Progesterone metabolism by major salivary glands of rat--I. Submandibular and sublingual glands

The metabolism of progesterone by the submandibular and sublingual salivary glands of female (nonpregnant and pregnant) and male rats was studied. The metabolism was in both sexes significantly greater in submandibular than in sublingual glands. Sex differences were not seen in sublingual glands but less metabolism was found in homogenates and microsomal fractions of female (nonpregnant and pregnant) submandibular glands compared to that of males. The metabolism did not differ between pregnant and nonpregnant female rats. The metabolites were mainly 5 alpha-pregnane-compounds. On the basis of the metabolites identified it can be concluded that rat submandibular and sublingual glands contain at least 3 alpha-, 3 beta-, 20 alpha- and 20 beta-hydroxysteroid dehydrogenase, 5 alpha- and 5 beta-steroid hydrogenase and 17 alpha-steroid hydroxylase activity. 5 alpha-steroid hydrogenase activity was significantly higher in all preparations of male submandibular glands than in females. In sublingual glands some enzyme activities showed pregnancy-related decreased.     

Studies on metabolism of vitamin A. The effect of vitamin A status on the secretion rates of some steroids into the ovarianvenous blood of pregnant rats

1. Rats raised on a vitamin A-deficient diet supplemented with either retinyl acetate or retinoic acid were mated and became pregnant. 2. The rates of secretion of progesterone, 20alpha-hydroxypregn-4-en-3-one, oestradiol-17beta and oestrone into the ovarian-venous blood of rats in these two groups were measured on days 9 and 15 of pregnancy. 3. Rates of secretion of progesterone and 20alpha-hydroxypregn-4-en-3-one, both on days 9 and 15, were lower for the rats given retinoic acid. No such differences were found in ovarian oestrogen secretion. 4. The implications of these results are discussed in the light of the previous demonstration that the activity of ovarian 3beta-hydroxy-Delta(5)-steroid dehydrogenase was markedly less in pregnant rats given retinoic acid.     

Solubilization and separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid-delta4-delta5-isomerase from bovine adrenal cortex microsomes.

A physical separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid delta4-delta5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3beta-ol-17-one, pregnen-3beta-ol-20-one and pregn-5-en-(3beta,17alpha)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.     

The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.     

Estrogen induction of 20alpha-hydroxysteroid dehydrogenase in mouse vaginal tissue

The conversion of progesterone to 20α-hydroxy-4-pregnen-3-one by 20α-hydroxysteroid dehydrogenase was measured in mouse vaginal tissue. The enzyme was confined to the 105,000 × g supernatant of tissue homogenates and the requirement for reduced NADP demonstrated. The Initial rates of 20α-hydroxysteroid dehydrogenase were determined in the cytosol of tissues from four-day estrogen-treated and untreated animals. The rate of 20α-hydroxy-4-pregnen-3-one formation per vagina was increased 15-fold by estrogen stimulation. This increase could not be accounted for on the basis of increased organ weight or increased availability of cofactor. These findings indicate that 20α-hydroxy steroid dehydrogenase induction in the mouse vaginae is under estrogen control.     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Early Steroid Metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris Embryos

Embryos of Xenopus laevis , Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos.
In Xenopus incubations progesterone was converted to 5α-pregnane-3,20-dione, 17α-hydroxy-4pregnen-3-one, 4-androstene-3,17-dione and 17α,20α:-dihydroxy-4-pregnen-3-one, indicating 5α-reductase, 17α-hydroxylase, 19–20-desmolase and 20α-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19–20-desmolase was found. In Rana oocytes were also not evidence of 17α-hydroxylase activity. All identified activities except 20α-hydroxylase were common to embryos of all three species.
It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthetized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.     

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.     

Attenuated hypotensive response to proadrenomedullin N-terminal 20 peptide in pregnant rats: modulation by steroid hormones

The hypotensive effect of proadrenomedullin N-terminal 20 peptide (PAMP) was examined in conscious pregnant (8, 14, and 20 days of pregnancy) and nonpregnant rats. Intravenous administration of PAMP (3–60 nmol/kg) produced a dose-dependent depressor response in both pregnant and nonpregnant rats. However, the maximum decrease in blood pressure was significantly attenuated in pregnant rats in mid- and late-gestation (14 and 20 days), but not in early gestation (8 days), than in nonpregnant rats. In ovariectomized rats, the depressor responses in 17β-estradiol (E2)-treated, progesterone (P)-treated, and E2+P-treated rats were significantly attenuated compared with the control rats. We also demonstrated that treatment of sex hormones reduces the depressor response to PAMP in 8-day pregnant rats. In addition, we showed that treatment of sex hormone receptor antagonists partially prevents the attenuation of the depressor response to PAMP in 20 day pregnant rats. These findings suggested that the hypotensive response to PAMP was more attenuated in pregnant rats in mid- and late-gestation than in nonpregnant rats, and that the changes in depressor response that occur at term in pregnant rats may be mediated by sex hormones. PAMP may play some important role in cardiovascular regulation during pregnancy.     

