special interest since changes in the activity of these enzymes are known hypophysial hormones, notably those stimulating the thyroid gland and the

Transcription

1 EFFECT OF HYPOPHYSECTOMY OR TREATMENT WITH THY- ROXINE OR TESTOSTERONE ON SECRETORY RESPONSES AND RESPIRATORY ENZYMES OF THE SUBMAXILLARY GLAND OF THE RAT. By P. OHLIN. From the Institute of Physiology, University of Lund, Sweden. (Received for publication 19th March 1965) The effect of hypophysectomy or of treatment with thyroxine or testosterone on the function of the submaxillary gland of rats was studied. (1) The dry weight of the submaxillary gland decreased by 46 per cent after hypophysectomy. The gland weight increased by 14 per cent after thyroxine and by 29 per cent after testosterone. (2) The maximal secretory response to pilocarpine was reduced after hypophysectomy. After hormonal treatment the maximal secretory response was increased when expressed per gland. It was also increased when calculated per unit weight after thyroxine, but not after testosterone. (3) The activity of succinic dehydrogenase, cytochrome oxidase and fumarase was not significantly changed after hormonal treatment when expressed per unit weight. The results are discussed and related to known morphological changes of the submaxillary gland of rats after hypophysectomy or after treatment with thyroxine or testosterone. THE relationship between hormones and salivary glands has attracted much attention since the discovery of the sexual dimorphism of the submaxillary gland of mice [Lacassagne, 194 a]. Most work in this field was done on mice and rats. The structure of the gland was found to be dependent on hypophysial hormones, notably those stimulating the thyroid gland and the gonads. Thus, the submaxillary gland of rats atrophied markedly after hypophysectomy [Gabe, 195; Sreebny, 1953], thyroidectomy [Leblond and Grad, 1948] or gonadectomy [Shafer and Muhler, 1953]. The atrophy concerned mainly the cells of the granular tubules. Treatment with thyroxine and testosterone prevented atrophic changes [Grad, 1949; Shafer et al., 1956]. Furthermore, the granular tubules increased in size when intact rats were treated with thyroid hormones or testosterone [Lacassagne, 194 b; Shafer and Muhler, 1953 and 1956]. To what extent these structural changes indicate modifications of the function of the submaxillary gland of rats is not known. In order to study functional aspects a method was used which made it possible to record the secretory responses to sialagogue drugs. As another indicator of the functional capacity, the activity of some respiratory enzymes, succinic dehydrogenase, cytochrome oxidase and fumarase, was studied. This seemed of special interest since changes in the activity of these enzymes are known to occur concomitantly with structural changes, e.g. after parasympathetic denervation [Nordenfelt et al., 196] and after hypophysectomy [Ohlin, 1962]. Since the parasympathetic nerve supply is important for the function of salivary glands some studies were also made in order to see whether parasympathetic denervation influenced the effects of hormonal treatment. 446

2 Hormonal Influences on -Rat's Submaxillary Gland 447 METHODS Female rats of a colony bred at this Institute were used. The breeding animals received a pelleted diet (Anticimex, 21); the experimental animals were given milk, wheat bread and barley ad libitum. Litters of four to six rats, when aged 4 to 5 months, were used; two rats of each litter were taken as controls. Three groups of animals were studied. In one group the rats were hypophysectomized and examined about 4 weeks later. Completeness of hypophysectomy was controlled as described by Jacobsohn [196]. The results after hypophysectomy refer to eleven rats. In these animals microscopic examination did not reveal any hypophysial cells other than small pars tuberalis cells lining the pituitary stalk. In two other groups intact rats were treated with either DL-thyroxine, 1,ug. daily subcutaneously, or testosterone propionate, -2 mg. in oil daily intramuscularly, for 3 weeks. Twenty-four rats were given