J. Physiol. (1971), 216, pp. 129-142 129With 4 text-figuresPrinted in Great Britain

FORMATION OF SALIVA AND POTASSIUM TRANSPORT INTHE PERFUSED CAT SUBMANDIBULAR GLAND

By 0. H. PETERSENFrom the Institute of Medical Physiology C,

University of Copenhagen, Denmark

(Received 27 November 1970)

SUMMARY

1. In the cat submandibular gland perfused with modified Locke solu-tions, salivary secretion during acetylcholine (ACh) infusion and K uptakefrom the perfusion fluid after the ACh-induced K loss were measured.

2. Strophanthin G (10-5 M) abolished K uptake, whereas salivarysecretion was unaffected.

3. Ethacrynic acid (10-4-2 x 10-4 M) hardly affected K uptake whereassalivary secretion was severely inhibited.

4. During perfusion with Ca-free solution K uptake was unaffected,whereas salivary secretion was severely reduced.

5. The presence or absence of C02/HCO3 in the perfusion fluid was of noimportance for the secretary process and the K uptake. Acetazolamide(2 x 10-4 10-3 M) did not inhibit these transport processes.

6. It is suggested that two kinds of Na transport occur in the acinarcells of the salivary glands: a Na extrusion coupled with K uptake, respon-sible for the maintenance of the concentration gradients across the cellmembrane; and a Na transport coupled with Cl transport into the acinarlumen, responsible for the formation of the primary secretion.

INTRODUCTION

The acinus is the site of the major outward fluid transport in the salivaryglands (Burgen, 1967). In the rat submandibular and parotid glands it hasbeen shown by micropuncture analysis that the primary acinar secretionhas a plasma-like composition with respect to osmolarity, concentration ofNa, K and Cl (Martinez, Holzgreve & Frick, 1966; Young & Schdgel, 1966;Mangos, Braun & Hamann, 1966). It is likely that the same is true for theprimary acinar secretion of the cat submandibular gland (Petersen &Poulsen, 1967). The ionic mechanisms responsible for this fluid formationare obscure. Lundberg (1958) proposed that the main step in the secretaryprocess was an active Cl transport through the contraluminal acinar cell

5-2

membranes. The results of recent work, however, are in disagreement withthis concept (Petersen, 1970b, c, d). The results of the present work showingthat K uptake into the salivary gland cells, coupled to Na extrusion(Petersen, 1970a), can be dissociated from the isotonic secretary process byvarious procedures and the fact that salivary secretion can be severelyreduced by the action of ethacrynic acid but not by strophanthin G,whereas the opposite is the case with the K uptake, suggest the existenceof two kinds of Na pumps in the acinar cells of the salivary glands, oneextruding Na and accumulating K while the other transports Na, Cl andwater into the gland lumen. Some of the findings have been published inpreliminary communications (Petersen, Poulsen & Thorn, 1968; Petersen1971).

METHODS

Cats anaesthetized with chloralose (80 mg/kg I.P.) were used. The main excretoryduct of the cat submandibular gland was cannulated with a polyethylene tube. Asdescribed by Petersen (1970a) all branches from the left common carotid arteryexcept the one supplying the submandibular gland were ligated. Likewise all tri-butaries to the left external jugular vein except the one draining the gland wereligated. Through a cannula inserted into the common carotid artery the gland wasperfused under a hydrostatic pressure of 80 mm Hg with modified Locke solutions.The venous perfusion fluid from the gland was collected from a cannula inserted intothe jugular vein and analysed for K by flame photometry (Petersen, 1970a). In someexperiments the Ca concentration of the effluent was also measured by using anatomic absorption spectrophotometer (Perkin-Elmer, model 290) as described byNielsen & Petersen (1970). The standard perfusion fluid (control solution) contained(mM): NaCl 140; KCl 4-0; NaH2PO4 0-6; Na2HPO4 2-4; CaCl2 2-2; MgCl2 1-0; glucose5.5. In some experiments a bicarbonate-containing solution was also employed(HCO3 solution); this solution contained (mM): NaCl 125; KCl 4-0; NaHCO3 25-0;CaCl2 2-2; MgCl2 1-0; glucose 5-5. The control solution was equilibrated with 02whereas the HCO3 solution was equilibrated with a mixture of 95% 02 and 5%CO2. The Ca-free solution was a control solution not containing CaCl2. Ethacrynicacid-(Edecrin, M.S.D.) and strophanthin G-(g-strophanthinum; Mecoben-zon,Copenhagen) containing solutions were control solutions to which were added theinhibitors to obtain the concentrations stated in the results section. The acetazol-amide-(Diamox, Lederle) containing solution was a HCO3 solution to which acetazol-amide was added to obtain the concentrations given in the Results section. Perfusionwas carried out at room temperature (23-25° C). The gland was stimulated to secreteby a continuous intra-arterial infusion ofACh (50 ,ug/min) lasting 1 min. The volumeof saliva secreted in the first minute after start of secretion, measured by collectingthe saliva into a tuberculin syringe, was taken as the salivary secretary rate.

