyrrolate significantly decreases salivary secretion, and has significantly fewer side effects than other muscarinic parasympathetic antagonists. Further controlled studies are required to determine the satety, efficacy and patient toler- ance of glycopyrrolate. (J Osaka Dent Univ 2009 ; 43 : 119-127)

Key words : Premedication ; Muscarinic receptor ; Salivary gland

rat submandibular binding and mo[ecular INTRODUCTION gland, ex- periments have indicated that different subtypes of

Parasympathetic antagonists are frequently used muscarinic receptors contribute to cholinergic re- 7 for premedication to reduce oral and respiratory se- sponses.S Currently, it is wondered whether mus- cretions and prevent bradycardia. Atropine is fre- carinic receptors other than muscarinic M3 recep- quently used as a premedication with both generai tors evoke secretory responses in the rat subman- and topical anesthesiat'2 to reduce bronchial secre- dibular gland. To investigate this issue, we used tions and prevent bradycardia,3 as well as to protect the muscarinic antagonists pirenzepine, methoctra-

against the deleterious effects of the instrumenta- mine, 4-diphenylacetoxy-N-methylpiperidine methio- tion on pulmonary mechanics,4 [t is genera"y dide <4-DAMP)S and glycopyrrolate. While 4-DAMP agreed that an increase in water secretion in re- shows a conspicuous and similar inhibitory potency sponse to muscarinic agonists is attributable to acti- on muscarinic M 1 and M3 receptors, pirenzepine vation of muscarlnic M 3 receptor subtypes.5 ln the has a selectivity profiie for the muscarinic M 1 re-

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120 S, Taniwa et al . Joumal of Osaka Denta1 University, April 2009

ceptor,g

Glycopyrrolate is said to have similar an- Chemicalassays

tisialagogue effects, but is [ess likely to cause sig- Analysis tor amylase activa'ty nificant tachycardia than atropine, Different anti- Saliva was collected in pre-weighed, jce-chilled vi- -30eC muscarinic receptor selectivity patterns could ex- als and then weighed, frozen and stored at plain the differences, and its selective and pro- until they were analyzed for amylase actMty. The Ionged effect of decreasing salivary gland secre- amylase activity of saliva was analyzed by an enzy- tion. Glycopyrrolate, a quaternary ammonium com- matic colorimetric test (Boehringer GmbH, pound availab[e in both orai and parenteral forms, Mannheim, Germany) using cr-4-nitropheny[malto- has been used in the fie[d of anesthesiology as a heptaoside (4 NP-G7) as substrate.13 One unit (U) pre-anesthetic agent because of its $elective and of catalytic activity of a-amylase is detined as the prolenged effect on decreasing salivary gland se- hydrolysis oi 1yM of 4NP-G7 per min per mL, cretion,iO This compound, which is structurally simi- which is equivalent to the definition of one interna- iar to atropine, affects the peripheral muscarinic re- tional unit. ceptors and has long-[asting inhibition of salivation

and respiratory tract secretions."" Analysis for protein concentTation This in vivo study attempted to examine whether The protein content of the fluid responses was ana-

muscarinic receptor subtypes other than muscarinic Iyzed by the method of Lowry.i"

M3 receptors exert exocrine functiona[ roles in the

rat submandibular gland. Since different antimus- MOasurement of biood pressure carinic receptor selectivity patterns could explain Blood pressure was measured continuously via a the differences, the effects of pirenzepine, methoc- catheter placed into the femoral artety, and heart tramine, 4-diphenylacetoxy-N-methylpiperidine-rate was measured using the tail-cuff method.'5

methiodine (4-DAMP) and glycopyrrolate were ex- amined on secretion from the major saljvary glands DrugsThe evoked by acetylcholine in pentobarbita]-anesthe- drugs employed were acetylcholine chloride tized rats. (Sigma, St. Louis, MO, US) ; pirenzepine dihydro- chloride (Wako, Osaka, Japan), methoctramine MATERIALS AND METHODS hemihydrate (Sigma), 4-diphenylacetoxy-N-methyl- Animals piperidine methiodide (4-DAMP ; Sigma), glycopyr- Male Wistar rats weighing 250-320g were used, rolate (Sigma), and NG-Nitro-L-arginine methyl es- Food but not water was withheld from the rats for ter, hydrochloride (L-NAME ; Sigma, St. Louis, US). 24h before each experiment. The present study A cannula placed in the femoral vein was used for

was conduced with approval from the Animal Study all drug administrations. Initial[y, five doses of ace-

Committee, and in accordance with the Osaka Den- ty[choline were administered. These secretory re-

tal University Animal Study Guidefiines (approvalsponses were used as control values for response$ number: 07-08002). obtained in the presence of muscarinic antagonists, and in the presence of the nitric oxide synthase in-

naeasurement of salivar]t flove rate hibitor L-NAME. In the first series of experiments,

Saliva samples, were collected from the submandi- acetylcholine was injected intravenously either in bular gland through polyethylene tubing (lntramedicsuccessively increasing doses, or before admini- PE-10; Becton & Dickinson, Franklin Lakes, NJ, stration of the mu$carinic antagonists.

