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Fluid and electrolyte secretion from the isolated, perfused submandibular and sublingual glands of the rat
OCO3-996W1
Anh.s oral Biol Vol. 26. pp. 555 10 561. 1981 Prmted IFI Great Bntaln
070555-0780200~0
Pergamon Prea
I id
FLUID AND ELECTROLYTE SECRETION FROM THE ISOLATED, PERFUSED SUBMANDIBULAR AND SUBLINGUAL GLANDS OF THE RAT J. COMPTON, J. R. MARTINEZ, A. MARINA MARTINEZ and J. A. YOUNG Departments of Child Health and Physiology, University of Missouri School of Medicine, Columbia, MO 65212, U.S.A. and Department of Physiology, University of Sydney, Sydney, Australia Summary-Isolated preparations of the rat submandibular gland secreted when perfused at a rate of 3 ml/min with physiological salt solutions containing glucose and either acetylcholine (1O-g to 10e4 M) or pilocarpine (lo-’ to toe3 M). The maximum secretory response was 250 ~1 g- ’ min- ’ with acetylcholine (lO-‘j M) and 130 ~1 g- ’ min- ’ with pilocarpine (lo- 6 M). Higher agonist concentrations usually resulted in smaller secretory responses. The response to continued stimulation showed a slow decline, although it was more sustained than with a similar preparation of the rabbit submandibular gland. The excretion curves for Na, K, Ca, Cl and HC03 evoked by the two parasympatho-mimetic agonists were similar to those reported from in-ciuo experiments. Ouabain (lo- 3 M), the substitution of Na by Li or the omission of K in the perfusion solution markedly inhibited the secretory response from the isolated, perfusedgland preparation. Partial substitution of the perfusate Na with choline caused only partial inhibition of secretion, but decreased Na and increased Ca and K concentrations in saliva. It is concluded that the perfused-gland preparation functions normally with respect to the secretion of fluid and electrolytes, and offers a suitable vehicle for investigation of the salivary secretory process. The sublingual gland secreted in response to pilocarpine. The maximum secretory rate was 13Oplg-’ min-’ and the saliva was poor in Na and Cl and rich in K, HC03 and Ca.
INTRODUCTION The composition of saliva is dependent not only on the activity of the secretory endpieces and ducts (Martinez, Holzgreve and Frick, 1966; Young and Van Lennep, 1979), but also on such factors as blood flow, blood pressure and blood composition. Although control of these factors can be achieved in isolated, perfused glands, only a few attempts have been made to study the composition of saliva formed under these conditions (Douglas and Poisner, 1963; Inada, Mello and Lima de Castro, 1976), largely because the secreTory response of isolated, perfused glands declines rapidly during vascular infusion of parasympathomimetic agonists. Better results have been obtained with a partially perfused preparation (Coroneo, Denniss and Young, 1979; Sprecher, Yoshida and Schneyer, 1967) and Case, Conigrave and Young (1977) .ind Case er u/. (1980) demonstrated secretion of water, protein and electrolytes for over 6 h in the fully isolated, perfused submandibular gland of the rabbit. We have developed an isolated, perfused-gland loreparation in the rat which is likely to be useful for :he following reasons: (1) many physiological, bio8:hemical and morphological studies on salivary glands and their secretions have been conducted using the rat (Young and Van Lennep, 1979); (2) in contrast to the rabbit submandibular gland, which lacks much sympathetic secretomotor innervation, the rat gland is well supplied with sympathetic secretomotor fibres and secretes vigorously in response both to ct- and fl-adrenergic agonists (Martinez et al., 1975a; Young and Van Lennep, 1979; Young and Martin, 1971);
(3) the rat salivary secretions in K.
are poor in Na and rich
METHODS Adult, male albino rats of the Sprague-Dawley strain weighing between 220 and 280g were used. A total of 88 submandibular glands and 49 sublingual glands from 53 animals was studied. The rats were fed a standard pellet diet (Purina Co.) and had access to water ad libitum. They were anaesthetized with sodium pentobarbital (6 mg per 1OOg body weight, intraperitoneally), the trachea was cannulated and the submandibular/sublingual gland complex exposed and dissected free. The external mandibular artery was dissected free down to the gland. The digastric muscle overlying the submandibular and sublingual main excretory ducts, was divided between ligatures at its tendinous insertion. Both ducts were cannulated and then cut and reflected away from the mandibular artery and its branch supplying the gland complex. Two loose ligatures were placed around the artery proximal to the glandular branch. Arterial branches from the main trunk to non-glandular structures were identified, dissected and cut between ligatures. The mandibular artery was then cannulated and the cannula tip advanced to within l-2mm of the junction with the glandular artery and tied in. As perfusion was begun, the gland became pale; one of the loose arterial ligatures was now tightened to occlude the blood supply. Leaks in the system and the compieteness of perfusion were checked simply by inspecting the colour of the gland and the colour and compo-
556
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sition of the venous effluent. When the perfusion conditions were satisfactory, the second ligature was tightened and the vessel divided. The gland was then removed and placed in the perfusion bath and the experiment begun. The gland complex was perfused at 3 ml min- ‘, using a BuchlerR roller pump and a perfusate with the following composition (in mM): Na = 146.0, K = 4.3, Ca = 2.5, Mg = 1.0, Cl = 129.3, HC03 = 25.0, so4 = 1.0, HP04/HzP0, = 1.0, glucose = 10.0 (Case et (II., 1980). The high perfusion rate was necessary for an adequate secretory response; decreasing the perfusion rate to 1.5 or 1.0 ml min- ’ caused a marked drop in the secretory rate. The glands became oedematous during perfusion, regardless of the perfusion rate. No secretion was observed from the isolated glands in the absence of secretagogue. Drugs in a similar perfusate were added via a threeway tap at 0.3 ml min- ‘. The glands were stimulated with solutions of acetylcholine chloride (Sigma, St. Louis, MO.) in concentrations of low9 to 10e4M, or pilocarpine nitrate (Sigma) in concentrations 10m7 to 10e3 M. The perfusate was warmed to 37S”C and gassed continuously with a mixture of 5 per cent CO2 and 95 per cent OZ. Any gas bubbles in the perfusion lines were trapped in a gravity-dependent bubble trap. The composition of the perfusate was modified in some experiments in order to assess some of the ionic requirements for salivary secretion. The following 300 Acetylcholine
( 10m6mol I-’ 1
et al.