RESULTS

The effect of strophanthin G. Fig. 1 shows the course of one of the fourexperiments carried out. In this experiment the secretary rate graduallydeclined in the course of the experiment, but it seemed that strophanthinG had no effect on the secretary rate. However, the K uptake occurring

0. H. PETERSEN130

SALIVARY SECRETION AND K TRANSPORTafter the ACh-induced K loss was abolished in the period of perfusion withthe strophanthin G-containing solution. In Table 1 are shown the resultsfrom all four experiments. The ability of the gland to accumulate K fromthe perfusion fluid is expressed as the maximal decrease in K concentrationin the venous perfusion fluid occurring after stimulation with ACh, i.e. thedifference between the K concentration immediately before ACh infusion

10 r

9

a,0

E

0Cc._

MCUC0U

8

7

6

5

4 _

3

c VuuC._

. 300

V 200

0X 100A-u0(n

ControlAChn

I Strophanthin G (10-3M)ACh I ACha I n

I'

10 30 50 ;

50

I

;~~~~~~~~~~~~~~~~~~~

10 30 5Time (min)

V0

70 90

I70 90

Fig. 1. The effect of strophanthin G on K transport and salivary secretion inexperiment no. 90. K concentration in the perfusion fluid coming out of thesubmandibular gland vein, as a function of time, is shown in the upper partof the Figure. The lower part shows the salivary secretary rate in the firstminute after start of ACh infusion.

and the minimal K concentration after ACh infusion. The secretary rateduring perfusion with the strophanthin G-containing solution was 81 %(ranging from 73 to 89 %) (n = 4) of the secretary rate in the preceding

ff- 11. . . .

131

,A^^n I-

I

0. H. PETERSEN

TABLE 1. The effect of strophanthin G on secretary rate andK accumulation

Secretory rate (,ul./min)

Strophanthin GControl (10-5 M)

560 500450 380300330 240390310 240

Maximal decrease in effluent[K] after ACh (m-mole/l.)

Control

1.91-71-62-01.01-0

Strophanthin G(10-5 M)

0-20-3

0

0

V

0E

4-0

C

0

Ethacrynic acid (2x 10-4M)AChn

E 300

02001

I-1000

10 30 50 70 90Time (min)

Fig. 2. The effect of ethacrynic acid on K transport and salivary secretion inexperiment no. 87. See Fig. 1 for further explanation.

control period. The K uptake during perfusion with strophanthin Gsolution was 7% (ranging from 0 to 18 %) of the K uptake in the precedingcontrol period. The flow of perfusion fluid through the gland during stimu-

132

Exp. no.

818288

90

SALIVARY SECRETION AND K TRANSPORT

lation with ACh was 4-8 ml./min + 0-6 (S.E., n = 6) in the control periodand 5-0 ml./min + 1-0 (n = 4) during perfusion with the strophanthin G-containing solution.

