Sympathetic submaxillary
control of Na, K transport main duct of rat
in perfused
L. H. SCHNEYER Department of Physiology and Biophysics, University of Alabama Medical Center, Birmingham, Alabama 35294
SCHNEYER, L. H. Sympathetic control of Na, K transport in perfused submaxillary main duct of rat. Am. J. Physiol. (230)2: 341-345. 1976. -The effect of stimulating the sympathetic innervation to rat submaxillary gland on ductal transport of Na, K, and water and on transepithelial PD was tested in the main excretory duct during perfusion through its lumen. Stimulation of the sympathetic nerve, supramaximally, caused a decrease of 3040% in net flux of Na from, and of K to, the lumen in ducts perfused with medium containing Na and K in isotonic concentrations. Net flux of water was unaffected. Transductal PD decreased by about 30% during supramaximal stimulation. Changes in PD and net cation fluxes were reversible. These effects of supramaximal stimulation of the sympathetics on ductal transport resemble those reported to occur after large doses of isoproterenol and suggest an adrenergic secretomotor innervation to the ducts. However, changes in PD evoked by supramaximal stimulation of the sympathetic nerve could not be suppressed with propranolol, but were with phenoxybenzamine, indicating that tu-adrenergic receptors are primarily involved in mediating at least the electrical responses of duct cells to sympathetic nerve stimulation.
salivary
secretion;
epithelial
transport;
autonomic
regulation
WELL ESTABLISHED that secretion of the fluid component of saliva is primarily a function of the acinar-intercalated duct complex of the salivary secretory unit and that it is at this level that fluid secretion is autonomically controlled (4, 20, 25). The broadest range of control is usually exerted through the parasympathetic innervation, but in many glands adrenergic sympathetic fibers also exert secretomotor control (1, 11, 15, 22). In either case, fluid secreted by the acinar-intercalated duct complex evidently is approximately isosmotic to plasma and contains sodium and potassium in essentially plasmalike concentrations (4, 7, 20, 24-26). In the ducts, generally, sodium is reabsorbed from the luminal fluid and potassium is secreted, so that a sodium-poor, potassium-rich saliva is ultimately elaborated (4, 7, 13, 20, 23,26). Autonomic control of reabsorption and secretion of ions by the duct cells, while not as firmly established as for secretion by acini, now also seems likely. Thus, it has recently been found that administration of cholinergic or P-adrenergic agonists can modify net electrolyte fluxes, and transmural electrical potential difference, in the terminal portion of the duct system of rat submaxillary gland (5, 6, 19). Only the terminal segment of the salivary duct system, i.e., the main excreIT IS NOW
tory duct, has been examined in this way because this is the only salivary ductal segment which is readily accessible for perfusion (23) and, hence, for measurement of transmural fluxes and potential differences. However, even with this duct segment, effects of direct stimulation of the innervation on transport parameters have not heretofore been investigated. It was the purpose of this investigation to examine effects on salivary transductal transport and PD of directly stimulating a branch of the autonomic innervation to the gland and to compare these effects with changes reported from the analogous use of autonomic drugs. For this, net fluxes of Na and K, and PD were measured across the luminally perfused main excretory duct of rat submaxillary gland while the cervical sympathetic nerve trunk was intermittently stimulated. METHODS
Long-Evans male rats, weighing 240-350 g, were anesthetized with Inactin (150 mg/kg, ip) and tracheotomized. One main submaxillary duct was cannulated at its oral opening, using Clay-Adams PE-10 tubing beveled at its tip. The ipsilateral submaxillary-sublingual gland complex was isolated from surrounding tissues, and the sublingual gland was deflected to uncover the submaxillary duct at its emergence from the hilus of the gland. The duct was cleared and cannulated at its hilar end, using polyethylene tubing (PE-10) drawn to a tip diameter of about 100 pm. The carotid sheath on the same side was isolated, and the vagus nerve and common carotid artery were freed for a length of at least 1 cm. The separated portion of vagus nerve was excised, and a narrow strip of Parafilm was placed under the carotid artery. Bipolar platinum electrodes were placed around the artery, so that the cervical sympathetic trunk which remained adherent to the dorsal arterial surface could be stimulated, using square-wave pulses (5 ms duration) at 10 or 20 Hz and 1.2-4.0 V (Grass Instrument Co. SD 5 stimulator), as previously described (14). Appearance of saliva from the intact ipsilatera1 parotid gland was used as a criterion of successful stimulation of the sympathetic trunk. In some experiments, atropine sulfate (1 mg/kg, ip) was administered before stimulating the sympathetic trunk to eliminate possible parasympathetic involvement from current spread (14). However, results did not differ whether pretreatment with atropine was used or omitted. Body
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342 temperature was maintained by keeping rats on a heated board during the experiments. Perfusion medium containing Na (140 mM), K (3.8 mM), Cl (124 mM), HCO:, (20 mM), and [13H]methoxyinulin (trace ccncentrations) with a total osmolarity of 270 mosM, as determined by freezing-point osmometer (Advanced Instruments), was delivered to the duct lumen at a rate of 940 nl/min by syringe pump (Harvard Apparatus, model 940). Details of the perfusion procedure, as well as the methods of recording transductal net fluxes of Na and K, and PD, have been previously described (12, 13, 23). In brief, net Na, K fluxes were determined from the change in concentration of the ions in the medium aftir perfusion, and the inulin ratio ([inulin] in perfused fluid/[inulin] in unperfused medium). Analyses were performed on samples of 3 ~1 volume, collected from the tip of the oral cannula in by disposashort lengths of PE-50 tubing and transferred ble micropipette. [Na] and [K] were determined by flame photometry (Instrumentation Laboratories, model 143), while the concentration of inulin was determined by radioactive counting, in a toluene-based “cocktail” (191, and expressed in terms of the counts per minute per microliter of medium. Inulin ratios were a.s well as unstimulated ducts. close to 1.0 in stimulated Therefore, net ion fluxes could be calculated directly i n unperfused and from the differences in concentration perfused medium and were expressed in units of nanoequivalents transferred per minute by the entire perfused segment of duct (neq/min x duct). Transductal PD was measured at the tip of the hilar cannula by attaching an agar bridge (polyethylene tubing containing 2% Difco agar in saturated KC1 solution) to the pump end of the hilar cannula by means of a multiport manifold (12). This agar bridge and another which contacted the serosal surface of the duct at the hilar end were led to vessels containing saturated KC1 solution, into each of which was placed one of two matched calomel electrodes (Markson). Voltage difference was recorded by using an electrometer volt meter with an input impedance of 1014 fl (Keithley Instruments, Inc., model 602). In some experiments, dibenzyline (phenoxybenzamine) and Inderal (propranolol) were used. These were injected intraperitoneally in amounts calculated to give approximately 3-4 mg/kg body wt. RESULTS
Changes in Na, K net fluxes and in&in ratio during sympathetic stimulation. Supramaximal stimulation of the cervical sympathetic nerve trunk at 10 or 20 Hz (3-4 V) during perfusion of the main duct with isotonic medium (containing Na, K, Cl, and HCO, in concentrations similar to those of plasma) resulted in appreciable decrease in transductal net flux of Na and K, but not in inulin ratio. Detailed data from two experiments are shown in Fig. 1, while values from nine rats are summarized in Table 1. As shown in Fig. 1, inhibition of Na, K fluxes usually beta .me apparent in the first sample of perfusate collected after the start of stimul .ation (3.5-
L. H. SCHNEYER
20Hz,3V
20Hz,3.4V 8 1
20Hz,3.2 t
t-N %T-10
V 1
,
20Hr.3.4
V*
c
-
-20
0
20
40
MINUTES
60
80
100
120
140
OF PERFUSION
FIG. 1. Changes in net fluxes of potassium and sodium, and inulin ratio, in luminally perfused main excretory duct, during periods of electrical stimulation of cervical sympathetic nerve (2 representative experiments). Negative values for the net flux indicate absorption from lumen; positive values indicate secretion into lumen.
