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The concentration of sodium, potassium and chloride in rabbit submandibular saliva during postnatal development
Archs oral Bid. Vol. 28, No. 9, pp. 879-883, 1983 Printed in Great Britain. All rights reserved
0003.9969/83 $03.00+0.00 Copyright CI 1983 Pergamon Press Ltd
THE CONCENTRATION OF SOJXUM, POTASSIUM AND CHLORIDE IN RABBIT SUBMANDIBULAR SALIVA DURING POSTNATAL DEVELOPMENT J. M. REYNOLDS and SYLVIA L. PULL Department
#ofPhysiology,
Royal Dental School, St George’s Hospital Medical School, Cranmer London SW17 ORE, England, U.K.
Terrace,
Summary-Saliva was collected from the cannulated ducts of submandibular glands of rabbits at various ages from birth to adulthood and analysed for sodium, potassium and chloride. Saliva was obtained at all ages by electrical stimulation of the parasympathetic innervation to the gland. On the day of birth (day I), salivary sodium was 108.5 + 3.4mM (n = 8) and chloride was 89.9 k 3.6 mM (n = 9) falling to adult-like concentrations of 5.7 + 0.6 mM (n = 13) and 16.5 k 1.2mM (n = 9) respectively by day 10. On Day 1, salivary potassium was 23.9 + 1.8mM (n = 7) and rose progressively to 76.8 + 2.5 mM (n = 8) on day 17. Adult potassium concentration was 30-40mM. A wide range of flows was obtained at all ages and at each age sodium and chloride concentrations were approximately constant at all flows. Therefore the changes in electrolyte concentration cannot be explained by differences in flow. The results suggest a maturation of ductal transport of sodium, potassium and chloride ions concurrent with the structural maturation #of striated duct cells.
INTRODUCTION
consistent with the structural changes which occur during the development of striated duct cells. The methods were demonstrated to the Physiological Society (Pull and Reynolds, 1979a) and preliminary results were reported also (Pull and Reynolds, 1979b; Pull, 1981).
We are studying the function and structure of the neonatal rabbit submandibular gland in order to gain insight into the processes involved in the development of secretory cells. This development could be expected to comprise several related factors e.g. cellular differentiation and division, functional maturation of newly-formed cells, the establishment of stimulus-secretion coupling, and could be subject to both genetic and environmental influences. Major salivary glands have two main secretory elements: acinar ceils which produce a primary secretion and striated ducts which can substantially modify that secretion. In ontogeny, the ducts develop first and in many speces acinar development has barely begun at birth (Jacoby and Leeson, 1959; Young and van Lennep, 1978). This is often interpreted incorrectly as indicating the absence of acinar cells during duct proliferation. In fact, a few acinar cells are present, at least as terminal buds, and it is probably more correct to describe duct growth as initially outstripping acinar development, the latter becoming predominant as duct growth nears completion. Previous studies of the function of developing salivary glands (Hall and Schneyer, 1969; Mangos, 1978) have been on rat glands but at ages when striated ducts were. already fully formed (Cutler and Chaudry, 1975). Studies of rat glands are complicated by the development of convoluted granular tubules between the striated and intercalated ducts. In contrast, the adult rabbit submandibular gland has short (two or three cells long) intercalated ducts. This suggests that the long ducts identified (LM) in prenatal and newborn rabbits by Oikawa et al. (1976) and Delattre and Yardin (1976) as “intercalated ducts” could be immature striated ducts. Preliminary observations confirm this (Reynolds and Webb, 1982), and we here argue that the functional characteristics of the new-born gland, and the functional changes occurring in the first ten days of life, are
MATERIALS AND METHODS
New Zealand White rabbits fed ad libitdm on a standard laboratory diet (RGP, Labsure, containing 0.7 per cent by weight NaCl) were used to provide litters. All offspring were left with their dams until required or until weaned (6 weeks). Anaesthetic doses (25 per cent urethane in 0.9 per cent saline) were 12ml/kg for animals up to 7 days, lOml/kg from 8 days to 1 month and 7 ml/kg thereafter. Initial anaesthetic administration was intraperitoneal in animals younger than 3 months old and intravenous in older animals. The post-mortem weights of five animals under 14 days were identical with their preexperimental weights, indicating no net fluid-loss as a result of the experimental procedures. The anaesthetized animals were placed supine on a heated operating table and body temperature maintained at 39°C (rectal probe). For administration of further anaesthetic, a femoral vein was cannulated using polythene tubing (Portex PP202) drawn down over an alcohol flame to outside diameters of 4s120pm (Evans et al., 1974). The trachea was not routinely cannulated in the very young animals (unless there was obvious respiratory difficulty) in order to leave the surgical area unobstructed. The submandibular duct was approached through the mylohyoid muscle and cannulated (see Pull and Reynolds 1979a,b for details). The chorda tympani (parasympathetic innervation) runs along the duct: cannulation therefore prevents any reflex parasympathetic stimulation. The duct was mounted on bipolar stimulating electrodes, the electrodes and duct 879
880
J. M. REYNOLDS and SYLVIA L. PULL Table
Age (days) (range)
I 2 3 4 5 6.75 (6-7) 10.8 (9-13) 17
1. Details
Body weight n
3 4 3
(g)
of new-born