TABLE 2. The effect of ethacrynic acid on secretary rate andK accumulation

Maximal decrease in effluentSecretory rate (#uL./min) [K] after ACh (m-mole/l.)

r~~~~~~~~AA

Ethacrynic acid Ethacrynic acidExp. no. Control (concn. M) Control (concn. M)

84 360 80 (10-4) 1-4 0-9 (10-4)85 560 3N0 (10-4) 1-5 1-4 (10-4)86 420 110 (2 x 10-4) 1-4 1-4 (2 x 10-4)87 280 1-1 -

320 170 (2 x 10-4) 1-3 1-0 (2 x 10-4)

The effect of ethacrynic acid. Fig. 2 shows the course of one of the fourexperiments carried out. In the control period the secretary rate remainedrelatively constant, but salivary secretion was depressed during perfusionwith the ethacrynic acid-containing solution. The K uptake was notaffected by this inhibitor. In Table 2 are shown all the results from thefour experiments of this kind. The secretary rate during perfusion with theethacrynic acid-containing solution was 42 % (ranging from 22 to 67 %)(n = 4) of the secretary rate in the preceding control period. The K uptakeduring perfusion with the ethacrynic acid solution was 85 % (ranging from64 to 100 /) (n = 4) of the K uptake in the preceding control period. In anadditional experiment in which an inhibitor concentration of 10-3 M wasemployed both secretion and K uptake were abolished. The flow of per-fusion fluid through the gland during ACh infusion was 7-3 ml./min + 1-2(n = 6) in the control periods and 5-2 ml./min + 1-0 (n = 5) during per-fusion with the ethacrynic acid-containing solution.The effect of omitting Ca from the perfusion fluid. In Fig. 3 is shown the

course of one of the three experiments carried out. Neither K release norK uptake after ACh stimulation was affected by omitting Ca from the per-fusion fluid in spite of a severe, but reversible, reduction in the secretaryability. About 10 min after introduction of the Ca-free solution the con-centration of Ca in the effluent was below 0-05 m-mole/l. in all the threeexperiments. The K release seen immediately after the reintroduction of thecontrol solution is probably due to a stimulating action of Ca on the para-sympathetic ganglionic cells perfused together with the gland. Concomi-tantly with the K release a small salivary secretion was observed. Thissecretion can be blocked by hexamethonium (McCarthy & Sheehan, 1966).

133

O. H. PETERSENDuring perfusion with the Ca-free solution (Fig. 3) a comparison was madebetween the effect of a rapid intra-arterial injection of ACh and a continu-ous infusion ofACh lasting 1 min. The amplitudes of the transient variationsin venousK concentration were the same but the duration of the transients

Time (min)

Fig. 3. The effect of lowering the perfusion fluid Ca concentration on Ktransport and salivary secretion in experiment no. 93. In addition to theparameters shown in Figs. 1 and 2 the Ca concentration in the perfusionfluid leaving the gland is shown. The arrow denotes an I.A. injection of10 jug ACh.

was different. Table 3 gives the results from all three experiments of thiskind. The secretary rate during the first ACh infusion after introductionof the Ca-free solution was 18 % (ranging from 15 to 21 %) (n = 3) of thesecretary rate in the preceding control period. The K uptake (Ca-free) was

134

SALIVARY SECRETION AND K TRANSPORT

TABLEu 3. The effect of omitting Ca from the perfusion fluid onsecretary rate and K accumulation

Secretory rate (,tl./min)

Control Ca-free

330 700

80270 40

040 -

320 60150

10 30 50

Time (min)

Maximal decrease in effluent[K] after ACh (m-mole/l.)

Control Ca-free

0-8 1-31-3

1-61.1 1-3

0-90-8 -

2-1 1-92-0

70 90 110

Fig. 4. The effect of HCO3 and acetazolamide on K transport and salivarysecretion in experiment no. 95. See Fig. 1 for further explanation.

123 % (ranging from 91 to 162 %) (n = 3) of the K uptake in the precedingcontrol period. The flow of perfusion fluid through the gland during in-fusion of ACh was 4-5 ml./min + 0-7 (n = 6) in the control period and6-3 ml./min + 1-2 (n = 5) during perfusion with Ca-free solution.The importance of HCO3/CO2 for the secretary process. Fig. 4 shows a

135

Exp. no.

91

92

93

.__

I,0E

0

0

0

C

0_.