min collection period) and reached a peak by 6-20 min. Since at a perfusion rate of 940 nl/min dead time for the duct segment may be between 2 and 3 min (16), flux changes undoubtedly occurred more rapidly after the start of stimulation than suggested by the data in Fig. 1. Inhibition of Na, K fluxes was generally sustained during the period of stimulation, even when this exceeded 20 min in duration, and was rapidly reversible upon cessation of stimulation (Fig. 1). The magnitude of the & inhi bition of net flux when it reached a steady value, was generally betwee n 30 and 4 0% for Na or K (mean 2 SE of % inhibition for Na flux was 31.6 2 2.67 and for K flux was 39.7 t 5.62). The actual net sodium flux was 30.2 ~fr 3.24 neq/min x duct before stimulation and decreased to 20.6 ~fr2.06 neq/min x duct during stimulation (9 rats). The difference, 9.7 t 1.40 neq/min x duct, was statistically significant (P < .Ol; Table 1). K flux in the same nine rats was 24.3 * 3.39 neq/min x duct before stimulation and decreased to 13.9 t 2.04 neq/min X duct during stimulation. The difference in net flux of K, of 10.4 t 2.34 neq/min x duct, was also significant (P < .Ol; Table 1). However, inulin ratio was unchanged during stimulation (. 967 t .0115 and .966 t .0157 during control and stimulated periods, respectively; P > .l> (Table 1). Evidently, transductal net m.ovement of water which was small in control conditions was not affected by stimulation of the sympathetic nerve. Changes in transductal PD during sympathetic stimuZation. Stimulation of the cervical sympathetic trunk, supramaximally, rapidly led to a decrease in transductal PD. As shown in Fig. 2, the decrease in PD during
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SYMPATHETIC
CONTROL
OF
SALIVARY
DUCTAL
343
TRANSPORT
stimulation not only appeared rapidly but usually approached its maximum rapidly, i.e., within 24 min, and remained near this maximum for as long as stimulation continued. The magnitude of the maximal decrease in PD during supramaximal stimulation was 17.6 t 1.88 mV (from 57.0 t 3.84 mV before to 39.4 t 3.99 mV during stimulation), which represented a 30.9 2 3.2% change from the unstimulated, control level (5 rats). This decrease was statistically significant (Table 2). Upon cessation of stimulation, the PD rose, usually to a level approaching that observed before stimulation started, as shown in Fig. 2. The individual records in Fig. 2 also show some less constant effects. Occasionally, the PD rose above the control value briefly at the start of the period of supramaximal stimulation (Fig. 24, last record; Fig. 2L3, last two records) and then declined as stimulation continued. Even less frequently, the early course or decline in PD was interrupted by a brief rise (Fig. 2B, fourth and fifth records). In addition, in some ducts, when stimulation was stopped, afterpotentials were sometimes seen. These usually took the
form of further decrease in the PD, which appeared early in the poststimulation period (Fig. 2A, second and third records; Fig. 2B, first two records), or an overshoot of the PD, above control which appeared later (Fig. 2B, second and fourth records; Fig. 2C, second and sixth records). In occasional cases during these experiments, lower stimulus voltages (1.6-2.6 V) were used. Such stimuli would be submaximal for secretion by the whole gland. In these cases, the most frequent finding was not a decrease, but an enhancement of transductal PD. Examples of this are seen in Fig. 2, B (third record) and C (third, fifth and seventh records). The effects of adrenergic antagonists on the decrease in PD evoked during supramaximal stimulation were examined only in a preliminary way. However, administration of phenoxybenzamine, but not propranolol, did give consistent effects. Thus, in three rats given phenoxybenzamine, the mean transductal PD, which was 54 t 4.06 mV before stimulation, decreased by only 5.7 t 2.08 mV during stimulation to reach a minimum value of 48.3 t 4.37 mV (Table 2). The magnitude of this de-
1. Effect of supramaximal stimulation of cervical sympathetic trunk Na and K, and in&in ratio, in perfused salivary main excretory duct
TABLE
Sodium
Mean+ SE
Flux*
Unstimulated
Stimulated
-30.2
-20.6
k3.24
Potassium Unstimulated
Difference
k2.06
P Negative $ Values
Inulin
Stimulated
L
STIMULUS VOLTS
-87--.;.o
i6
Difference
Stimulated
Difference
.966
.OOl
24.3
13.9
10.4
.967
+ 1.40
k3.39
22.04
k2.34
2.0115
M
-60
* .0157
c.01 net movement rats.
from
Sfimuhs
Frequency
= 2OLsec
Stlinulus
Frequency
= ZO/.sec
, 2.6
Ratio+
Unstimulated
9.7
sign indicates are from nine
STIMULUS VOLTS
-80r
of
Flux*
c.01
* Units are neq/min x duct. perfused/unperfused medium.
on net fluxes
i-”
,
3.8
0B
, 3.0
Xl
t Counts/min
lumen.
