Oral Physiology: Proceedings of the International Symposium Held in Wenner-Gren Center, Stockholm, August 1971

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OPENING ADDRESS

YNGVE ZOTTERMAN

WHEN in the 1920s, as a junior assistant in Physiology at Karolinska Institutet, I had to assist the professor in his teaching of the odontology students, I clearly understood that this teaching needs its own specialists in oral physiology. It subsequently became obvious that the odontologists had great difficulties in finding proper advice and facilities for their own basic research which could not be met without the building up of special centres devoted to oral physiology. Institutes for oral physiology have been erected at several places in Japan, U.S.A., and a few other countries, but most dental schools in the world like those in Sweden still lack such departments under the guidance of experts in oral physiology.

For these reasons we considered it important to invite a number of physiologists active within this field of research to this symposium to discuss common problems and trends. The information given at this meeting will not only be useful for the researchers and teachers of oral physiology, but will, I hope, also promote the understanding that oral physiology should be entitled to receive more extensively its own departments to the benefit not only of this important branch of physiology but also to odontology and medicine in general.

In an era when physiologists become more and more specialized I believe that it is important that we should come together and discuss our problems in the presence of specialists in other fields of the science. For example, the study of the function of the nervous system should not only be limited to neurologists pursuing electrophysiological studies. It is necessary to face the problem from different angles and it is of greatest importance that scientists and clinicians should speak the same scientific language.

It is the aim of this conference and this volume to spread some information about what is done in oral physiology which spans over a wide field where the problems have been approached from different angles and been attacked by different methods. This adds more variety and colour to our discussions. Also we may come to different opinions. But it is not the possession of truth but the seeking of truth which characterizes cultured men.

CONTROL OF SALIVARY GLANDS

NILS EMMELIN,     Institute of Physiology, University of Lund, Sweden

Publisher Summary

The function of the salivary gland system is characterized by the continuous resting secretion upon which intermittently an enormously increased activity is superimposed. The saliva manufactured by a single gland is highly variable. The gland consists of a number of different types of effector cells, concerned with the production and discharge of the saliva. The various cells of the acini are divided into serous and mucous cells. This chapter discusses the way the activity of these various effectors is regulated. In some salivary glands, the secretory machinery is permanently active, continuously producing and discharging saliva, even in the absence of extraneous stimuli. The salivary glands provide one of the best examples of a control mechanism exerted by a local hormone. The kinins of interest for the regulation of the blood flow, kallidin and bradykinin, have no secretory effect in the glands in reasonable dosage. The nerves play the main part in the control of salivary glands. After denervation or decentralization, many of the glands show considerable atrophy. As a long-term function, the nerves regulate the sensitivity of the gland cells to secretory stimuli, and it applies to the parasympathetic innervations.

THE conception generally expressed in textbooks of physiology that the activity of salivary glands is controlled by nerves from both divisions of the autonomic nervous system, parasympathetic nerves causing a lively flow of thin saliva and sympathetic nerves sparse amounts of viscous fluid, originates from classical experiments largely on the submaxillary gland of the dog carried out by Ludwig, Bernard, Heidenhain and others in the latter half of the previous century. It would be equally correct to state that the sympathetic nerves cause no secretion at all, as in the parotid of the dog (Babkin, 1950) or a copious flow of watery saliva, as in the submaxillary gland of the cat (Langley, 1898). This may serve to illustrate the important fact that in the case of the salivary glands there are great species variations, and also considerable differences between the glands in the same species.

The existence of a rather elaborate and complicated control of the salivary glands can be suspected from the fact that the composition of the saliva, poured into the mouth, can be varied over a wide range, and according to the views of Pavlov, also expressed by later investigators (Baxter, 1933; Montgomery and Stuart, 1936), it can be varied in a seemingly purposeful way, adapted to the manifold digestive, protective and other functions of the saliva. The more precise mechanism through which such an adaptation of the secretion to the quality of the stimulus can take place is unknown. On the receptor side it may be noted that secretion of saliva can be elicited by stimulation of a great variety of receptors, gustatory, olfactory, tactile, nociceptive. Similarly, on the effector side the conditions for great variability in the composition of the saliva are fulfilled. There is a large number of different salivary glands, producing juices of different composition, and the contributions of all the glands which together form the mixed saliva may vary considerably. Furthermore, the saliva manufactured by a single gland is highly variable. Partly at least this is due to the fact that the gland consists of a great number of different types of effector cells, concerned with the production and discharge of the saliva: the various cells of the acini, which in a probably very primitive way are divided into serous and mucous cells; the cells lining the duct systems, the importance of which for the formation of saliva is increasingly recognized; the smooth muscles of the blood vessels, which supply the raw material for the juice; and the myoepithelial cells, assumed to contribute to the flow of saliva. It is the purpose of the present paper to examine how the activity of these various effectors is regulated.