0U0:

U)

1(n

typical experiment. The secretary rate was independent of the presence orabsence of HC03/C02 in the perfusion fluid, and acetazolamide did notinhibit secretion. The pH of all three perfusion solutions was close to 7-3.Table 4 shows the results from all three experiments carried out. Thesecretary rate during perfusion with HCO3 solution was 91 % (rangingfrom 83 to 100 %) (n = 5) of the secretary rate in the preceding controlperiod. The K uptake, during perfusion with HCO3 solution, was 111 %(ranging from 75 to 140%) (n = 3) of the K uptake in the preceding controlperiod. The secretary rate during perfusion with acetazolamide solution

TABLE 4. The effect of C02/HCO3 and acetazolamide on secretary rateand K accumulation

Maximal decrease in effluentSecretory rate (jtl./min) [K] after ACh (m-mole/l.)

Acetazolamide AcetazolamideExp. no. Control HCO3 (concn. M) Control HCO3 (concn. M)

94 340 340 1-6 1*2 -300 250 210 (2x 10-4) 1.1 13 1.0 (2x 10-4)

150 - 0 995 270 230 0-8

230 200 170 (10-3) 0 5 0*7 0-5 (10-3)- 140 -- 0*5 05(-)

0 5 -

96 - 250 1*3 -290 200 190 (10-3) 1.1 0.8 (10-3)

150 1.0 (10-3)- - - - 1*-1

was 88% (ranging from 84 to 95 %) (n = 3) of the secretary rate duringperfusion with HCO3 solution in the preceding period. The K uptake(acetazolamide) was 72 and 77 % of the K uptake in the preceding (HC03)period. The flow of perfusion fluid through the gland during infusion ofACh was 5-7 ml./min + 0 5 (n = 5) during perfusion with control solution,4.7 ml./min + 0 3 (n = 9) during perfusion with HC03 solution and 5-2 ml./min + 0*2 (n = 3) during perfusion with acetazolamide-containing solution.

DISCUSSION

Diamond (1968) has proposed that the energy-requiring process in theformation of the isotonic absorbate in the gall-bladder is an active electro-neutral transport of NaCl from the intracellullar to the intercellular spaces,water following passively after. Yoshimura, Inoue, Imai & Yoshimura(1962) have shown by electronmicroscopy of the dog submandibular gland

136 0. H. PETERSEN

SALIVARY SECRETION AND K TRANSPORT

that intercellular canaliculi communicating with the acinar lumen exist,and that these intercellular spaces are distended after stimulation withpilocarpine but are collapsed in the resting state. However, Rutberg (1961),in an electronmicroscopical investigation on the mouse parotid gland,showed that the intercellular spaces communicated most widely with theinterstitial fluid and not with the lumen. Probably this discrepancy is dueto the fact that the dog submandibular, as the cat submandibular, ismainly a mucous gland whereas the mouse parotid is a serous gland.In the proximal tubular cells of the kidney Whittembury & Fishmann

(1969), Whittembury & Proverbio (1970) and Proverbio, Robinson &Whittembury (1970) have proposed that two kinds ofNa pumps exist. Onepump extrudes Na and accumulates K and is inhibited by strophanthin G,the other extrudes Na together with Cl and is inhibited by ethacrynic acid.In the perfused cat submandibular gland active K uptake from theinterstitial fluid can readily be studied by following the K concentration inthe effluent after a brief stimulation with ACh, which evokes a markedrelease ofK from the gland cells soon followed by a marked uptake (Figs.1-4). It is probable that this K uptake is linked to Na extrusion (Petersen,1970a). Petersen (1970a) discussed from which glandular elements theACh-inducedK loss, and consequently the subsequentK uptake, originated.It was concluded that the acinar cells did at least participate in thisexchange and probably were responsible for the major part of the Ktransport, but it could not be excluded that other cell types were alsoinvolved. Burgen (1956), who first studied the stimulation-induced K lossand uptake, found that after 3 min continuous stimulation of the dogsubmandibular gland 42% of the gland's initial K store was lost. Blair-West, Coghlan, Denton, Nelson, Wright & Yamauchi (1969) found that inthe sheep parotid gland 80% of the lobular space was occupied by aciniwhereas only 6% was occupied by the ducts. These figures taken togetherindicate that by far the greatest amount of K released upon stimulationand therefore also taken up again originates from the acini. It is thereforeassumed that the K release and K uptake studied in this work mostlyreflect processes occurring across the acinar cell membranes.The experiments with strophanthin G and ethacrynic acid (Figs. 1 and