I 3.0
2.0089
x
~1 (from
]3H]methoxyinulin)
of
J 1
FIG. 2. Changes in transductal PD of perfused main excretory duct, in response to stimulation of cervical sympathetic nerve truck (3 experiments). Start of stimulation is denoted by head of each arrow, while duration of stimulation is given by length of its horizontal limb. Sign of potential is with regard to interstitial side.
4
V
-4o-
. 1 0
I IO
1
1 20
I
1 30
I
1 40
I
1 50
I
I 60
MINUTES
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344
L. H. SCHNEYER
2. Effect of supramaximal stimulation of cervical sympathetic trunk on transmural PD
of the low dose (19). K flux is decreased by isoproterenol at low, as at high, dose levels , when Na is present in the perfusion medium (19). Net movement of water across the perfused duct, as indicated by the inulin ratio, is unchanged by administering isoproterenol in high or low dose (6, 19). When Na is omitted from the perfusion medium, administration of large doses of isoproterenol Without adrenergic antagonist leads to enhancement of secretion of potassium and, Meant 57.0 39.4 17.6 30.9 SE k3.84 k3.99 21.88 23.27 again, of bicarbonate (6). The present data show that P co1 CO1 actual stimulation of the sympathetic innervation to the With phenoxybenzamine submaxillary gland leads to alterations in Na, K transMean* 54.0 48.3 5.7 10.6 port and transepithelial PD in the main duct, and that SE k4.06 k4.37 22.08 23.81 the effects of supramaximal sympathetic stimulation on P9 CO5 CO5 transpo rt and PD resemble those observ redafter admi nis* Lumen is negative with respect to serosal side. t $ Values tration of large doses of isoproterenol. In j addition, the are from five and three rats, respectively. 9 While a decrease in transductal hyperpolarization, rather than depolarizaPD may still occur with phenoxybenzamine, this decrease is signifition, which appeared in preliminary experiments with cantly reduced (p < .Ol). submaximal levels of sympathetic stimulation, is at least consistent with previous observations (19) of an crease was significantly less (P < .Ol; Table 2) than that enhancement of Na flux by threshold dosesof isoprotereobtained during stimulation in the absence of phenoxynol. However , further examination of these threshold benzamine. effects is needed before a general similarity between them can be established. DISCUSSION The mechanisms which underlie the effects on ductal While the influence of the sympathetic secretomotor transport of maximally stimulating the sympathetic ininnervation to salivary gland has been extensively stud- nervation, or administering large dosesof isoproterenol, ied in relation to functions of acinar cells, relatively are so far not delineated. These effects are probably not little attention has been given to sympathetic secretomo- primarily related to accompanying vascular changes for tor effects on the activity of ductal cells. In early work, at least two reasons. First, while vasoconstriction does in which reabsorption of sodium was proposed as a result from stimulation of the glandular sympathetic prominent feature of the salivary duct system, Thaysen innervation, its effect on secretory activity seems to (21) avoided assumptions about a possible role of secreto- require appreciable delay (2), whereas changes i n transmotor nerves on ductal transport. However, Lundberg ductal PD, at least, occur very rapidly after the start of (3) noted soon after that transmembrane potentials in sympathetic stimulation. Second, the decrease in transsome salivary cells are distinctively higher than in aci- ductal PD which characteristically accompanies sympanar cells and that these cells, which were presumed to thetic, or sympathomimetic, stimulation has also been be ductal, do respond to sympathetic or parasympaobserved after addition of isoproterenol to ducts perthetic stimulation and become depolarized. In that fused in vitro (5), and in these conditions vascular work, submaxillary gland of cat was used, but it was changes could not greatly affect the cellular responses. later shown that in submaxillary gland of rat also highThe effects observed in this study, where the sympapotential cells are present and that these are quite thetic innervation to th .e gland was directly stimulated, definitely ductal (17, 18). and in earlier studies, where the P-adrenergic agent, Further clarification of the effects of adrenergic or isoproterenol, was administered, together strongly sugcholinergic stimulation on ductal secretory function has gest that the ducts in rat submaxillary do receive a come mainly from work on perfused main excretory secretomotor sympathetic innervation. This is indicated duct. These investigations have dealt only with the also by another recent study, in which a different segactions of autonomic drugs, with isoproterenol as the ment of the rat submaxillary duct system, the granular main adrenergic drug used (5, 6, 19); however, they duct, showed histological alterations indicative of secrehave provided important information concerning tion in response to stimulation of the sympathetic innerchanges in electrolyte transport which accompany stim- vation (8). Since Norberg and Olson (9) had earlier ulation of ducta 1 autonomic receptors. First, it has be- reported observations from fluorescent microscopic excome clear that cholinergic and p-adrenergic agents do amination of rat submaxillary which made it doubtful affect transport of electrolytes by duct cells, at least in that there is an adrenergic innervation to duct cells, all the main duct segment of rat submaxillary (5, 6, 19, 20, of these newer findings are of importance in resolving 25). The ,&adrenergic agent, isoproterenol, for example, this doubt. when administered to the rat in large doses, was found The histochemical changes that have been reported to to depress Na, K transport and transepithelial PD, but occur in granular ducts after stimulation of the sympaenhance HCO,, secretion, in the main duct when the thetic innervation appear to be mediated bY cw-adrenerperfusion medium contained 140 meq/liter Na (6). Un- gic receptors (8). In the main duct, also, the effects of der similar conditions of perfusion, threshold doses of supramaximal stimulation of the sympathetic nerve, on isoproterenol (13 pg/kg, ip) produce somewhat different the transductal PD at least, seem principally to involve effects on net Na flux, which is enhanced after injection alpha receptors. Thus, in several preliminary experiTABLE
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SYMPATHETIC
CONTROL
OF
SALIVARY
DUCTAL
345
TRANSPORT
ments, administration of phenoxybenzamine significantly reduced the degree of depolarization during sympathetic stimulation, while with propranolol no clear effects on PD were observed. It must be tentatively concluded that while the effects of isoproterenol indicate that P-receptor activity can modify transport in the main duct, it is a-receptors that are of importance in mediating the changes, at least in transepithelial PD,
when thetic
the ducts a re stimulated inn .ervation
through
their
sympa-
Assistance by Mr. H. Forrest and Mrs. F. M. Thomas during the experiments is gratefully acknowledged. The work was supported by National Institutes of Health Grant DE 02110 and Career Development Award 5-K6-DE 3341. Received
for publication
30 July
1975.