modifications were used: (1) the replacement of the perfusate Na by either Li or choline; (2) the omission of K from the perfusion solution; (3) the addition of ouabain (lo-3 M) to the perfusate. The osmolality of the perfusate remained constant when ionic substitutions were made. At the conclusion of each experiment, the gland complex was removed from the bath, blotted dry and separated into its component glands, each of which was weighed separately. The average weight of the submandibular glands was 0.266 g (SD = 0.05) and of the sublingual glands, 0.047 g (SD = 0.02). The saliva was collected in tared polyethylene tubes and the sample volumes calculated from the weights, assuming a sample specific gravity of unity. Samples were stored refrigerated under paraffin oil and analysis was performed within 24 h. The samples were assayed for Na and K with an ILK flame photometer with Li internal standard (Instrumentation Laboratories, Lexington, Mass.). Calcium was determined with a Corning” Ca analyzer (Corning Instruments Co., Medfield, Mass.) and Cl by coulombo-metric titration using a BuchlerR chloride titrator. The data were stored on computer floppy discs and sorted into groups according to flow rate; the group sizes were chosen to be integral multiples of and, as far as possible, each group 5plg-‘min-’ contained equal numbers of samples. Graphic representation followed the conventional form as discussed by Young (1979). In samples where Na, K, Ca and Cl had all been determined, a residual anion concentration (Na + K + Ca - Cl) was computed. In rat saliva, virtually all of this residue is attributable to bicarbonate (Young and Van Lennep, 1979).
RESULTS
Submandibular glands
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60
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100
( min 1
Fig. 1. Flow response of the isolated mandibular gland of the rat, perfused continuously with acetylcholine (10m6 M) or pilocarpine (1O-5 M). Six individual experiments are depicted.
In every experiment only one parasympatho-mimetic agonist was employed, but two or three different concentrations were usually infused. Because Case et a/. (1980) reported, for the isolated rabbit submandibular gland, that differing concentrations of infused acetylcholine can cause differences in the salivary concentrations of K and HCO, we examined our data to determine whether a similar effect was present. Although we were unable to detect any such effect of drug concentration on the salivary concentration of any of the electrolytes studied, the majority of our data were obtained at a single concentration for each drug (10m6 M for acetylcholine and 10m5 M for pilocarpine) and small differences caused by various doses of secretagogue may have been overlooked. Our data were also subdivided into those obtained on the first occasion that a gland was stimulated and those obtained on subsequent stimulations, but no difference was found. Therefore, the results obtained for each agonist have been pooled, regardless of the drug concentration employed or the time during the experiment at which the stimulation took place. Flow responses Figure 1 shows the flow responses of a number of glands in which stimulation with acetylcholine (10m6 M) or pilocarpine (lo- 5 M) was continued for
Secretion by isolated, perfused rat submandibular gland
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Fig. 2. Electrolyte excretion curves for the rat mandibular gland, isolated and perfused in vitro with acetylcholine (ACh) or pilocarpine. Most experiments were carried out using acetylcholine concentrations of 1O-6 M and pilocarpine concentrations of 10e5 M, but some data have been obtained using other concentrations (see text). Each point is the mean +SEM of 7-51 samples collected from 16-20 glands. periods of 50-100 min. As Case ef ul. (1977, 1980) observed in the rabbit submandibular gland, flow rate was high initially but decreased with time. In general, the rate of decline followed a slower time course in the rat, and in some experiments showed no tendency to decline at all. The first series of experiments (36 glands, 171 samples) was carried out using pilocarpine nitrate as the stimulating agonist. However, because the largest flow rates observed were not as great as those obtained in Guo using carbachol (Young and Martin, 1971) or acetylcholine (Coroneo et al., 1979), additional experiments were performed (31 glands, 129 samples) using acetylcholine. With pilocarpine, the maximum secretory rates averaged 130 ~1 g- ’ min- I. Secretion at measurable rates was observed at agonist concentrations between lo-’ and 10m3 M, but the glands gave a maximal response at concentrations of iOe5 M and the use of higher agonist concentrations commonly caused the secretory rate to stabilize at lower levels. With acetylcholine, the maximum secretory rates were about 250 ~1 g-’ min-’ and secretion at measurable rates was observed with infusion of acetylcholine in concentrations between 1O-9 and ;0e4 M. The glands gave a maximal response at a concentration of 10m6 M and, as for pilocarpine, higher concentrations of agonist often resulted in lower secretory rates. Hectrolyte
excretion
All the results are expressed as means and SEM. With pilocarpine the sodium concentrations (Fig. 2) were high at low secretory rates (2.5.0mM, + 1.3, )I = 47) and fell rapidly to a mean plateau value
of 11.8 mM (k1.3, n = 120) between flow rates of 2&12Opl g-l min-‘. At the highest flow rates encountered, the concentration rose significantly (p < 0.001) to a mean value of 23.3 mM ( + 1.1, n = 5). Potassium concentrations (Fig. 2) were also high at low flow rates (56.0 mM, _t 1.6, n = 111) and fell about 10mM as flow rate approached its maximum. Calcium was present only in low concentrations (Fig. 3) but the excretion curve showed clear flowdependence, with a relatively high concentration at low flow rates (0.89 mequiv l- ‘, -&0.06, n = 86) which fell to a plateau value of 0.46 mequiv I-’ (_tO.O4. n = 58) at flow rates in excess of 50 ~1 g- ’ min- ‘. The major salivary anion was chloride. Its concentration ranged from 40 to 50mM and tended to fall slightly with increasing secretory rate (Fig. 2). Comparison of the summed cation concentrations (in mequiv l- ‘) with the Cl concentration indicated the presence of at least one other anion, presumably HCO,, in a concentration of 2&30 mequiv I-‘. The concentration of this residual anion, like that of Cl, fell slightly with increasing flow rate (Fig. 2). With acetylcholine, the Na concentration observed (Fig. 2) was 3G50mM higher than with pilocarpineevoked saliva and the excretion curve showed an approximately linear rise from a mean of 42.6 mM (k2.8, n = 51) at low flow rates, to 68.9 mM (k 1.0, n = 10) at the highest rates. In contrast, the K content of acetylcholine-evoked saliva was 12-l 5 mM lower than in pilocarpine-evoked saliva, although the form of the excretion curve (Fig. 2) was similar. At the lowest flow rates, the mean concentration was 45.5 mM (&- 1.6, n = 51) and at higher flow rates a plateau value of 38.5 mM (kO.8, n = 49) was reached.
J. Compton ef al.
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Fig. 3. The concentration of free calcium in the saliva of the rat mandibular gland, isolated, and perfused in uitro with acetylcholine or pilocarpine (from the same experiments as are depicted in Fig. 2).
