however, reduced after parasympathetic denervation [Nordenfelt et al., 1960]. opposite to those caused by parasympathetic denervation.

Transcription

1 CHOLINE ACETYLASE IN SALIVARY GLANDS OF THE CAT AFTER SYMPATHETIC DENERVATION. By IVAR NORDENFELT. From the Institute of Physiology, University of Lund, Sweden. (Received for publication 20th April 1964) The choline acetylase activity of the cat's submaxillary and parotid glands has been found to increase after excision of the superior cervical ganglion. Both concentration and total activity were significantly changed. The enzyme increase did not occur if only the preganglionic sympathetic trunk had been sectioned. The increase of choline acetylase was found to be dependent on the presence of intact pre- and postganglionic parasympathetic neurones. The enzyme change appeared, however, even if the glandular cells were made inactive with the atropine-like substance Hoechst DEGENERATION of the postganglionic sympathetic fibres of the salivary glands of the cat causes certain changes in the glands, many of which are opposite to those caused by parasympathetic denervation. There is an increase in weight of the glands [Bradford, 1888] in contrast to the wellknown atrophy induced by section of the parasympathetic fibres. The maximal secretory capacity of the submaxillary gland (secretion evoked by pilocarpine) is slightly enhanced after sympathetic but decreased after parasympathetic denervation [Emmelin et al., 1960]. Degeneration of the sympathetic neurones of the glands causes also increase of both the concentration and the total amount of respiratory enzymes, the activities of which are, however, reduced after parasympathetic denervation [Nordenfelt et al., 1960]. The synthesis of acetylcholine (ACh) in the cat's salivary glands is reduced after postganglionic parasympathetic denervation, due to degeneration of the cholinergic nerves [Nordenfelt, 1963]. In the present investigation the choline acetylase (ChAc) activity has been studied in the cat's submaxillary and parotid glands after removal of the superior cervical ganglion. The ChAc activity of the glands has been found to increase after this denervation and an attempt has been made to analyse this increase of ChAc activity. METHODS Thirty-one cats were used in the experiments. The following operations were carried out under pentobarbitone anaesthesia (Nembutal, Abbott Ltd., 30 mg./kg. intraperitoneally). The superior cervical ganglion was excised on one side in most of the animals. In a few cats instead of removing the ganglion the cervical sympathetic was cut on one side. In five cats both preganglionic parasympathetic and postganglionic sympathetic denervation of the submaxillary gland was achieved by section of the chorda lingual nerve and excision of the sympathetic ganglion on the same side. In another series of animals the auriculotemporal nerve was cut [Stromblad, 1955] and the superior cervical ganglion removed on the same side, thereby obtaining both postganglionic parasympathetic and sympathetic denervation of the parotid gland. 57

2 58 Nordenfelt On the same day as the superior cervical ganglion was excised, treatment with an atropine-like substance Hoechst 9980 (aa-diphenyl-y-piperidinobutyramide) was started in some cats. This drug was injected subcutaneously once a day (1 mg./kg. for days followed by 2 mg./kg. for 9-12 days). A varying time (7-118 days) after the ganglionectomy the cats were killed by ether and the submaxillary and parotid glands removed, cleaned and weighed. The ChAc activity of the glands was thein determined by the method devised by Hebb [Nordenfelt, 1963]. The contralateral, non-denervated gland was used as a control in all experiments. RESIULTS Postganglionic Sympathetic Denervation. - Postganglionic sympathetic denervation caused an increase in ChAc activity of both submaxillary and parotid glands when compared with the enzyme activity of the contralateral TABLE I. CHOLINE ACETYLASE ACTIVITY OF THE SUBMAXILLARY GLAND OF THE CAT AFTER EXCISION OF THE SUPERIOR CERVICAL GANGLION ON ONE SIDE. THE MEANS ( ± S.E. OF MEAN) ARE ALSO GIVEN. Enzyme activity in,ig. Enzyme activity in aig. ACh/hr./g. acetone ACh/hr./wlhole gland powder Days - D/C, D/C after Denervated Contralateral percentage Denervated Contralateral percentage operation * * ± ± Mean weight of fresh gland (g.) Denervated : 1-36 ±0-11 (n =11). Contralateral: 1-27 ±0-12 (n=li). glands (Tables I and II). The enzyme change was slightly more pronounced in the parotid than in the submaxillary glands. The concentration (calculated per g. acetone powder) of ChAc increased. In the case of the submaxillary glands the concentration was 118 ±1.9 per cent (n = 11) and in the parotid glands 124 ± 2.4 per cent (n = 11) of the contralateral glands. Since the glands also increased in weight [see Nordenfelt et al., 1960], the total increase in ChAc was larger. In the submaxillary glands the total activity (calculated per whole gland) was 122 ±3.4 per cent (n = 11) and in the parotid glands 130 ± 4.8 per cent (n = 11) of their control glands. Both the increase in concentration and in total activity is significant (p < 0.001) for the two kinds of glands compared with the normal percentage variation in activity between right and left glands [submaxillary glands, conc.: 100 ±2.4 (n=7); total 101 ±2.4 (n=7); parotid glands, conc.:

