A pilot study identifying type V collagenolytic activity in human amniotic fluid.

Amniorrhexis complicates pregnancies if it occurs in a preterm pregnancy or remote from the onset of labor in a term pregnancy. There are different collagen types (I-V) that create the extracellular matrix of the amnion. Collagenases specific to these collagen types, with the exception of type V collagen, are found in human amniotic fluid, fibroblasts, polymorphonuclear leukocytes and bacteria. Type V collagen is a major component of the amniotic basement membrane and is responsible for maintaining a barrier to bacteria and to the loss of amniotic fluid. We sought to find evidence of type V collagenolytic activity in human amniotic fluid obtained from pregnancies in different clinical states.

Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: influence of thyroid and ovarian hormones.

The glucocorticoid receptor capacity Ro and the dissociation constant Kd were determined in the liver of Xenopus laevis by Scatchard analysis. In 5-year-old female toads Ro was about three times higher than that in males (153.9, 54.3 fmol/mg protein) and Kd was similar in both sexes (4.0, 4.1 nM). Some of the animals used had abnormal enlarged thyroid glands, atrophic ovaries, or both defects in connection with different levels of Ro, but not of Kd, compared to those of normal animals. Females with ovarian atrophy showed significantly lower Ro values, in the same range as in normal males, and a high liver weight. In male and female toads with enlarged thyroid glands and in animals with both defects a significantly higher Ro occurred compared to that of the corresponding group without this abnormality. To study the influence of thyroid hormones on glucocorticoid receptors, young toads (2-3 years old) received injections of 4-phenyl-2-thiouracil, T3, or T4 on 7 consecutive days. Ro and Kd were determined on the following day. Doses of 50 and 500 ng T3 and of 500 and 5000 ng T4 per gram of body weight and day resulted in an increase of Ro up to 250% of the controls. Injections of T3 were more efficient in males than in females. The effect of thyroxine was about the same in both sexes. These observations suggest that thyroid and ovarian hormones exert an influence on glucocorticoid receptor capacity and may belong to the factors which regulate glucocorticoid receptors.

The role of decidual tissue in the development of the dependency on luteinizing hormone in the rat corpus luteum.

In pregnant rats, treatment with an antiserum to LH (LHAS) on days 2-5 inclusive (early LHAS treatment; day 1 = insemination) delayed implantation by about 4 days. The appearance of the dependency of the corpora lutea on LH for the maintenance of progesterone secretion (LH dependency), as determined by the rate of fall in progesterone secretion after a single test injection of LHAS, was also delayed by 4 days. Treatment with a small amount of estradiol on either day 4 or days 4-9 prevented the delay in both implantation and LH dependency. Implantation thus prevented early LHAS treatment from delaying LH dependency, but its effect seems to have been due to decidualization, which accompanies implantation in the rat. In decidual tissue (DT)-bearing pseudopregnant rats, early LHAS treatment delayed LH dependency for only 1 day, while it delayed LH dependency for at least 3 days in ordinary pseudopregnant rats and for at least 4 days in hysterectomized pseudopregnant rats. Estrogen itself seems to have prevented the delay in LH dependency only by inducing implantation, since it had no effect in the DT-bearing pseudopregnant rats. How DT affects the corpus luteum's dependency on LH is unknown, but it may be related to whatever effect DT has on prostaglandin production in the endometrium.

Influence of glucocorticoids on postnatal lung development in the rat: possible modulation by thyroid hormone.

Between the 4th and 10th days of postnatal life in the rat, serum corticosterone levels were low and basal, while the rate of [3H]thymidine incorporation into lung DNA was maximal. From day 13, serum corticosterone levels began to rise significantly, and the lung [3H]thymidine incorporation rate began to fall dramatically; however, these events were obtunded by propylthiouracil-induced hypothyroidism. When 6- to 8-day-old euthyroid pups were given a single sc injection of 10 micrograms dexamethasone, the rate of DNA synthesis in the lung fell by 96.7% of the initial rate at 24 h. This steroidal effect was blunted in hypothyroid pups and restored by exogenous thyroid hormone. The thyroid status of the pup did not modify the response patterns of lung phosphodiesterase and cytosolic glucocorticoid receptor levels to dexamethasone treatment, although both parameters were influenced by thyroid hormone availability. Radiocholine incorporation into lung phospholipids, which was altered in hypothyroidism, was unaffected by dexamethasone treatment. An in vivo assessment of radiothymidine incorporation into DNA of various tissues in 5-day-old euthyroid pups given 10 micrograms dexamethasone 24 h earlier revealed that of the several tissues in which inhibition of DNA synthesis was demonstrable, the developing lung was the most sensitive to the anti-mitogenic steroidal effect. When considered in the light of existing evidence, these observations suggest that glucocorticoids play an important role in triggering lung cytodifferentiation during the third postnatal week in the rat, and that preconditioning of the lung by thyroid hormone optimizes this developmental effect of glucocorticoids.