Gland
animals
weight (g)
used and their maximum
Gland
weight
Body weight
%
secretion
rates
Maximum secretion rate
Maximum secretion rate (pl.min-‘.g-‘) of
(pl.min-I)
gland tissue
4
64.3 _+5.3 67.0 + 5.4 63.7 + 6.0 (I 17.0) 79.5 i: 10.4
0.0702 + 0.0070 0.0907 + 0.0 196 0.0898 * 0.0255 (0.1284) 0.1002 + 0.0178
0.11 0.14 0.14 (0. I I) 0.13
3.77 * 2.21 7.28 i 2.4 5.35 k 2.29 (8.9) 6.69 f 1.39
49.19k24.85 91.27 k 39.30 56.19 + 16.21 (69.5) 66.27 i 12.53
4
121.5 + 15.2
0.1312 _+0.0233
0.11
3.62 k I .06
30.86 &-10.21
5 2
180.8 + 23.6 490
0.1808 k 0.0360 0.3896
0.10 0.08
3.15 i 0.82 9.19( + 0.33)
20.92 k 6.71 31.45 k4.15
1
All values are mean + SE. From other experiments, the size of the submandibular gland, relative to body weight, continues to decline with age. The maximum secretion rate for each age group is the mean of the highest flows from each individual gland. Note, these are not instantaneous flows: the minimum collection time was 5 min.
being electrically isolated from surrounding tissue by Klingfilm and flooded with liquid paraffin. The adult
rabbit submandibular gland does not secrete in response to sympathetic stimulation. Preliminary experiments showed this also in neonates. Sectioning the sympathetic trunk made no measurable difference to the saliva electrolytes and in most experiments the trunk was left intact. The stimulus was an isolated (Neurolog: NL800) square wave (1 ms, maximum 10 V, 1 mA) of 0.5-20 Hz frequency. Electrical stimulation invariably commenced at 20 Hz (1 ms sq. wave). In some older animals, low flows were obtained by decreasing the stimulus frequency. Even in cases where stimulus frequency was reduced, all glands were stimulated continuously in order to avoid the possibility of ion transients (Burgen and Emmelin, 1961). Attempts to induce transients by increasing stimulus frequency produced no detectable change in potassium or sodium concentrations in the neonatal animals. Saliva was collected into pre-weighed microcentrifuge vials either directly or after passing through a capillary tube. The flow measured by timing the movement of an air bubble along the capillary was similar to the gravimetrically determined flow. Secretion rates varied between 167nl,min-’ and 13.1 pl.rnin-‘. Fifteen to twenty microlitres was required to measure the three ions and in some cases therefore only one or two of the ions could be determined if collection time was not to be unreasonably long. Sodium and potassium concentrations were measured with an EEL Flame Photometer using procedures similar to those described by Bradbury et al. (1972). Chloride concentrations were measured by a chloridometer (Buchler Instruments, Searle Analytic). RESULTS Saliva flow was elicited by electrical stimulation from cannulated submandibular glands from the day of birth (day 1). As with the isolated gland (Case et al., 1980), flow decreased with duration of a constant
stimulus. A slow (co.1 pl.min.g-’ gland wt) unstimulated secretion was noticed as early as the
second day of life, but all the results reported here are for stimulated saliva, The maximum secretion rates obtained for each age group (Table 1) showed no age dependence. It must be stressed that these maximal flows were (a) not instantaneous, (b) may reflect developmental differences in stimulus-secretion coupling, and (c) may reflect differences in the number of fibres actually stimulated. The sodium (and chloride) concentrations showed little flow dependence at any age (Fig. 1) including the adult (Pull and Reynolds, 1979b), although in the youngest animals there was a suggestion of lower concentrations at higher flows. However, there was a marked difference in the ion concentrations between the age groups, high concentrations at birth declining to adult concentrations by the ninth day. Saliva electrolyte concentration is often flow- and timedependent (Young, 1979) but, clearly, these sources of variation cannot account for the changes in sodium concentration observed during the neonatal period. On the first day of life (day l), saliva sodium concentration was 108.5 + 3.4 mM (n = 8), declining to less than half this figure by day 4 and to an adult-like concentration of 5.7 f 0.6 mM (n = 13) by days 9-l 1 (Fig. 2). During the first 5 days of life body weight and gland weight were almost constant, suggesting that the changes in sodium concentration during this period were not related to changes in body or gland size. Similarly, body weight increased rapidly after day 11 but no further changes in sodium concentration occurred. The time course for the change in chloride concentration with age (Fig. 3) was similar to the time course for the change in sodium. Chloride concentration was 89.9 + 3.6 mM (n = 9) on day 1 and on days 9-l 1 was 16.5 k 1.2 (n = 9). Potassium concentration was much more variable even though precautions were taken to avoid transients. Thus for potassium there is a much less clearly-defined difference between age groups than for sodium and chloride (Fig. 4). As with sodium and chloride, the differences between ages cannot be explained in terms of changes of concentration with secretion rate. It is surprising that the progressive rise
Saliva composition l-2 A
days
4-6
A
A
881
in neonatal development
IO-I? days
days
A
I
I
I
I
I
40
60
0
40
60
Secretion
rate (~1. min-‘, g-l gland
wt
1
Fig. 1. Sodium concentrations in rabbit submandibular saliva at l-2, 46 and lb-17 days of age. Saliva flow was elicited by continuous stimulation of the parasympathetic innervation to the cannulated gland. Flow is expressed per unit gland weight and each point represents a single sample.