3 Choline Acetylase and Sympathetic Denervation 104 ±3.2 (n=7); total 102 ±3.5 (n=7)]. These figures for normal glands are mainly taken from an earlier investigation [Nordenfelt, 1963]. Time Course of Enzyme Change. - The increase in ChAc activity of the glands after sympathetic denervation is not significant until after about 2 weeks. Two cats examined 1 week after denervation showed no increase in total activity of the denervated gland compared with the contralateral one (submaxillary gland: 103 and 100 per cent; parotid gland: 93 and 106 per cent of the control). These figures should be compared with the results TABLE II. CHOLINE ACETYLASE ACTIVITY OF THE PAROTID GLAND OF THE CAT AFTER EXCISION OF THE SUPERIOR CERVICAL GANGLION ON ONE SIDE. THE MEANS ( ±S.E. OF MEAN) ARE ALSO GIVEN Enzvme activity in jig. Enzyme activity in,lig. ACh/hr./g. acetone ACh/hr./whole gland powder Days sa D/(:C D./C after Denervated Contralateral percentage Denervated Contralateral percentage operatioml * *9 37* * ± ± ± ± ±4-8 Mean weight of fresh gland (g.) Denervated : (n= 1). Contralateral: 0-87 i0-08 (n =11). obtained after about two weeks (Tables I and II). The increase in enzyme activity which is observed at 2 weeks does not progress further (studied up to 118 days). Preganglionic Sympathetic Denervation. - As distinguished from postganglionic denervation, section of the preganglionic sympathetic fibres did not influence the ChAc content of the glands. The total ChAc activity (estimated days after the denervation) of preganglionically denervated submaxillary glands in per cent of those of the contralateral glands were 111, 97 and 93; corresponding figures for denervated parotids were 92 and 104. Postganglionic Sympathetic Denervation Combined with Pre- or Postganglionic Para-sympathetic Denervation. - The influence of degeneration of preor postganglionic parasympathetic nerves on the increase in ChAc activity after excision of the sympathetic ganglion in the neck was studied in ten experiments. It was found earlier [Nordenfelt, 1963] that section of the auriculotemporal nerve causes a profound fall in ChAc activity of the parotid gland. The total activity of the gland found after a few weeks was 9 ± 2.7 per cent (n = 5) of that of the control gland. The low remaining activity is attributed to the presence of cholinergic neurones in the parenchyma of the gland which remain intact after the operation. When, as in the present 59