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15
30
45
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Except for the wider range of flow rates evoked by acetylcholine, the Ca excretion curves for the two agonists were almost identical (Fig. 3). In acetylcholine-evoked saliva, the Ca concentration at low flow rates was 0.78 mequiv 1-l (fO.lO, n = 43) and the plateau concentration seen at higher flow rates was 0.44mequiv 1-l (f0.02, n = 71). The salivary anions in acetylcholine-evoked saliva exhibited excretion curves (Fig. 2) similar in form to those seen in pilocar-
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Fig. 4. Flow response of the isolated mandibular gland of the rat perfused continuously with perfusates containing either ouabain or Li. The perfusates also contained acetylcholine (10m6 M). (1) the perfusate Na was partially (75-82 per cent) replaced with Li; (2) all the Na was replaced by Li; (3) ouabain (10e3 M) was present in the perfusate.
g-’ min-‘1
Flow
I I5
were
Effects of changes in the perfusate
Substitution of the perfusate Na with Li caused a rapid and significant decline in the rate of flow (Fig. 4). This effect was evident whether the perfusate Na was substituted completely or partially by Li. The secretion rate decreased to such an extent in these experiments that it was not possible to collect enough fluid for the assessment of changes in saliva
5
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4
-
3
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to--
60
o
Ca
I5
30
45
60
Time (min)
Fig. 5. Flow changes and electrolyte excretion curves for the isolated rat mandibular gland perfused in vitro with K+-free solutions in the presence of acetylcholine (1Om6M).
Secretion
by isolated,
perfused
rat submandibular
559
gland
Co
Flow mte
T
T
100
No
2 0 Time (min)
Fig. 6. Flow and electrolyte excretion patterns in the isolated rat mandibular with a solution where Na was partially (75-82%) replaced by choline chloride. the presence of acetylcholine (10m6 M).
composition. Figure 4 also shows that the addition of ouabain (10m3 M) to the unsubstituted perfusate caused a rapid decline in rate of flow. A similar effect was observed when K was omitted from the perfusate (Fig. 5). Neither the addition of ouabain nor the omission of K from the perfusate resulted in marked changes in the Na or K concentrations of saliva. This i.; shown for the K-free perfusate in Fig. 5. In both cases, however, the saliva Ca concentration, which did not change during perfusion with the substituted perfusate, increased when the gland was subsequently perfused with regular perfusate (Fig. 5). Substitution of the perfusate Na with choline caused a partial inhibition of salivary flow, associated with a marked decrease in Na concentration and increases in the K and Ca concentrations of saliva (Fig. 6). Xuhlingual glands The results of a few experiments (8 glands, 32 samples) in which we studied the electrolyte content of sublingual saliva evoked in response to pilocarpine infusion are shown in Table 1. The small size of the
Table 1. The composition of saliva collected from the isolated. perfused sublingual gland of the rat stimulated to secrete with pilocarpine (Mean flow rate 62.0 + 5.8 cclgg ’ min-‘, n = 32; n = number of samples)
Na (mmol I- ‘) K (mmol 1.. ‘) Ca (mequiv l- ‘) Cl (mm01 1~‘) Residual anions*
Mean
SEM
n
65.0 46.4 2.41 79.9 33.9
5.1 1.8 0.34 4.4 5.9
32 32 21 28 18
* Na + K + Ca - Cl in mequiv 1-r.
gland
perfused
in Gtro
Experiments were done in
glands and the correspondingly small volumes of saliva secreted resulted in a smaller number of data. The secretory rates were maximal during infusion of pilocarpine (lo- 5 mol l- ‘) and seldom exceeded 13Oplg-’ min-‘. The saliva was relatively poor in Na and Cl, and particularly rich in K, HCO, and Ca (Table 1). DISCUSSION
A method for the isolation and the in-vitro perfusion of the rat submandibular and sublingual glands has been developed. The preparation gives a stable secretory response to parasympatho-mimetic stimulation which, in terms of both the volume and the electrolyte content of the saliva, is similar to that obtained when the gland is stimulated in uiuo (Young and Martin, 1971; Martinez et al., 1975b; Coroneo et al., 1979; Young and Van Lennep, 1979). In fact, the maximum flow response in vitro during acetylcholine infusion is greater than that elicited by close arterial infusions of the agonist in uiuo (Coroneo et al., 1979), perhaps deriving from a slower rate of hydrolysis of acetylcholine in the isolated gland preparation. The Na and K excretion curves evoked by acetylcholine infusion resemble closely those reported from in-uioo studies by Coroneo et al. (1979). Similar in-uiuo studies on acetylcholine-stimulated HC03 excretion are not available, but data from experiments with the closely related agonist, carbachol, show similar excretion patterns for the common monovalent electrolytes, including HC03 (Martinez et al., 1975a,b; Young and Martin, 1971). The available evidence indicates that cholinergic drugs act on the endpiece cells (and perhaps the intercalated ducts) to increase the rate of formation of an isotonic fluid with a plasma-like electrolyte content. This fluid is then modified during its passage through the glandular duct system by active transport mechanisms for Na