thyroxine and thirty-two testosterone. Studies were performed on the submaxillary gland of different groups of rats as follows: (1) after hypophysectomy or hormonal treatment the secretory responses to sialagogue drugs were estimated; (2) after hormonal treatment the activity of respiratory enzymes was studied. The effect of hypophysectomy on enzymic activity had been investigated previously [Ohlin, 1962]; (3) the effect of hormonal treatment on parasympathetically denervated glands was studied. (1) To estimate the secretory responses of the submaxillary gland the following technique was developed. The rats were anaesthetized with ether and a thin polythene tube connected to an injection needle was inserted into an exposed femoral vein. Chloralose (1 mg./kg. body weight) was then given intravenously and the ether administration was discontinued. After intubation of the trachea the salivary ducts were exposed in the neck. After opening the submaxillary duct, medial to the sublingual duct, a fine glass cannula was inserted. Cannulse giving 8 drops out of 1 ml. of distilled water were used. Secretion appearing at the tip of the cannula was noted and registered on a smoked drum. The following drugs were injected intravenously to determine a threshold dose: acetylcholine chloride, -2-2 pg./kg.; methacholine chloride, -2-1,ug./kg.; and adrenaline chloride, -2-2,ug./kg., and the flow of saliva was recorded in each case for the doses above threshold. To record a maximal secretory response, pilocarpine was injected intravenously every 3-6 sec. in increasing doses from 5-1 and finally 2 jug./kg. The flow of saliva was recorded, using an ordinate recorder on a smoked drum. The maximal secretory response was usually reached within 1-15 min. after a total dose of about 2 mg. pilocarpine/kg. The maximal secretory response after pilocarpine was calculated and expressed in u1l. saliva per min. per gland or per mg. of glandular tissue (dry weight). To make this calculation possible the volume of one drop of saliva was determined. For this purpose saliva was collected when the gland was secreting at a maximal rate. The saliva was transferred as soon as possible from a pipette through the cannula used, the rate being kept equal to the maximal flow rate of the gland. At the end of the experiments on secretory responses to sialagogue drugs including pilocarpine, the submaxillary glands were removed, cleaned and weighed (wet weight). The dry weight of the glands was determined after heating to C. for 48 hr. The wet weight of the right submaxillary gland had previously been found to be the same as that of the left gland [Ohlin, 1962]. In six rats the left submaxillary gland was excised in advance, and the right gland was extirpated after estimation of secretory responses. The wet weight of the right gland exceeded that of the left one in all these animals, the average difference being 13 per cent. The dry weight of the right gland was, on the other hand, about the same as that of the left gland, 98±4-2 (6) * per cent. The difference observed in the wet weight was probably due to a * Mean ± standard error of mean (number of observations)

3 448 IOhlin slight cedema of the right gland after secretory activity. For this reason the gland weight is given as the dry weight after experiments on secretory responses. (2) For the enzymic estimations the animals were killed by cervical dislocation. The submaxillary glands were immediately dissected out, cleaned, dried between filter papers, weighed and homogenized in redistilled water. The activity of succinic dehydrogenase and cytochrome oxidase was estimated manometrically, and that of fumarase was determined spectrophotometrically [Nordenfelt et al., 196; Ohlin, 1962]. The activity of respiratory enzymes is expressed in units per gland, which indicates enzyme changes irrespective of weight changes, and in units per g. of glandular tissue (wet weight). For succinic dehydrogenase and cytochrome oxidase, 1 unit of activity corresponds to a utilization of 1,sl. oxygen in 3 min. at 37 C. For fumarase, 1 unit is defined as the amount of enzyme causing a