USA) inserted into the oral opening of the main

duct. Fluid weight of coliected saliva was then Ftinctional studies

Measured,i2 The animals were anaesthetized with sodium pen-

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Vol. 43, No. 1 Effect of premedication on salivary secretlon I2i

1OO tobarbital (50 mg kg i.p.) followed by supplementary i6Eo

doses injected intravenously as required. The tra-

chea was cannulated and the body temperature 80o([Eb

was maintained at 38'C, The duct of the submandi-

bular vvas cannulated, All saliva secreted in ' gland 60a response to adminlstration of acety[choline was col-

lected and weighed. The saliva was collected in

small trays, which were immediately weighed in or-

der to minimize evaporation. In additional experi- ments, the effects of p[renzepine, methoctramine, ili;:1 8 4-DAMP and glycopyrrolate were examined on '/1OO acetylcholine-evoked salivary flow rate. In each ani- o 200 300 400 500 mal, only one gland of each pair was tested. A can- Acetylcho[ineconcentration(ltglkg} nula placed in the femoral vein was used for all Fig. 1 Salivary secretlon of rat submandibular gland evoked drug administrations. In the experiments, acetyl- by intravenous administration of acetylcholine of between 1OO choline was injected repeatedly beginning at 100 ;tg and 500"g, The secretory flow rate was calculated as the salivary flow rate measured in the units oi mgfkg and the dose was increased by another 100 "gfminllOO mg. The data are presented as mean values ± SE. mgfkg each time until 500 mglkg was reached. In-

itially, five doses of acetylcholine were adm[nistered 1oo in the absence of the muscarinic antagonist. These

AaE secretoty responses were u$ed as contror values 1 80oog.gE'." for responses obtained in the presence of the mus-

carinic antagonist. Pirenzepine, methoctoramine, 4-

-･-100 mg DAMP'6 and glycopyrrolate were used at doses that 60'vx --- 1O mg were above the threshold dose for etfect, ln order +1.0 mg

to collect adequate amounts of saliva for analysis, 9N ÷ O] mg 40)8k +Centrol several responses were occasionally pooled. Ace- tylcholine was given at least twice in $uccession in 2okco order to evacuate the dead space of the cannula

and avoid contamination with secretion elicited by --T 1 any previous procedure. The second sample was o- T 100 200 300 400 500 analyzed for protein. Acetylcolineconcentration("gJkg)

RESULTS Fig,2 re- sponse to intravenous Injection of ACh at various doses after intraveneus lnjection ot pirenzepine. Sa"vary fiow rate

The muscarinic agonjst ACh was infused intrave- nously, under specific muscarinic receptor block- pendent and ranged from 21.6-83.6ug/min/100

ade, in anesthetized rat$ at various dose levels, mg. Salivary flow rates reached a maximum at 400

The intravenous injections of Ach at the differentptglkg of acetylcholine. doses elicited successively increased secretoty re- Methoctramine inhibited the Ach-evoked re-

sponses. 1 shows the submandibular Figure gland sponses (Fig, 3), 4-DAMP (Fig. 4) and glycopyrro- salivary flow rates evoked by acetylcholine stimula- late (Fig. 5) markedly inhibjted the acetylcholine- tion. In the absence of stimulation,the submandibu- evoked fluid responses in the submandibular gland, lar gland secreted virtually no fluid, The salivary Pirenzepine showed significantly lower inhibitory flow rate submandibular was from the dose de- potency than 4-DAMP and glycopyrrolate (Fig. 2),

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et al, 722 S.Taniwa Journal of Osaka Dentai University,April 2009

1OO 100 ]"U')E 1"CJ',g

80 so-i.O'2 i 1'Ex. -- 1 OO mg 60avoN +1O mg

+1,O mg -x- O.1 mg +O-di -x-O.1 mg 40Lg9-.tsts 4o. Ll -k- --te-Control Control

uoit

20zdico 20- ;t -T -1 i;IEIIIiF, o l- 1- oL ii-, 100 200 300 400 500 100 200 300 400 500 Acetylcolineconcentration(Ftgtkg) Acetylcolineconcentration("glkg)

Fig. 3 Secretion of submandibular gland saiiva in rats in re- Fig, 5 Secretion of submandibular gland saiiva in rats jn re- sponse to intravenous injection of ACh at various doses after sponse to intravenous inlection of ACh at various doses after

intravenous injection of methoctoramine, intravenous injection of glycopyrrolate.