560
J. Compton et (11.
re-absorption and K and HC03 secretion. The transport rates across the duct are affected by autonomic agonists, e.g. cholinergic drugs normally reduce Na transport quite markedly (Martin and Young, 1971b; Young and Martin, 1971). In view of the close similarity between the electrolyte composition of cholinergically evoked saliva in our in-vitro study and in the earlier in-uiuo ones, it seems reasonable to conclude that both ducts and endpieces were functioning effectively in the isolated gland preparation. However, the K levels in both acetylcholine- and pilocarpine-stimulated saliva were higher in the present study than those reported by Young and Van Lennep (1979). This might be related to the higher K levels of the perfusate compared to rat plasma, or to the diet, but, more likely, indicates some alteration in gland function. In this study, pilocarpine evoked a smaller secretory response than acetylcholine, and the saliva was markedly poorer in Na and richer in K. In the two in-riuo studies available for comparison (Martinez et al., 1975a,b; Young and Schogel, 1966) pilocarpine evoked smaller flow responses than acetylcholine and the saliva was also poor in Na. On the other hand, the K content of our pilocarpine-evoked saliva was higher than in saliva collected in uiuo by Young and Schiigel (1966). As pilocarpine and other chohnergic agents evoke primary saliva of approximately the same composition (Young and Schiigel, 1966; Young and Martin, 1971), it follows that differences in the electrolyte excretion patterns evoked by these agonists indicate differences in the ductal modification of the primary saliva. If, as we have already suggested above, carbachol and acetylcholine inhibit ductal Na transport and stimulate K secretion more markedly than does pilocarpine (Martin and Young, 1971b; Coroneo et al., 1979; Young and Van Lennep, 1979; Case et al., 1980), then no further explanation is needed to account for the present findings. The Ca excretion patterns seen following stimulation with acetylcholine and pilocarpine (Fig. 3) were similar to those reported for glands stimulated in uiuo by Martinez et al. (1975a) although the plateau concentration in Go averaged 0.25 and 1.0 mequiv l- ‘, respectively, whereas in-oitro they averaged, respectively, 0.44 and 0.46 mequiv I- I. Although these values were lower than that of the perfusate (5.0 mequiv I- ‘), without a knowledge of the primary-fluid Ca concentration, it is difficult to determine whether or not the ducts transport Ca. In only one study, on the rat parotid gland, has the primary fluid Ca content been measured (Mangos et al., 1978). The value reported (2.9 mequiv l- ‘) has been interpreted as indicating that the salivary ducts reabsorb Ca since the Ca concentration in final saliva is usually less than this. Because more than half the salivary Ca is bound to secreted protein (Young and Van Lennep, 1979), it is difficult to interpret Ca excretion curves without a knowledge of the protein content of the saliva. The mechanisms responsible for salivary secretion are only partially understood, but it is generally accepted that stimulation with parasympatho-mimetic agents causes an influx of Na and an efflux of K from the salivary cells, resulting in the activation of ionic pumps and the generation of an osmotic gradient for the movement of water (Peterson, 1972; Young and
Van Lennep, 1979). In our experiment, the replacement of Na in the perfusion fluid resulted in a marked inhibition of the secretory response to acetylcholine, a finding which suggests that availability of external Na is indeed essential for secretion. However, the observation that the response to acetylcholine was also reduced during perfusion with K-free solutions suggests that an incrase in intracellular Na is not enough, by itself, to activate the secretory process. Substitution of most of the perfusate Na with choline only caused a partial inhibition of salivary flow; this effect can be best interpreted as due to a gradient limitation in Na secretion at the primary level. Studies on the isolated cat submandibular gland suggest that the transport mechanism responsible for the formation of saliva is different from the Na+, K+-ATPase system (Petersen, 1971; Laugesen, Dich Nielsen and Poulsen, 1976). Because ouabain in the perfusate also inhibited the secretory response, it seems likely that the maintenance of normal intracellular ion concentrations by the activity of a Na+, K+-ATPase, particularly after the stimulation-induced K efflux, is a requisite for sustained secretion to occur. Another factor which has been shown to be required for a sustained secretory response is extracellular Ca (Martinez and Petersen, 1971). Although the small size of the rat sublingual gland has limited investigation of its electrolyte excretion patterns, flow curves obtained in ciuo during stimulation with carbachol and acetylcholine are available (Martin and Young, 1971a; Coroneo et al., 1979). Our data, obtained during stimulation only with pilocarpine agree well with these earlier in-viva studies and do not suggest any major differences in the actions of the three drugs on sublingual ducts, at least under conditions of maximal stimulation.
Acknowledgements-We thank the Post Graduate Medical Foundation of the University of Sydney for the award of a travel grant to one of us (J.C.). The work was supported by a grant from the Public Health Service (NIH grant AM 18150).
REFERENCES
Case R. M., Conigrave A. D. and Young J. A. 1977. The isolated, saline-perfused rabbit mandibular gland. Proc. Amt. physiol. p
Douglas W. W. and Poisner A. M. 1963. The influence of calcium on the secretory response of the submaxillary gland to acetylcholine or to noradrenaline. J. PhysioL, Lond. 165, 528-541. Inada M., Mello A. and Lima-de-Castro A. 1976. Influencia dos ions calcio e potassio na responsta secretora da glandula submandibular isolada e perfundida. a estimulacao parassimpatica. Rhuta bras. Pesq. med. hiol. 9, 2633271. Laugesen L. P., Dich Nielsen 3. 0. and Poulsen J. H. 1976. Partial dissociation between salivary secretion and active
Secretion
by isolated,
perfused
potassium transport in the perfused cat submandibular gland. P&yers Arch. Eur. J. Physiol. 364, 167-173. Mangos J. A., Garrish M. T., Wells R., Farnham W. and Bouchlas G. 1978. A micropuncture study of the handling of calcrum by the rat parotid. J. dent. Res. 57, 818-825. Martin C. J. and Young J. A. 1971a. Electrolyte concenrations m primary and final saliva of the rat sublingual sland studied by micropuncture and catheterization techniques. t’jiiiyer Arch. Eur. J. Physiol. 324, 344360. Martin C. J. and Young J. A. 1971b. A microoerfusion Investigation of the effects of a sympathomime;ic and a parasympathomimetic drug on water and electrolyte fluxes in the main duct of the rat submaxillary gland. Pjliigrrs Arch. Eur. J. Phvsiol. 327, 303-323. Martinez J. R.. Holzgreve H. and Frick A. 1966. Micropuncture study of submaxillary glands of adult rats. Pfliigers Arch. yes. Physiol. 290, 124-133. Martinez J. R. and Petersen 0. H. 1971. The importance of extracellular calcium for acetylcholine-evoked salivary secretion. Experientiu 28, 167-168. Martinez J. R., Quisseli D. 0.. Wood D. L. and Giles M. 1975a. Abnormal secretory response to parasympathomimetic and sympathomimetic stimulation from the submaxillary gland of rats treated with reserpine. J. PharWK. exp. Ther. 194, 384-395. Martinez J. R., Adshead P. C., Quissell D. 0. and Barber0 G. J. 197% The chronically reserpinized rat as a poss-
rat submandibular
gland
S61
ible model for cystic fibrosis. II. Composition and cilioinhibitory effects of submaxillary saliva. Pediutric Res. 9, 47S-475. Petersen 0. H. 1971. Formation of saliva and potassium transport in the perfused cat submandibular gland. J. Physiol., Land. 216, 129-142. Petersen 0. H. 1972. Acetylcholine-induced ion transport involved in the formation of saliva. Am physiol. scund. Suppl. 381, l-58. Sprecher R. L., Yoshida Y. and Schneyer L. H. 1967. Partial isolation and perfusion of rat submaxillary gland. Proc. Sac. exp. Biol. Med. 125, 1227-1229. Young J. A. 1979. Salivary secretion of inorganic electrolytes. In: International Reciew of Phvsioloqv, Gastroinirstinal Physiology III (Edited by Crane R. l?.), Vol. 19, pp. l-58. University Park Press. Baltimore. Young J. A. and-Van Lennep E. W. 1979. Transport in salivary and salt glands: salivary glands. In: bfemhrune Trunsport in Biology (Edited by Giebisch G.). Vol. 4. pp. 5633674. Springer, Berlin, Young J. A. and Martin C. J. 1971. The effect of a sympatho- and a parasympathomimetic drug on the electrolyte concentrations of primary and final saliva of the rat submaxillary gland. Pfliigers Arch. Eur. J. Physiol. 327, 285-302. Young J. A. and Schogel E. 1966. Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pfriigers Arch. yes. Physioi. 291, 85-98.