I THE SECRETORY CELLS OF THE GLANDS

The function of the salivary gland system is characterized by the continuous resting secretion upon which intermittently an enormously increased activity is superimposed. The resting flow, which may be very slow for instance during sleep, serves to keep the mucosa of the mouth and throat moist; the lively secretion occurs particularly during meals and in some species, such as dogs and rats, as part of a thermoregulatory response also. Because of the very brief time period which the food spends in the mouth a regulatory mechanism is obviously required which is able to raise the secretory activity very rapidly to a high level, and there can be no doubt that secretory nerves are responsible for the control of this function of the salivary glands. A more prolonged activity, for the non-digestive functions of the saliva, could be maintained by some other mechanism. A number of different ways in which the secretory cells can be made to enter into activity may now be considered.

1 Spontaneous Secretion

It should first be emphasized that in some salivary glands the secretory machinery is permanently active, continuously producing and discharging saliva, even in the absence of extraneous stimuli. Babkin (1950) proposed the term spontaneous secretion for this type of activity. It should not be used as a synonym for resting secretion which may have a component of spontaneous activity but is usually to a great extent of reflex origin, the dryness of the oral or pharyngeal mucosa serving as stimulus. In a well-anaesthetized animal most salivary glands are silent, but from some glands a flow can be recorded which cannot be abolished by the ordinary pharmacological blockers of salivary secretion and which persists even in an isolated gland. When a number of species are studied it can be observed that examples of spontaneous secretion can be found in parotid, submaxillary, sublingual and small mucosal glands; so far no histological or electrophysiological features characteristic of such glands have been described. The French physiologist Colin seems to have been the first to report that in the ox the parotid differs from the submaxillary gland in secreting continuously (see Milne-Edwards, 1860), and later investigations have shown that this gland secretes spontaneously in ruminants (Eckhard, 1867, 1893; Brettel, 1869; Moussu, 1890; Coats et al., 1956; Kay, 1958). In cats, dogs and rats the sublingual gland is responsible for the spontaneous flow, and in rabbits the submaxillary gland (Emmelin, 1953; Nordenfelt and Ohlin, 1957; Ohlin and Perec, 1965). Figure 1 shows spontaneous secretion from a sublingual gland of a cat under chloralose anaesthesia. In such a cat there is no submaxillary or parotid secretion, but from the small sublingual gland saliva is discharged at a slow, constant rate. A brief period of stimulation of the secretory nerves, parasympathetic or sympathetic, greatly accelerates the secretion.

FIG. 1 Spontaneous secretion from sublingual glands of two cats under chloralose anaesthesia. Each column represents millilitres of saliva collected during 10 min into a pipette, connected to a cannula in the sublingual duct. The high columns show the effect of nerve stimulation in the beginning of a collection period. Left: chorda stimulation during 30 sec. Right: sympathetic stimulation during 1 min. (From Emmelin, Acta physiol. scand. Suppl. 111, 1953, 34–58.)

2 Endocrine Control of Salivary Glands

Attempts have been made to demonstrate a hormonal control mechanism of salivary glands analogous to those acting on the gastric and pancreatic glands, but they have met with no success (Sachs and Kim, 1929). The only hormones known to cause salivation are the catecholamines, which have a secretory effect on certain salivary glands, but normally their concentration in the blood is far below the threshold for salivary secretion. This should by no means be taken to imply that hormones do not play an important role in the function of salivary glands. There is a comprehensive literature dealing with the effect of hormones, and of lack of hormones, on salivary glands (see Ohlin, 1966; Blair-West et al., 1967). For instance, hypophysectomy greatly reduces the secretory capacity of the glands and lowers their sensitivity to sialogogue drugs. Testosterone and thyroxine have the opposite effect. Thus, even if there is no evidence showing that hormones released in the course of physiological events can evoke salivary secretion, hormones may well be assumed to modulate the responses of the glandular cells to the normal sialogogue stimuli. Hypersalivation sometimes seen in pregnancy may be due to such a modulating effect of hormones at a central or glandular level; cases have also been described in which women suffer from xerostomia except during pregnancy.