change in optical density of.1/min. at 24/m,. (3) To study the effect of treatment with thyroxine or testosterone on parasympathetically denervated glands, the chorda-lingual nerve was cut under ether anawsthesia. The hormonal treatment was started on the day of denervation and continued for 3 weeks. The secretory responses to sialagogue drugs and the dry weight of the glands were estimated as described above. The effect of parasympathetic denervation will be described separately in detail [Ohlin, 1965]. RESULTS Gland Weight. - Within 4 weeks after hypophysectomy, the dry weight of the submaxillary gland decreased markedly. After treatment with thyroxine or testosterone, on the other hand, the dry weight increased (Table I). The decrease in gland weight after hypophysectomy and the increase after hormonal treatment were significant (P < -1). Similar changes of the wet weight were found after experiments on enzymic estimations. After treatment with thyroxine or testosterone the wet weight of the submaxillary gland increased from 153 h 4.2 (16) mg. to 175 i 3.9 (16) mg. after thyroxine and to (21) mg. after testosterone. The changes in gland weight after hypophysectomy and hormonal treatment were marked even when related to body weight (Table I). At 4 weeks after hypophysectomy the dry weight of the submaxillary gland had decreased by 46 per cent, but the body weight was reduced by 14 per cent only. After treatment with thyroxine the dry weight of the glands was 14 per cent higher than in the controls; the body weight was increased by 6 per cent less than that of the controls. After treatment with testosterone the body weight increased by 5 per cent. The dry weight of the glands increased 29 per cent more than in the controls. Secretory Responses. - The threshold doses of acetylcholine, methacholine and adrenaline in the different control glands can be seen in Table II. When the doses of methacholine and adrenaline were increased above threshold a marked secretory response was evoked. The secretory response to methacholine, 1 pug./kg., was about one to three drops, and that to adrenaline, 2 pug./kg., was about one to two drops. Acetylcholine, on the other hand, evoked small secretory responses, usually less than one drop of saliva, even when large doses were given. Pilocarpine elicited a marked flow of saliva

4 Hormonal Influences on Rat's Submaxillary Gland 449 ceglo o, oq g- p4e4 Ei ' m WVtV 4-- cq tn Em E E'o. 1 Q ^ ]Weq eq e mxoo t4-1~ 6 eq c E-h4 Z11 e 4 Pr Ho eq H - m -I -X4 - e Meq - eq eo ~~o m -o m PEO i-4. Fi- eq eq - -

5 45 Ohlin, which usually started after 5 pug. pilocarpine/kg. The maximal secretory response of a control gland to pilocarpine was (24) jul. per min. or, expressed in,ul. per min. per mg. (dry weight),.46 E.31 (Table I). The effect of hypophysectomy on the threshold doses of the secretory drugs. studied can be seen in Table II. The threshold dose of acetylcholine seemed slightly increased, whereas the threshold doses of methacholine and adrenaline were not different from those of the controls. The effect of hypophysectomy on the secretory responses to doses above threshold was most clearly seen when the maximal secretory response to pilocarpine was determined. Under TABLE II. THRESHOLD DosEs (pg./kg.) OF ACETYCHOLINE (ACH), METHACHOLINE (MECH) AND ADRENALINE (ADR) IN THE SUBMAXILLARY GLAND; (1) OF UNTREATED RATS (=NTROL); (2) AFTER HYPOPHYSECTOMY; (3) OF INTACT RATS GIVEN THYROXINE OR; (4) TESTOSTERONE. THE FIGURES GIVE THL NUMBER OF RATS SHOWING THE INDICATED THRESHOLD DOSE. Control Hypophysectomy Thyroxine Testosterone ACh * *.. 