Anh.s oral Biol Vol. 26. pp. 555 10 561. 1981 Prmted IFI Great Bntaln
070555-0780200~0
Pergamon Prea
I id
FLUID AND ELECTROLYTE SECRETION FROM THE ISOLATED, PERFUSED SUBMANDIBULAR AND SUBLINGUAL GLANDS OF THE RAT J. COMPTON, J. R. MARTINEZ, A. MARINA MARTINEZ and J. A. YOUNG Departments of Child Health and Physiology, University of Missouri School of Medicine, Columbia, MO 65212, U.S.A. and Department of Physiology, University of Sydney, Sydney, Australia Summary-Isolated preparations of the rat submandibular gland secreted when perfused at a rate of 3 ml/min with physiological salt solutions containing glucose and either acetylcholine (1O-g to 10e4 M) or pilocarpine (lo-’ to toe3 M). The maximum secretory response was 250 ~1 g- ’ min- ’ with acetylcholine (lO-‘j M) and 130 ~1 g- ’ min- ’ with pilocarpine (lo- 6 M). Higher agonist concentrations usually resulted in smaller secretory responses. The response to continued stimulation showed a slow decline, although it was more sustained than with a similar preparation of the rabbit submandibular gland. The excretion curves for Na, K, Ca, Cl and HC03 evoked by the two parasympatho-mimetic agonists were similar to those reported from in-ciuo experiments. Ouabain (lo- 3 M), the substitution of Na by Li or the omission of K in the perfusion solution markedly inhibited the secretory response from the isolated, perfusedgland preparation. Partial substitution of the perfusate Na with choline caused only partial inhibition of secretion, but decreased Na and increased Ca and K concentrations in saliva. It is concluded that the perfused-gland preparation functions normally with respect to the secretion of fluid and electrolytes, and offers a suitable vehicle for investigation of the salivary secretory process. The sublingual gland secreted in response to pilocarpine. The maximum secretory rate was 13Oplg-’ min-’ and the saliva was poor in Na and Cl and rich in K, HC03 and Ca.
INTRODUCTION The composition of saliva is dependent not only on the activity of the secretory endpieces and ducts (Martinez, Holzgreve and Frick, 1966; Young and Van Lennep, 1979), but also on such factors as blood flow, blood pressure and blood composition. Although control of these factors can be achieved in isolated, perfused glands, only a few attempts have been made to study the composition of saliva formed under these conditions (Douglas and Poisner, 1963; Inada, Mello and Lima de Castro, 1976), largely because the secreTory response of isolated, perfused glands declines rapidly during vascular infusion of parasympathomimetic agonists. Better results have been obtained with a partially perfused preparation (Coroneo, Denniss and Young, 1979; Sprecher, Yoshida and Schneyer, 1967) and Case, Conigrave and Young (1977) .ind Case er u/. (1980) demonstrated secretion of water, protein and electrolytes for over 6 h in the fully isolated, perfused submandibular gland of the rabbit. We have developed an isolated, perfused-gland loreparation in the rat which is likely to be useful for :he following reasons: (1) many physiological, bio8:hemical and morphological studies on salivary glands and their secretions have been conducted using the rat (Young and Van Lennep, 1979); (2) in contrast to the rabbit submandibular gland, which lacks much sympathetic secretomotor innervation, the rat gland is well supplied with sympathetic secretomotor fibres and secretes vigorously in response both to ct- and fl-adrenergic agonists (Martinez et al., 1975a; Young and Van Lennep, 1979; Young and Martin, 1971);
(3) the rat salivary secretions in K.
are poor in Na and rich
METHODS Adult, male albino rats of the Sprague-Dawley strain weighing between 220 and 280g were used. A total of 88 submandibular glands and 49 sublingual glands from 53 animals was studied. The rats were fed a standard pellet diet (Purina Co.) and had access to water ad libitum. They were anaesthetized with sodium pentobarbital (6 mg per 1OOg body weight, intraperitoneally), the trachea was cannulated and the submandibular/sublingual gland complex exposed and dissected free. The external mandibular artery was dissected free down to the gland. The digastric muscle overlying the submandibular and sublingual main excretory ducts, was divided between ligatures at its tendinous insertion. Both ducts were cannulated and then cut and reflected away from the mandibular artery and its branch supplying the gland complex. Two loose ligatures were placed around the artery proximal to the glandular branch. Arterial branches from the main trunk to non-glandular structures were identified, dissected and cut between ligatures. The mandibular artery was then cannulated and the cannula tip advanced to within l-2mm of the junction with the glandular artery and tied in. As perfusion was begun, the gland became pale; one of the loose arterial ligatures was now tightened to occlude the blood supply. Leaks in the system and the compieteness of perfusion were checked simply by inspecting the colour of the gland and the colour and compo-
556
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sition of the venous effluent. When the perfusion conditions were satisfactory, the second ligature was tightened and the vessel divided. The gland was then removed and placed in the perfusion bath and the experiment begun. The gland complex was perfused at 3 ml min- ‘, using a BuchlerR roller pump and a perfusate with the following composition (in mM): Na = 146.0, K = 4.3, Ca = 2.5, Mg = 1.0, Cl = 129.3, HC03 = 25.0, so4 = 1.0, HP04/HzP0, = 1.0, glucose = 10.0 (Case et (II., 1980). The high perfusion rate was necessary for an adequate secretory response; decreasing the perfusion rate to 1.5 or 1.0 ml min- ’ caused a marked drop in the secretory rate. The glands became oedematous during perfusion, regardless of the perfusion rate. No secretion was observed from the isolated glands in the absence of secretagogue. Drugs in a similar perfusate were added via a threeway tap at 0.3 ml min- ‘. The glands were stimulated with solutions of acetylcholine chloride (Sigma, St. Louis, MO.) in concentrations of low9 to 10e4M, or pilocarpine nitrate (Sigma) in concentrations 10m7 to 10e3 M. The perfusate was warmed to 37S”C and gassed continuously with a mixture of 5 per cent CO2 and 95 per cent OZ. Any gas bubbles in the perfusion lines were trapped in a gravity-dependent bubble trap. The composition of the perfusate was modified in some experiments in order to assess some of the ionic requirements for salivary secretion. The following 300 Acetylcholine
( 10m6mol I-’ 1
et al.
