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Progressive secretory dysfunction in the rat submandibular gland after excretory duct ligation
0003-9969 82 060443.08503.00 0 CopyrIght 0 19X? Pergamon Press Ltd
PROGRESSIVE SECRETORY DYSFUNCTION IN THE RAT SUBMANDIBULAR GLAND AFTER EXCRETORY DUCT LIGATION J. R. MARTINEZ, D. B. BYLUND and N. CASSITY Departments of Child Health. Physiology and Pharmacology, University School of Medicine, Columbia, MO 65212, U.S.A.
of Missouri,
Summary-Unilateral ligation of the main excretory duct of the rat submandibular gland caused a progressive deterioration of secretory function characterized by: (1) the secretion of progressively smaller volumes of saliva in response to a standard, maximal intraperitoneal (i.p.) dose of pilocarpine. Saliva volume was reduced 69.4, 88.8 and 95.9 per cent, respectively, 1, 3 and 7 days after duct ligation. By 2 weeks, the contralateral, non-ligated gland had an enhanced response to pilocarpine and the ligated gland a 96.8 per cent reduction in the volume of saliva secreted; (2) a progressive reduction in the maximum flow rates attained upon stimulation with pilocarpine, which were 23.4, 10.1 and 5.1 per cent of those attained in the contralateral gland at, respectively, 1, 3 and 7 days after ligation; (3) a progressive increase in the sodium concentrations of saliva, which became plasma-like 2 weeks after ligation; (4) a significantly reduced secretory response to standard doses of isoproterenol, phenylephrine, methoxamine and substance P 2 weeks after ligation. In addition, both cholinergic and adrenergic receptors were significantly reduced in number 2 weeks after ligation. Administration of daily injections of pilocarpine (10 mg, i.p.) after duct ligation reduced the gland atrophy observed 3 days (by 17 per cent) and 1 week (by 9 per cent) later, but did not prevent the reduction in volume or flow rate observed after duct ligation alone. Thus ligation of the main excretory duct causes a progressive dysfunction in acinar and duct cells in the rat submandibular gland and alters their responsiveness to physiological regulators of secretion. The alterations in glandular secretory function and in the composition of saliva which occur in diseases causing partial or complete obstruction to the flow of saliva are likely to be similar to the changes described.
INTRODUCTION L,igation of the excretory ducts of both human and animal salivary glands causes marked morphological changes in acinar and duct cells, leading to acinar cell atrophy and to loss of granularity in convoluted granular tubules (Tamarin, 1967, 1971a, b; Shiba, Hamada and Kawakatsu, 1972; Emmelin, Garrett and Ohlin, 1974; Harrison and Garrett, 1976; Garrett and Parsons, 1979). Functional abnormalities also develop after duct ligation, including the production of smaller volumes of saliva in response to parasympathomimetic stimulation (Schneyer and Schneyer, 1961) and changes in saliva composition (Smaje, 1974). Little is known, however, about the time course of the functional changes caused by duct ligation, or about the response of the ligated gland to various physiological regulators of secretion, particularly sympathomimetic agents. This study was undertaken, therefore, to assess these two aspects of the functional capacity of the ligated submandibular gland of the rat. The time course of the alterations in saliva volume and composition was investigated by using pilocarpine as the stimulant of salivary secretion. The response of the ligated gland to t(- and fi-adrenergic agents and to the stimulatory peptide substance P was correlated with changes in autonomic receptors as determined by radio-ligand binding procedures. As questions have been raised about the possibility that ligation in 443
the rat submandibular gland involves a parasympathetic denervation (Harrison and Garrett, 1976), the effects of repeated injections of pilocarpine on the post-ligation changes were also assessed.
MATERIALS AND METHODS Adult, male albino rats of the Sprague-Dawley strain were used. The animals weighed between 175 and 25Og at the time of the experiment and were given a standard pelleted diet and water ad libitum. The main excretory duct was carefully dissected on one side of the neck under pentobarbital anaesthesia (7-8 mg/lOOg body wt) and securely tied with triple-O surgical suture at a point 6-8 mm distal to the gland hilum. The skin incision was closed with surgical clamps and the animals were allowed to recover from anaesthesia in a cage maintained under a high-intensity lamp for warmth. Aseptic conditions were used throughout this procedure of duct ligation to reduce the risk of infection. After the animals had fully recovered from the anaesthetic, they were returned to the animal quarters and were watched carefully until the second phase of the experiment was carried out. In all animals, the contralateral, nonligated gland was used as the control for the different studies performed.
444
J. R. Martinez, D. B. Bylund and N. Cassity
The rats were subsequently studied 1, 3, 7 and 14 days after the duct has been surgically ligated. They were anaesthetized with sodium pentobarbital (7-8 mg/lOO g body wt) and the trachea was intubated with a small plastic cannulae (Clay Adams tubing, PE205). The submandibular glands and ducts were carefully dissected and the latter were cannulated with short lengths of polyethylene tubing (Clay Adams, PElO), pulled at the end over a microflame to a tip diameter of 25530pm. The animals were then stimulated by means of intraperitoneal (i.p.) injections of the different agents used (see below) and the appearance of salivary secretion in the ductal cannula was observed. The fluid secreted in the first 3-4min was usually discarded, as it represents saliva contained within the glandular duct system prior to stimulation. Samples were subsequently collected in pre-weighed plastic microsample tubes for timed intervals. Saliva collection was continued for 9&100 min and the individual tubes were re-weighed as they were collected, in order to obtain a gravimetric estimate of the volume of saliva secreted in each collection period. The glands were excised at the end of the experiment, gently blotted in tissue paper and weighed. The animals were kept on a heated operating table throughout the experiment. The saliva samples obtained throughout the collection period were analysed for sodium and potassium in an Instrumentation Laboratories flame photometer with lithium internal standard. A 10~1 sample was diluted 200 times for this determination. Calcium concentrations in 20 ~1 portions of the collected samples were analysed in duplicate in a Corning Instruments Calcium Analyzer. This method uses a fluorimetric procedure for the measurement of this divalent cation. For the receptor-binding experiments, the ligated and control glands were removed, weighed and homogenized in 50mM tris-HCI buffer, pH 8.0, in a Tissumizer, setting 80, for 30 s. The homogenate was centrifuged for 10 min at 49,OOOg, resuspended in buffer and centrifuged again. The pellet, a crude particulate fraction, was then resuspended for the receptor assays in 50 mM tris-HCl at a final tissue concentration of 5-10 mg of original wet-tissue weight per ml. Total binding was determined in tubes containing the membrane suspension and increasing concentrations of radio-ligand and non-specific binding in similar tubes containing appropriate concentrations of an unlabelled drug. Specific binding is the difference between total and non-specific binding. After 25 min of incubation at 22°C the suspensions were filtered through GF/B glass-fibre filter-paper using a manifold (Brandel Cell Harvester) which filters 24 samples simultaneously. The tubes and filter paper were washed with cold buffer and the radioactivity retained on the filter paper was determined in each sample by scintillation spectroscopy. The following radio-ligands were used: (1) [3H]-dihydroalprenolol (DHA) which specifically labels /I-adrenergic receptors; (2) [3H]-WB4101, which binds to a,-adrenergic receptors; (3) [3H]-quinuclylidyl benzylate (QNB), which labels muscarinic cholinergic receptors. To assess the possibility that a parasympathetic denervation caused by the duct ligature is responsible for the observed changes, a group of animals was treated with daily i.p. injections of pilocarpine nitrate
Submandibular
gland function
44s
after duct ligation
Table 2. Volumes of saliva and maximum rates of flow in control and ligated glands following stimulation with pilocarpine*
Time ligated
n
24 h 3 day 1 week 2 week
8 7 7 19
Volume of saliva secreted in 60 min (mg) Ligated n Control 114f3 39 * 10 15+5 17+3
7 7 7 18
372 345 374 536
f f + f
n 8 7 7 19
33 39 83 88
Maximum flow rate (mg/min g wet wt) Ligated n Control 18.3 + 7.5 f 2.7 + 3.8 k
6.8 2.2 0.8 0.7
7 7 7 18
78.3 74.0 52.0 65.5
f & f k
9.3 12.2 16.7 10.2
* Pilocarpine was administered i.p. in a dose of 10 mg/kg body wt. Values are means f SD of the mean. (10 mg/day) starting on the day that the duct was ligated. Three days or one week later, the animals were anaesthetized as already indicated and the submandibular glands were removed for estimation of weight changes or prepared for duct cannulation and saiiva collection as described above. Secretion of saliva from both the ligated and control submandibular glands was induced by the i.p. injection of pilocarpine (lOmg/kg body wt). In some experiments, the glands were removed at the end of the secretory period and prepared for morphological studies indicated above.