2) show that the K uptake and the secretion can be separated from eachother. The finding that strophanthin G can inhibit K uptake withoutreducing salivary secretion was actually made previously (Petersen, 1970a(Fig. 3)) but the phenomenon was not studied further. These results aremost easily interpreted by the hypothesis of Whittembury. In the acinarcells there must exist a pump, extruding Na and accumulating K, whichcan be inhibited by strophanthin G. There must also exist an activetransport mechanism responsible for the formation ofthe isotonic secretion.

137

It is probable that this could be a Na pump moving Na, C1 and water intothe lumen or possibly the intercellular space. This pump is inhibited byethacrynic acid. The results of the present work cannot show whether thelatter pump is an electroneutral NaCl pump as has been suggested in thecase of the gall-bladder (Diamond, 1962) or whether it is an electrogenic Napump as has been shown in the Necturus proximal tubule (Whittembury,1971). The present results do not show that the secretary mechanismcannot be inhibited by strophanthin G. Petersen (1970a) noted that thesensitivity of the secretary mechanism for strophanthin G varied fromgland to gland. Also it is very likely that greater inhibitor concentrationswould have depressed salivary secretion. The objective of the present workwas only to show that the K uptake can be separated from the secretarymechanism and that the K uptake is more sensitive to the action ofstrophanthin G than is the secretary mechanism.In both the gall-bladder (Diamond, 1964) and the proximal kidney

tubule (Rumrich & Ullrich, 1968) the presence of bicarbonate in the extra-cellular fluid is needed for the isotonic water reabsorption. However, this isnot the case with the acinar cells of the cat submandibular gland. The samesecretary rate was obtained with and without CO2/HCO3 in the perfusionfluid (Fig. 4). In view of this finding it is not surprising that the carbonicanhydrase inhibitor acetazolamide had no effect on the salivary secretaryprocess. In previous in vivo experiments in which salivary secretion waselicited by electrical stimulation of the chorda-lingual nerve, large doses ofacetazolamide given i.v. or close I.A. to the gland were shown to inhibitsalivary secretion (Petersen & Poulsen, 1966). Although this could possiblybe due to inhibition of the synaptic transmission in the parasympatheticganglia, it cannot be excluded that acetazolamide in large doses has someinhibitory effect on one or more of the processes, occurring after attach-ment of ACh to the basal acinar cell membrane, leading to the productionof the primary secretion. Petersen & Poulsen (1966) showed that acet-azolamide reduced the size of the transmembrane secretary potentials. Asthe mechanism of this potential change probably is an increase in Kpermeability of the basal acinar cell membrane (Petersen, 1970b) it couldbe postulated that ACh in the large doses used in the study of Petersen &Poulsen (1966) had affected this permeability change induced by ACh. Inthe present study, under more controlled conditions, carbonic anhydraseinhibitor in concentrations comparable to those effective in inhibitingisotonic water transport of the rabbit gall-bladder (Wheeler, Ross & King,1969) and the cat pancreas (Case, Scratcherd & Wynne, 1970) had noeffect on K permeability in the acinar cells as judged from the K releasephenomenon occurring after stimulation with ACh (Fig. 4). One mightobject that acetazolamide was not allowed sufficient time to inhibit the

O.H.PETERSEN138

SALIVARY SECRETION AND K TRANSPORT

secretary process in the present experiments. However, in the perfused catpancreas (Case, Scratcherd & Wynne, 1970) 2 x 10-4 M acetazolamidealready inhibited the secretary rate markedly in the first 10 min periodafter the introduction of the inhibitor, whereas in the present experiments,10-3 M acetazolamide was without effect after 12 min of perfusion with theinhibitor.