4 MeCh * Adr 1* * these conditions a reduction by 56 per cent was found when the maximal secretory response was expressed per gland. When calculated per unit weight the decrease was 17 per cent, which was not significant (Table I). After treatment with thyroxine the threshold doses of acetylcholine and methacholine seemed slightly decreased (Table II). The threshold dose of acetylcholine was, for instance, in seven out of ten treated animals less than the lowest threshold dose in the controls. The threshold dose of adrenaline was not influenced however. The maximal secretory response to pilocarpinewas increased by 77 per cent when calculated per gland, and by 57 per cent when calculated per unit weight (Table I). After treatment with testosterone the threshold doses were influenced in. the same way as after thyroxine, though to a lesser degree (Table II). Ir. some rats lower threshold doses of acetylcholine and methacholine were found. The threshold dose of adrenaline did not differ from that of controls. After treatment with testosterone the maximal secretory response to pilocarpine was found increased by 41 per cent when expressed per gland

6 Hormonal Influences on Rat's Submaxillary Gland 451 Significant changes were not obtained after calculation per unit weight (Table I). The increase of the maximal secretory response to pilocarpine, expressed per gland, appeared to follow the increase of the gland weight. Respiratory Enzymes. - Succinic Dehydrogenase. The activity of this enzyme (and of cytochrome oxidase and fumarase) in the submaxillary gland of the two groups of controls to rats treated with thyroxine or testosterone was similar (Table III). On the other hand, the activity of succinic dehydrogenase was lower in these controls than found previously (Ohlin, 1962). The rats used in the present and previous studies did not belong to the same strain and they had received different diets. After treatment with thyroxine or testosterone the activity of succinic dehydrogenase per gland was slightly increased. When expressed per g. of glandular tissue (wet weight) the activity was found similar to that of the control glands (Table III). Cytochrome Oxidase and Fumarase. - The activity of cytochrome oxidase and fumarase seemed increased after treatment with thyroxine or testosterone when expressed per gland. This increase corresponded to the enlargement of the submaxillary gland after hormonal treatment. The activity was similar to that of the controls when expressed per g. of glandular tissue. Denervated Glands. - The dry weight of denervated glands was found to be 27 h 1-2 (13) mg. After treatment with thyroxine or testosterone the dry weight increased to 33 i 1-7 (7) mg., and 36 : 1.2 (12) mg., respectively. After section of the chorda-lingual nerve the threshold doses of acetylcholine, methacholine and adrenaline were lowered. Treatment with thyroxine or testosterone did not affect the threshold doses of these secretory drugs in the denervated glands. The maximal secretory response to pilocarpine increased, when calculated per gland, from 24 i 1.7 (12) 1l. per min. to (6) after thyroxine and to 31 i 4.5 (7) after testosterone. When expressed per mg. dry weight, the maximal secretory response was practically unchanged both after thyroxine and testosterone, which is presumably due to the increase in gland weight. DIscuSSION The sexual dimorphism of the submaxillary gland is morphologically not very marked in rats. The cells of the granular tubules are slightly more developed in male than in female rats [Grad and Leblond, 1949; Sreebny, 1953; Jacoby and Leeson, 1959]. This and other differences are believed to be due to actions of testicular hormones. Since it is not known whether the sexual dimorphism affects the parameters studied in the present investigation, females only were used. Most workers have investigated the effect of hormonal treatment on salivary glands after at least 2 weeks of treatment. The duration of the present experiments was 3 weeks. The amounts of hormones given in the present study were for thyroxine about double the physiological dose [Griesbach et al., 1949] and for testosterone about equal to the physiological dose. It should be mentioned here that bigger doses, thirty times for