REFERENCES 1. EMMELIN, N. Control of salivary glands. In: Oral PhysioZogy, edited by N. Emmelin and Y. Zotterman. Oxford: Pergamon, 1972, p. 1-16. 2. EMMELIN, N., AND J. ENGSTROM. On the existence of specific secretory sympathetic fibers for the cat’s submaxillary gland. J. Physiol., London 153: l-8, 1960. 3. LUNDBERG, A. The electrophysiology of the submaxillary gland of the cat. Acta Physiol. Stand. 35: l-25, 1955. 4. MANGOS, J. A., N. R. MCSHERRY, AND S. N. ARVANITAKIS. Autonomic regulation of secretion and transductal fluxes of ions in the rat parotid. Am. J. Physiol. 225: 683-688, 1973. 5. MARTIN, C. J., A. R. DENNISS, 2. H. ENDRE, AND J. A. YOUNG. Mechanism of action of neurotransmitters and their analogues on salivary duct electrolyte transport. In: Secretory Mechanism ofExocrine GZands, edited by N. A. Thorn and 0. H. Petersen. Copenhagen: Munksgaard, 1974, p. 549-569. 6. MARTIN, C. J., AND J. A. YOUNG. A microperfusion investigation of the effects of a sympathomimetic and a parasympathomimetic drug on water and electrolyte fluxes in the main duct of the rat submaxillary gland. Pfluegers Arch. 327: 303-323, 1971. 7. MARTINEZ, J. R., H. HOLZGREVE, AND A. FRICK. Micropuncture study of submaxillary glands of adult rats. Pfluegers Arch. 290: 124-133, 1966. 8. MATTHEWS, R. W. Studies of the granular convoluted tubule in the rat submandibular gland (special issue). J. Dental Res. 53: 143, 1974. 9. NORBERG, K.-A., AND L. OLSON. Adrenergic innervation of the salivary glands in the rat. 2. ZeZlforsch. 68: 183-189, 1965. 10. SCHNEYER, C. A. Salivary gland changes after isoproterenolinduced enlargement. Am. J. Physiol. 203: 232-236, 1962. 11. SCHNEYER, C. A., AND H. D. HALL. Autonomic pathways involved in a sympathetic-like action of pilocarpine on salivary composition. Proc. Sot. Exptl. Biol. Med. 121: 96-100, 1966. 12. SCHNEYER, L. H. Secretory processes in perfused excretory duct of rat submaxillary gland. Am. J. Physiol. 215: 664-670, 1968. 13. SCHNEYER, L. H. Secretion of potassium by perfused excretory duct of rat submaxillary gland. Am. J. Physiol. 217: 1324-1329, 1969. 14. SCHNEYER, L. H. Secretion of K and fluid by rat submaxillary during sympathetic nerve stimulation. Am. J. Physiol. 229: 1056-1061, 1975.
15. SCHNEYER, L. H., AND N. EMMELIN. Salivary secretion. In: MTP International Review of Science. Gastrointestinal Physiology. London: Butterworths, 1974, PhysiologySer. 1, vol. 4, chapt. 6, p. 183-226. 16. SCHNEYER, L. H., AND R. F. FLATLAND. Evaluation of a reservoir in the main excretory duct of rat submaxillary gland. J. Appl. Physiol. 39: 519-522, 1975. 17. SCHNEYER, L. H., AND C. A. SCHNEYER. Membrane potentials of salivary gland cells of rat. Am. J. Physiol. 209: 1304-1310, 1965. 18. SCHNEYER, L. H., C. A. SCHNEYER, AND Y. YOSHIDA. Membrane . potentials of developing cells in immature rat submaxillary gland. Am. J. Physiol. 215: 1146-1150, 1968. 19. SCHNEYER, L. H., AND T. THAVORNTHON. Isoproterenol-induced stimulation of sodium absorption in perfused salivary duct. Am. J. PhysioZ. 224: 136-139, 1973. 20. SCHNEYER, L. H., J. A. YOUNG, AND C. A. SCHNEYER. Salivary secretion of electrolytes. PhysioZ. Rev. 52: 720-777, 1972. 21. THAYSEN, J. H. Handling of alkali metals by exocrine glands other than kidney. In: Handbuch der Experimentellen Pharmakologie. Berlin: Springer, 1960, part II, vol. 13, chapt. 5, p. 424-507. 22. YOSHIDA, Y., R. L. SPRECHER, C. A. SCHNEYER, AND L. H. SCHNEYER. Role of P-receptors in sympathetic regulation of electrolytes in rat submaxillary saliva. Proc. Sot. Exptl. BioL. Med. 126: 912-916, 1967. 23. YOUNG, J. A., E. FR~MTER, E. S~H~GEL, AND K. F. HAMANN. A microperfusion investigation of sodium resorption and potassium secretion by the main excretory duct of the rat submaxillary gland. Pfluegers Arch. 295: 157-172, 1967. 24 YOUNG, J. A., AND C. J. MARTIN. The effect of a sympathoand a parasympathomimetic drug on the electrolyte concentrations of primary and final saliva of the rat submaxillary gland. PfZuegers Arch. 327: 285-302, 1971. 25 YOUNG, J. A., AND C. J. MARTIN. Electrolyte transport in the excurrent duct system of the submaxillary gland. I. Studies on the intact gland. In: Oral Physiology, edited by N. Emmelin and Y. Zotterman. Oxford: Pergamon, 1972, p. 99-113. 26 YOUNG, J. A., AND E. SCH~GEL. Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pfluegers Arch. 291: 85-98, 1966.
