Saliva and Salivation: Satellite Symposium of the 28th International Congress of Physiological Sciences, Székesfehérvár, Hungary, 1980

Advances in Physiological Sciences, Volume 28: Saliva and Salivation covers the proceedings of the Saliva and Salivation satellite symposium, which is a pre-congress meeting connected to the 28th International Congress of Physiological Sciences. The book discusses a wide variety of studies that are relevant to the function of salivary system. This variety includes denervation as a method to produce a prolonged stimulation of salivary glands and reflex activation of the preganglionic fibers innervating the submandibular gland. This text also explains reflex discharge recorded from rat submandibular ganglion cells in vivo and effects of acetylcholine and adrenergic agonist on potassium transport in the salivary glands in vitro. This monograph will be of great interest to professionals and researchers, including dentists and zoologists, whose work concerns oral physiology.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483147536

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proceedings.

IMPRESSIONS on the Saliva and Salivation Symposium (Székesfehérvár, 10–12 July, 1980)

At the suggestion of the Commission on Oral Physiology, under the chairmanship of professor Kawamura, a satellite symposium on Saliva and Salivation was organized, and it took place in the ancient and beautiful city of Székesfehérvár, a former capital of Hungary. For the hard work with all the arrangements a local committee was responsible from the Research Group of Oral Biology of the Semmelweis University Medical School, Budapest. I strongly feel, I should emphasize that the practical arrangements were superb, that it all worked very smoothly and we were overwhelmed by the endless patience, helpfulness, friendliness and hospitality of our hosts. Social programmes, including a magnificent reception and a sightseeing tour to Budapest with a visit to the laboratories of the research group gave ample opportunity for informal discussions and formation of new ties of friendship.

The conference was opened by professor Kawamura and the president of the Hungarian Dental Association, Dr. Orsós. About 120 scientists from many corners of the world attended. The great number of the colleagues from Japan was striking, reflecting the great importance attributed in that country to oral physiology in medical and dental schools, which I regard as an example to be followed in Europe, but certainly in my country. No less than 61 communications were delivered, in part as posters. It is obviously an impossible task to try to give a fair account for all these presentations, partly for the simple physical reason that some papers were given simultaneously in different lecture halls but mainly because one brain cannot absorb and digest that vast quantity of specialized information. Fortunately, the papers–in form of a book–will soon be published by the Publishing House of the Hungarian Academy of Sciences, so if I leave out important papers or, unintentionally, misinterpret others, the damage is not irreparable. Just at the very beginning let me mention, very briefly, some of the earlier contributions which have brought us to the present state of research on salivary glands, suggesting that a really fruitful work has been going on, as confirmed also by the present symposium.

Salivary glands have for a long time been favourite objects for physiologists, readily accessible, with ducts easy to cannulate, blood vessels to isolate, nerves to excite and cut, sensitive to stimulating and inhibitory agents. During the second half of the previous century they attracted the attention of many great physiologists and a remarkable number of observations of general importance were made. Ludwig had discovered the secretory nerves and made his fundamental work on the secretory process. Claude Bernard added another new category of nerves (the vasodilator nerve), and his paralytic secretion gave an early hint of a long-term regulatory function of glandular nerves. Eckhard described the spontaneous secretion and the motor effects of salivary nerves. Heidenhain’s studies on flow rate and electrolyte content in saliva became the basis of our present concept of how saliva formed, and his atropine-resistant vasodilation was to play an important part in the discussion on non-cholinergic, non-adrenergic nerves. He and Langley examined the relationship between secretory granules and protein secretion. Langley’s classical work on the autonomic nervous system seems to have been greatly influenced by his early experiments on the physiology and pharmacology of salivation.

At the beginning of this century Pavlov wrote his fundamental work on conditioned reflexes, and Pavlov’s and Babkin’s (his pupil) school greatly contributed to our knowledge of salivary secretion. The impact of the new ideas about chemical transmission of nerve impulses during that period was obvious; in fact, many early findings in this field were made on salivary glands. There followed the development of the old receptor concept, and the discoveries of polypeptides, active on secretory cells and blood vessels. Still, there is much to be learnt about the glandular nerves; how they are reflexly activated, how they are distributed within the glands, their transmitters and modulators, the relationship between control of secretion and blood supply, and the long-term regulation of growth and development of salivary glands.