I&l
1
24
hour
RESULTS
Gland
weighrs
The initial effect of excretory duct ligation upon the rat submandibular gland appeared to involve a slight increase in gland weight during the first 24 h. Thus, the ligated-gland to control-gland weight-ratio increased to 115 per cent (Table 1). However, 3 days after duct ligation, the weight of the ligated gland began to decrease so that the weight ratio to the control gland was only 81 + 2 per cent; by 1 week following ligation it was significantly reduced, both in
ligature
3 day
ligature
Ido-
Control 0 Ligated
ml-
l
gland glond
loo-
m I week
ligature
Flow Fig. 1. Relationship control glands
2 week
rote
(mg/min.g
ligature
wet weight)
between salivary Na+-concentrations and rate of flow in submandibular saliva from (0) and from ligated glands (0) at different times after excretory duct ligature.
J. R. Martinez, D. B. Bylund and N. Cassity
446
IM-
24 hour ligature
ltQ-’
3 day ligature
Control gland 0 Ligated gland
Ml-,
l
0 ,M2
t g
MO_
140
I week ligature
u
+ Y 120-
0
Izo
Flow
0
2 week ligature
rate ( mg/mln *g wet weight)
Fig. 2. Relationship between salivary K+-concentrations and flow rate in submandibular
saliva from
control (0) and ligated glands (0) at different times after duct ligature.
absolute terms and in terms of the ratio of ligatedgland/control-gland weights (Table 1). A further reduction in weight occurred in the ligated gland between 1 and 2 weeks post-ligation and, as expected, the control gland showed an increased weight at this time. Table 1 shows the values obtained for absolute gland weight and for the weight ratios at the different times. The progressive decrease in gland weight observed after duct ligation evidently results from the progressive glandular atrophy observed at the morphological level (Tamarin, 1967, 1971a, b; Shiba et al., 1972). As gland and body weights normally increase with age, it is difficult to assess if a certain degree of compensatory hypertrophy took place in the non-ligated gland as the ligated gland atrophied. Secretory
response to pilocarpine
The secretory response to a standard, supramaxima1 i.p. dose of pilocarpine nitrate was characterized by a progressive reduction in the volume of saliva secreted by the ligated gland and by progressive changes in its sodium concentration, which gradually increased until it became plasma-like 2 weeks after ligation. The findings regarding volumes are shown in Table 2. Twenty-four hours after duct ligation, the ligated gland secreted less than one-third of the volume of saliva secreted by the contralateral, control gland. Three days after ligation, the volume secreted
by the ligated gland was only 11 per cent of that secreted by the non-ligated gland. One week after ligation, the volume of saliva produced by the ligated gland was only 4 per cent of that secreted by the control gland. Two weeks after ligation it became further reduced to 3 per cent of the volume secreted by the normal gland; the non-ligated gland secreted a larger volume of saliva than corresponding control glands at earlier periods. The progressive secretory dysfunction in the ligated gland is also shown by a comparison of the maximum flow rates attained after pilocarpine stimulation (Table 2). Salivary sodium concentrations increased progressively in the post-ligation period. Figure 1 shows the relationship between sodium concentrations and rates of salivary flow in submandibular saliva from ligated and control glands at the different time periods. Twenty-four hours after duct ligation the sodium concentrations in saliva collected from the ligated gland were significantly elevated in comparison to those observed in saliva of the control gland. Three days after ligation, salivary sodium concentrations were still higher; 1 or 2 weeks after ligation they became plasma-like. The findings (Fig. 1) represent true increases in salivary sodium concentrations, as the values were standardized against flow rate and the possible effects of discrepancies in the latter between the control and ligated glands have been eliminated. Salivary potassium concentrations tended to be
Submandibular gland function after duct ligation Table 3. Saliva secretion
from ligated and control glands in response gues and to substance P
447 to adrenergic
secretago-
n
Volume of saliva secreted in 60 min (mg) Control Ligated* n
n
Ligated*
n
Control
7
9.6 f 1.9
6
462 k 55
7
1.8 f 0.6
6
149 _t 8
Isoproterenol 10 mg/rat (i.p.)
6
9.8 k 7.5
6
7OIfr16
6
1.8 k 1.2
6
6+2
Methoxamine 5 mg/kg (ip.)
3
5.2 + 4.1
3
193 & 55
3
0.9 + 0.7
3
22 * 9
Phenylephrine 5 mg/kg (i.p.)
5
0.7 f 0.5
5
73 k 21
6
0.1 k 0.1
6
11 *7
Stimulant Substance P 1 pg/kg/min
Maximum
flow rate
(mg/min . g wet wt)
(iv.)?