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control of Na, K transport main duct of rat
in perfused
L. H. SCHNEYER Department of Physiology and Biophysics, University of Alabama Medical Center, Birmingham, Alabama 35294
SCHNEYER, L. H. Sympathetic control of Na, K transport in perfused submaxillary main duct of rat. Am. J. Physiol. (230)2: 341-345. 1976. -The effect of stimulating the sympathetic innervation to rat submaxillary gland on ductal transport of Na, K, and water and on transepithelial PD was tested in the main excretory duct during perfusion through its lumen. Stimulation of the sympathetic nerve, supramaximally, caused a decrease of 3040% in net flux of Na from, and of K to, the lumen in ducts perfused with medium containing Na and K in isotonic concentrations. Net flux of water was unaffected. Transductal PD decreased by about 30% during supramaximal stimulation. Changes in PD and net cation fluxes were reversible. These effects of supramaximal stimulation of the sympathetics on ductal transport resemble those reported to occur after large doses of isoproterenol and suggest an adrenergic secretomotor innervation to the ducts. However, changes in PD evoked by supramaximal stimulation of the sympathetic nerve could not be suppressed with propranolol, but were with phenoxybenzamine, indicating that tu-adrenergic receptors are primarily involved in mediating at least the electrical responses of duct cells to sympathetic nerve stimulation.
salivary
secretion;
epithelial
transport;
autonomic
regulation
WELL ESTABLISHED that secretion of the fluid component of saliva is primarily a function of the acinar-intercalated duct complex of the salivary secretory unit and that it is at this level that fluid secretion is autonomically controlled (4, 20, 25). The broadest range of control is usually exerted through the parasympathetic innervation, but in many glands adrenergic sympathetic fibers also exert secretomotor control (1, 11, 15, 22). In either case, fluid secreted by the acinar-intercalated duct complex evidently is approximately isosmotic to plasma and contains sodium and potassium in essentially plasmalike concentrations (4, 7, 20, 24-26). In the ducts, generally, sodium is reabsorbed from the luminal fluid and potassium is secreted, so that a sodium-poor, potassium-rich saliva is ultimately elaborated (4, 7, 13, 20, 23,26). Autonomic control of reabsorption and secretion of ions by the duct cells, while not as firmly established as for secretion by acini, now also seems likely. Thus, it has recently been found that administration of cholinergic or P-adrenergic agonists can modify net electrolyte fluxes, and transmural electrical potential difference, in the terminal portion of the duct system of rat submaxillary gland (5, 6, 19). Only the terminal segment of the salivary duct system, i.e., the main excreIT IS NOW
tory duct, has been examined in this way because this is the only salivary ductal segment which is readily accessible for perfusion (23) and, hence, for measurement of transmural fluxes and potential differences. However, even with this duct segment, effects of direct stimulation of the innervation on transport parameters have not heretofore been investigated. It was the purpose of this investigation to examine effects on salivary transductal transport and PD of directly stimulating a branch of the autonomic innervation to the gland and to compare these effects with changes reported from the analogous use of autonomic drugs. For this, net fluxes of Na and K, and PD were measured across the luminally perfused main excretory duct of rat submaxillary gland while the cervical sympathetic nerve trunk was intermittently stimulated. METHODS
Long-Evans male rats, weighing 240-350 g, were anesthetized with Inactin (150 mg/kg, ip) and tracheotomized. One main submaxillary duct was cannulated at its oral opening, using Clay-Adams PE-10 tubing beveled at its tip. The ipsilateral submaxillary-sublingual gland complex was isolated from surrounding tissues, and the sublingual gland was deflected to uncover the submaxillary duct at its emergence from the hilus of the gland. The duct was cleared and cannulated at its hilar end, using polyethylene tubing (PE-10) drawn to a tip diameter of about 100 pm. The carotid sheath on the same side was isolated, and the vagus nerve and common carotid artery were freed for a length of at least 1 cm. The separated portion of vagus nerve was excised, and a narrow strip of Parafilm was placed under the carotid artery. Bipolar platinum electrodes were placed around the artery, so that the cervical sympathetic trunk which remained adherent to the dorsal arterial surface could be stimulated, using square-wave pulses (5 ms duration) at 10 or 20 Hz and 1.2-4.0 V (Grass Instrument Co. SD 5 stimulator), as previously described (14). Appearance of saliva from the intact ipsilatera1 parotid gland was used as a criterion of successful stimulation of the sympathetic trunk. In some experiments, atropine sulfate (1 mg/kg, ip) was administered before stimulating the sympathetic trunk to eliminate possible parasympathetic involvement from current spread (14). However, results did not differ whether pretreatment with atropine was used or omitted. Body
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342 temperature was maintained by keeping rats on a heated board during the experiments. Perfusion medium containing Na (140 mM), K (3.8 mM), Cl (124 mM), HCO:, (20 mM), and [13H]methoxyinulin (trace ccncentrations) with a total osmolarity of 270 mosM, as determined by freezing-point osmometer (Advanced Instruments), was delivered to the duct lumen at a rate of 940 nl/min by syringe pump (Harvard Apparatus, model 940). Details of the perfusion procedure, as well as the methods of recording transductal net fluxes of Na and K, and PD, have been previously described (12, 13, 23). In brief, net Na, K fluxes were determined from the change in concentration of the ions in the medium aftir perfusion, and the inulin ratio ([inulin] in perfused fluid/[inulin] in unperfused medium). Analyses were performed on samples of 3 ~1 volume, collected from the tip of the oral cannula in by disposashort lengths of PE-50 tubing and transferred ble micropipette. [Na] and [K] were determined by flame photometry (Instrumentation Laboratories, model 143), while the concentration of inulin was determined by radioactive counting, in a toluene-based “cocktail” (191, and expressed in terms of the counts per minute per microliter of medium. Inulin ratios were a.s well as unstimulated ducts. close to 1.0 in stimulated Therefore, net ion fluxes could be calculated directly i n unperfused and from the differences in concentration perfused medium and were expressed in units of nanoequivalents transferred per minute by the entire perfused segment of duct (neq/min x duct). Transductal PD was measured at the tip of the hilar cannula by attaching an agar bridge (polyethylene tubing containing 2% Difco agar in saturated KC1 solution) to the pump end of the hilar cannula by means of a multiport manifold (12). This agar bridge and another which contacted the serosal surface of the duct at the hilar end were led to vessels containing saturated KC1 solution, into each of which was placed one of two matched calomel electrodes (Markson). Voltage difference was recorded by using an electrometer volt meter with an input impedance of 1014 fl (Keithley Instruments, Inc., model 602). In some experiments, dibenzyline (phenoxybenzamine) and Inderal (propranolol) were used. These were injected intraperitoneally in amounts calculated to give approximately 3-4 mg/kg body wt. RESULTS