.
P
.Z
I
d.0.b. 2 0
I
I
I
,
2
4
6
8
r:
. IO
I
,
1
I2
l 14
I I6
I6
..
47-56
I
Adult
Age ( doys 1 Fig. 2. Sodium concentrations in rabbit submandibular saliva during development. Day 1 = date of birth. Saliva flow was elicited by continuous stimulation of the parasympathetic innervation to the cannulated gland. As the sodium concentration showed little flow dependence (Fig. I), points are plotted irrespective of flow. N.B. Some points overlap, e.g. 8 samples are represented at day 1.
: I
5
n
.
2
.
.
n
0
:
II)
2 b i u
4c-
.
.
:
. .
I . I
05
do
a *
4 C
.
b
i
: I
::
l
I
I
I
I
I
I
I
I
2
4
6
8
IO
12
14
16
I8
;
, %Fk-x+
Age (days I Fig. 3. Chlorde concentrations in rabbit submandibular saliva during development. The range of chloride concentrations at 47-56 days corresponds to a wide range of potassium concentrations found in juvenile but not adult animals.
J. M.
882 l-2 f-
and
REYNOLDS
4-6
days
SYLVIA
L.
PULL IO - 17days
days
120
5 :
.
c : 80 8
I
E I : ,D g 40 , .
I:, 0
.
40
.
(II 00
. #
SecretIon
rate
( ~1
. .
mine’
7
. l:l 80
40
0
.
.
I m
. 9.
. .
.. -+’
P _ 0 vl
l
‘.
1 0
g-’ gland wt
40
80
)
Fig. 4. Potassium concentration in rabbit submandibular saliva at 1-2, 4-6 and l&17 days of age. Flow is per unit gland weight and each point represents a single sample collected under continuous stimulation. The range of potassium concentrations found in juvenile animals does not appear to be due to transients.
in potassium concentration was up to nearly 80 mM at 17 days when the adult concentration is almost always 3&40 mM. Thus the pattern of development for potassium secretion is quite different from that for sodium and chloride. Reynolds (1981) suggested that protein concentration may exert a profound effect on potassium concentration in rabbit submandibular saliva. In adult rabbits, this is reflected by a marked deficit (up i.e. 150mM) of saliva anions to Na + K 9 Cl + HCO; . In experiments with neonates, small sample size precluded the measurement of bicarbonate and total protein, but the charge difference between the measured cations (Na+ and K + ) and anion (chloride) varied only between 28 and 62mM. Assuming that a proportion of this was bicarbonate, the anion deficit was not large enough to suggest that protein is heavily influencing the ionic composition of neonatal saliva. DISCUSSION
When this study began it was expected that the ion concentraticns would reflect age differences in the relative cell populations of acini and striated ducts (Burgen and Emmelin, 1961). Thus, as the ducts develop first, it was expected that ductal function (the removal of sodium and the addition of potassium) would be most marked early in life and new-born saliva would have a high potassium and low sodium concentration. This would have given the opportunity to study (a) the development of acinar cell function and (b) the functions of the striated duct alone. The results actually obtained suggest that the most notable functional changes are affecting the ducts and not the acini. The sodium, potassium and chloride concentrations in whole saliva at birth were similar to the concentrations in the primary secretions from adult acinar cells (Young, 1979; Endre and Young, 1981). Assuming that a given cell type does not reverse its ion fluxes, it seems most probable that at birth the final saliva is an almost unmodified primary secretion
despite the higher proportion of duct cells to acinar cells than later in life. If the acinar cells at birth are able to produce an adult-type primary secretion, it follows that any changes in the secondary modifications of saliva must reflect a change in duct function. In that case, certainly for sodium and chloride, the capacity of the ducts to transport these ions increases during the first nine days of life. This could be due to changes in duct response to stimulation, but preliminary studies (Reynolds and Webb, 1982) indicate no striations and few mitochondria in striated ducts at birth, suggesting that the duct cells themselves are maturing during this period. Oikawa et al. (1976) remarked on the absence of striae in the ducts of 21-day rabbit fetuses. Oikawa et al. appear to have mistakenly identified striated ducts from neonatal rabbits as intercalated ducts, an indication of how immature-looking are the neonatal ducts. Cutler and Chaudry (1975) describe in detail the changes in striated-duct cell structure in the rat fetus. At 18 days, there are few basal infoldings, lateral projections and mitochondria whereas, at 20 days, the cells have a much more adult-like striated appearance due to many basal infoldings and mitochondria. It appears that in the rabbit these changes occur postnatally. The potassium results can also be explained in terms of increasing duct function. It is clear, however, that the time-course of events for potassium is quite different from that of sodium. This could indicate that the duct-handling of sodium and potassium is by separate mechanisms. The apparent developmental changes are not due simply to increased body weight or gland weight as periods of growth do not correspond with periods of marked change in saliva electrolytes. It seems possible that the changes are genetically determined but factors such as tooth development (Yardin, 1968), diet or hormones have yet to be excluded. For example, Hall and Schneyer (1969) showed that salivary gland growth is inhibited in rats fed on a liquid diet. In contrast to the above interpretation of salivary gland development, Schneyer and Hall (1968) and