In the fifties two important contributions greatly accelerated investigations in salivary physiology. Lundberg applied for the first time in glandular physiology the technique of intracellular electrical recording to the salivary glands, which had been so rewarding in nerve and muscle research; and Thyasen, Thorn and Schwartz set up the two-stage hypothesis to explain secretion. Utilizing the electrophysiological methods, and the methods for micropuncture, microperfusion and microanalysis of electrolytes, methods largely borrowed from renal physiology, a tremendous amount of work has been carried out on the transport of ions in acinar and ductal cells, to elucidate how saliva is formed and gets its composition and how the processes in acini and ducts are controlled. At the same time experiences gained during synthesis, storage and export of protein in the pancreas has been applied to the salivary glands. The ideas of stimulus-secretion coupling and of secondary messengers in these processes in secretion of fluid and macromolecules has evoked much work on the glands. Special books and special symposia are now devoted to saliva and salivation and also the present symposium confirms the impression that still there are a great deal to be done in the branches of salivary physiology, I have tried to refer to.

Considering, in the first instance, the control of the activity of salivary glands, several papers were devoted to problems of salivary reflexes. Matsuo and Kawamura from Osaka University described experiments on rabbits in which electrical recording was made from single preganglionic parasympathetic fibres of the chorda tympani. Most of these fibres responded reflexly to electrical stimulation of the cutaneous tissues of the oral region, and different fibres were characterized. Records of secretion suggested that central inhibition because of nociceptive stimulation of the skin might be a complicating factor. Suzuki, also from Japan, had managed to isolate in rats single postganglionic parasympathetic ganglion cells on the submandibular duct and define two types of cells; one synaptically connected to a single preganglionic axon and reflexly responsive particularly to taste stimuli to the tongue, the other with contacts from several preganglionic fibres and responding reflexly to heat rather than to gustatory stimuli. This raises the interesting question whether the former type of postganglionic neurons may represent the secretory pathway of Ludwig and the other type Bernard’s vasodilator nerve. In recent years some physiologists have liked to think that one and the same parasympathetic axon may control, by en passant contacts, different types of effectors such as secretory cells and blood vessels, providing an improved blood supply for the glandular cells when they start to secrete. Suzuki’s experiment may support the old concept of specific secretory and vasodilator nerves.

Gjörtstrup (Lund) had worked out a fistula preparation for chronic observetions on parotid secretion in conscious rabbits which at the same time allowed him to introduce unobtrusively different taste stimuli into the mouth. The two secretory pathways could be activated reflexly independently of each other. Chewing tasteless pellets evoked a parasympathetic secretion of fluid and a sympathetic secretion of amylase, and a separate paper together with Anderson (Bristol) indicated this to be a masticatory–salivary reflex. Sugary taste, on the other hand, activated exclusively the sympathetic pathway causing secretion of amylase and so little fluid that the effect could only be revealed by providing a parasympathetic background flow with eserine injected through the duct.

The classical physiology of salivary secretion was based on experiments on dogs and rabbits. Later, morphologists and biochemists preferred the rat. It took some time until the physiologists began to cannulate the ducts of these animals, so that salivary flow responses and amylase secretion could be correlataed with structural and biochemical data. Recently in a series of papers and posters a large group of colleagues from Amsterdam described numerous observations on mice. The distribution of cholinergic and adrenergic nerves in the glands had been explored histochemically. Saliva had not been collected or nerves stimulated so far, but the glands had been exposed to sympathomimetic and parasympathomimetic drugs and a great number of structural and biochemical changes indicative of secretion were analyzed. Mice have, of course, been used for many years in other contexts, for instance, to study sexual dimorphism or the presence of a multitude of strange biologically active compounds in salivary glands, and in vitro experiments by Nishiyama as well as by Petersen and his group (Dundee) when nerves had to be stimulated. Gallacher (Dundee) described observations in which field stimulation caused secretory potentials, to use Lundberg’s term, which disappeared in sodium-free solution or after tetrodoxin, showing that axons were stimulated. They could also be completely abolished by atropine, since (a puzzling observation) not only cholinergic but also adrenergic nerves surround the acini and adrenaline added to the superfusing medium evoked the characteristic electrical responses.