1OOAag

so:'Ex. ,･liii g:. DAcetylchoLineonly -.-1OO mg ,olg EPi;enzepine 60' --- 1O mg -Methoctramine 6018 +1.0 mg M4-DAMPasGlycopyrrolate -x-O.1 mg ,o 4ol9m +Controf g 2 1k 2o-3.osco : / o 3e 6o go 12o tso

'"" Time (min) Ot"1 T 1'7 100 200 300 400 500 Fig, 6 Comparison ot changes in mean blood pressure after Acetylcolineconcentration("gtkg) intravenous injection of various muscarinic antagonists,

Fig. 4 Secretion of submandibular gland saliva in rats in re- sponse to intravenous injection of ACh at various doses after

jntravenous injection of 4-DAMP, Blood pressure and heart rate Mean arterial blood pressure during ACh was rela- while methoctramine exerted an even lower inhibi- tively stable and averaged 82 ± 4mmHg (Fig. 6). tory effect. The reduction of the protein concentra- During infusion of the anti-muscarinic drugs, tion in submandibular saliva caused by 4-DAMP, methoctramine and 4-DAMP, there was a signifi- glycopyrrolate and pirenzepjne were inhibited by L- cant increase in b]ood pressure to an average NAME, Thus, in addition to muscarinic M3 recep- mean valve of 122± 6mmHg and 118 ± 8mmHg

tors, other muscarinic receptors contributed to in respectively. GIycopyrrolate increased the blood vivo functional responses in the rat submandibular pressure moderately. In each case, recovery to gland. These other receptors were muscarinic M 1 within the normal range occurred within 150min receptors, (data not shown), ACh stimulation was relative]y

stable after infusion of anti-muscarinic drugs, Mean

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Vol. 43, No. 1 Effect of premedication on salivary secretion 123

ri40- A140Tvi8i2ottlOOLt9

120- '''' '' ['E '''' ' 100) ''/ '''' ' '' DAcety[chellneenly DCentrelMPirenzepine '''' ts80Dv EPirenzepine soU.9 ' -Methoctramine lMethoctramine 6ol ''''''''''' g4-DAMPssGlycopyrrolate 60reli8 M4-DAMPasGlycopyrroiate :,1/- '' ' 8 4o-= '' '' 4o88 ' '''' 20 2o･[g,i /' ''' o

O 30 60 90 120 150 O 30 60 90 120 150 Time {min} Time (min)

Fig.7 Comparison of changes in mean heartrate in- Fig, 8 Comparison of changes in protejn concentration over travenous injection of various muscarinic antagonists,after time in submandibular gland secretory responses in rats to intravenous inject]on of acetylchoiine in the presence of vari- ous muscarinic antagonists.

heart rate during ACh was relatively stable and av- eraged 88 ± 3/min (Fig, 7), During infusion of the 70 anti-muscarinic drugs, methoctramine and 4-DAMP,

there was a sjgnjficant increase in heart rate to an 60 average of 122 ± 6 lmin and 118 lmin, respective[y. gso)rt-4o･->--8 Glycopyrrolate decreased the average to 94 ± 61 MCentrolEPirenzepine min, lMethoctramine 3oee M4-DAMPgeGlycopyrrolate

Protein secnetion 20Eh< The protein concentration in submandibular saliva 10 was obtained in response to ACh in the absence of muscarinic antagoni$ts, Pirenzepine reduced the o O 30 60 90 120 150 protein secretion, Sjmjiar to pirenzepine, administra- Time (min) tion of 4-DAMP reduced the concentration of pro- Fig. 9 Compari$on of the changes in amylase activity over tein by about 20%, while reduced it glycopyrroiate time in submandibular gland secretory responses in rats to by about 28%. Methoctramine had no effect on the intravenous injection of acetylcholine in the presence of vari- ous muscarinic antagonists. protein concentration (Fig. 8).