Saliva composition in neonatal development (1978) reported that the sodium concentration in rat parotid saliva (stimulated) increased over the first months of life. This change was attributed to an age-dependent decrease in transductal sodium flux. No such change was observed in rat submandibular saliva (Schneyer and Schneyer, 1961). At the ages studied, both these rat glands had morphologically-distinct striated ducts (Jacoby and Leeson, 1959; Schneyer and Hall, 1969) but developmental changes were still occurring in the acini and convoluted granular tubules. It seems unlikely that this acinar development can explain the low sodium in immature paroticl saliva as, if anything, the primary sodium concentration is likely to be greater than in the adult (Holzgreve, Martinez and Vogel, 1966; Mangos, 1978). Denniss and Young (1975) showed that the transductal sodium flux across the adult rabbit submandibular secretory duct is reduced by acetylcholine. If this were to apply to striated duct cells from adults but not to the new-born, salivary sodium concentration would increase with age as a result of a changed response to stimulation, not as a result of changes in .jtriated duct cell structure per se. Some support for this interpretation can be gleaned from Mangos (1973) by noting that, at low flows, with presumably minimal inhibition of transductal sodium flux, immature parotid sodium concentration was higher than the adult at the same flows. The situation is complicated by the use of pilocarpine as the stimulant in most of these experiments on rats. Mangos
REFERENCES
Bradbury M. W. B., Crowder J., Desai S., Reynolds J. M., Reynolds M. and Saunders N. R. (1972) Electrolytes and water in the brain and cerebrospinal fluid of the foetal sheep and guinea-pig. J. Physiol. 227, 591-610. Burgen A. S. V. and Emmelin N. G. (1961) Physiology of the Salivary Glands (Edward) Arnold, London. Case R. M., Conigrave A. D., Novak I. and Young J. A. (1980) Electrolyte and protein secretion by the perfused rabbit mandibular gland stimulated with acetylchohne or catechoiamines. J. Physiol. 300, 467488. Cutler L. S. and Chaudry A. P. (1975) Cytodifferentiation of striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland. Am. J. Anal. 143, 201-218. Delattre J. P. and Yardin M. (1976) Examen histomorphologique de la glande sous-maxillaire du lapin (Oryctolagus cunictlus). J. Biol. buccale 4, 77-86.
Denniss A. R. and Young J. A. (1975) The action of neurotransmitter hormones and analogues and cyclic nucleotides and theophylline on electrolyte transport by
the excretory
duct of the rabbit mandibular
883 gland.
Pfliigers Arch. 357, 77-89.
Endre Z. H. and Youne J. A. (1981) Electrolvte content of primary and final siliva of rabbit mandibular glands studied in vivo. Adv. Physiol. Sci. 28, 29-34. Evans C. A. N., Reynolds J. M., Reynolds M. L., Saunders N. R. and Segal M. B. (1974) The development of a blood-brain barrier mechanism in foetal sheep. J. Physiol. 238, 371-386.
Hall H. D. and Schneyer C. A. (I 969) Physiological activity and regulation of growth of developing parotid. Proc. Sot. exp. Biol. Med. 131, 1288-1291. Holzgreve H., Martinez J. R. and Vogel A. (1966) Micropuncture and histologic study of submaxillary glands of young rats. Pfiigers Arch. 290, 134143. Jacoby F. and Leeson C. R. (1959) The postnatal development of the rat submaxillary gland. J. Anat. 93, 201-216.
Mangos J. A. (1978) Micropuncture study of post-natal functional maturation of the rat parotid. J. denr. Res. 57, 826833. Oikawa M.. Higuchi K., Mori T. and Okano T. (1976) Embryological and histochemical investigations on the submandibular glands in the rabbit fetuses. Report I. Histogenesis and carbohydrates in the submandibular gland of the fetuses. Aichi-Gakuin J. dent. Sci. 13, 349-364. Pull S. L. (1981) Chloride secretion by the neonatal rabbit submandibular gland. J. dent. Res. 60(B), 202.
Pull S. L. and Reynolds J. M. (1979a) The collection of saliva in the newborn rabbit, guinea-pig and rat. J. Physiol. 289, l-2P. Pull S. L. and Reynolds J. M. (1979b) Electrolyte secretion in the newborn rabbit submandibular gland. J. Physiol. 289, 88P.