This has now brought us to the neuro-glandular junction where the transmitters are released and act via their receptors. The general opinion is of course that acetylcholine and noradrenaline are the transmitters and that activation of muscarinic receptors and α-adrenoceptors triggers mechanisms causing movement of ions and as a consequence transepithelial flow of water in processes in which calcium ions play an important second messenger role, and that activation of β-adrenoceptors results in exocytosis of granules and secretion of amylase. In these events cyclic AMP and calcium ions may be involved. The β-adrenoceptors seem to be mainly of the β1-subgroup, as discussed by Danielsson and co-workers (Umeå). Incidentally, this is a rather schematic picture, worked out originally in rodents. In the dog, for instance, secretion of fluid on sympathetic stimulation seems to be exclusively β-receptor mediated. The picture has now also been complicated by the appearance of the polypeptides, peptides present in glandular nerves of some species, acting as transmitters of modulators in special peptidergic nerves or perhaps in cholinergic nerves. Thus Substance P occurs in glands of rats evoking secretion and several experiments showed that the secretion on chorda stimulation may have an atropine-resistant component. The Dundee group reported that acetylcholine, adrenaline by an α-adrenoceptor action and Substance P similarly affect the membrane potential and the membrane permeability of the acinar cells. From a paper by Kato, Togari and Nagatsu on the effects of a series of substance P-analogues on the secretion of amylase, it was apparent that the effects were essentially of a type similar to that produced by cholinergic stimulation. Martinez (Missouri) gave an account of the secretion of fluid and amylase caused by Substance P itself and of interesting modulating effects on the responses to acetylcholine.

Participation of polypeptides in the control of granular activity has been discussed ever since Hilton and Lewis in the fifties suggested that bradykinin might regulate the blood flow in the gland. After the discovery that in most species kallikrein seems to be present in the ducts, attempts have been made to assign other important tasks to this system, and interest regarding blood flow regulation has shifted to other active substances, particularly to the vasoactive intestinal peptide VIP, playing a role in the atropine-resistant vasodilation on parasympathetic stimulation. A biologically active substance, the histamine, was suggested to be a physiological vasodilator. Erjavec (Yugoslavia) produced a chain of arguments in support of the view that histamine might be released from mast cells in the glands on parasympathetic stimulation and by way of H1 and H2-receptors contribute to the vasodilation. For those who want to study glandular blood flow, I recommend the poster of Fazekas describing a method to do so in rats: he has worked out a sulphanilamide uptake method, compared it with rubidium uptake and microsphere methods and found it useful. Among other things a number of contributions of methodological interest could thus be well illustrated and discussed at leisure, for instance those by Zelles and his group on the preparation of secretory granules, such as separation of zymogen granules from solube enzymes by gel filtration and separating mature and immature granules by density gradient centrifugation.

So far my discussion had dealt mainly with acinar cells. In the duct system the primary acinar fluid is modified by absorptive and secretory processes, and the control of this has in recent years been extensively studied by Young and his group (Sydney), at a time in co-operation with the late professor Leon Schneyer. The ductal transport of ions may be influenced not only by acetylcholine and noradrenaline but also by agents like Substance P and VIP, which may be present in nerves of the ducts. This group now reports work on the electrolyte transport from the stage of the formation of the primary saliva to that of the final saliva, partly utilizing a new and promising perfusion preparation of the rabbit’s submandibular gland.

In addition to their immediate effects on the glands, the nerves exert long-term actions, as I suggested in my introduction. Charlotte Schneyer gave a review of her work over many years on growth and development of salivary glands showing the importance of the nerves in these connections. The role of the nerves, and at a very early stage particularly of the sympathetic nerves, was emphasized in a poster by Donáth.

Even if nerves seem to dominate in the immediate and long-term control of salivary glands, the importance of hormonal regulatory mechanisms is obvious, for instance in the ion transport in the ducts or in the growth of the glands. In this symposium the role of the hormones was illustrated in a poster by Zelles and co-workers showing the effect of cortisone treatment on synthesis and intracellular distribution of amylase.