Changes in Na, K net fluxes and in&in ratio during sympathetic stimulation. Supramaximal stimulation of the cervical sympathetic nerve trunk at 10 or 20 Hz (3-4 V) during perfusion of the main duct with isotonic medium (containing Na, K, Cl, and HCO, in concentrations similar to those of plasma) resulted in appreciable decrease in transductal net flux of Na and K, but not in inulin ratio. Detailed data from two experiments are shown in Fig. 1, while values from nine rats are summarized in Table 1. As shown in Fig. 1, inhibition of Na, K fluxes usually beta .me apparent in the first sample of perfusate collected after the start of stimul .ation (3.5-
L. H. SCHNEYER
20Hz,3V
20Hz,3.4V 8 1
20Hz,3.2 t
t-N %T-10
V 1
,
20Hr.3.4
V*
c
-
-20
0
20
40
MINUTES
60
80
100
120
140
OF PERFUSION
FIG. 1. Changes in net fluxes of potassium and sodium, and inulin ratio, in luminally perfused main excretory duct, during periods of electrical stimulation of cervical sympathetic nerve (2 representative experiments). Negative values for the net flux indicate absorption from lumen; positive values indicate secretion into lumen.
min collection period) and reached a peak by 6-20 min. Since at a perfusion rate of 940 nl/min dead time for the duct segment may be between 2 and 3 min (16), flux changes undoubtedly occurred more rapidly after the start of stimulation than suggested by the data in Fig. 1. Inhibition of Na, K fluxes was generally sustained during the period of stimulation, even when this exceeded 20 min in duration, and was rapidly reversible upon cessation of stimulation (Fig. 1). The magnitude of the & inhi bition of net flux when it reached a steady value, was generally betwee n 30 and 4 0% for Na or K (mean 2 SE of % inhibition for Na flux was 31.6 2 2.67 and for K flux was 39.7 t 5.62). The actual net sodium flux was 30.2 ~fr 3.24 neq/min x duct before stimulation and decreased to 20.6 ~fr2.06 neq/min x duct during stimulation (9 rats). The difference, 9.7 t 1.40 neq/min x duct, was statistically significant (P < .Ol; Table 1). K flux in the same nine rats was 24.3 * 3.39 neq/min x duct before stimulation and decreased to 13.9 t 2.04 neq/min X duct during stimulation. The difference in net flux of K, of 10.4 t 2.34 neq/min x duct, was also significant (P < .Ol; Table 1). However, inulin ratio was unchanged during stimulation (. 967 t .0115 and .966 t .0157 during control and stimulated periods, respectively; P > .l> (Table 1). Evidently, transductal net m.ovement of water which was small in control conditions was not affected by stimulation of the sympathetic nerve. Changes in transductal PD during sympathetic stimuZation. Stimulation of the cervical sympathetic trunk, supramaximally, rapidly led to a decrease in transductal PD. As shown in Fig. 2, the decrease in PD during
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CONTROL
OF
SALIVARY
DUCTAL
343
TRANSPORT
stimulation not only appeared rapidly but usually approached its maximum rapidly, i.e., within 24 min, and remained near this maximum for as long as stimulation continued. The magnitude of the maximal decrease in PD during supramaximal stimulation was 17.6 t 1.88 mV (from 57.0 t 3.84 mV before to 39.4 t 3.99 mV during stimulation), which represented a 30.9 2 3.2% change from the unstimulated, control level (5 rats). This decrease was statistically significant (Table 2). Upon cessation of stimulation, the PD rose, usually to a level approaching that observed before stimulation started, as shown in Fig. 2. The individual records in Fig. 2 also show some less constant effects. Occasionally, the PD rose above the control value briefly at the start of the period of supramaximal stimulation (Fig. 24, last record; Fig. 2L3, last two records) and then declined as stimulation continued. Even less frequently, the early course or decline in PD was interrupted by a brief rise (Fig. 2B, fourth and fifth records). In addition, in some ducts, when stimulation was stopped, afterpotentials were sometimes seen. These usually took the
form of further decrease in the PD, which appeared early in the poststimulation period (Fig. 2A, second and third records; Fig. 2B, first two records), or an overshoot of the PD, above control which appeared later (Fig. 2B, second and fourth records; Fig. 2C, second and sixth records). In occasional cases during these experiments, lower stimulus voltages (1.6-2.6 V) were used. Such stimuli would be submaximal for secretion by the whole gland. In these cases, the most frequent finding was not a decrease, but an enhancement of transductal PD. Examples of this are seen in Fig. 2, B (third record) and C (third, fifth and seventh records). The effects of adrenergic antagonists on the decrease in PD evoked during supramaximal stimulation were examined only in a preliminary way. However, administration of phenoxybenzamine, but not propranolol, did give consistent effects. Thus, in three rats given phenoxybenzamine, the mean transductal PD, which was 54 t 4.06 mV before stimulation, decreased by only 5.7 t 2.08 mV during stimulation to reach a minimum value of 48.3 t 4.37 mV (Table 2). The magnitude of this de-
1. Effect of supramaximal stimulation of cervical sympathetic trunk Na and K, and in&in ratio, in perfused salivary main excretory duct
TABLE
Sodium
Mean+ SE
Flux*
Unstimulated
Stimulated
-30.2
-20.6
k3.24
Potassium Unstimulated
Difference
k2.06
P Negative $ Values
Inulin
Stimulated
L
STIMULUS VOLTS
-87--.;.o
i6
Difference
Stimulated
Difference
.966
.OOl
24.3
13.9
10.4
.967
+ 1.40
k3.39
22.04
k2.34
2.0115
M
-60
* .0157
c.01 net movement rats.
from
Sfimuhs
Frequency
= 2OLsec
Stlinulus
Frequency
= ZO/.sec
, 2.6
Ratio+
Unstimulated
9.7
sign indicates are from nine
STIMULUS VOLTS
-80r
of
Flux*
c.01
* Units are neq/min x duct. perfused/unperfused medium.
on net fluxes
i-”
,
3.8
0B
, 3.0
Xl
t Counts/min
lumen.