3 Control by Local Hormones

The salivary glands provide one of the best examples of a control mechanism exerted by a local hormone: the local production in a secreting gland of a kinin dilating the vascular bed of the gland and causing functional hyperaemia, which promotes secretion (see Gautvik in this volume). It does not seem unreasonable to hypothesize that a similar mechanism operates to maintain the flow of saliva, but so far there is little evidence to support this view. The kinins of interest for the regulation of the blood flow, kallidin and bradykinin, have no secretory effect in the glands in reasonable dosage. There are, however, some other polypeptides which are very powerful as sialogogues; in some salivary glands they are, in fact, the most active secretory agents known. Admittedly, the first discovered peptides of this group, the endecapeptides physalaemin and eledoisin, are not known as constituents of the mammalian body, the one originating from a South American amphibian, the other from a mollusc (Erspamer and Erspamer, 1962; Bertaccini and de Caro, 1965). Later, however, it has been found that a more familiar polypeptide, substance P, has a similar effect (Lembeck and Starke, 1968), and from bovine hypothalamus a sialogogue polypeptide, probably related to substance P, has been extracted (Leeman and Hammerschlag, 1967). Since all these polypeptides have very marked vascular effects in doses which cause salivation, they do not seem suitable as true hormones; but it does not appear too far-fetched to consider the possibility that a polypeptide of this type may act as a local hormone, causing secretion or modifying the effect of normal stimuli. In this connexion species differences should be kept in mind. The polypeptides now mentioned evoke secretion of saliva in dogs and rats, and also in hens, but not in cats, rabbits or guinea-pigs; their effect in humans is unknown. An interesting feature is that these compounds cause a flow of saliva which cannot be abolished by the ordinary blockers of salivary secretion, parasympatholytics, or by blockers of α- or β-adrenoreceptors (Bertaccini and de Caro, 1965; Emmelin and Lenninger, 1967); it was formerly taken for granted that drugs can evoke secretion of saliva only by activating the secretory nerves or imitating the action of their transmitters. Figure 2 shows the secretory effect of physalaemin on a submaxillary gland of a dog. The drug was injected through the salivary duct into the gland to avoid general circulatory disturbances. For comparison the effects of methacholine and of the β-receptor stimulating isoprenaline are demonstrated. After a β-receptor blocking drug, INPEA [D-(—)-N-isopropyl-p-nitrophenyl ethanolamine] the effect of isoprenaline disappeared, whereas that of methacholine and physalaemin remained. Atropine would have abolished the response to methacholine but left that to physalaemin intact.

FIG. 2 Submaxillary secretion in a dog under chloralose-urethane anaesthesia. Records from above: Time in minutes; signal to mark injections of drugs; drops of saliva before; about 10 min after intravenous injection of INPEA 5 mg/kg; and about 10 min after a further dose of INPEA 5 mg/kg. Sections to the left: injections of isoprenaline 2 μg/kg intravenously. To the right: methacholine 1 μg/kg intravenously. Intermediate sections: physalaemin, 1 μ-g/kg in 0.5 ml saline solution injected through the salivary duct into the gland. (From Emmelin and Lenninger, Br. J. Pharmac. Chemother. 1967, 30, 676–680.)

4 Control by Secretory Nerves

There can be no doubt that nerves play the main part in the control of salivary glands. After denervation or decentralization many of the glands show considerable atrophy. As a long-term function the nerves regulate the sensitivity of the gland cells to secretory stimuli, and this applies particularly to the parasympathetic innervation (see Emmelin, 1952, 1961a, 1965). Increased stimulation of the glands, for instance by repeated injections of pilocarpine, carbachol or eserine, lowers the sensitivity of the glandular cells to secretory stimuli. It rises, on the other hand, if the influence of the nerves on the gland is diminished or abolished. This can be achieved in different ways: by section of afferent nerves from the mouth, which reduces the inflow of impulses to the salivary nuclei and thus the outflow of secretory impulses to the glands; by section of preganglionic secretory nerves and even more by section of postganglionic nerves, which not only deprives the gland of secretory stimuli but also of randomly leaking transmitter; by injection into the gland of botulinum toxin, which interferes with the release of acetylcholine; or by prolonged treatment with ganglion blocking or atropine-like drugs, which abolishes the action of the transmitter at the ganglionic or neuroglandular level, respectively. Supersensitivity to secretory agents has even been demonstrated in humans treated for a long time period with atropine (Mózsik et al., 1967).