Reynolds J. M. (1981) Anions in rabbit submandibular saliva. J. dent. Res. 60(B), 136. Reynolds J. M. and Webb B. W. (1982) Developmental changes in the structure and function of striated duct of rabbit submandibular gland. J. Physiol. 330, 32P. Schneyer C. A. and Hall D. H. (1968) Time course and autonomic regulation of development of secretory function of rat parotid. Am. J. Physiol. 214, 8088813. Schneyer C. A. and Hall D. H. (1969) Growth pattern of postnatally developing rat parotid gland. Proc. Sot. exp. Biol. Med. 130, 603-607. Schneyer C. A. and Schneyer L. H. (1961) Secretion by salivary glands deficient in acini. Am. J. Physiol. 201, 939-942.
Yardin M. (1968) Evolution des dents du jeune lapin. Mammalia 32, 677-689. Young J. A. (1979) Salivary secretion of inorganic electrolytes. In: MTP International Review of Physiology. Gastrointestinal Physiology III (Edited by Crane R. K.) Vol. 19, pp. l-58. University Park Press, Baltimore. Young J. A. and van Lennep E. W. (1978) The Morphology of Salivary Glands. Academic Press, London.
0003.9969/83 $03.00+0.00 Copyright CI 1983 Pergamon Press Ltd
THE CONCENTRATION OF SOJXUM, POTASSIUM AND CHLORIDE IN RABBIT SUBMANDIBULAR SALIVA DURING POSTNATAL DEVELOPMENT J. M. REYNOLDS and SYLVIA L. PULL Department
#ofPhysiology,
Royal Dental School, St George’s Hospital Medical School, Cranmer London SW17 ORE, England, U.K.
Terrace,
Summary-Saliva was collected from the cannulated ducts of submandibular glands of rabbits at various ages from birth to adulthood and analysed for sodium, potassium and chloride. Saliva was obtained at all ages by electrical stimulation of the parasympathetic innervation to the gland. On the day of birth (day I), salivary sodium was 108.5 + 3.4mM (n = 8) and chloride was 89.9 k 3.6 mM (n = 9) falling to adult-like concentrations of 5.7 + 0.6 mM (n = 13) and 16.5 k 1.2mM (n = 9) respectively by day 10. On Day 1, salivary potassium was 23.9 + 1.8mM (n = 7) and rose progressively to 76.8 + 2.5 mM (n = 8) on day 17. Adult potassium concentration was 30-40mM. A wide range of flows was obtained at all ages and at each age sodium and chloride concentrations were approximately constant at all flows. Therefore the changes in electrolyte concentration cannot be explained by differences in flow. The results suggest a maturation of ductal transport of sodium, potassium and chloride ions concurrent with the structural maturation #of striated duct cells.
INTRODUCTION
consistent with the structural changes which occur during the development of striated duct cells. The methods were demonstrated to the Physiological Society (Pull and Reynolds, 1979a) and preliminary results were reported also (Pull and Reynolds, 1979b; Pull, 1981).
We are studying the function and structure of the neonatal rabbit submandibular gland in order to gain insight into the processes involved in the development of secretory cells. This development could be expected to comprise several related factors e.g. cellular differentiation and division, functional maturation of newly-formed cells, the establishment of stimulus-secretion coupling, and could be subject to both genetic and environmental influences. Major salivary glands have two main secretory elements: acinar ceils which produce a primary secretion and striated ducts which can substantially modify that secretion. In ontogeny, the ducts develop first and in many speces acinar development has barely begun at birth (Jacoby and Leeson, 1959; Young and van Lennep, 1978). This is often interpreted incorrectly as indicating the absence of acinar cells during duct proliferation. In fact, a few acinar cells are present, at least as terminal buds, and it is probably more correct to describe duct growth as initially outstripping acinar development, the latter becoming predominant as duct growth nears completion. Previous studies of the function of developing salivary glands (Hall and Schneyer, 1969; Mangos, 1978) have been on rat glands but at ages when striated ducts were. already fully formed (Cutler and Chaudry, 1975). Studies of rat glands are complicated by the development of convoluted granular tubules between the striated and intercalated ducts. In contrast, the adult rabbit submandibular gland has short (two or three cells long) intercalated ducts. This suggests that the long ducts identified (LM) in prenatal and newborn rabbits by Oikawa et al. (1976) and Delattre and Yardin (1976) as “intercalated ducts” could be immature striated ducts. Preliminary observations confirm this (Reynolds and Webb, 1982), and we here argue that the functional characteristics of the new-born gland, and the functional changes occurring in the first ten days of life, are
MATERIALS AND METHODS
New Zealand White rabbits fed ad libitdm on a standard laboratory diet (RGP, Labsure, containing 0.7 per cent by weight NaCl) were used to provide litters. All offspring were left with their dams until required or until weaned (6 weeks). Anaesthetic doses (25 per cent urethane in 0.9 per cent saline) were 12ml/kg for animals up to 7 days, lOml/kg from 8 days to 1 month and 7 ml/kg thereafter. Initial anaesthetic administration was intraperitoneal in animals younger than 3 months old and intravenous in older animals. The post-mortem weights of five animals under 14 days were identical with their preexperimental weights, indicating no net fluid-loss as a result of the experimental procedures. The anaesthetized animals were placed supine on a heated operating table and body temperature maintained at 39°C (rectal probe). For administration of further anaesthetic, a femoral vein was cannulated using polythene tubing (Portex PP202) drawn down over an alcohol flame to outside diameters of 4s120pm (Evans et al., 1974). The trachea was not routinely cannulated in the very young animals (unless there was obvious respiratory difficulty) in order to leave the surgical area unobstructed. The submandibular duct was approached through the mylohyoid muscle and cannulated (see Pull and Reynolds 1979a,b for details). The chorda tympani (parasympathetic innervation) runs along the duct: cannulation therefore prevents any reflex parasympathetic stimulation. The duct was mounted on bipolar stimulating electrodes, the electrodes and duct 879