modifications were used: (1) the replacement of the perfusate Na by either Li or choline; (2) the omission of K from the perfusion solution; (3) the addition of ouabain (lo-3 M) to the perfusate. The osmolality of the perfusate remained constant when ionic substitutions were made. At the conclusion of each experiment, the gland complex was removed from the bath, blotted dry and separated into its component glands, each of which was weighed separately. The average weight of the submandibular glands was 0.266 g (SD = 0.05) and of the sublingual glands, 0.047 g (SD = 0.02). The saliva was collected in tared polyethylene tubes and the sample volumes calculated from the weights, assuming a sample specific gravity of unity. Samples were stored refrigerated under paraffin oil and analysis was performed within 24 h. The samples were assayed for Na and K with an ILK flame photometer with Li internal standard (Instrumentation Laboratories, Lexington, Mass.). Calcium was determined with a Corning” Ca analyzer (Corning Instruments Co., Medfield, Mass.) and Cl by coulombo-metric titration using a BuchlerR chloride titrator. The data were stored on computer floppy discs and sorted into groups according to flow rate; the group sizes were chosen to be integral multiples of and, as far as possible, each group 5plg-‘min-’ contained equal numbers of samples. Graphic representation followed the conventional form as discussed by Young (1979). In samples where Na, K, Ca and Cl had all been determined, a residual anion concentration (Na + K + Ca - Cl) was computed. In rat saliva, virtually all of this residue is attributable to bicarbonate (Young and Van Lennep, 1979).
RESULTS
Submandibular glands
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40
60
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100
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Fig. 1. Flow response of the isolated mandibular gland of the rat, perfused continuously with acetylcholine (10m6 M) or pilocarpine (1O-5 M). Six individual experiments are depicted.
In every experiment only one parasympatho-mimetic agonist was employed, but two or three different concentrations were usually infused. Because Case et a/. (1980) reported, for the isolated rabbit submandibular gland, that differing concentrations of infused acetylcholine can cause differences in the salivary concentrations of K and HCO, we examined our data to determine whether a similar effect was present. Although we were unable to detect any such effect of drug concentration on the salivary concentration of any of the electrolytes studied, the majority of our data were obtained at a single concentration for each drug (10m6 M for acetylcholine and 10m5 M for pilocarpine) and small differences caused by various doses of secretagogue may have been overlooked. Our data were also subdivided into those obtained on the first occasion that a gland was stimulated and those obtained on subsequent stimulations, but no difference was found. Therefore, the results obtained for each agonist have been pooled, regardless of the drug concentration employed or the time during the experiment at which the stimulation took place. Flow responses Figure 1 shows the flow responses of a number of glands in which stimulation with acetylcholine (10m6 M) or pilocarpine (lo- 5 M) was continued for
Secretion by isolated, perfused rat submandibular gland
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Fig. 2. Electrolyte excretion curves for the rat mandibular gland, isolated and perfused in vitro with acetylcholine (ACh) or pilocarpine. Most experiments were carried out using acetylcholine concentrations of 1O-6 M and pilocarpine concentrations of 10e5 M, but some data have been obtained using other concentrations (see text). Each point is the mean +SEM of 7-51 samples collected from 16-20 glands. periods of 50-100 min. As Case ef ul. (1977, 1980) observed in the rabbit submandibular gland, flow rate was high initially but decreased with time. In general, the rate of decline followed a slower time course in the rat, and in some experiments showed no tendency to decline at all. The first series of experiments (36 glands, 171 samples) was carried out using pilocarpine nitrate as the stimulating agonist. However, because the largest flow rates observed were not as great as those obtained in Guo using carbachol (Young and Martin, 1971) or acetylcholine (Coroneo et al., 1979), additional experiments were performed (31 glands, 129 samples) using acetylcholine. With pilocarpine, the maximum secretory rates averaged 130 ~1 g- ’ min- I. Secretion at measurable rates was observed at agonist concentrations between lo-’ and 10m3 M, but the glands gave a maximal response at concentrations of iOe5 M and the use of higher agonist concentrations commonly caused the secretory rate to stabilize at lower levels. With acetylcholine, the maximum secretory rates were about 250 ~1 g-’ min-’ and secretion at measurable rates was observed with infusion of acetylcholine in concentrations between 1O-9 and ;0e4 M. The glands gave a maximal response at a concentration of 10m6 M and, as for pilocarpine, higher concentrations of agonist often resulted in lower secretory rates. Hectrolyte
excretion
All the results are expressed as means and SEM. With pilocarpine the sodium concentrations (Fig. 2) were high at low secretory rates (2.5.0mM, + 1.3, )I = 47) and fell rapidly to a mean plateau value
of 11.8 mM (k1.3, n = 120) between flow rates of 2&12Opl g-l min-‘. At the highest flow rates encountered, the concentration rose significantly (p < 0.001) to a mean value of 23.3 mM ( + 1.1, n = 5). Potassium concentrations (Fig. 2) were also high at low flow rates (56.0 mM, _t 1.6, n = 111) and fell about 10mM as flow rate approached its maximum. Calcium was present only in low concentrations (Fig. 3) but the excretion curve showed clear flowdependence, with a relatively high concentration at low flow rates (0.89 mequiv l- ‘, -&0.06, n = 86) which fell to a plateau value of 0.46 mequiv I-’ (_tO.O4. n = 58) at flow rates in excess of 50 ~1 g- ’ min- ‘. The major salivary anion was chloride. Its concentration ranged from 40 to 50mM and tended to fall slightly with increasing secretory rate (Fig. 2). Comparison of the summed cation concentrations (in mequiv l- ‘) with the Cl concentration indicated the presence of at least one other anion, presumably HCO,, in a concentration of 2&30 mequiv I-‘. The concentration of this residual anion, like that of Cl, fell slightly with increasing flow rate (Fig. 2). With acetylcholine, the Na concentration observed (Fig. 2) was 3G50mM higher than with pilocarpineevoked saliva and the excretion curve showed an approximately linear rise from a mean of 42.6 mM (k2.8, n = 51) at low flow rates, to 68.9 mM (k 1.0, n = 10) at the highest rates. In contrast, the K content of acetylcholine-evoked saliva was 12-l 5 mM lower than in pilocarpine-evoked saliva, although the form of the excretion curve (Fig. 2) was similar. At the lowest flow rates, the mean concentration was 45.5 mM (&- 1.6, n = 51) and at higher flow rates a plateau value of 38.5 mM (kO.8, n = 49) was reached.
J. Compton ef al.
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Fig. 3. The concentration of free calcium in the saliva of the rat mandibular gland, isolated, and perfused in uitro with acetylcholine or pilocarpine (from the same experiments as are depicted in Fig. 2).