The most conspicuous effect of eliminating the influence of the nerves on the glands is the immediate cessation of salivary secretion, showing that the main control mechanism is nervous. However, this does not apply to all salivary glands, or to all situations. Some secretion may persist or reappear under certain conditions, as shown by the following examples.

(a) Glands endowed with the ability to secrete spontaneously continue after denervation to discharge saliva at a constant rate, as already mentioned.

(b) About a day after section of postganglionic parasympathetic nerves all salivary glands are found to be in a state of secretory activity, which lasts for one or two days. This is the so-called degeneration secretion of saliva, which is due mainly to a temporary increase in the leakage of acetylcholine from the degenerating nerves (Emmelin and Strömblad, 1958; Emmelin, 1962, 1967a; Ohlin, 1963; Nordenfelt, 1964). Figure 3 shows the onset and gradual increase of the degeneration secretion in a submaxillary gland of a cat. Under certain conditions this phenomenon can be seen shortly after sympathetic denervation of salivary glands also; the secretion is now elicited by noradrenaline released from the degenerating postganglionic sympathetic neurones (Coats and Emmelin, 1962).

FIG. 3 Onset of degeneration secretion from a submaxillary gland of a cat under chloralose anaesthesia. Above each section is shown the time in hours that had passed since (partial) postganglionic parasympathetic denervation had been carried out; this operation had been made using a short-acting barbiturate, and the cat had then been allowed to wake up. Upper record: time in minutes; lower record: drops of saliva. Three weeks before the experiment the preganglionic parasympathetic fibres had been cut. This operation increases the degeneration secretion because it sensitizes the gland cells, but it is not necessary for the phenomenon to appear. (From Emmelin, J. Physiol. 1962, 162, 270–281.)

(c) The paralytic secretion once described by Claude Bernard (1862, 1864) is, likewise, a consequence of nerve section. It appears, however, only under exceptional conditions: catecholamines circulating in the blood evoke the secretion, and the phenomenon can, therefore, only be obtained in salivary glands with adrenoreceptors for secretion. Furthermore, these glands have to acquire a pronounced supersensitivity to the catecholamines, as may be the case two or three weeks after parasympathetic decentralization or denervation, and even then secretion is only obtained if there is a very marked release of the hormones from the adrenal medulla (Emmelin, 1952).

(d) In experiments on paralytic secretion Heidenhain (1868) observed not only secretion from a decentralized submaxillary gland, but a very slow flow from the contralateral gland also. This antilytic secretion (Langley, 1885) had very likely the same cause as the paralytic secretion: catecholamines acting on supersensitive gland cells. Sensitization of the normal gland was obtained when the other gland was decentralized by section of the chorda-lingual nerve, which also entails section of afferent fibres from the mouth (Emmelin, 1961b).

(e) It is quite common that some secretion can still be obtained reflexly after attempted denervation of salivary glands for the simple reason that the denervation was incomplete; we are namely remarkably ignorant about the anatomy of the secretory nerves of salivary glands (see Holmberg in this monograph). Under such conditions the reflexly induced response may increase with time because supersensitivity develops even in cells only partly denervated. In addition, the effect may rise because of collateral regeneration from the remaining secretory axons.

Parasympathetic secretory nerves

seem to be distributed to all salivary glands, and by way of these nerves the central nervous system can vary the flow rate and the composition of the saliva over a wide range. The salivary flow rate rises with the frequency of the impulse discharge in these nerves up to a maximum, which in the submaxillary gland of the dog is reached at 10–12 impulses/sec (Emmelin and Holmberg, 1967a). This is shown in Fig. 4. In a number of anaesthetized dogs the secretory rate was estimated at different frequencies of stimulation of the chorda-lingual nerves and expressed as apercentage of the maximum obtainable at nerve stimulation. Earlier the same dogs had been examined without anaesthesia and with polyethylene tubes in their submaxillary ducts; under these conditions the highest flow rates that could be elicited by feeding had been estimated. As shown in the figure the physiological discharge frequency in the secretory nerves scarcely exceeds 8 impulses/sec.

FIG. 4 Frequency-response relations in submaxillary glands of seven dogs under chloralose-urethane anaesthesia. Abscissa: shocks/sec applied to the chorda-lingual nerve. Ordinates: secretory rate in percent of maximum obtainable by stimulating the nerve. The vertical lines show the range within which secretion had been obtained when the glands were maximally activated by feeding. (From Emmelin and Holmberg, J. Physiol. 1967, 191, 205–214.)