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8 Hormonal Influences on Rat's Submaxillary Gland 453 thyroxine [Wells and Munson, 1,958] and five times for testosterone [Argonz and Corral Saleta, 1961], did not seem further to increase the glandular enlargement. The weight of the submaxillary gland of. rats decreased after hypophysectomy but increased after treatment with thyroxine or testosterone. The structural changes after hypophysectomy and hormonal treatment are mainly found in the cells of the granular tubules [Lacassagne, 194 b; Gabe, 195; Shafer and Muhler, 1956]. The maximal secretory response to pilocarpine, expressed per gland, decreased after hypophysectomy and increased after hormonal treatment. Though some changes occur in glandular cells other than those of the granular tubules, e.g. after hypophysectomy in the acinar cells [Bixler et al., 1959], it seems reasonable to assume that the cells of the granular tubules are more important to the maximal secretory response to pilocarpine than the acinar cells. This would seem to agree with the finding in rats that pilocarpine.elicits a considerable flow of saliva from the cut ducts of salivary glands lacking acinar cells [Schneyer and Schneyer, 1961]. The maximal secretory response to pilocarpine, expressed per unit weight, was reduced after hypophysectomy and enhanced after treatment with thyroxine but unchanged after testosterone. It seems likely that the secretory capacity of the granular tubules is under the control of thyroxine and thus of thyrotropin; this does not exclude hypophysial factors other than thyrotropin. The present findings agree with the observations that the total amount of saliva appearing in the mouth after stimulation with pilocarpine is decreased after radiothyroidectomy [Shafer' et al., 1958]. Histochemically the activity of suepinlic dehydrogenase is observed to be high in the cells of the granular tubules and in the duct cells, but low in the acinar cells of the submaxillary gland of rats [Padykula, 1952]. Cytochrome oxidase has been studied only in rabbits. It is found to be rather evenly distributed in the cells of the submaxillary gland, though the content appears lower in the acinar cells than in the duet cells [Schneider and Person, 196]. After hypophysectomy the weight 9f the submaxillary gland of rats and the activity of succinic dehydrogenase, cytochrome oxidase and fumarase, when expressed per gland, decreased [Ohlin, 1962]; in the present study the reverse was observed in intact rats treated with hormones. It seems likely that the changes in enzyme activity are confined mainly to the cells of the granular tubules. When the enzyme activity is modified in one glandular structure only, the possibility of demonstrating changes 'is dependent on the enzyme activity in other glandular cells, especially when the activity is expressed per g. of total gland. When the activity of the respiratory enzymes was expressed per unit weight, the activity of succinic dehydrogenase decreased after hypophysectomy, but that of cytochrome oxidase and fumarase was unchanged [Ohlin, 1962]; on the other hand, the activity of all enzymes appeared unchanged after treatment with thyroxine or testosterone. The findings on enzyme activity per unit weight after hypophysectomy may indicate that the activity of succinic dehydrogenase decreased markedly in the cells of the granular tubules. It cannot 'be excluded, however, that the

9 454 Ohlin activity of cytochrome oxidase and fumarase also decreased in the tubuilar cells. The enzyme concentration did not seem to increase in the granular tubules after treatment with thyroxine or testosterone. The threshold doses of acetylcholine and methacholine seemed slightly decreased after treatment with thyroxine or testosterone. A tendency of the threshold dose of acetylcholine to increase was seen after hypophysectomy. The changes of the threshold doses of parasympathomimetic agents seem closely linked to structural modifications of the granular tubules. On the other hand, the threshold dose of adrenaline seemed to be independent of this structure since it was unaffected under the various experimental conditions. In this connection it should be recalled that sympathetic denervation of the submaxillary gland of rats affects only the acinar cells [Snell and Garrett, 1958] and that treatment with a catecholamine, isoprenaline, causes an enlargement of these cells [Selye et al., 1961]. Section of the parasympathetic nerve of the submaxillary gland of rats caused marked changes in glandular function [Ohlin, 1965]. In spite of this, after treatment with thyroxine or testosterone the weight of the gland and the maximal secretory response to pilocarpine, when expressed per gland, still increased. Treatment with thyroxine did not result in an increase of the maximal secretory response of the denervated glands when expressed per unit weight. ACKNOWLEDGEMENT This work was supported by a grant from the Faculty of Medicine in Lund. REFERENCES ARGONZ, J. J. and RRAL SALETA, J. M. DE (1961). 