Leo M. Sreebny

Morphologic and biochemical observations on the development of the rat parotid gland.

Litters of rats were sacrificed at 2–5-day intervals from 18 days in utero to 40 days postnatally, and their parotid glands were subjected to biochemical and light and electron microscopic analyses. Biochemical assays were employed in homogenates of the glands for DNA, RNA, protein, and the secretory digestive enzymes α-amylase, alkaline RNase, and DNase I. The results revealed that acinar differentiation in the rat parotid gland is primarily postnatal. Prior to and at birth, the parenchyma of the gland is rudimentary in regard to the enzymes and structures which are characteristic of the acini of the mature gland. One day after birth, the secretory digestive enzymes begin to increase above the levels found in adjacent, nonparenchymal tissues. At the same time, secretory granules appear in the clusters of cells at the terminal ends of the tubular apparatus. The appearance of PAS-positive material in the ductal lumens suggests that functional (secretory) activity begins at this time. There are two periods of rapid growth and differentiation, from about birth to 12 and 15–25 days. The proportional increases of the enzymes in the gland are much higher in the first period, but the absolute amounts gained are higher in the second. Cell size, as indicated by the concentration of DNA per unit of tissue, appears to decrease slightly during the first period, and increase markedly in the second, while the number of cells (DNA per gland) increases rapidly in both periods. At 25 days, the gland has nearly achieved the adult (100-day) levels of all parameters measured except weight. Increases in the amounts of secretory granules and rough endoplasmic reticulum and the development of active Golgi complexes appear to be well correlated with increases in the secretory enzymes and in RNA. The secretory digestive enzymes assayed increase in a noncoordinate manner, suggesting that the control of their synthesis may be asynchronous during the course of acinar differentiation. The onsets of the first and second periods of rapid differentiation coincide respectively with the onsets of suckling and weaning. This suggests that substrates in the food and secretory stimulation may have inductive significance in the differentiation of the gland.

Experimental protein-calorie malnutrition in rats: Biochemical and ultrastructural studies

Abstract Young rats (100–130 gm) fed a low-protein diet which simulates the protein value of diets eaten by children in areas where protein-calorie malnutrition is prevalent developed many of the features associated with kwashiorkor. Among the findings were marked loss in body weight, atrophy of many visceral organs, fatty liver, hypoproteinemia, edema, and profound changes in serum amino acid patterns. Associated with the alterations in serum amino acid levels were extreme disproportions in the fasting levels of amino acids in liver. This experimental model of rodent kwashiorkor showed marked reduction in liver protein and RNA contents, but DNA remained remarkably constant indicating loss of cellular contents rather than decrease in cell population. The decreased RNA content of the liver was consistent with the observations of massive breakdown of the membranes of the endoplasmic reticulum as well as the elevated activity of alkaline ribonuclease assayed in the absence of PCMB. The polysomes showed extensive disaggregation resulting in increased monomer and dimer peaks. The significance of these biochemical and anatomical changes as well as their possible relevance to kwashiorkor in children was discussed.

Effect of food consistency and starvation on the diurnal cycle of the rat parotid gland.

Abstract The parotid glands of rats fed pelleted chow (Purina) were shown to demonstrate a diurnal cycle which is correlated with food intake. To determine the effect of mastication of food on the regulation of this cycle, rats were starved or fed liquid chow. The effects on the gland of starvation or of feeding liquid chow were similar. Without mastication the secretory products (e.g., amylase) which had accumulated in the gland during non-eating periods were not expelled during the eating cycle. Without expulsion further accumulation ceased. Within 4–6 days the gland became atrophic. If these atrophic glands were stimulated by iso proterenol, expulsion of the remaining stores occurred. This was then followed by a reaccumulation of secretory products. These results suggest that mastication of food stimulates expulsion of stored secretory products from the parotid gland and that such expulsion is a prime factor in the regulation of the diurnal secretory cycle.

Effect of Increasing the Bulk Content of the Diet on the Rat Parotid Gland and Saliva

Parotid gland enlargement was induced in rats by incorporating non-nutritive bulk into the stock diet. The magnitude of gland enlargement was directly correlated to the percentage of bulk in the diet. The protein concentration of parotid saliva was increased, but the composition of secretory proteins was not noticeably affected.