* Glands studied 2 weeks after ligation. ? i.v., Intravenously. Values are means + SD of the mean.
slightly lower in the saliva from ligated glands when measured 24 h after ligation of the duct (Fig. 2). At later times after ligation, however, the concentrations of this ion were indistinguishable in the salivas of ligated and control glands (Fig. 2). The small volumes of saliva produced by the ligated glands did not permit the measurements of many salivary calcium concentrations. In the few samples where this ion was measured, however, no differences in concentration (related to flow rates) were observed in the saliva obtained from the ligated gland (not shown). Response to other stimulants of secretion Table 3 compares the volumes of saliva secreted by the ligated glands and by the contralateral control gland following stimulation with substance P and with tl- and /?-adrenergic agents. The values shown in this table correspond to glands studied 2 weeks after hgation and show that the volumes of saliva secreted in response to the various stimulants listed were markedly reduced in the ligated gland. Thus, the volume of saliva secreted by the ligated gland in response to isoproterenol stimulation was 14 per cent of that secreted by the control gland. Likewise, the volumes of saliva obtained from the ligated glands following stimulation with the a-adrenergic agents phenylephrine and methoxamine were, respectively, 1 and 2-3 per cent of those secreted by the non-ligated control gland. As for pilocarpine, the maximum flow rates attained by the ligated gland after stimulation with adrenergic secretagogues were also significantly smaller than those of the control glands (Table 3). Of particular interest is the markedly reduced secretory response to substance P, an undecapeptide which effectively causes saliva secretion from the rat submandibular gland (see Table 3 and Martinez and Martinez, 1981). The volumes of saliva secreted by the ligated glands after stimulation with these various agents were reduced to such an extent that repeated measurements of saliva electrolyte composition were not poss-
ible. However, in a few samples where measurements were possible, the Na+ concentration was always elevated, reaching plasma-like values (> 100 mequiv/l) in most samples. Autonomic receptors A comparison of the characteristics of autonomic receptors present in the ligated gland 2 weeks after ligature and in the contralateral control glands is shown in Table 4, which presents the affinity (K, in nM) and the number of receptors (B,,, in pmol/g tissue, fmol/mg protein and pmol/gland) for the bindof C3H]-WB4101 (cr-adrenergic receptors), ing C3H]-DHA (/?-adrenergic receptors) and [3H]-QNB (muscarinic cholinergic receptors) in crude membrane preparations from ligated and control glands. Fourteen days after main excretory-duct ligation, all three receptors were significantly decreased in the ligated gland. Thus, t(i receptors were reduced 70 per cent, B-receptors 67 per cent and muscarinic receptors 53 per cent in the ligated gland, when expressed per g tissue. As shown in Table 4, the reduction in the number of muscarinic cholinergic receptors amounted to 30 per cent one day after duct ligation and to 60 per cent one week later. As saliva excretion is expressed as the volume secreted per gland, a comparison with receptor number may be most appropriately made with the data expressed as pmol/gland. Expressed in this way, the reductions are even more striking, as for all three receptors there is a 90 per cent decrease in the number of receptors in the ligated glands. On the other hand, there were no significant changes in the Kr, values, indicating that the affinity characteristics of the receptors were unchanged. Eflects of chronic pilocarpine administration Administration of pilocarpine on a daily basis after duct ligature resulted in a smaller degree of glandular weight loss than that caused by ligature alone. Thus, the ratio of the weight of the ligated gland to that of
448
J. R. Martinez, D. B. Bylund and N. Cassity the contralateral control gland was 95 per cent after 3 days (as opposed to 81 per cent after ligature alone) and 49 per cent after 1 week (in contrast to 41 per cent with duct ligature alone). These results are shown in Table 5, which also summarizes the volume of saliva and the maximum secretory flow rate observed in these experiments. It can be seen that the smaller degree of atrophy observed after 3 days of pilocarpine administration did not result in an improvement in secretory capacity. Thus, the volume of saliva secreted by the ligated gland was only 11 per cent of that secreted by the control gland (Table 5). By one week after duct ligation and pilocarpine administration, the volume of saliva secreted by the ligated gland was only 2 per cent of that produced by the contralateral control gland (Table 5). This value was lower than that observed in ligated without pilocarpine administration (Table 2).
DISCUSSlON
Our findings indicate that ligation of the main excretory duct of the rat submandibular gland causes a progressive deterioration of its secretory capacity, characterized by the production of smaller volumes of saliva containing increasingly higher concentrations of Na+. By studying saliva secretion at various times after duct ligation, we have been able to document that the response to a standard maximal dose of pilocarpine becomes progressively more abnormal in regards to these two parameters, in such a way that 2 weeks after ligation the gland secretes only 34 per cent of the volume of saliva produced by the contralateral control gland. This fluid, furthermore, has plasma-like Na+-concentrations. The reduction in secretory response is likely to be the result of the progressive glandular atrophy caused by duct ligation (Schneyer and Schneyer, 1961; Tamarin, 1967, 1971a, b; Shiba et al., 1972). Parallel experiments to the ones reported here, in which the structural and ultrastructural changes induced by duct ligation were studied, confirm previous observations indicating that the ligated gland shows a regressive atrophy (Shiba et al., 1972). As in the salivary glands of the cat (Emmelin et al., 1974), the reduction in secretory capacity was more marked, however, than could be expected from the decrease in gland size. Salivary volumes were also significantly reduced 24 h after ligation, when the degree of atrophy is not marked (Tamarin, 1967, 1971a b; Shiba et al., 1972). These findings suggest that, initially, duct ligation alters secretory function by mechanisms other than cell atrophy, probably involving a metabolic lesion of the secretory cells. The observations on flow rate indirectly support this view, by showing that the 24 h post-ligated gland secretes less saliva per unit of tissue mass than the control gland. The decreased secretory capacity in the ligated gland may be related to the reduction in rough endoplasmic reticulum, which has been suggested as a possible pathway for fluid secretion in this gland (Simson et al., 1978). A similar progressive decrease in secretory response to adrenaline was described in the rat submandibular gland within the first 6 days after duct ligation (Ohlin and Perec, 1967).
Submandibular Table 5. Effect of chronic
Duration of ligature 3 days 1 week
Days of pilocarpine administration* 3 days 1 week
pilocarpine
449
gland function after duct ligation administration
n
Ligated gland wt (mg)/control gland weight (mg) x 100
8 9
95 + 4 49 f 2
on the changes caused by duct ligation Volume of saliva secreted in 60 min (mg) Ligated Control
Ligated
Control
4Okll 6*2
7k2.0 1 * 0.4
54k4.0 58 _t 5.0
360f42 345 f 45
Maximum rate
flow
(mg/min.d
* Pilocarpine was administered daily i.p. in a dose of 10 mg/rat. Values are means + SD of the mean.