I 3.0
2.0089
x
~1 (from
]3H]methoxyinulin)
of
J 1
FIG. 2. Changes in transductal PD of perfused main excretory duct, in response to stimulation of cervical sympathetic nerve truck (3 experiments). Start of stimulation is denoted by head of each arrow, while duration of stimulation is given by length of its horizontal limb. Sign of potential is with regard to interstitial side.
4
V
-4o-
. 1 0
I IO
1
1 20
I
1 30
I
1 40
I
1 50
I
I 60
MINUTES
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344
L. H. SCHNEYER
2. Effect of supramaximal stimulation of cervical sympathetic trunk on transmural PD
of the low dose (19). K flux is decreased by isoproterenol at low, as at high, dose levels , when Na is present in the perfusion medium (19). Net movement of water across the perfused duct, as indicated by the inulin ratio, is unchanged by administering isoproterenol in high or low dose (6, 19). When Na is omitted from the perfusion medium, administration of large doses of isoproterenol Without adrenergic antagonist leads to enhancement of secretion of potassium and, Meant 57.0 39.4 17.6 30.9 SE k3.84 k3.99 21.88 23.27 again, of bicarbonate (6). The present data show that P co1 CO1 actual stimulation of the sympathetic innervation to the With phenoxybenzamine submaxillary gland leads to alterations in Na, K transMean* 54.0 48.3 5.7 10.6 port and transepithelial PD in the main duct, and that SE k4.06 k4.37 22.08 23.81 the effects of supramaximal sympathetic stimulation on P9 CO5 CO5 transpo rt and PD resemble those observ redafter admi nis* Lumen is negative with respect to serosal side. t $ Values tration of large doses of isoproterenol. In j addition, the are from five and three rats, respectively. 9 While a decrease in transductal hyperpolarization, rather than depolarizaPD may still occur with phenoxybenzamine, this decrease is signifition, which appeared in preliminary experiments with cantly reduced (p < .Ol). submaximal levels of sympathetic stimulation, is at least consistent with previous observations (19) of an crease was significantly less (P < .Ol; Table 2) than that enhancement of Na flux by threshold dosesof isoprotereobtained during stimulation in the absence of phenoxynol. However , further examination of these threshold benzamine. effects is needed before a general similarity between them can be established. DISCUSSION The mechanisms which underlie the effects on ductal While the influence of the sympathetic secretomotor transport of maximally stimulating the sympathetic ininnervation to salivary gland has been extensively stud- nervation, or administering large dosesof isoproterenol, ied in relation to functions of acinar cells, relatively are so far not delineated. These effects are probably not little attention has been given to sympathetic secretomo- primarily related to accompanying vascular changes for tor effects on the activity of ductal cells. In early work, at least two reasons. First, while vasoconstriction does in which reabsorption of sodium was proposed as a result from stimulation of the glandular sympathetic prominent feature of the salivary duct system, Thaysen innervation, its effect on secretory activity seems to (21) avoided assumptions about a possible role of secreto- require appreciable delay (2), whereas changes i n transmotor nerves on ductal transport. However, Lundberg ductal PD, at least, occur very rapidly after the start of (3) noted soon after that transmembrane potentials in sympathetic stimulation. Second, the decrease in transsome salivary cells are distinctively higher than in aci- ductal PD which characteristically accompanies sympanar cells and that these cells, which were presumed to thetic, or sympathomimetic, stimulation has also been be ductal, do respond to sympathetic or parasympaobserved after addition of isoproterenol to ducts perthetic stimulation and become depolarized. In that fused in vitro (5), and in these conditions vascular work, submaxillary gland of cat was used, but it was changes could not greatly affect the cellular responses. later shown that in submaxillary gland of rat also highThe effects observed in this study, where the sympapotential cells are present and that these are quite thetic innervation to th .e gland was directly stimulated, definitely ductal (17, 18). and in earlier studies, where the P-adrenergic agent, Further clarification of the effects of adrenergic or isoproterenol, was administered, together strongly sugcholinergic stimulation on ductal secretory function has gest that the ducts in rat submaxillary do receive a come mainly from work on perfused main excretory secretomotor sympathetic innervation. This is indicated duct. These investigations have dealt only with the also by another recent study, in which a different segactions of autonomic drugs, with isoproterenol as the ment of the rat submaxillary duct system, the granular main adrenergic drug used (5, 6, 19); however, they duct, showed histological alterations indicative of secrehave provided important information concerning tion in response to stimulation of the sympathetic innerchanges in electrolyte transport which accompany stim- vation (8). Since Norberg and Olson (9) had earlier ulation of ducta 1 autonomic receptors. First, it has be- reported observations from fluorescent microscopic excome clear that cholinergic and p-adrenergic agents do amination of rat submaxillary which made it doubtful affect transport of electrolytes by duct cells, at least in that there is an adrenergic innervation to duct cells, all the main duct segment of rat submaxillary (5, 6, 19, 20, of these newer findings are of importance in resolving 25). The ,&adrenergic agent, isoproterenol, for example, this doubt. when administered to the rat in large doses, was found The histochemical changes that have been reported to to depress Na, K transport and transepithelial PD, but occur in granular ducts after stimulation of the sympaenhance HCO,, secretion, in the main duct when the thetic innervation appear to be mediated bY cw-adrenerperfusion medium contained 140 meq/liter Na (6). Un- gic receptors (8). In the main duct, also, the effects of der similar conditions of perfusion, threshold doses of supramaximal stimulation of the sympathetic nerve, on isoproterenol (13 pg/kg, ip) produce somewhat different the transductal PD at least, seem principally to involve effects on net Na flux, which is enhanced after injection alpha receptors. Thus, in several preliminary experiTABLE
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ments, administration of phenoxybenzamine significantly reduced the degree of depolarization during sympathetic stimulation, while with propranolol no clear effects on PD were observed. It must be tentatively concluded that while the effects of isoproterenol indicate that P-receptor activity can modify transport in the main duct, it is a-receptors that are of importance in mediating the changes, at least in transepithelial PD,
when thetic
the ducts a re stimulated inn .ervation
through
their
sympa-
Assistance by Mr. H. Forrest and Mrs. F. M. Thomas during the experiments is gratefully acknowledged. The work was supported by National Institutes of Health Grant DE 02110 and Career Development Award 5-K6-DE 3341. Received
for publication
30 July
1975.