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30
45
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Except for the wider range of flow rates evoked by acetylcholine, the Ca excretion curves for the two agonists were almost identical (Fig. 3). In acetylcholine-evoked saliva, the Ca concentration at low flow rates was 0.78 mequiv 1-l (fO.lO, n = 43) and the plateau concentration seen at higher flow rates was 0.44mequiv 1-l (f0.02, n = 71). The salivary anions in acetylcholine-evoked saliva exhibited excretion curves (Fig. 2) similar in form to those seen in pilocar-
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Fig. 4. Flow response of the isolated mandibular gland of the rat perfused continuously with perfusates containing either ouabain or Li. The perfusates also contained acetylcholine (10m6 M). (1) the perfusate Na was partially (75-82 per cent) replaced with Li; (2) all the Na was replaced by Li; (3) ouabain (10e3 M) was present in the perfusate.
g-’ min-‘1
Flow
I I5
were
Effects of changes in the perfusate
Substitution of the perfusate Na with Li caused a rapid and significant decline in the rate of flow (Fig. 4). This effect was evident whether the perfusate Na was substituted completely or partially by Li. The secretion rate decreased to such an extent in these experiments that it was not possible to collect enough fluid for the assessment of changes in saliva
5
-
4
-
3
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to--
60
o
Ca
I5
30
45
60
Time (min)
Fig. 5. Flow changes and electrolyte excretion curves for the isolated rat mandibular gland perfused in vitro with K+-free solutions in the presence of acetylcholine (1Om6M).
Secretion
by isolated,
perfused
rat submandibular
559
gland
Co
Flow mte
T
T
100
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Fig. 6. Flow and electrolyte excretion patterns in the isolated rat mandibular with a solution where Na was partially (75-82%) replaced by choline chloride. the presence of acetylcholine (10m6 M).
composition. Figure 4 also shows that the addition of ouabain (10m3 M) to the unsubstituted perfusate caused a rapid decline in rate of flow. A similar effect was observed when K was omitted from the perfusate (Fig. 5). Neither the addition of ouabain nor the omission of K from the perfusate resulted in marked changes in the Na or K concentrations of saliva. This i.; shown for the K-free perfusate in Fig. 5. In both cases, however, the saliva Ca concentration, which did not change during perfusion with the substituted perfusate, increased when the gland was subsequently perfused with regular perfusate (Fig. 5). Substitution of the perfusate Na with choline caused a partial inhibition of salivary flow, associated with a marked decrease in Na concentration and increases in the K and Ca concentrations of saliva (Fig. 6). Xuhlingual glands The results of a few experiments (8 glands, 32 samples) in which we studied the electrolyte content of sublingual saliva evoked in response to pilocarpine infusion are shown in Table 1. The small size of the
Table 1. The composition of saliva collected from the isolated. perfused sublingual gland of the rat stimulated to secrete with pilocarpine (Mean flow rate 62.0 + 5.8 cclgg ’ min-‘, n = 32; n = number of samples)
Na (mmol I- ‘) K (mmol 1.. ‘) Ca (mequiv l- ‘) Cl (mm01 1~‘) Residual anions*
Mean
SEM
n
65.0 46.4 2.41 79.9 33.9
5.1 1.8 0.34 4.4 5.9
32 32 21 28 18
* Na + K + Ca - Cl in mequiv 1-r.
gland
perfused
in Gtro
Experiments were done in
glands and the correspondingly small volumes of saliva secreted resulted in a smaller number of data. The secretory rates were maximal during infusion of pilocarpine (lo- 5 mol l- ‘) and seldom exceeded 13Oplg-’ min-‘. The saliva was relatively poor in Na and Cl, and particularly rich in K, HCO, and Ca (Table 1). DISCUSSION
A method for the isolation and the in-vitro perfusion of the rat submandibular and sublingual glands has been developed. The preparation gives a stable secretory response to parasympatho-mimetic stimulation which, in terms of both the volume and the electrolyte content of the saliva, is similar to that obtained when the gland is stimulated in uiuo (Young and Martin, 1971; Martinez et al., 1975b; Coroneo et al., 1979; Young and Van Lennep, 1979). In fact, the maximum flow response in vitro during acetylcholine infusion is greater than that elicited by close arterial infusions of the agonist in uiuo (Coroneo et al., 1979), perhaps deriving from a slower rate of hydrolysis of acetylcholine in the isolated gland preparation. The Na and K excretion curves evoked by acetylcholine infusion resemble closely those reported from in-uioo studies by Coroneo et al. (1979). Similar in-uiuo studies on acetylcholine-stimulated HC03 excretion are not available, but data from experiments with the closely related agonist, carbachol, show similar excretion patterns for the common monovalent electrolytes, including HC03 (Martinez et al., 1975a,b; Young and Martin, 1971). The available evidence indicates that cholinergic drugs act on the endpiece cells (and perhaps the intercalated ducts) to increase the rate of formation of an isotonic fluid with a plasma-like electrolyte content. This fluid is then modified during its passage through the glandular duct system by active transport mechanisms for Na