The characteristics of the salivary Na+-concentration pattern in the saliva obtained from ligated glands suggest that duct ligation impairs Na+ reabsorption in the salivary ducts. Previous findings have demonstrated that duct ligation causes a reduction in succinate dehydrogenase activity in the ducts within 3-5 days, accompanied by the disappearance of basal striations or unfoldings and by a decrease in the number of mitochondria (Tamarin, 1967; Shiba et al., 1972). There is likely to be, therefore, a reduced energy available for the active transport of Na+ across the duct epithelium. A similar increase in saliwas observed after duct vary Na+ concentrations ligation in the submandibular gland of the rat by Schneyer and Schneyer (1961) and rabbit by Smaje (1974). Increased salivary Na+ concentrations in rat saliva after duct ligation are not accompanied by changes in salivary Kf concentrations (Schneyer and Schneyer, 1961). This is in contrast to the observations in rabbit submandibular gland, where K+-concentrations were reduced (Smaje, 1974). The is, as is the increase in salivary Na+ concentrations reduction in flow rate, a progressive phenomenon (Fig. 1). It is clear from our results that duct ligation affects the secretory response of the rat submandibular gland to all types of secretagogues. This suggests that the damage produced by the obstruction resulting from duct ligature is a generalized lesion affecting the physiological function of both acinar and duct cells. The reduced secretory response in all cases seems to be related to a decrease in the number of autonomic receptors (Table 4). In view of the acinar cell atrophy, the decrease in the number of autonomic receptors suggests that a moderate proportion of these receptors are localized in acinar cells. However, the receptors do not disappear entirely even 2 weeks after liga1:ion and it can be speculated that the remaining receptors, if localized in the atrophied acinar cells, have a decreased coupling to the secretory process. Some of these receptors could be localized in duct cells and also show a less efficient coupling. The alterations in the number of autonomic receplors that we found after duct ligation are interesting in view of previous suggestions that ligature of the main excretory duct in the rat submandibular gland usually includes the chorda tympani nerve and thus involves a parasympathetic denervation (Harrison and Garrett, 1976). However, section of the chordalingual nerve in rabbits did not cause changes in saliva compositions (Smaje, 1974); Ohlin and Perec (1967) found that duct ligation in rats did not alter the
total activity of choline acetylase in the gland and so concluded that ligation atrophy was not due to changes in parasympathetic neurons. Peronace ef al. (1964), on the other hand, concluded that the atrophy observed after sectioning of the parasympathetic nerves was due only to the denervation. Our present findings on the effects of the chronic administration of pilocarpine after duct ligation support the view that both ligation and parasympathetic denervation contribute to the glandular atrophy. Previous studies have shown that the administration of pilocarpine to normal rats causes, after 6 days, some hypertrophy of the submandibular and other salivary glands (Sturgess and Reid, 1973). We limited our studies to 3 days and 1 week regimens, as longer treatments with pilocarpine were shown by Sturgess and Reid to cause reductions in the size of acinar cells. During the first 3 days, the drug treatment reduced the glandular atrophy caused by duct ligation. By 1 week, however, the gland was reduced in size to about half of the control gland. Parasympathetic denervation thus appears to be an important factor initially, while the reduced secretory activity secondary to duct ligation may become the critical factor in the later stages of atrophy. Varying degrees of obstruction to the free flow of saliva occur in certain diseases affecting the salivary glands, including sialolithiasis and cystic fibrosis (Mandel and Wotman, 1976). It is possible that the alterations in secretory function and in saliva composition observed in such diseases are the result of partial or complete obstruction to salivary flow, which causes similar morphological and metabolic alterations to the ones described and discussed here. If part of the observed changes are due, on the other hand, to parasympathetic denervation, the possibility that alterations in the regulatory role of this branch of the autonomic nervous system play a role in the salivary gland pathology of cystic fibrosis should be considered, particularly in view of suspected autonomic dysfunction in this disease (Barber0 and Chernick, 1964). Acknowledgement-Supported by grant AM-18150 from the U.S.P.H.S., National Institutes of Health. REFERENCES
Barber0 G. J. and Chernick W. S. 1964. The role of the autonomic nervous system in cystic fibrosis. In: Research on the Parhogenesis of Cystic Fibrosis (Edited by di Sant’Agnese P. A.) pp. 208-222. National Institute of Arthritis and Metabolic Diseases, Bethesda, U.S.A.
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Emmelin N., Garrett J. R. and Ohlin P. 1974. Secretory activity and the myoepithelial cells of salivary glands after duct ligation in cats. Archs oral Biol. 19, 275-283. Garrett J. R. and Parsons P. A. 1979. Changes in the main submandibular salivary duct of rabbits resulting from ductal ligations. 2. mikrosk-tmar. Forsch. 93, 593-608. Harrison J. D. and Garrett J. R. 1976. Histological effects of duct ligation of salivary glands of the cat. J. Path. 118, 245-254. Mandel I. D. and Wotman S. 1976. The salivary secretions in health and disease. Oral Sci. Rev. 8, 25547. Martinez J. R. and Martinez A. M. 1981. Stimulatory and inhibitory effects of substance P on rat submandibular secretion. J. dent. Res. 60, 1031-1038. Ohlin P. and Perec C. 1967. Secretory responses and choline acetylase of the rat’s submaxillary gland after duct ligation. Experienria 23, 248-249. Peronace A. V., Davison T. A., Houssay A. B. and Perec C. J. 1964. Alterations in submandibular and retrolingual glands following parasympathetic denervation in rats. Anat. Rec. 150, 25-34. Schneyer C. A. and Schneyer L. H. 1961. Secretion by salivary glands deficient in acini. Am J. Physiol. 201, 939-942. Shiba R., Hamada T. and Kawakatsu K. 1972. Histochemical and electron microscopical studies on the effect of
duct ligation of rat salivary glands. Archs oral Biol. 17, 299-309. Simson J. A. V., Spicer S. S., Setser M. E. and Martinez J. R. 1978. Histochemistry and ultrastructure of rat submandibular acinar cells: effects of chronic reserpine on secretion. Lab. Invest. 39, 157-166. Smaje L. H. 1974. Spontaneous secretion in the rabbit submaxillary gland. In: Secretory Mechanisms of Exocrine Glands (Edited by Thorn N. A. and Petersen 0. H.) pp. 608-622. Benzon Symposium VII, Munksgaard, Copenhagen. Sturgess J. and Reid L. 1973. The effect of isoprenahne and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen. Br. J. exp. Path. 54, 388403. Tamarin A. 1967. Secretory cell alterations associated with submaxillary gland duct ligation. In: Secretory Mechanisms of Salivary Glands (Edited by Schneyer L. H. and Schneyer C. A.) pp. 22Ck235. Academic Press, New York. Tamarin A. 1971a. Submaxillary gland recovery from obstruction. I. Overall changes and electron microscopic alterations of granular duct cells. J. Ultrastract. Res. 34, 27&287.