I am well aware of the fact that this has been a very superficial and fragmentary account of the Symposium. I have not gone much into the problems of amylase secretion or the general composition of saliva as described for instance by Ferguson, nor have I touched upon papers with clinical implications such as Shannon’s recipes for treatment of xerostomia. To all these colleagues thus neglected I apologize.

Finally, let me once more express thanks to our hosts. We are immensely grateful for the magnificent way in which they have brought together what one of my young co-workers once called the Salivation army.

Nils Emmelin

DENERVATION AS A METHOD TO PRODUCE A PROLONGED STIMULATION OF SALIVARY GLANDS

N. Emmelin,     Institute of Physiology, University of Lund, Sweden

Publisher Summary

This chapter discusses the role of denervation as a method to produce a prolonged stimulation of salivary glands. The chapter describes an experiment in which drops of saliva falling from cannulae in the two parotid ducts of an anaesthetized cat were marked on the smoked drum using an electron magnetic pen. The supersensitivity produced by the nerve section disappeared after repeated injections of pilocarpine or carbachol for two or three days, supporting the view that acetylcholine is of importance in the physiological control of the sensitivity of the glandular cells. The subsequent partial postganglionic denervation further raised the supersensitivity, but this was preceded by a temporary lowering of the level of sensitivity. This dip in the curve, which could be increased by injecting serine and prevented by injecting atropine during the period corresponding to the degeneration secretion, was at its lowest three days after the dissection of the chorda.

Some time after section of postganglionic autonomic nerves a period of activity in the denervated organ appears, called degeneration activity (see Emmelin and Trendelenburg 1972). This phenomenon is mainly due to a temporary increase in the release of the neurotransmitter into the junctional cleft. It was first observed in the parotid gland of the anaesthetized cat after section of the auriculo-temporal nerve (Emmelin and Strömblad 1958), which contains most of the parasympathetic postganglionic nerves of the gland (Ekström and Emmelin 1974). Fig. 1 shows an example of a degeneration secretion of parotid saliva, starting about 30 hours after denervation; such a flow continues for one day up to one and a half increasing gradually in rate to a maximumi then slowly declining. A similar secretion can be produced in the submandibular and sublingual glands; the difficulty in these cases is the brief postganglionic parasympathetic neurone, but a partial postganglionic denervation can be attained by tracing under the dissecting microscope the chorda into the hilum of the glands and cutting it as far distally as possible (Emmelin 1960, 1962). Parasympathetic degeneration secretion from the salivary glands has also been observed in dogs (Coats and Emmelin 1962a), rabbits (Ohlin 1963, Nordenfelt 1964) and rats (Delfs and Emmelin 1979). In glands well supplied with sympathetic secretory nerves a sympathetic degeneration secretion can be produced; the phenomenon can thus under favourable conditions be detected in the submandibular gland of the cat after removal of the superior cervical ganglion (Coats and Emmelin 1962b, Emmelin 1968a), and it can easily be obtained from this gland of the rat (Delfs and Emmelin 1974, Stefano et al. 1974, Almgren et al. 1976). The time scale of the phenomenon depends on many factors such as the anaesthesia and the type of nerve cut, sympathetic or parasympathetic. It varies with the species, as shown in Fig. 2, which schematically summarized observations on the parasympathetic degeneration secretion of submandibular saliva in four species. In the same gland of a certain species it varies with the level at which the postganglionic nerve is cut (Emmelin 1968b, Almgren et al. 1976). This is illustrated in Fig. 1, showing an experiment in which the auriculo-temporal nerve was cut on one side near its exit from the skull (proximally), on the other side close to the gland (distally). This experiment illustrates that the phenomenon can be made use of as an indicator to show when the degeneration of the nerve has reached a certain stage. In this case the importance of the length of the nerve stump left in connexion with the effector is demonstrated. In a similar way the degeneration secretion has been used to study effects of drugs on the progress of the nerve degeneration (Emmelin 1972, Almgren and Lundberg 1976, Arbilla et al. 1977, Perec et al. 1977). Particularly, however, the phenomenon seems useful for the following reason. Since the secretion is due mainly to an increased transmitter release which at a certain stage of the degeneration becomes supraliminal for activation of the effector, rises to a maximum and then slowly decreases and ceases as the process of degeneration proceeds, we are provided with a preparation where the effector cells for many hours, up to one or two days, depending on the gland and species chosen, are exposed to the physiological excitant in amounts sufficient to cause secretion, imitating prolonged nerve stimulation. Once the nerve has been cut this can take place in the anaesthetized or non-anaesthetized animal and even under in vitro conditions. The use of this preparation may be illustrated by the following four examples.