The glandular cells seem to be well supplied with parasympathetic axons. Lundberg (1958) inserted a microelectrode into acinar submaxillary cells of cats and found that a single shock applied to the chorda-lingual nerve, containing the preganglionic parasympathetic fibres of the gland, caused hyperpolarization. When the strength of the shocks was increased gradually the potentials could be seen to increase stepwise (Fig. 5) and from such observations it was concluded that there is a marked convergence in the parasympathetic pathway of the gland. This can be further studied in another type of experiment (Ekström and Emmelin, 1971). The hypoglossal nerve is sewn to the chorda-lingual—the two nerves are cut and the central ends of both are connected to the peripheral end of the chorda-lingual nerve—and time is left for regeneration. The sensitivity of the gland cells to chemical stimuli is estimated at monthly intervals and used as an indicator of regeneration. Decentralization of the gland by chorda-lingual nerve section causes supersensitivity, but it is found to disappear gradually as reinnervation is established, and after some months a preparation has been obtained in which two nerves produce secretion on electrical stimulation central to the nerve suture: the chorda-lingual and the hypoglossal nerves. The secretory responses to both are abolished by ganglionic blocking drugs and by atropine, showing that regenerated chorda and hypoglossal fibres have reinnervated the postganglionic, parasympathetic neurone. If the two nerves are stimulated together at frequencies which are just below threshold for each nerve, no secretion is obtained; or, when one nerve is excited at a frequency which causes secretion, the response cannot be augmented by additional stimulation of the other nerve at a subliminal frequency. Thus, no facilitation is obtained, and the implication seems to be that at the ganglionic level there is very little overlap between the fields of distribution of the preganglionic axons. These findings contrast to those obtained when both nerves are stimulated at frequencies high enough to set up impulses in the postganglionic pathway reinnervated by each nerve. Pronounced facilitation can then be observed; when the frequencies are chosen so that each nerve causes just perceptible flow of saliva, the combined stimulation evokes a rapid salivation, indicating that there is marked convergence of postganglionic parasympathetic neurones onto the glandular cells. This corresponds to the finding by Garrett (1966) in electron microscopical work that several axons can be seen near the single gland cell. If, on the other hand, each nerve is stimulated at a frequency producing largest possible effect it can often be observed that this maximum is about the same for both nerves, and of the same magnitude as the maximum obtainable by injection of pilocarpine. It can then be shown that the response to simultaneous stimulation of the two nerves is not larger than that to each nerve separately. This is illustrated in Fig. 6, in which the rate of salivary flow was recorded using an ordinate recorder, each ordinate measuring the time interval between two drops of saliva. Stimulation of the hypoglossal nerve caused rapid secretion, but the flow rate characteristically soon decreased. The chorda-lingual nerve was then stimulated central to the suture. This evoked a secretion as fast as that seen during hypoglossal stimulation, and the flow rate was well maintained. Finally the hypoglossal nerve was excited for a long period, and on three occasions the chorda-lingual nerve was stimulated as well. It can be seen that the most rapid flow caused by the hypoglossal nerve could not be accelerated by additional chorda-lingual stimulation; this was possible only when the hypoglossal secretion had slowed down. It is obvious that at the high frequencies of stimulation both sets of nerves are able to activate maximally the same secretory cells. The morphological basis may be that the axons, according to Garrett (1966), form en-passant contacts with many glandular cells. The possibility, suggested by Burgen (1967), that activity may spread from cell to cell within an acinus may also have to be considered; nexuses have been described in the submaxillary gland of the rat (Dewey and Barr, 1964).

FIG. 5 Electrical responses obtained with intracellular electrode in acinar cell of a submaxillary gland of a cat under chloralose anaesthesia. Single shocks of increasing strength were applied to the chorda. (From Lundberg, Acta physiol. scand. 1955, 35, 1–25.)

FIG. 6 Submaxillary secretion in a cat under chloralose anaesthesia. Nerve anastomosis had been carried out 9 months earlier as described in the text. Records from above: time in minutes, signal to mark nerve stimulation, flow of saliva recorded with an ordinate writer. A: hypoglossal stimulation at 80/sec. B: chorda-lingual stimulation at 20/sec. During C the hypoglossal nerve was stimulated for the whole period showing secretion, but on three occasions (signal upwards) the chorda-lingual nerve was stimulated as well. (From Ekström and Emmelin, J. Physiol 1971, 213,