Tamarin A. 197lb. Submaxillary gland recovery from obstruction. II. Electron microscopic alterations of acinar cells. J. Ultrastruct. Res. 34, 288-302.
PROGRESSIVE SECRETORY DYSFUNCTION IN THE RAT SUBMANDIBULAR GLAND AFTER EXCRETORY DUCT LIGATION J. R. MARTINEZ, D. B. BYLUND and N. CASSITY Departments of Child Health. Physiology and Pharmacology, University School of Medicine, Columbia, MO 65212, U.S.A.
of Missouri,
Summary-Unilateral ligation of the main excretory duct of the rat submandibular gland caused a progressive deterioration of secretory function characterized by: (1) the secretion of progressively smaller volumes of saliva in response to a standard, maximal intraperitoneal (i.p.) dose of pilocarpine. Saliva volume was reduced 69.4, 88.8 and 95.9 per cent, respectively, 1, 3 and 7 days after duct ligation. By 2 weeks, the contralateral, non-ligated gland had an enhanced response to pilocarpine and the ligated gland a 96.8 per cent reduction in the volume of saliva secreted; (2) a progressive reduction in the maximum flow rates attained upon stimulation with pilocarpine, which were 23.4, 10.1 and 5.1 per cent of those attained in the contralateral gland at, respectively, 1, 3 and 7 days after ligation; (3) a progressive increase in the sodium concentrations of saliva, which became plasma-like 2 weeks after ligation; (4) a significantly reduced secretory response to standard doses of isoproterenol, phenylephrine, methoxamine and substance P 2 weeks after ligation. In addition, both cholinergic and adrenergic receptors were significantly reduced in number 2 weeks after ligation. Administration of daily injections of pilocarpine (10 mg, i.p.) after duct ligation reduced the gland atrophy observed 3 days (by 17 per cent) and 1 week (by 9 per cent) later, but did not prevent the reduction in volume or flow rate observed after duct ligation alone. Thus ligation of the main excretory duct causes a progressive dysfunction in acinar and duct cells in the rat submandibular gland and alters their responsiveness to physiological regulators of secretion. The alterations in glandular secretory function and in the composition of saliva which occur in diseases causing partial or complete obstruction to the flow of saliva are likely to be similar to the changes described.
INTRODUCTION L,igation of the excretory ducts of both human and animal salivary glands causes marked morphological changes in acinar and duct cells, leading to acinar cell atrophy and to loss of granularity in convoluted granular tubules (Tamarin, 1967, 1971a, b; Shiba, Hamada and Kawakatsu, 1972; Emmelin, Garrett and Ohlin, 1974; Harrison and Garrett, 1976; Garrett and Parsons, 1979). Functional abnormalities also develop after duct ligation, including the production of smaller volumes of saliva in response to parasympathomimetic stimulation (Schneyer and Schneyer, 1961) and changes in saliva composition (Smaje, 1974). Little is known, however, about the time course of the functional changes caused by duct ligation, or about the response of the ligated gland to various physiological regulators of secretion, particularly sympathomimetic agents. This study was undertaken, therefore, to assess these two aspects of the functional capacity of the ligated submandibular gland of the rat. The time course of the alterations in saliva volume and composition was investigated by using pilocarpine as the stimulant of salivary secretion. The response of the ligated gland to t(- and fi-adrenergic agents and to the stimulatory peptide substance P was correlated with changes in autonomic receptors as determined by radio-ligand binding procedures. As questions have been raised about the possibility that ligation in 443
the rat submandibular gland involves a parasympathetic denervation (Harrison and Garrett, 1976), the effects of repeated injections of pilocarpine on the post-ligation changes were also assessed.
MATERIALS AND METHODS Adult, male albino rats of the Sprague-Dawley strain were used. The animals weighed between 175 and 25Og at the time of the experiment and were given a standard pelleted diet and water ad libitum. The main excretory duct was carefully dissected on one side of the neck under pentobarbital anaesthesia (7-8 mg/lOOg body wt) and securely tied with triple-O surgical suture at a point 6-8 mm distal to the gland hilum. The skin incision was closed with surgical clamps and the animals were allowed to recover from anaesthesia in a cage maintained under a high-intensity lamp for warmth. Aseptic conditions were used throughout this procedure of duct ligation to reduce the risk of infection. After the animals had fully recovered from the anaesthetic, they were returned to the animal quarters and were watched carefully until the second phase of the experiment was carried out. In all animals, the contralateral, nonligated gland was used as the control for the different studies performed.
444
J. R. Martinez, D. B. Bylund and N. Cassity
The rats were subsequently studied 1, 3, 7 and 14 days after the duct has been surgically ligated. They were anaesthetized with sodium pentobarbital (7-8 mg/lOO g body wt) and the trachea was intubated with a small plastic cannulae (Clay Adams tubing, PE205). The submandibular glands and ducts were carefully dissected and the latter were cannulated with short lengths of polyethylene tubing (Clay Adams, PElO), pulled at the end over a microflame to a tip diameter of 25530pm. The animals were then stimulated by means of intraperitoneal (i.p.) injections of the different agents used (see below) and the appearance of salivary secretion in the ductal cannula was observed. The fluid secreted in the first 3-4min was usually discarded, as it represents saliva contained within the glandular duct system prior to stimulation. Samples were subsequently collected in pre-weighed plastic microsample tubes for timed intervals. Saliva collection was continued for 9&100 min and the individual tubes were re-weighed as they were collected, in order to obtain a gravimetric estimate of the volume of saliva secreted in each collection period. The glands were excised at the end of the experiment, gently blotted in tissue paper and weighed. The animals were kept on a heated operating table throughout the experiment. The saliva samples obtained throughout the collection period were analysed for sodium and potassium in an Instrumentation Laboratories flame photometer with lithium internal standard. A 10~1 sample was diluted 200 times for this determination. Calcium concentrations in 20 ~1 portions of the collected samples were analysed in duplicate in a Corning Instruments Calcium Analyzer. This method uses a fluorimetric procedure for the measurement of this divalent cation. For the receptor-binding experiments, the ligated and control glands were removed, weighed and homogenized in 50mM tris-HCI buffer, pH 8.0, in a Tissumizer, setting 80, for 30 s. The homogenate was centrifuged for 10 min at 49,OOOg, resuspended in buffer and centrifuged again. The pellet, a crude particulate fraction, was then resuspended for the receptor assays in 50 mM tris-HCl at a final tissue concentration of 5-10 mg of original wet-tissue weight per ml. Total binding was determined in tubes containing the membrane suspension and increasing concentrations of radio-ligand and non-specific binding in similar tubes containing appropriate concentrations of an unlabelled drug. Specific binding is the difference between total and non-specific binding. After 25 min of incubation at 22°C the suspensions were filtered through GF/B glass-fibre filter-paper using a manifold (Brandel Cell Harvester) which filters 24 samples simultaneously. The tubes and filter paper were washed with cold buffer and the radioactivity retained on the filter paper was determined in each sample by scintillation spectroscopy. The following radio-ligands were used: (1) [3H]-dihydroalprenolol (DHA) which specifically labels /I-adrenergic receptors; (2) [3H]-WB4101, which binds to a,-adrenergic receptors; (3) [3H]-quinuclylidyl benzylate (QNB), which labels muscarinic cholinergic receptors. To assess the possibility that a parasympathetic denervation caused by the duct ligature is responsible for the observed changes, a group of animals was treated with daily i.p. injections of pilocarpine nitrate
Submandibular
gland function
44s
after duct ligation
Table 2. Volumes of saliva and maximum rates of flow in control and ligated glands following stimulation with pilocarpine*
Time ligated
n
24 h 3 day 1 week 2 week
8 7 7 19
Volume of saliva secreted in 60 min (mg) Ligated n Control 114f3 39 * 10 15+5 17+3
7 7 7 18
372 345 374 536
f f + f
n 8 7 7 19
33 39 83 88
Maximum flow rate (mg/min g wet wt) Ligated n Control 18.3 + 7.5 f 2.7 + 3.8 k
6.8 2.2 0.8 0.7
7 7 7 18
78.3 74.0 52.0 65.5
f & f k
9.3 12.2 16.7 10.2
* Pilocarpine was administered i.p. in a dose of 10 mg/kg body wt. Values are means f SD of the mean. (10 mg/day) starting on the day that the duct was ligated. Three days or one week later, the animals were anaesthetized as already indicated and the submandibular glands were removed for estimation of weight changes or prepared for duct cannulation and saiiva collection as described above. Secretion of saliva from both the ligated and control submandibular glands was induced by the i.p. injection of pilocarpine (lOmg/kg body wt). In some experiments, the glands were removed at the end of the secretory period and prepared for morphological studies indicated above.