Salivaty amylase actiw'ty Efiiect of L-IVAnaE

The mean amylase activity in response to acety[- Administration of the nitric oxide synthase inhibitor choline at 400mgfkg was 65,4± 1.6 Umgi, Amy- L-NAME increased the protein concentration in the iase activity was decreased by the addition of mus- ACh-evoked submandjbular saljva during admini-

carinic antagonists with acetylcholine, lnparticular, stration of the muscarinic antagonists 4-DAMP and 4-DAMP and glycopyrrolate significantly inhibited glycopyrrolate, without affecting the fluid response. concentration both protein and amylase activity in Pirenzepine caused no further reduction of the sub- the acetylcholine-evoked submandibular saliva, mandibular protein concentration in the presence of while methoctramine did not affect the responses, L-NAME (Fig, 1O). The reduction of amy[ase activity Methoctramine also had no effect on the protein in submandibular saliva caused by pirenzepine, 4- concentration 9), (Fig. DAMP and glycopyrrolate were inhibited by L- NAME (Fig. 11). The protein concentrations and

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f24 S.Taniwa etat. Joumat of Osaica Dentai Cinivetsity, April 2009

T140-vEirmal20-'t muscarinic agonists has generally been attributed to activation of muscarinic receptors solely of the M

['EIOo 3 subtype,i'

DAcetylcholineonly The general concept that muscarinic M3 recep- e4-DAMP-4-DAMP+L-NAME tors exclusively mediate the cholinergic stimulation of rat salivary glands has lately been challenged in i/z:1b[g IGIycopyrrolate ssGlycopyrrolate a number of studies, Generally, the cholinergic re- 4o-=1ov. +L-NAME sponses in the rat submandibular g[and were con-

20t6.di sidered to be mediated via activation ofthe mus- -i carinic M 3 receptors,iS in spite of earlier reports in- o o 30 60 90 120 150 dicating involvement of muscarinic Ml receptor Time (min) subtypes as well,"g'20 Our re$u[ts thus confirm previ-

Fig. 10 Effect L-NAME the ot on concentration responsepresenceous conclusions that the muscarinic receptor popu- over time in ACh-induced sal[vary secretion in the lation in the rat submandibular consists of a of various muscarinic antagonists, gland mixture of muscarinjc M 1 and M3 receptors. The

concept that the muscarinic receptor in 70･ population $alivary glands consists homogeneous[y of the

muscarinic M 3 subtype has also been further chal- a [enged by data from binding studies in the subman- Sso ZAcetylcholineonly g4-DAMP dibular suggesting that a complementary tS4o'e,,omS.. gland, 14-DAMP+L-NAME muscarinic receptor is the muscarinic M5 recep- NGIyco I asGlyco+L-NAME tor.2i Functional experiment$ on isolated rat sub- 20E mandibular gland fragments also support the idea

that the co-receptor is not the muscarinic M 1 re- io ceptor subtype,22 4-DAMP seems to have a Iower ..1 o affinity in the submandibular g]and than in the pa- O 30 60 90 l20 /50 rotid ; it has 40 times and 380 times higher affinity Time (min) than pirenzepine and methoctramine, respectjvely,23 Fig, 11in Effect of L-NAME on the amylase activity over time ACh-induced $alivary secretion inthe presence ot various Our results indicate that glycopyrrolate might have muscarinic antagonists, a higher affinity than 4-DAMP in the $ubmandibular gland. amylase activity in the fluid responses were not sig- The fact that pirenzepine affects submandibular nificantly affected by methoctramine, protein secretion as much as 4-DAMP confirms its

inhibition of mu$carinic M 1 receptor-mediated se- DISCUSSION cretion. The findings may be related to and in ac-

The effects of muscarinic receptor antagoni$ts on cordance with the reports demonstrating the exis-

responses to acetylcholine were determined in tence of nitric oxide synthase2` and the differential pentobarbital-anesthetized rats, The M1-selective cho]inergic activation of nitric oxide synthase in rat antagonist pirenzepine reduced the fluid response salivary glands.25'2G lt is noteworthy that nitric oxide by 20%, but did not reduce the protein output. The has been shown to be very important for parasym- M 2-selective antagonist methoctramine had no et- pathetic nerve-evoked protein secretion in salivary fect on fluid responses or protein output. The con- glands.27 Since the blockage of nitric oxide syn- tribution of muscarinic M3 receptors to the fluid re- thase reduces acetylcholine-elicited protein secre- sponse was greater than that of the muscarinic M 1 tion, it is temping to suggest that the muscarinic M receptors. The increase in sa[ivary fiow evoked by 1 receptor via nitric oxide indirectly evokes protein