STUDIES OF SALIVARY GLAND PROTEASES

Proteases of salivary gland origin have been known since the time of Hufner in 1873. Later, Willstiitter et al. (1929) described proteolytic enzymesin the salivary glands of the dog, pig, horse, and man. These investigators were concerned with placing the salivary glands in the general context of the digestive glands. The direction of interest in salivary proteases changed with Lacassagnes (1940) discovery of morphologic sex differences in the submaxillary glands of mice. Lacassagnes findings focused interest on the hormonal control of salivary glands in rodents. Hormonal control of salivary proteases in mice was first described by Junqueira et al. in 1949. Since then protein-splitting enzymes have been found in the submaxillary glands of other rodents. Our studies were concerned with the characterization of the proteolytic activity of the submaxillary gland in the adult rat, with its developmental history and with its hormonal control. Parallel studies were conducted on the pancreas in order to compare the physiological role of the protea5es of the two organs, and to compare a gland that exhibits sex differences with one that does not.

HORMONES, INANITION AND SALIVARY GLANDS

ABSTRACT This report will deal with the effect of the thyroid gland on enzyme activities of exocrine glands of the rat, and with the habitat, cell of origin, and function of the proteolytic enzyme system of the submaxillary gland. It will show that hypothyroidism reduces cell size and number, and thereby the weight, of the exocrine glands. It seems to affect the acini as well as the tubules. This uniform change in weight is not accompanied by a uniform effect on the activity of the digestive enzymes of these organs. This effect is selective: Hypothyroidism reduces the amylase of the pancreas and the parotid but has no effect on the proteases of the pancreas or the submaxillary gland. Castration reduces submaxillary proteases but does not influence those of the pancreas. Chronic inanition decreases the proteolytic activity of the submaxillary gland but has no effect on pancreatic protease. Evidence is presented which localizes the proteolytic enzyme of the submaxillary gland to the convoluted tubules. The site and mechanism of synthesis and the function of the enzyme system remain a matter of speculation.

Preservation of the secretory granules of rat parotid gland for electron microscopy

Abstract Fixation of rat parotid gland for electron microscopy with osmium tetroxide, in several different buffer systems, results in imperfect preservation of the acinar secretory granules. This is obviated by prior fixation in glutaraldehyde. In the latter instance, two types of secretory granules can be discerned: one associated with the Golgi regions, and the other located primarily in the apical cytoplasm. The findings raise the question as to whether osmium tetroxide, without prior fixation with aldehydes, brings about the solubilization of the contents of the secretory granules.

On the nature of the molecular heterogeneity of rat parotid amylase

Abstract Amylase was isolated from rat parotid tissue and its multiple forms were studied utilizing analytical, SDS-, and isoelectric focusing polyacrylamide gel electrophoresis. Investigations on total amylase, and on fractions prepared by preparative polyacrylamide electrophoresis revealed a major amylase isozyme which appears able to convert into two other isoenzyme forms, and a fourth minor isozyme apparently unrelated to the others.

THE EFFECT OF INANITION ON THE SUBMANDIBULAR SALIVARY GLANDS AND EXOCRINE PANCREAS OF THE RAT

The object of this study was to determine the relative reactivity of three different exocrine organs of the rat to the effects of total starvation. The three organs are the major sublingual gland (SL), the submaxillary gland (SM), and the exocrine pancreas (P). The sublingual gland is mainly a mucus-producing organ; the pancreas is a digestive enzyme-producing organ showing no mucinous moiety; the submaxillary gland has characteristics of both, for it can be shown that mucinous as well as enzymatic components are produced.

The Effect of Castration on the Submaxillary Gland of Hunt-Hoppert Caries-Resistant and Caries-Susceptible Rats

IN 1944, Hunt, Hoppert, and Erwin1 reported the development of two strains of albino rats which differed from the common strains in their susceptibility to dental caries. When placed on a cariogenic diet, one strain showed a late and low incidence of caries; the other an abnormally early and high incidence. The caries-susceptible strain is difficult to maintain and shows signs of low vitality. Assays of the proteolytic enzyme secretion of the submaxillary gland have shown the protease activity of the susceptible animals to be 4 times that of the resistant strain.2 It was found, however, that the proteolytic activity in the caries-susceptible strain was of the same order as in Sprague-Dawley rats3 which are of intermediate caries susceptibility. The proteolytic activity in the caries-resistant strain, on the other hand, was comparatively low. In Sprague-Dawley rats, the proteolytic activity of the submaxillary gland has been shown to be under partial control by the male sex hormone.4 A similar control mechanism presumably exists in other strains of rats. The present study was undertaken to examine the role of the male sex hormone in male rats of the Hunt-Hoppert caries-susceptible and caries-resistant strains. The two strains were compared with respect to the time course of postnatal development and to the effects of castration.