I&l
1
24
hour
RESULTS
Gland
weighrs
The initial effect of excretory duct ligation upon the rat submandibular gland appeared to involve a slight increase in gland weight during the first 24 h. Thus, the ligated-gland to control-gland weight-ratio increased to 115 per cent (Table 1). However, 3 days after duct ligation, the weight of the ligated gland began to decrease so that the weight ratio to the control gland was only 81 + 2 per cent; by 1 week following ligation it was significantly reduced, both in
ligature
3 day
ligature
Ido-
Control 0 Ligated
ml-
l
gland glond
loo-
m I week
ligature
Flow Fig. 1. Relationship control glands
2 week
rote
(mg/min.g
ligature
wet weight)
between salivary Na+-concentrations and rate of flow in submandibular saliva from (0) and from ligated glands (0) at different times after excretory duct ligature.
J. R. Martinez, D. B. Bylund and N. Cassity
446
IM-
24 hour ligature
ltQ-’
3 day ligature
Control gland 0 Ligated gland
Ml-,
l
0 ,M2
t g
MO_
140
I week ligature
u
+ Y 120-
0
Izo
Flow
0
2 week ligature
rate ( mg/mln *g wet weight)
Fig. 2. Relationship between salivary K+-concentrations and flow rate in submandibular
saliva from
control (0) and ligated glands (0) at different times after duct ligature.
absolute terms and in terms of the ratio of ligatedgland/control-gland weights (Table 1). A further reduction in weight occurred in the ligated gland between 1 and 2 weeks post-ligation and, as expected, the control gland showed an increased weight at this time. Table 1 shows the values obtained for absolute gland weight and for the weight ratios at the different times. The progressive decrease in gland weight observed after duct ligation evidently results from the progressive glandular atrophy observed at the morphological level (Tamarin, 1967, 1971a, b; Shiba et al., 1972). As gland and body weights normally increase with age, it is difficult to assess if a certain degree of compensatory hypertrophy took place in the non-ligated gland as the ligated gland atrophied. Secretory
response to pilocarpine
The secretory response to a standard, supramaxima1 i.p. dose of pilocarpine nitrate was characterized by a progressive reduction in the volume of saliva secreted by the ligated gland and by progressive changes in its sodium concentration, which gradually increased until it became plasma-like 2 weeks after ligation. The findings regarding volumes are shown in Table 2. Twenty-four hours after duct ligation, the ligated gland secreted less than one-third of the volume of saliva secreted by the contralateral, control gland. Three days after ligation, the volume secreted
by the ligated gland was only 11 per cent of that secreted by the non-ligated gland. One week after ligation, the volume of saliva produced by the ligated gland was only 4 per cent of that secreted by the control gland. Two weeks after ligation it became further reduced to 3 per cent of the volume secreted by the normal gland; the non-ligated gland secreted a larger volume of saliva than corresponding control glands at earlier periods. The progressive secretory dysfunction in the ligated gland is also shown by a comparison of the maximum flow rates attained after pilocarpine stimulation (Table 2). Salivary sodium concentrations increased progressively in the post-ligation period. Figure 1 shows the relationship between sodium concentrations and rates of salivary flow in submandibular saliva from ligated and control glands at the different time periods. Twenty-four hours after duct ligation the sodium concentrations in saliva collected from the ligated gland were significantly elevated in comparison to those observed in saliva of the control gland. Three days after ligation, salivary sodium concentrations were still higher; 1 or 2 weeks after ligation they became plasma-like. The findings (Fig. 1) represent true increases in salivary sodium concentrations, as the values were standardized against flow rate and the possible effects of discrepancies in the latter between the control and ligated glands have been eliminated. Salivary potassium concentrations tended to be
Submandibular gland function after duct ligation Table 3. Saliva secretion
from ligated and control glands in response gues and to substance P
447 to adrenergic
secretago-
n
Volume of saliva secreted in 60 min (mg) Control Ligated* n
n
Ligated*
n
Control
7
9.6 f 1.9
6
462 k 55
7
1.8 f 0.6
6
149 _t 8
Isoproterenol 10 mg/rat (i.p.)
6
9.8 k 7.5
6
7OIfr16
6
1.8 k 1.2
6
6+2
Methoxamine 5 mg/kg (ip.)
3
5.2 + 4.1
3
193 & 55
3
0.9 + 0.7
3
22 * 9
Phenylephrine 5 mg/kg (i.p.)
5
0.7 f 0.5
5
73 k 21
6
0.1 k 0.1
6
11 *7
Stimulant Substance P 1 pg/kg/min
Maximum
flow rate
(mg/min . g wet wt)
(iv.)?