Influence of streptozotocin-induced diabetes on hexokinase activity of rat salivary glands

The influence of diabetes on the enzyme hexokinase (HK) was examined in the salivary glands of rats. Diabetes was induced by an intraperitoneal injection of streptozotocin (60 mg/Kg body weight) in overnight fasted rats (180-200 g). The animals were killed 48 hours and 30 days after the induction of diabetes and the submandibular and parotid salivary glands extracted for use. Hyperglycemia was evaluated by determining the blood sugar. The area occupied by each intralobular component, acini, ducts, total parenchyma and stroma was measured, and no differences were observed compared with control. In the soluble fraction of the submandibular gland, no difference in the specific activity of HK was observed, between the diabetic and control animals, however, the activity per gland and per g of tissue showed lower values than control. The specific activity of the bound form was reduced in the diabetic gland. The results obtained for the parotid gland were different from the submandibular. The specific activity of both the soluble and bound forms were increased in the diabetic animals. The DEAE-cellulose column chromatography of the soluble and bound forms of the enzyme from both glands showed a first peak appearing during the washing of the column and two other peaks were eluted by the gradient. Thus, three isoenzymes in the submandibular and parotid salivary glands for the control and diabetic rats have been found.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Alteration of Ca2+‐ATPase activity in the homogenate,plasma membrane and microsomes of the salivary glands of streptozotocin‐induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca²⁺‐ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin‐induced diabetes. We have also examined the influence of the acidosis state on this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH₄Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca²⁺‐ATPase (total, independent, and dependent) was determined in the homogenate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be higher in the diabetic animals. Ca²⁺‐ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative developmental analysis of the parotid, submandibular and sublingual glands in the neonatal rat.

Analysis of the soluble protein fractions from the rat parotid, submandibular and sublingual glands by polyacrylamide-gel electrophoresis reveals similarities in overall patterns of protein synthesis at birth. Tissue-specific changes in protein and glycoprotein synthesis occur shortly after birth and again at the time of weaning, 21--28 days later. Incorporation of [3H]thymidine into DNA was at its highest after birth and gradually decreased in both the parotid and submandibular gland, whereas [3H]thymidine incorporation in the sublingual gland was low throughout the time of neonatal development. [14C]Leucine incorporation into total protein increased in all glands with age after birth, showing an accelerated rate 21--28 days later. Trichloroacetic acid/phosphotungstic acid-precipitable [3H]fucose in glycoproteins declined over the time of neonatal development in the parotid and submandibular gland, but its incorporation remained higher in the sublingual gland. alpha-Amylase (EC 3.2.1.1) in the salivary glands increased at the time of weaning, as judged by detectability in sodium dodecyl sulphate/polyacrylamide gels and by immune precipitation. Two membrane-bound enzymes, UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) and UDP-galactose:2-acetamido-2-deoxy-D-galactosaminyl-protein 3 beta-galactosyltransferase (no EC number), undergo tissue-specific change rather than changes induced by physiological stimulation of the salivary glands.     

Immunocytochemical localization of nerve growth factor in mouse salivary glands

Summary The submandibular glands of female mice and the sublingual and parotid glands of adult male and female mice have been examined by light microscopical immunocytochemistry for nerve growth factor (NGF). In female submandibular glands, staining for NGF was observed in granular convoluted tubule and striated duct cells. Sublingual glands of the mouse contained relatively few granular cells staining for NGF compared with submandibular glands. However, such granular cells appeared to be more numerous in male sublingual glands than in female glands. The remainder of the intralobular duct cells in both male and female sublingual glands exhibited apical subluminal staining for NGF as well as light basal plasmalemmal staining. Parotid glands in both male and female mice exhibited a similar pattern of staining for NGF in striated duct cells. However, the glands did not contain granular cells nor did they exhibit any pattern of staining which reflected a sexual dimorphism. Immunodot staining of salivary gland extracts confirmed the presence of immunoreactivity for NGF in all three of the major salivary glands.     

Characteristics of sialidase in the rat salivary glands

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.     

Sex difference in L-glutamine D-fructose-6-phosphate aminotransferase activity of mouse submandibular gland

L-Glutamine D-fructose-6-phosphate aminotransferase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino transferring), EC 5.3.1.19) activities in the three main salivary glands of male and female mice were measured. It was found that the activity in the submandibular gland was about 10 times more in females than in males, whereas the activities in the sublingual and parotid glands of males and females were similar. The activity in the submandibular gland of female mice was not affected appreciably by ovariectomy but it decreased to the level in males on injection of testosterone. The activity in males was not affected appreciably by injection of progesterone or 17β-estradiol, but it increased to the level in females after castration. The increased acitivity in castrated male mice was decreased again to the normal level by testosterone injection. Thus, this sex difference is caused by androgen, not by female hormones. On the basis of in vivo experiments using actinomycin D, it was suggested that testosterone produced an “enzyme inhibitor”, which suppressed the enzyme activity in the submandibular glands of androgen-rich animals.     

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.