880
J. M. REYNOLDS and SYLVIA L. PULL Table
Age (days) (range)
I 2 3 4 5 6.75 (6-7) 10.8 (9-13) 17
1. Details
Body weight n
3 4 3
(g)
of new-born
Gland
animals
weight (g)
used and their maximum
Gland
weight
Body weight
%
secretion
rates
Maximum secretion rate
Maximum secretion rate (pl.min-‘.g-‘) of
(pl.min-I)
gland tissue
4
64.3 _+5.3 67.0 + 5.4 63.7 + 6.0 (I 17.0) 79.5 i: 10.4
0.0702 + 0.0070 0.0907 + 0.0 196 0.0898 * 0.0255 (0.1284) 0.1002 + 0.0178
0.11 0.14 0.14 (0. I I) 0.13
3.77 * 2.21 7.28 i 2.4 5.35 k 2.29 (8.9) 6.69 f 1.39
49.19k24.85 91.27 k 39.30 56.19 + 16.21 (69.5) 66.27 i 12.53
4
121.5 + 15.2
0.1312 _+0.0233
0.11
3.62 k I .06
30.86 &-10.21
5 2
180.8 + 23.6 490
0.1808 k 0.0360 0.3896
0.10 0.08
3.15 i 0.82 9.19( + 0.33)
20.92 k 6.71 31.45 k4.15
1
All values are mean + SE. From other experiments, the size of the submandibular gland, relative to body weight, continues to decline with age. The maximum secretion rate for each age group is the mean of the highest flows from each individual gland. Note, these are not instantaneous flows: the minimum collection time was 5 min.
being electrically isolated from surrounding tissue by Klingfilm and flooded with liquid paraffin. The adult
rabbit submandibular gland does not secrete in response to sympathetic stimulation. Preliminary experiments showed this also in neonates. Sectioning the sympathetic trunk made no measurable difference to the saliva electrolytes and in most experiments the trunk was left intact. The stimulus was an isolated (Neurolog: NL800) square wave (1 ms, maximum 10 V, 1 mA) of 0.5-20 Hz frequency. Electrical stimulation invariably commenced at 20 Hz (1 ms sq. wave). In some older animals, low flows were obtained by decreasing the stimulus frequency. Even in cases where stimulus frequency was reduced, all glands were stimulated continuously in order to avoid the possibility of ion transients (Burgen and Emmelin, 1961). Attempts to induce transients by increasing stimulus frequency produced no detectable change in potassium or sodium concentrations in the neonatal animals. Saliva was collected into pre-weighed microcentrifuge vials either directly or after passing through a capillary tube. The flow measured by timing the movement of an air bubble along the capillary was similar to the gravimetrically determined flow. Secretion rates varied between 167nl,min-’ and 13.1 pl.rnin-‘. Fifteen to twenty microlitres was required to measure the three ions and in some cases therefore only one or two of the ions could be determined if collection time was not to be unreasonably long. Sodium and potassium concentrations were measured with an EEL Flame Photometer using procedures similar to those described by Bradbury et al. (1972). Chloride concentrations were measured by a chloridometer (Buchler Instruments, Searle Analytic). RESULTS Saliva flow was elicited by electrical stimulation from cannulated submandibular glands from the day of birth (day 1). As with the isolated gland (Case et al., 1980), flow decreased with duration of a constant
stimulus. A slow (co.1 pl.min.g-’ gland wt) unstimulated secretion was noticed as early as the
second day of life, but all the results reported here are for stimulated saliva, The maximum secretion rates obtained for each age group (Table 1) showed no age dependence. It must be stressed that these maximal flows were (a) not instantaneous, (b) may reflect developmental differences in stimulus-secretion coupling, and (c) may reflect differences in the number of fibres actually stimulated. The sodium (and chloride) concentrations showed little flow dependence at any age (Fig. 1) including the adult (Pull and Reynolds, 1979b), although in the youngest animals there was a suggestion of lower concentrations at higher flows. However, there was a marked difference in the ion concentrations between the age groups, high concentrations at birth declining to adult concentrations by the ninth day. Saliva electrolyte concentration is often flow- and timedependent (Young, 1979) but, clearly, these sources of variation cannot account for the changes in sodium concentration observed during the neonatal period. On the first day of life (day l), saliva sodium concentration was 108.5 + 3.4 mM (n = 8), declining to less than half this figure by day 4 and to an adult-like concentration of 5.7 f 0.6 mM (n = 13) by days 9-l 1 (Fig. 2). During the first 5 days of life body weight and gland weight were almost constant, suggesting that the changes in sodium concentration during this period were not related to changes in body or gland size. Similarly, body weight increased rapidly after day 11 but no further changes in sodium concentration occurred. The time course for the change in chloride concentration with age (Fig. 3) was similar to the time course for the change in sodium. Chloride concentration was 89.9 + 3.6 mM (n = 9) on day 1 and on days 9-l 1 was 16.5 k 1.2 (n = 9). Potassium concentration was much more variable even though precautions were taken to avoid transients. Thus for potassium there is a much less clearly-defined difference between age groups than for sodium and chloride (Fig. 4). As with sodium and chloride, the differences between ages cannot be explained in terms of changes of concentration with secretion rate. It is surprising that the progressive rise
Saliva composition l-2 A
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A
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Fig. 1. Sodium concentrations in rabbit submandibular saliva at l-2, 46 and lb-17 days of age. Saliva flow was elicited by continuous stimulation of the parasympathetic innervation to the cannulated gland. Flow is expressed per unit gland weight and each point represents a single sample.