* Glands studied 2 weeks after ligation. ? i.v., Intravenously. Values are means + SD of the mean.
slightly lower in the saliva from ligated glands when measured 24 h after ligation of the duct (Fig. 2). At later times after ligation, however, the concentrations of this ion were indistinguishable in the salivas of ligated and control glands (Fig. 2). The small volumes of saliva produced by the ligated glands did not permit the measurements of many salivary calcium concentrations. In the few samples where this ion was measured, however, no differences in concentration (related to flow rates) were observed in the saliva obtained from the ligated gland (not shown). Response to other stimulants of secretion Table 3 compares the volumes of saliva secreted by the ligated glands and by the contralateral control gland following stimulation with substance P and with tl- and /?-adrenergic agents. The values shown in this table correspond to glands studied 2 weeks after hgation and show that the volumes of saliva secreted in response to the various stimulants listed were markedly reduced in the ligated gland. Thus, the volume of saliva secreted by the ligated gland in response to isoproterenol stimulation was 14 per cent of that secreted by the control gland. Likewise, the volumes of saliva obtained from the ligated glands following stimulation with the a-adrenergic agents phenylephrine and methoxamine were, respectively, 1 and 2-3 per cent of those secreted by the non-ligated control gland. As for pilocarpine, the maximum flow rates attained by the ligated gland after stimulation with adrenergic secretagogues were also significantly smaller than those of the control glands (Table 3). Of particular interest is the markedly reduced secretory response to substance P, an undecapeptide which effectively causes saliva secretion from the rat submandibular gland (see Table 3 and Martinez and Martinez, 1981). The volumes of saliva secreted by the ligated glands after stimulation with these various agents were reduced to such an extent that repeated measurements of saliva electrolyte composition were not poss-
ible. However, in a few samples where measurements were possible, the Na+ concentration was always elevated, reaching plasma-like values (> 100 mequiv/l) in most samples. Autonomic receptors A comparison of the characteristics of autonomic receptors present in the ligated gland 2 weeks after ligature and in the contralateral control glands is shown in Table 4, which presents the affinity (K, in nM) and the number of receptors (B,,, in pmol/g tissue, fmol/mg protein and pmol/gland) for the bindof C3H]-WB4101 (cr-adrenergic receptors), ing C3H]-DHA (/?-adrenergic receptors) and [3H]-QNB (muscarinic cholinergic receptors) in crude membrane preparations from ligated and control glands. Fourteen days after main excretory-duct ligation, all three receptors were significantly decreased in the ligated gland. Thus, t(i receptors were reduced 70 per cent, B-receptors 67 per cent and muscarinic receptors 53 per cent in the ligated gland, when expressed per g tissue. As shown in Table 4, the reduction in the number of muscarinic cholinergic receptors amounted to 30 per cent one day after duct ligation and to 60 per cent one week later. As saliva excretion is expressed as the volume secreted per gland, a comparison with receptor number may be most appropriately made with the data expressed as pmol/gland. Expressed in this way, the reductions are even more striking, as for all three receptors there is a 90 per cent decrease in the number of receptors in the ligated glands. On the other hand, there were no significant changes in the Kr, values, indicating that the affinity characteristics of the receptors were unchanged. Eflects of chronic pilocarpine administration Administration of pilocarpine on a daily basis after duct ligature resulted in a smaller degree of glandular weight loss than that caused by ligature alone. Thus, the ratio of the weight of the ligated gland to that of
448
J. R. Martinez, D. B. Bylund and N. Cassity the contralateral control gland was 95 per cent after 3 days (as opposed to 81 per cent after ligature alone) and 49 per cent after 1 week (in contrast to 41 per cent with duct ligature alone). These results are shown in Table 5, which also summarizes the volume of saliva and the maximum secretory flow rate observed in these experiments. It can be seen that the smaller degree of atrophy observed after 3 days of pilocarpine administration did not result in an improvement in secretory capacity. Thus, the volume of saliva secreted by the ligated gland was only 11 per cent of that secreted by the control gland (Table 5). By one week after duct ligation and pilocarpine administration, the volume of saliva secreted by the ligated gland was only 2 per cent of that produced by the contralateral control gland (Table 5). This value was lower than that observed in ligated without pilocarpine administration (Table 2).
DISCUSSlON
Our findings indicate that ligation of the main excretory duct of the rat submandibular gland causes a progressive deterioration of its secretory capacity, characterized by the production of smaller volumes of saliva containing increasingly higher concentrations of Na+. By studying saliva secretion at various times after duct ligation, we have been able to document that the response to a standard maximal dose of pilocarpine becomes progressively more abnormal in regards to these two parameters, in such a way that 2 weeks after ligation the gland secretes only 34 per cent of the volume of saliva produced by the contralateral control gland. This fluid, furthermore, has plasma-like Na+-concentrations. The reduction in secretory response is likely to be the result of the progressive glandular atrophy caused by duct ligation (Schneyer and Schneyer, 1961; Tamarin, 1967, 1971a, b; Shiba et al., 1972). Parallel experiments to the ones reported here, in which the structural and ultrastructural changes induced by duct ligation were studied, confirm previous observations indicating that the ligated gland shows a regressive atrophy (Shiba et al., 1972). As in the salivary glands of the cat (Emmelin et al., 1974), the reduction in secretory capacity was more marked, however, than could be expected from the decrease in gland size. Salivary volumes were also significantly reduced 24 h after ligation, when the degree of atrophy is not marked (Tamarin, 1967, 1971a b; Shiba et al., 1972). These findings suggest that, initially, duct ligation alters secretory function by mechanisms other than cell atrophy, probably involving a metabolic lesion of the secretory cells. The observations on flow rate indirectly support this view, by showing that the 24 h post-ligated gland secretes less saliva per unit of tissue mass than the control gland. The decreased secretory capacity in the ligated gland may be related to the reduction in rough endoplasmic reticulum, which has been suggested as a possible pathway for fluid secretion in this gland (Simson et al., 1978). A similar progressive decrease in secretory response to adrenaline was described in the rat submandibular gland within the first 6 days after duct ligation (Ohlin and Perec, 1967).
Submandibular Table 5. Effect of chronic
Duration of ligature 3 days 1 week
Days of pilocarpine administration* 3 days 1 week
pilocarpine
449
gland function after duct ligation administration
n
Ligated gland wt (mg)/control gland weight (mg) x 100
8 9
95 + 4 49 f 2
on the changes caused by duct ligation Volume of saliva secreted in 60 min (mg) Ligated Control
Ligated
Control
4Okll 6*2
7k2.0 1 * 0.4
54k4.0 58 _t 5.0
360f42 345 f 45
Maximum rate
flow
(mg/min.d
* Pilocarpine was administered daily i.p. in a dose of 10 mg/rat. Values are means + SD of the mean.