REFERENCES 1. EMMELIN, N. Control of salivary glands. In: Oral PhysioZogy, edited by N. Emmelin and Y. Zotterman. Oxford: Pergamon, 1972, p. 1-16. 2. EMMELIN, N., AND J. ENGSTROM. On the existence of specific secretory sympathetic fibers for the cat’s submaxillary gland. J. Physiol., London 153: l-8, 1960. 3. LUNDBERG, A. The electrophysiology of the submaxillary gland of the cat. Acta Physiol. Stand. 35: l-25, 1955. 4. MANGOS, J. A., N. R. MCSHERRY, AND S. N. ARVANITAKIS. Autonomic regulation of secretion and transductal fluxes of ions in the rat parotid. Am. J. Physiol. 225: 683-688, 1973. 5. MARTIN, C. J., A. R. DENNISS, 2. H. ENDRE, AND J. A. YOUNG. Mechanism of action of neurotransmitters and their analogues on salivary duct electrolyte transport. In: Secretory Mechanism ofExocrine GZands, edited by N. A. Thorn and 0. H. Petersen. Copenhagen: Munksgaard, 1974, p. 549-569. 6. MARTIN, C. J., AND J. A. YOUNG. A microperfusion investigation of the effects of a sympathomimetic and a parasympathomimetic drug on water and electrolyte fluxes in the main duct of the rat submaxillary gland. Pfluegers Arch. 327: 303-323, 1971. 7. MARTINEZ, J. R., H. HOLZGREVE, AND A. FRICK. Micropuncture study of submaxillary glands of adult rats. Pfluegers Arch. 290: 124-133, 1966. 8. MATTHEWS, R. W. Studies of the granular convoluted tubule in the rat submandibular gland (special issue). J. Dental Res. 53: 143, 1974. 9. NORBERG, K.-A., AND L. OLSON. Adrenergic innervation of the salivary glands in the rat. 2. ZeZlforsch. 68: 183-189, 1965. 10. SCHNEYER, C. A. Salivary gland changes after isoproterenolinduced enlargement. Am. J. Physiol. 203: 232-236, 1962. 11. SCHNEYER, C. A., AND H. D. HALL. Autonomic pathways involved in a sympathetic-like action of pilocarpine on salivary composition. Proc. Sot. Exptl. Biol. Med. 121: 96-100, 1966. 12. SCHNEYER, L. H. Secretory processes in perfused excretory duct of rat submaxillary gland. Am. J. Physiol. 215: 664-670, 1968. 13. SCHNEYER, L. H. Secretion of potassium by perfused excretory duct of rat submaxillary gland. Am. J. Physiol. 217: 1324-1329, 1969. 14. SCHNEYER, L. H. Secretion of K and fluid by rat submaxillary during sympathetic nerve stimulation. Am. J. Physiol. 229: 1056-1061, 1975.
15. SCHNEYER, L. H., AND N. EMMELIN. Salivary secretion. In: MTP International Review of Science. Gastrointestinal Physiology. London: Butterworths, 1974, PhysiologySer. 1, vol. 4, chapt. 6, p. 183-226. 16. SCHNEYER, L. H., AND R. F. FLATLAND. Evaluation of a reservoir in the main excretory duct of rat submaxillary gland. J. Appl. Physiol. 39: 519-522, 1975. 17. SCHNEYER, L. H., AND C. A. SCHNEYER. Membrane potentials of salivary gland cells of rat. Am. J. Physiol. 209: 1304-1310, 1965. 18. SCHNEYER, L. H., C. A. SCHNEYER, AND Y. YOSHIDA. Membrane . potentials of developing cells in immature rat submaxillary gland. Am. J. Physiol. 215: 1146-1150, 1968. 19. SCHNEYER, L. H., AND T. THAVORNTHON. Isoproterenol-induced stimulation of sodium absorption in perfused salivary duct. Am. J. PhysioZ. 224: 136-139, 1973. 20. SCHNEYER, L. H., J. A. YOUNG, AND C. A. SCHNEYER. Salivary secretion of electrolytes. PhysioZ. Rev. 52: 720-777, 1972. 21. THAYSEN, J. H. Handling of alkali metals by exocrine glands other than kidney. In: Handbuch der Experimentellen Pharmakologie. Berlin: Springer, 1960, part II, vol. 13, chapt. 5, p. 424-507. 22. YOSHIDA, Y., R. L. SPRECHER, C. A. SCHNEYER, AND L. H. SCHNEYER. Role of P-receptors in sympathetic regulation of electrolytes in rat submaxillary saliva. Proc. Sot. Exptl. BioL. Med. 126: 912-916, 1967. 23. YOUNG, J. A., E. FR~MTER, E. S~H~GEL, AND K. F. HAMANN. A microperfusion investigation of sodium resorption and potassium secretion by the main excretory duct of the rat submaxillary gland. Pfluegers Arch. 295: 157-172, 1967. 24 YOUNG, J. A., AND C. J. MARTIN. The effect of a sympathoand a parasympathomimetic drug on the electrolyte concentrations of primary and final saliva of the rat submaxillary gland. PfZuegers Arch. 327: 285-302, 1971. 25 YOUNG, J. A., AND C. J. MARTIN. Electrolyte transport in the excurrent duct system of the submaxillary gland. I. Studies on the intact gland. In: Oral Physiology, edited by N. Emmelin and Y. Zotterman. Oxford: Pergamon, 1972, p. 99-113. 26 YOUNG, J. A., AND E. SCH~GEL. Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pfluegers Arch. 291: 85-98, 1966.
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