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Age ( doys 1 Fig. 2. Sodium concentrations in rabbit submandibular saliva during development. Day 1 = date of birth. Saliva flow was elicited by continuous stimulation of the parasympathetic innervation to the cannulated gland. As the sodium concentration showed little flow dependence (Fig. I), points are plotted irrespective of flow. N.B. Some points overlap, e.g. 8 samples are represented at day 1.
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Age (days I Fig. 3. Chlorde concentrations in rabbit submandibular saliva during development. The range of chloride concentrations at 47-56 days corresponds to a wide range of potassium concentrations found in juvenile but not adult animals.
J. M.
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and
REYNOLDS
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Fig. 4. Potassium concentration in rabbit submandibular saliva at 1-2, 4-6 and l&17 days of age. Flow is per unit gland weight and each point represents a single sample collected under continuous stimulation. The range of potassium concentrations found in juvenile animals does not appear to be due to transients.
in potassium concentration was up to nearly 80 mM at 17 days when the adult concentration is almost always 3&40 mM. Thus the pattern of development for potassium secretion is quite different from that for sodium and chloride. Reynolds (1981) suggested that protein concentration may exert a profound effect on potassium concentration in rabbit submandibular saliva. In adult rabbits, this is reflected by a marked deficit (up i.e. 150mM) of saliva anions to Na + K 9 Cl + HCO; . In experiments with neonates, small sample size precluded the measurement of bicarbonate and total protein, but the charge difference between the measured cations (Na+ and K + ) and anion (chloride) varied only between 28 and 62mM. Assuming that a proportion of this was bicarbonate, the anion deficit was not large enough to suggest that protein is heavily influencing the ionic composition of neonatal saliva. DISCUSSION
When this study began it was expected that the ion concentraticns would reflect age differences in the relative cell populations of acini and striated ducts (Burgen and Emmelin, 1961). Thus, as the ducts develop first, it was expected that ductal function (the removal of sodium and the addition of potassium) would be most marked early in life and new-born saliva would have a high potassium and low sodium concentration. This would have given the opportunity to study (a) the development of acinar cell function and (b) the functions of the striated duct alone. The results actually obtained suggest that the most notable functional changes are affecting the ducts and not the acini. The sodium, potassium and chloride concentrations in whole saliva at birth were similar to the concentrations in the primary secretions from adult acinar cells (Young, 1979; Endre and Young, 1981). Assuming that a given cell type does not reverse its ion fluxes, it seems most probable that at birth the final saliva is an almost unmodified primary secretion
despite the higher proportion of duct cells to acinar cells than later in life. If the acinar cells at birth are able to produce an adult-type primary secretion, it follows that any changes in the secondary modifications of saliva must reflect a change in duct function. In that case, certainly for sodium and chloride, the capacity of the ducts to transport these ions increases during the first nine days of life. This could be due to changes in duct response to stimulation, but preliminary studies (Reynolds and Webb, 1982) indicate no striations and few mitochondria in striated ducts at birth, suggesting that the duct cells themselves are maturing during this period. Oikawa et al. (1976) remarked on the absence of striae in the ducts of 21-day rabbit fetuses. Oikawa et al. appear to have mistakenly identified striated ducts from neonatal rabbits as intercalated ducts, an indication of how immature-looking are the neonatal ducts. Cutler and Chaudry (1975) describe in detail the changes in striated-duct cell structure in the rat fetus. At 18 days, there are few basal infoldings, lateral projections and mitochondria whereas, at 20 days, the cells have a much more adult-like striated appearance due to many basal infoldings and mitochondria. It appears that in the rabbit these changes occur postnatally. The potassium results can also be explained in terms of increasing duct function. It is clear, however, that the time-course of events for potassium is quite different from that of sodium. This could indicate that the duct-handling of sodium and potassium is by separate mechanisms. The apparent developmental changes are not due simply to increased body weight or gland weight as periods of growth do not correspond with periods of marked change in saliva electrolytes. It seems possible that the changes are genetically determined but factors such as tooth development (Yardin, 1968), diet or hormones have yet to be excluded. For example, Hall and Schneyer (1969) showed that salivary gland growth is inhibited in rats fed on a liquid diet. In contrast to the above interpretation of salivary gland development, Schneyer and Hall (1968) and