It has been shown by Douglas & Poisner (1963) that extracellular Ca isneeded in order to evoke salivary secretion by ACh stimulation. Petersen,Poulsen & Thorn (1967) showed that the sizes of the transmembranesecretary potentials were unchanged during perfusion with Ca-free solution.The present results (Fig. 3) showed that lack of extracellular Ca had noeffect on K release and uptake occurring after ACh stimulation in spite ofa severe inhibition of the salivary secretion. The effect of a low Ca con-centration in the perfusion fluid was very similar to that produced byethacrynic acid. This does not necessarily imply that lack of extracellularCa inhibits the pump forming the primary secretion, though this is onepossibility. In the proximal convolution of the rat kidney, peritubularcapillary perfusion with a Ca-free solution did not affect the isotonic waterreabsorption, but the ability of the tubule to generate a concentrationdifference for Na across the wall under conditions of no water reabsorptionwas abolished (Ullrich, Baldamus, Uhlich & Rumrich, 1969). This seemsto indicate that the permeability of the tubule wall to Na was enhancedduring perfusion with Ca-free solution. It has very recently been shown byDiamond (1971) that the high permeability of cations through the gall-bladder epithelium is mainly due to a shunt path represented by theso-called tight junctions between the cells. It is thus likely that the effect ofa low external Ca concentration is to loosen the junctional complexbetween cells, as has been shown by Sedar & Forte (1964) to occur with theoxyntic cells of gastric glands. It is not clear whether such an effect couldalone explain why salivary secretion stopped in the absence of external Ca,but the fact that isotonic water reabsorption continued in the proximalkidney convolution during perfusion with Ca-free fluid may indicate thatthis is not a sufficient explanation. However, when comparing data fromkidney and salivary gland perfusion studies it should be remembered thatthe direction of the water transport is different in these two organs. In theproximal tubule water is transported into the artificial perfusion fluid, butin the salivary glands water is transported from the artificial perfusionfluid into the lumen. The fact that both K release (Fig. 3) and secretarypotentials (Petersen et al. 1967) were uninfluenced after removal ofextracellular Ca shows that the processes at the basal acinar cell membraneafter stimulation with ACh occur normally in the absence of Ca. It ispossible that Ca entry to the acinar cytoplasm, evoked either directly by

139

the action of ACh or indirectly via the increased intracellular Na con-centration occurring as a consequence of the ACh-induced Na entry coupledto K release (Petersen, 1970a,b) is critically important for the activation ofthe secretary mechanism proper. The latter possibility is attractive, sinceBaker, Blaustein, Hodgkin & Steinhardt (1969) and Glitsch, Reuter &Scholz (1970) have shown that Ca influx increases with increasing intra-cellular Na concentration in the squid axon and the guinea-pig atria. AlsoPetersen (1970d) has shown that the salivary secretary rate is highlydependent on the extracellular Na concentration and the size of themembrane potential during stimulation with ACh, parameters determiningthe size of the Na influx occurring after ACh stimulation.

The excellent technical assistance of G. Pedersen is gratefully acknowledged.The work was supported by Johann and Hanne Weimann's legacy.

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BLAIR-WEST, J. R., COGHLAN, J. P., DENTON, D. A., NELSON, J., WRIGHT, R. D. &YAMAUCHI, A. (1969). Ionic, histological and vascular factors in the reaction of thesheep's parotid to high and low mineralocorticoid status. J. Phy8iol. 205, 563-579.

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DIAMOND, J. M. (1971). The route of transepithelial ion permeation in the gall-bladder. In Electrophysiology of Epithelial Cell,8, ed. GIEBISCH, G. & FROMTER, E.Stuttgart; New York: Schattauer Verlag (in the press).

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LUNDBERG, A. (1958). Electrophysiology of salivary glands. Physiol. Rev. 38, 21-40.MANGOS, J. A., BRAUN, G. & HAMANN, K. F. (1966). Micropuncture study of sodiumand potassium excretion in the rat parotid saliva. Pflugera Arch. ge8. Physiol. 291,99-106.

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SALIVARY SECRETION AND K TRANSPORT 141MARTINEZ, J. R., HOLZGREVE, H. & FRICK, A. (1966). Micropuncture study of sub-

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