'Action de diverses hormones sur la glande sous-maxillaire du rat', C.R. Soc. Biol., Pari, 155, 172. BIXLER, D., MUHLER, J. C. and SHAFER, W. G. (1959). 'Growth hormone and thyroxine: effects on submaxillary gland of hypophysectomized rat', Proc. Soc. exp. Biol., N. Y. 1, GABE, M. (195). 'Action de la thyroxine sur la glande sous-maxillaire du rat hypophysectomise', C.R. Acad. Sci., Pari8, 23, GRAD, B. (1949). 'Joint actions of thyroxine and testosterone in thyroidectomizedcastrated albino rats', Anat. Rec. 13, 458. GRAD, B. and LEBLOND, C. P. (1949). 'The necessity of testis and thyroid hormones for the maintenance of the serous tubules of the submaxillary gland in the male rat', Endocrinology, 45, GRIESBACH, W. E., KENNEDY, T. H. and PURVES, H. D. (1949). 'The physiological activities of the stereoisomers of thyroxine', Endocrinology, 44, JABY, F. and LEESON, C. R. (1959). 'The post-natal development of the rat submaxillary gland', J. Anat., Lond. 93, JABSOHN, D. (196). 'Effects of thyroxine on growth of mammary glands, whole body, heart and liver in hypophysectomized rats treated with insulin, cortisone and ovarian steroids', Acta endocr., Copenhagen, 85,

10 Hormonal Influences on Rat's Submaxillary Gland 455 LACASSAGNE, A. (194 a). 'Dimorphisme sexuel de la glande sous-maxillaire chez la souris', C.R. Soc. Biol., Paris, 133, LACASSAGNE, A. (194 b). 'Reactions de la glande sous-maxillaire a l'hormone male, chez la souris et le rat', (C.R. Soc. Biol., Paris, 133, LEBLOND, C. P. and GRAD, B. (1948). 'Control of the serous acini of the rat submaxillary gland by the thyroid hormone', Anat. Rec. 1, 75. NORDENFELT, I., OHLIN, P. and STROMBLAD, B. C. R. (196). 'Effect of denervation on respiratory enzymes in salivary glands', J. Physiol. 155, OHLIN, P. (1962). 'The effect of hypophysectomy on respiratory enzymes in the submaxillary gland of the rat', Quart. J. exp. Physiol. 47, OHLIN, P. (1965). To be published. PADYKULA, H. (1952). 'The localization of succinic dehydrogenase in tissue sections of the rat', Amer. J. Anat. 91, SCHNEIDER, R. M. and PERSON, P. (196). 'The isolation of submaxillary gland acini and duct segments', Exp. Cell Res. 2, SCHNEYER, C. A. and SCHNEYER, L. H. (1961). 'Secretion by salivary glands deficient in acini', Amer. J. Physiol. 21, SELYE, H., VEILLEUX, R. and CANTIN, M. (1961). 'Excessive stimulation of salivary gland growth by isoproterenol', Science, 133, SHAFER, W. G. and MUHLER, J. C. (1953). 'Effect of gonadectomy and sex hormones on the structure of the rat salivary glands', J. dent. Res. 32, SHAFER, W. G., CLARK, P. G. and MUHLER, J. C. (1956). 'The inhibition of hypophysectomy-induced changes in the rat submaxillary glands', Endocrinology, 59, SHAFER, W. G. and MUHLER, J. C. (1956). 'The effect of desiccated thyroid, propylthiouracil, testosterone, and fluorine on the submaxillary glands of the rat', J. dent. Res. 35, SHAFER, W. G., CLARK, P. G., BIXLER, D. and MUHLER, J. C. (1958). 'Salivary gland function in rats. II. Effect of thyroid function on salivary flow and viscosity', Proc. Soc. exp. Biol., N.Y. 98, SNELL, R. S. and GARRETT, J. R. (1958). 'The effect of postganglionic sympathectomy on the structure of the submandibular and major sublingual salivary glands of the rat', Z. Zellforsch. 48, SREEBNY, L. M. (1953). 'Histologic and enzymatic differences in the submaxillary glands of normal and hypophysectomized male and female white rats', J. dent. Res. 32, 686. WELLS, H. and MUNSON, P. L. (1958). 'Effect of thyroxine on the weight of submaxillary salivary glands', J. dent. Res. 37, 55.