Saliva composition in neonatal development (1978) reported that the sodium concentration in rat parotid saliva (stimulated) increased over the first months of life. This change was attributed to an age-dependent decrease in transductal sodium flux. No such change was observed in rat submandibular saliva (Schneyer and Schneyer, 1961). At the ages studied, both these rat glands had morphologically-distinct striated ducts (Jacoby and Leeson, 1959; Schneyer and Hall, 1969) but developmental changes were still occurring in the acini and convoluted granular tubules. It seems unlikely that this acinar development can explain the low sodium in immature paroticl saliva as, if anything, the primary sodium concentration is likely to be greater than in the adult (Holzgreve, Martinez and Vogel, 1966; Mangos, 1978). Denniss and Young (1975) showed that the transductal sodium flux across the adult rabbit submandibular secretory duct is reduced by acetylcholine. If this were to apply to striated duct cells from adults but not to the new-born, salivary sodium concentration would increase with age as a result of a changed response to stimulation, not as a result of changes in .jtriated duct cell structure per se. Some support for this interpretation can be gleaned from Mangos (1973) by noting that, at low flows, with presumably minimal inhibition of transductal sodium flux, immature parotid sodium concentration was higher than the adult at the same flows. The situation is complicated by the use of pilocarpine as the stimulant in most of these experiments on rats. Mangos
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Denniss A. R. and Young J. A. (1975) The action of neurotransmitter hormones and analogues and cyclic nucleotides and theophylline on electrolyte transport by
the excretory
duct of the rabbit mandibular
883 gland.
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Endre Z. H. and Youne J. A. (1981) Electrolvte content of primary and final siliva of rabbit mandibular glands studied in vivo. Adv. Physiol. Sci. 28, 29-34. Evans C. A. N., Reynolds J. M., Reynolds M. L., Saunders N. R. and Segal M. B. (1974) The development of a blood-brain barrier mechanism in foetal sheep. J. Physiol. 238, 371-386.
Hall H. D. and Schneyer C. A. (I 969) Physiological activity and regulation of growth of developing parotid. Proc. Sot. exp. Biol. Med. 131, 1288-1291. Holzgreve H., Martinez J. R. and Vogel A. (1966) Micropuncture and histologic study of submaxillary glands of young rats. Pfiigers Arch. 290, 134143. Jacoby F. and Leeson C. R. (1959) The postnatal development of the rat submaxillary gland. J. Anat. 93, 201-216.
Mangos J. A. (1978) Micropuncture study of post-natal functional maturation of the rat parotid. J. denr. Res. 57, 826833. Oikawa M.. Higuchi K., Mori T. and Okano T. (1976) Embryological and histochemical investigations on the submandibular glands in the rabbit fetuses. Report I. Histogenesis and carbohydrates in the submandibular gland of the fetuses. Aichi-Gakuin J. dent. Sci. 13, 349-364. Pull S. L. (1981) Chloride secretion by the neonatal rabbit submandibular gland. J. dent. Res. 60(B), 202.
Pull S. L. and Reynolds J. M. (1979a) The collection of saliva in the newborn rabbit, guinea-pig and rat. J. Physiol. 289, l-2P. Pull S. L. and Reynolds J. M. (1979b) Electrolyte secretion in the newborn rabbit submandibular gland. J. Physiol. 289, 88P.
Reynolds J. M. (1981) Anions in rabbit submandibular saliva. J. dent. Res. 60(B), 136. Reynolds J. M. and Webb B. W. (1982) Developmental changes in the structure and function of striated duct of rabbit submandibular gland. J. Physiol. 330, 32P. Schneyer C. A. and Hall D. H. (1968) Time course and autonomic regulation of development of secretory function of rat parotid. Am. J. Physiol. 214, 8088813. Schneyer C. A. and Hall D. H. (1969) Growth pattern of postnatally developing rat parotid gland. Proc. Sot. exp. Biol. Med. 130, 603-607. Schneyer C. A. and Schneyer L. H. (1961) Secretion by salivary glands deficient in acini. Am. J. Physiol. 201, 939-942.
Yardin M. (1968) Evolution des dents du jeune lapin. Mammalia 32, 677-689. Young J. A. (1979) Salivary secretion of inorganic electrolytes. In: MTP International Review of Physiology. Gastrointestinal Physiology III (Edited by Crane R. K.) Vol. 19, pp. l-58. University Park Press, Baltimore. Young J. A. and van Lennep E. W. (1978) The Morphology of Salivary Glands. Academic Press, London.