560
J. Compton et (11.
re-absorption and K and HC03 secretion. The transport rates across the duct are affected by autonomic agonists, e.g. cholinergic drugs normally reduce Na transport quite markedly (Martin and Young, 1971b; Young and Martin, 1971). In view of the close similarity between the electrolyte composition of cholinergically evoked saliva in our in-vitro study and in the earlier in-uiuo ones, it seems reasonable to conclude that both ducts and endpieces were functioning effectively in the isolated gland preparation. However, the K levels in both acetylcholine- and pilocarpine-stimulated saliva were higher in the present study than those reported by Young and Van Lennep (1979). This might be related to the higher K levels of the perfusate compared to rat plasma, or to the diet, but, more likely, indicates some alteration in gland function. In this study, pilocarpine evoked a smaller secretory response than acetylcholine, and the saliva was markedly poorer in Na and richer in K. In the two in-riuo studies available for comparison (Martinez et al., 1975a,b; Young and Schogel, 1966) pilocarpine evoked smaller flow responses than acetylcholine and the saliva was also poor in Na. On the other hand, the K content of our pilocarpine-evoked saliva was higher than in saliva collected in uiuo by Young and Schiigel (1966). As pilocarpine and other chohnergic agents evoke primary saliva of approximately the same composition (Young and Schiigel, 1966; Young and Martin, 1971), it follows that differences in the electrolyte excretion patterns evoked by these agonists indicate differences in the ductal modification of the primary saliva. If, as we have already suggested above, carbachol and acetylcholine inhibit ductal Na transport and stimulate K secretion more markedly than does pilocarpine (Martin and Young, 1971b; Coroneo et al., 1979; Young and Van Lennep, 1979; Case et al., 1980), then no further explanation is needed to account for the present findings. The Ca excretion patterns seen following stimulation with acetylcholine and pilocarpine (Fig. 3) were similar to those reported for glands stimulated in uiuo by Martinez et al. (1975a) although the plateau concentration in Go averaged 0.25 and 1.0 mequiv l- ‘, respectively, whereas in-oitro they averaged, respectively, 0.44 and 0.46 mequiv I- I. Although these values were lower than that of the perfusate (5.0 mequiv I- ‘), without a knowledge of the primary-fluid Ca concentration, it is difficult to determine whether or not the ducts transport Ca. In only one study, on the rat parotid gland, has the primary fluid Ca content been measured (Mangos et al., 1978). The value reported (2.9 mequiv l- ‘) has been interpreted as indicating that the salivary ducts reabsorb Ca since the Ca concentration in final saliva is usually less than this. Because more than half the salivary Ca is bound to secreted protein (Young and Van Lennep, 1979), it is difficult to interpret Ca excretion curves without a knowledge of the protein content of the saliva. The mechanisms responsible for salivary secretion are only partially understood, but it is generally accepted that stimulation with parasympatho-mimetic agents causes an influx of Na and an efflux of K from the salivary cells, resulting in the activation of ionic pumps and the generation of an osmotic gradient for the movement of water (Peterson, 1972; Young and
Van Lennep, 1979). In our experiment, the replacement of Na in the perfusion fluid resulted in a marked inhibition of the secretory response to acetylcholine, a finding which suggests that availability of external Na is indeed essential for secretion. However, the observation that the response to acetylcholine was also reduced during perfusion with K-free solutions suggests that an incrase in intracellular Na is not enough, by itself, to activate the secretory process. Substitution of most of the perfusate Na with choline only caused a partial inhibition of salivary flow; this effect can be best interpreted as due to a gradient limitation in Na secretion at the primary level. Studies on the isolated cat submandibular gland suggest that the transport mechanism responsible for the formation of saliva is different from the Na+, K+-ATPase system (Petersen, 1971; Laugesen, Dich Nielsen and Poulsen, 1976). Because ouabain in the perfusate also inhibited the secretory response, it seems likely that the maintenance of normal intracellular ion concentrations by the activity of a Na+, K+-ATPase, particularly after the stimulation-induced K efflux, is a requisite for sustained secretion to occur. Another factor which has been shown to be required for a sustained secretory response is extracellular Ca (Martinez and Petersen, 1971). Although the small size of the rat sublingual gland has limited investigation of its electrolyte excretion patterns, flow curves obtained in ciuo during stimulation with carbachol and acetylcholine are available (Martin and Young, 1971a; Coroneo et al., 1979). Our data, obtained during stimulation only with pilocarpine agree well with these earlier in-viva studies and do not suggest any major differences in the actions of the three drugs on sublingual ducts, at least under conditions of maximal stimulation.
Acknowledgements-We thank the Post Graduate Medical Foundation of the University of Sydney for the award of a travel grant to one of us (J.C.). The work was supported by a grant from the Public Health Service (NIH grant AM 18150).
REFERENCES
Case R. M., Conigrave A. D. and Young J. A. 1977. The isolated, saline-perfused rabbit mandibular gland. Proc. Amt. physiol. p
Douglas W. W. and Poisner A. M. 1963. The influence of calcium on the secretory response of the submaxillary gland to acetylcholine or to noradrenaline. J. PhysioL, Lond. 165, 528-541. Inada M., Mello A. and Lima-de-Castro A. 1976. Influencia dos ions calcio e potassio na responsta secretora da glandula submandibular isolada e perfundida. a estimulacao parassimpatica. Rhuta bras. Pesq. med. hiol. 9, 2633271. Laugesen L. P., Dich Nielsen 3. 0. and Poulsen J. H. 1976. Partial dissociation between salivary secretion and active
Secretion
by isolated,
perfused
potassium transport in the perfused cat submandibular gland. P&yers Arch. Eur. J. Physiol. 364, 167-173. Mangos J. A., Garrish M. T., Wells R., Farnham W. and Bouchlas G. 1978. A micropuncture study of the handling of calcrum by the rat parotid. J. dent. Res. 57, 818-825. Martin C. J. and Young J. A. 1971a. Electrolyte concenrations m primary and final saliva of the rat sublingual sland studied by micropuncture and catheterization techniques. t’jiiiyer Arch. Eur. J. Physiol. 324, 344360. Martin C. J. and Young J. A. 1971b. A microoerfusion Investigation of the effects of a sympathomime;ic and a parasympathomimetic drug on water and electrolyte fluxes in the main duct of the rat submaxillary gland. Pjliigrrs Arch. Eur. J. Phvsiol. 327, 303-323. Martinez J. R.. Holzgreve H. and Frick A. 1966. Micropuncture study of submaxillary glands of adult rats. Pfliigers Arch. yes. Physiol. 290, 124-133. Martinez J. R. and Petersen 0. H. 1971. The importance of extracellular calcium for acetylcholine-evoked salivary secretion. Experientiu 28, 167-168. Martinez J. R., Quisseli D. 0.. Wood D. L. and Giles M. 1975a. Abnormal secretory response to parasympathomimetic and sympathomimetic stimulation from the submaxillary gland of rats treated with reserpine. J. PharWK. exp. Ther. 194, 384-395. Martinez J. R., Adshead P. C., Quissell D. 0. and Barber0 G. J. 197% The chronically reserpinized rat as a poss-
rat submandibular
gland
S61
ible model for cystic fibrosis. II. Composition and cilioinhibitory effects of submaxillary saliva. Pediutric Res. 9, 47S-475. Petersen 0. H. 1971. Formation of saliva and potassium transport in the perfused cat submandibular gland. J. Physiol., Land. 216, 129-142. Petersen 0. H. 1972. Acetylcholine-induced ion transport involved in the formation of saliva. Am physiol. scund. Suppl. 381, l-58. Sprecher R. L., Yoshida Y. and Schneyer L. H. 1967. Partial isolation and perfusion of rat submaxillary gland. Proc. Sac. exp. Biol. Med. 125, 1227-1229. Young J. A. 1979. Salivary secretion of inorganic electrolytes. In: International Reciew of Phvsioloqv, Gastroinirstinal Physiology III (Edited by Crane R. l?.), Vol. 19, pp. l-58. University Park Press. Baltimore. Young J. A. and-Van Lennep E. W. 1979. Transport in salivary and salt glands: salivary glands. In: bfemhrune Trunsport in Biology (Edited by Giebisch G.). Vol. 4. pp. 5633674. Springer, Berlin, Young J. A. and Martin C. J. 1971. The effect of a sympatho- and a parasympathomimetic drug on the electrolyte concentrations of primary and final saliva of the rat submaxillary gland. Pfliigers Arch. Eur. J. Physiol. 327, 285-302. Young J. A. and Schogel E. 1966. Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pfriigers Arch. yes. Physioi. 291, 85-98.