The characteristics of the salivary Na+-concentration pattern in the saliva obtained from ligated glands suggest that duct ligation impairs Na+ reabsorption in the salivary ducts. Previous findings have demonstrated that duct ligation causes a reduction in succinate dehydrogenase activity in the ducts within 3-5 days, accompanied by the disappearance of basal striations or unfoldings and by a decrease in the number of mitochondria (Tamarin, 1967; Shiba et al., 1972). There is likely to be, therefore, a reduced energy available for the active transport of Na+ across the duct epithelium. A similar increase in saliwas observed after duct vary Na+ concentrations ligation in the submandibular gland of the rat by Schneyer and Schneyer (1961) and rabbit by Smaje (1974). Increased salivary Na+ concentrations in rat saliva after duct ligation are not accompanied by changes in salivary Kf concentrations (Schneyer and Schneyer, 1961). This is in contrast to the observations in rabbit submandibular gland, where K+-concentrations were reduced (Smaje, 1974). The is, as is the increase in salivary Na+ concentrations reduction in flow rate, a progressive phenomenon (Fig. 1). It is clear from our results that duct ligation affects the secretory response of the rat submandibular gland to all types of secretagogues. This suggests that the damage produced by the obstruction resulting from duct ligature is a generalized lesion affecting the physiological function of both acinar and duct cells. The reduced secretory response in all cases seems to be related to a decrease in the number of autonomic receptors (Table 4). In view of the acinar cell atrophy, the decrease in the number of autonomic receptors suggests that a moderate proportion of these receptors are localized in acinar cells. However, the receptors do not disappear entirely even 2 weeks after liga1:ion and it can be speculated that the remaining receptors, if localized in the atrophied acinar cells, have a decreased coupling to the secretory process. Some of these receptors could be localized in duct cells and also show a less efficient coupling. The alterations in the number of autonomic receplors that we found after duct ligation are interesting in view of previous suggestions that ligature of the main excretory duct in the rat submandibular gland usually includes the chorda tympani nerve and thus involves a parasympathetic denervation (Harrison and Garrett, 1976). However, section of the chordalingual nerve in rabbits did not cause changes in saliva compositions (Smaje, 1974); Ohlin and Perec (1967) found that duct ligation in rats did not alter the
total activity of choline acetylase in the gland and so concluded that ligation atrophy was not due to changes in parasympathetic neurons. Peronace ef al. (1964), on the other hand, concluded that the atrophy observed after sectioning of the parasympathetic nerves was due only to the denervation. Our present findings on the effects of the chronic administration of pilocarpine after duct ligation support the view that both ligation and parasympathetic denervation contribute to the glandular atrophy. Previous studies have shown that the administration of pilocarpine to normal rats causes, after 6 days, some hypertrophy of the submandibular and other salivary glands (Sturgess and Reid, 1973). We limited our studies to 3 days and 1 week regimens, as longer treatments with pilocarpine were shown by Sturgess and Reid to cause reductions in the size of acinar cells. During the first 3 days, the drug treatment reduced the glandular atrophy caused by duct ligation. By 1 week, however, the gland was reduced in size to about half of the control gland. Parasympathetic denervation thus appears to be an important factor initially, while the reduced secretory activity secondary to duct ligation may become the critical factor in the later stages of atrophy. Varying degrees of obstruction to the free flow of saliva occur in certain diseases affecting the salivary glands, including sialolithiasis and cystic fibrosis (Mandel and Wotman, 1976). It is possible that the alterations in secretory function and in saliva composition observed in such diseases are the result of partial or complete obstruction to salivary flow, which causes similar morphological and metabolic alterations to the ones described and discussed here. If part of the observed changes are due, on the other hand, to parasympathetic denervation, the possibility that alterations in the regulatory role of this branch of the autonomic nervous system play a role in the salivary gland pathology of cystic fibrosis should be considered, particularly in view of suspected autonomic dysfunction in this disease (Barber0 and Chernick, 1964). Acknowledgement-Supported by grant AM-18150 from the U.S.P.H.S., National Institutes of Health. REFERENCES
Barber0 G. J. and Chernick W. S. 1964. The role of the autonomic nervous system in cystic fibrosis. In: Research on the Parhogenesis of Cystic Fibrosis (Edited by di Sant’Agnese P. A.) pp. 208-222. National Institute of Arthritis and Metabolic Diseases, Bethesda, U.S.A.
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Emmelin N., Garrett J. R. and Ohlin P. 1974. Secretory activity and the myoepithelial cells of salivary glands after duct ligation in cats. Archs oral Biol. 19, 275-283. Garrett J. R. and Parsons P. A. 1979. Changes in the main submandibular salivary duct of rabbits resulting from ductal ligations. 2. mikrosk-tmar. Forsch. 93, 593-608. Harrison J. D. and Garrett J. R. 1976. Histological effects of duct ligation of salivary glands of the cat. J. Path. 118, 245-254. Mandel I. D. and Wotman S. 1976. The salivary secretions in health and disease. Oral Sci. Rev. 8, 25547. Martinez J. R. and Martinez A. M. 1981. Stimulatory and inhibitory effects of substance P on rat submandibular secretion. J. dent. Res. 60, 1031-1038. Ohlin P. and Perec C. 1967. Secretory responses and choline acetylase of the rat’s submaxillary gland after duct ligation. Experienria 23, 248-249. Peronace A. V., Davison T. A., Houssay A. B. and Perec C. J. 1964. Alterations in submandibular and retrolingual glands following parasympathetic denervation in rats. Anat. Rec. 150, 25-34. Schneyer C. A. and Schneyer L. H. 1961. Secretion by salivary glands deficient in acini. Am J. Physiol. 201, 939-942. Shiba R., Hamada T. and Kawakatsu K. 1972. Histochemical and electron microscopical studies on the effect of
duct ligation of rat salivary glands. Archs oral Biol. 17, 299-309. Simson J. A. V., Spicer S. S., Setser M. E. and Martinez J. R. 1978. Histochemistry and ultrastructure of rat submandibular acinar cells: effects of chronic reserpine on secretion. Lab. Invest. 39, 157-166. Smaje L. H. 1974. Spontaneous secretion in the rabbit submaxillary gland. In: Secretory Mechanisms of Exocrine Glands (Edited by Thorn N. A. and Petersen 0. H.) pp. 608-622. Benzon Symposium VII, Munksgaard, Copenhagen. Sturgess J. and Reid L. 1973. The effect of isoprenahne and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen. Br. J. exp. Path. 54, 388403. Tamarin A. 1967. Secretory cell alterations associated with submaxillary gland duct ligation. In: Secretory Mechanisms of Salivary Glands (Edited by Schneyer L. H. and Schneyer C. A.) pp. 22Ck235. Academic Press, New York. Tamarin A. 1971a. Submaxillary gland recovery from obstruction. I. Overall changes and electron microscopic alterations of granular duct cells. J. Ultrastract. Res. 34, 27&287.
Tamarin A. 197lb. Submaxillary gland recovery from obstruction. II. Electron microscopic alterations of acinar cells. J. Ultrastruct. Res. 34, 288-302.