Fig. 1 Parotid degeneration secretion in a cat after section of the auriculo-temporal nerve, proximally on one side (P) and distally on the other (D). The numerals give the hour after denervation. Uppermost: minute marks. Drops of saliva falling from cannulae in the two parotid ducts of the anaesthetized cat were marked on the smoked drum using an electronmagnetic pen (Emmelin 1968).

Fig. 2 Submandibular degeneration secretion after partial parasympathetic denervation by dissection of the chorda tympani. The abscissa gives the time in hours after the operation. The beginning, maximum and end of the phenomenon in rats, rabbits, cats and dogs are shown schematically.

1 Control of the chemo-sensitivity of salivary glands

The supersensitivity to chemical excitants which gradually develops in e.g. the submandibular gland of the cat after section of the chorda-lingual nerve, a parasympathetic decentralization, can be further increased by the partial postganglionic denervation described above (Emmelin 1960). This is in agreement with The law of denervation (Cannon 1939). Supersensitivity of similar kind can be created by injections of atropine and other drugs with atropine action, repeated over two or three weeks, or by administration of botulinum toxin (Emmelin and Muren 1952, Emmelin 1961a). In the presence of atropine the supersensitivity can be demonstrated using for instance adrenaline as an excitant. This sensitization, produced by pharmacological denervation (Emmelin 1961b) was considerably larger than that caused by section of the chorda-lingual nerve, a decentralization, and also exceeded that seen after the surgical denervation; this seems reasonable in view of the fact that this latter denervation was far from complete. From these and other observations (Emmelin 1965) it has been concluded that supersensitivity develops in glands for some time deprived of some action of acetylcholine. To this action contributes not only acetylcholine released by impulses from the central nervous system, as shown when preganglionic fibres are cut, but in addition acetylcholine continuously released from the postganglionic neurones, as demonstrated by the increased effect of surgical or pharmacological denervation. The supersensitivity produced by nerve section disappears after repeated injections of pilocarpine or carbachol for two or three days, supporting the view that acetylcholine is of importance in the physiological control of the sensitivity of the glandular cells (Emmelin and Muren 1952). These drugs, which were given subcutaneously may, however, exert various actions, and in imitating the actions of acetylcholine on the gland they can obviously not distinguish between effects of transmitter released by nerve impulses and of acetylcholine randomly liberated even from a decentralized neurone.

Observations during parasympathetic degeneration secretion may throw some light on this question, since under those conditions the gland cells are exposed for a long period to increased amounts of acetylcholine released from the decentralized postganglionic neurone. Fig. 3 shows such an experiment on the submandibular gland of the cat (Emmelin 1964a). The sensitivity of the gland, expressed as the secretory response to a standard dose of adrenaline, was determined repeatedly in the course of two months using the following method (Emmelin 1964b). The cat was anaesthetized with ether followed by a short-acting barbiturate, which was given intracardially. The submandibular duct was cannulated from its orifice in the mouth, and secretory drugs were injected through the needle in the heart. This needle and the salivary cannula were then removed and the cat was left to wake up. In this way many observations could be made in the same animal with intervals of a few days. The sensitivity of the gland cells was found to remain at a constant level over long time periods in each animal under standardized conditions. As can be seen in Fig. 3 the sensitivity increased markedly after section of the chorda-lingual nerve and reached a maximum within three weeks and stayed there. The subsequent partial postganglionic denervation further raised the supersensitivity but this was preceded by a temporary lowering of the level of sensitivity. This dip in the curve, which could be increased by injecting eserine and prevented by injecting atropine during the period corresponding to the degeneration secretion, was at its lowest three days after the dissection of the chorda. These observations seem to support the opinion that acetylcholine released locally, continuously and for a long time from the postganglionic neurone, exerts an action on the chemo-sensitivity of the gland cells; and this occurs even when the neurone is disconnected from the central nervous