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Vol, 43, No. 1 Effect of premedication on salivary secretion 125

secretion in the rat submandibu[ar gland, ported, in vivo as well in vitro, in the rabbit $ubman- lt is generaliy agreed that the increases in the dibular gland,30'32 The significance of muscarinic M

flow of saliva evoked by cholinergic $timulation are 1 as well as M5 receptors has been reported for

mediated by activation of muscarinic M3 recep- the rat submandibular gland.22 ln salivary glands, tors,"9･28 However, this view has been questioned in parasympathetic nerve transmission may involve

a number of studies showing that muscarinic M 1, not only the classical autonomic transmitter acetyl-

M 2, M 3 and M 4 receptors are present in the rab- choline, but also nonadrenergic, noncholinergic bit submandibular gland29 and that muscarinic M 1 transmitters.3B Parasympathetically nerve-evoked receptors elicit functional effects in this gland.30 Fur- vasodilatation, which is evoked by nonadrenergic, thermere, in the rat sublingual gland, muscarinic M nonchoiinergic transmitters, is particularly resistant 1 receptor mRNA has been shown to be present in to atropine.39 However, a secretory response also substantial amounts,20 and its corresponding recep- persists after full atropinization in salivary glands of

tor has been shown to elicit functional effects when several species, including the submandibular studied in vitro3i and in vlvo,32 ln the rat submand[- gland,4o bular gland, both muscarinic M 1 and possibly M 5 Endothelium-derived nitric oxide plays an impor- 22 receptors seem to play functional roles,9･'i while tant role in regulation of the contraction and tone of in the ovine submandibular g]and, muscarinic M 1 vascular smooth muscle." Endothelium-derived ni-

receptors contribute to the secretory responses, tric oxide is synthesized from L-arg[nine by nitric particuiarly at [ow intensity stimulation of the para- oxide synthase (NOS) in the endethe]ium, and acti- SYmpathetic nerve,33 vates soluble guanylyl cyclase in vascular smooth Our in vivo study provided evidence for mus- muscle. Guanylyl cyclase is an enzyme found only carinic M3 receptors mediating a large part of the in the luminal aspect of intestinal epithelium. The parasympathetic fluid response. It has been shown receptor has an extracellular ligand-binding domain,

to be effective at decreasing the drooling that oc- a sjngle transmembrane region, a region with a se-

curs in children with cerebral palsy in an open-labefi quence similar to that of protein kinases, and a C- trial.3` ln addition, the study showed that glycopyrro-terminal guanylyl cyclase domain. Tyrosine kinase late had a much smaller proportion of side effects activity mediates the guanylyl cyclase signaling than other anticholinergic agents, Unlike other anti- pathway within the cell. Guanylyl cyclase is a key cholinergic drugs, glycopyrrolate does not cross the receptor for heat-stable enterotoxins which are re-

blood-brain barrier, has a rapid onset of action, and sponsib]e for acute secretory diarrhea. Heat-$table

has a strong specificity for the salivary glands,35 ln enterotoxins are produced by pathogens such as addition, we found that these anti-muscarinic drugs dscherichia coli. Knockout mice deficient in the cause less elevation in heart rate and blood pres- guanylyl cyclase gene do not show secretory diar-

sure than other anti-mus ¢ arinic drugs. Furthermore, rhea on infection with E. cofi, though they do with

glycopyrrolate causes a [ower Incidence of dys- cholera toxin. Thi$ demonstrates the specificity of

rhythmias,36 lt is commonly used to inhibit salivation the guanylyl cycla$e receptor. during the induction of anesthesia in the North The synthesis of endothelium-derived nitric oxide America and Europe,35 lt dose not cross the b]ood- can be blocked by L-NAME,42 ln the present study, brain barrier, has a rapid onset of action, and has a the effect of acetylcholine on submandibular protein strong specificity for salivary glands. Giycopyrrolateand amylase secretion in the rat was tested in the causes less heart rate elevation and has a lower in- presence of the nen-selective NO synthase inhibitor cidence of dysrhythmias compared with other anti- L-NAME, Since the volume of saliva secreted in re-

cholinergic medications.36 sponse to acety]choline was the same with or with-

The functional significance of muscarinic M 1 re- out the inhibitors of NO synthase, any change in ceptors for the secretory response has been re- concentration of protein or amylase occurring repre-

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126 S. Taniwa etal. Joumal of Osaka DentaJ University, April 2009

sented a change in total output of protein and amy- This study was presented at the 516th meeting of the Osaka Odontelogicai Society, De¢ ember 13, 2008, lase. The present study showed that the major part Hirakata, Japan. (60-70%) of the acetylcholine-induced increase in the concentratjon of protein and amylase activity of REFERENCE$

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Vol. 43, No. 1 Effect of premedication on salivary secretion 127

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