4 60 Nordenfelt experiments, both the postganglionic sympathetic and parasympathetic fibres (auriculotemporal nerve) were cut, the decrease in enzyme activity of the parotid gland after days was very similar, down to 8 ± 3.1 per cent (n = 5) of the contralateral gland. Parasympathetic denervation of the submaxillary gland (section of the chorda lingual nerve) has been shown to cause a fall of its ChAc activity. After some weeks the activity (per whole gland) is per cent (n = 10) compared with the control glands, probably mainly due to degeneration of preganglionic fibres in the gland tissue [Nordenfelt, 1963]. When the postganglionic sympathetic fibres were also allowed to degenerate, as in the present investigation, the remaining enzyme activity after days was almost exactly the same, 59 ± 3.7 per cent (n = 5). Sympathetic Postganglionic Denervation and Treatment with Hoechst The increase in ChAc activity after ganglionectomy thus did not appear when the preganglionic parasympathetic neurones also were sectioned. This result might be due, not to loss of impulse flow in the postganglionic parasympathetic fibres, but to the fact that the denervation inactivates the gland cells. A similar condition is obtained if the long-acting drug Hoechst 9980 is given once a day in the doses used in this investigation, when probably all effect of released ACh on the gland cells is abolished. The glands treated with Hoechst 9980 after removal of the sympathetic ganglion, showed, however, an increase of the total ChAc activity of the same order as that observed in untreated animals: the mean of the submaxillary gland was 125 ± 3-5 per cent (n = 5) and of the parotid gland 136 ± 4.9 per cent (n = 5) of their control glands. DIscusSION The ChAc of the salivary glands is most likely confined to the cholinergic neurones there. A fact speaking in favour of this is that over 90 per cent of the enzyme activity disappears from the gland after degeneration of the postganglionic parasympathetic fibres [Nordenfelt, 1963]. Also the ChAc increase found in the present investigation after excision of the superior cervical ganglion does not occur after section of the postganglionic parasympathetic nerve, showing it to be dependent on intact cholinergic fibres in the gland. The ChAc increase must be induced by the degeneration of the postganglionic sympathetic fibres in the glands and not by loss of impulse flow in the sympathetic nerves, because preganglionic denervation did not cause any change of enzyme activity. It was found that preganglionic parasympathetic denervation of the gland prevents the enzyme increase after removal of the sympathetic ganglion. Treatment with the atropine-like substance Hoechst 9980 did not, however, influence the ChAc change after ganglionectomy. The enzyme increase seems thus to be dependent not on normally secreting gland cells but on a normal impulse flow in the parasympathetic nerves.

5 Choline Acetylase and Sympathetic Denervation 61 As the ChAc is probably confined to the cholinergic neurones, the change of enzyme activity after excision of the superior cervical ganglion must be due either to an increase of the enzyme content of the present cholinergic neurones, to further growth of their processes or to an increase in number of the fibres, i.e. collateral sprouting from the neurones. There are certain facts which speak in favour of the last explanation, i.e. collateral regeneration. Collateral sprouting has been shown to occur in the autonomic nervous system by Murray and Thompson [1957]. The time course of the ChAc increase fits reasonably well with these investigators' finding of collateral regeneration in the superior cervical ganglion. According to them the sprouts appeared after a few days and were fully developed after about four weeks. The sprouting process would probably reach a certain stage before it would be possible to detect any significant change in ChAc, and the first appearing sprouts may not contain any ChAc until later in their development. Degeneration of the postganglionic sympathetic fibres in the glands might thus induce sprouting from adjacent parasympathetic fibres. This hypothesis could, however, only be verified by further investigation, using histological or histochemical methods. ACKNOWLEDGMENT This work was supported by a grant from the Faculty of Medicine in Lund. REFERENCES BRADFORD, J. R. (1888). 'Some points in the physiology of gland nerves', J. Physiol. 9, 287. EMMELIN, N., MALM, L. and STROMBLAD, B. C. R. (1960). 'Effect of denervation on the maximal secretory capacity of salivary glands', Quart. J. exp. Physiol. 45, 349. MURRAY, J. G. and THOMPSON, J. W. (1957). 'The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat', J. Phy8iol. 135, 133. NORDENFELT, I. (1963). 'Choline acetylase in normal and denervated salivary glands', Quart. J. exp. Physiol. 48, 67. NORDENFELT, I., OHLIN, P. and STROMBLAD, B. C. R. (1960). 'Effect of denervation on respiratory enzymes in salivary glands', J. Physiol. 152, 99. STROMBLAD, R. (1955). 'Sensitivity of the normal and denervated parotid gland to chemical agents', Acta physiol. scand. 33, 83.