Thyroid hormone influences glucocorticoid receptor levels in the neonatal rat lung.

In the early postnatal rat, the level of glucocorticoid receptors in the lung increases during the first week of life (5) when thyroid hormone ontogenesis occurs (6). Inhibition of thyroid function in neonatal rat pups by combined pre- and postnatal propylthiouracil treatment abolished the developmental rise in pulmonary glucocorticoid receptor levels. A single injection of 1 microgram T3 to these pups caused an elevation in receptor concentration to euthyroid values in 48 h. These findings support the hypothesis that thyroid hormone promotes tissue receptivity to glucocorticoids in the developing lung of the infant rat.

Endocrine influences on aspects of lung biochemistry.

Evidence is presented that the postnatal rat lung is receptive to several hormones. Experimental diabetes caused a significant depression in lung glucose oxidation rate which was normalized by exogenous insulin therapy. Diabetes also depressed the rate of amino acid incorporation into lung protein; in vitro, insulin stimulated synthesis of selective protein species by the diabetic lung. These results, together with the demonstration of pulmonary receptor sites for insulin and the observation that diabetes selectively affects the ultrastructure of only two cell types in the lung, suggest that insulin may be an important regulator of lung function. That androgens and oestrogens may also exert an effect on the lung is suggested by the presence of distinct receptor activities for these hormones, which appear to be influenced by serum titres of the gonadal steroids. The lung is capable of generating a considerable amount of cyclic GMP in vitro. This ability appears to be dependent upon continued protein synthesis and is influenced by the endocrine state of the animal. Certain steroid hormones are capable of altering cyclic GMP production by the lung when introduced in vitro.

Thyroid activity in a hypometabolic primate, the owl monkey (Aotus trivirgatus).

Serum levels of triiodothyronine (T3) and tetraiodothyronine (T4) were significantly lower in owl monkeys than in long-tailed macaques. These observations were considered to be consistent with the lower metabolic rate of the owl monkey. However, the absence of a significant difference in the levels of thyroid stimulating hormone (TSH) between the two species suggested a lower thyroid sensitivity to TSH in the owl monkeys. There was an inverse relation between levels of T3 and TSH in the owl monkeys at night and during the day.

Alterations in the ultrastructure of nonciliated bronchiolar epithelial (Clara) cells by streptozotocin-induced diabetes in rats.

Recent studies showed marked alterations in type II pneumocyte ultrastructure by streptozotocin-induced diabetes. To see whether other pulmonary epithelium is affected, we examined terminal bronchioles by transmission electron microscopy. Ten adult male rats received one intrajugular injection of streptozotocin (75 mg/kg of body weight) and were killed 14 days later. As control subjects, 5 additional rats received only saline, and 5 rats given streptozotocin injections received protamine zinc insulin (1 unit/rat/day). As compared to lungs of control animals, the bronchiolar epithelial cell type most altered in diabetic rat lungs was the Clara cell. The numbers of ovoid secretory granules decreased significantly (29 of 57 cells were without granules versus 0 of 78 in control animals), and the number of rod-shaped granules increased. Administration of exogenous insulin alleviated the changes. These findings show that the changes in pulmonary metabolic function associated with diabetes mellitus result from alterations not only in granular pneumocytes, but also in nonciliated, bronchiolar secretory epithelial (Clara) cells.

Na+ pump activity and nuclear T3 receptors in tissues of genetically obese (ob/ob) mice.

A dramatic reduction in ouabain-sensitive tissue respiration of obese mouse muscle and liver was observed, suggesting that Na+-transport-dependent calorigenesis is impaired in these animals. Additionally, a significantly depressed nuclear triiodothyronine binding capacity in liver and lung tissue was exhibited by obese mice. These data support the suggestion that the hypometabolism and hypothermia of the genetically obese mouse is a result of reduced Na+-pump-related thermogenesis; and further, provides evidence that this may be the consequence of reduced nuclear binding of triiodothyronine.

Receptors for androgen and estrogen in the rat lung.

By the use of sucrose density gradient and protamine sulfate analyses, the cytosol fraction of rat lung has been found to contain binding activities for both androgenic and estrogenic steroids. These activities are present in lungs of rats of both sexes and at all ages examined. Both activities interacted stereospecifically with, and possessed high affinity for, the respective ligand, and sedimented in the 8-9S region of low salt sucrose density gradients. The androgen-binding activity appeared to bind 5alpha-dihydrotestosterone (DHT) and testosterone with similar affinities, but in vitro, whole lung incubation studies demonstrated that DHT is the perdominant androgen found in lung nuclei. In the immature male rat, removal of endogenous androgen by castration-adrenalectomy had no effect on DHT-binding activity in lung cytosol. In the adult, however, this procedure led to a marked cycloheximide-sensitive increase in binding activity. During postnatal development in male rats, relatively constant levels of both androgen- and estrogen-binding activities were found in the lung until the peripubertal period, from which time a striking increase in DHT binding and a decline in that of estradiol were observed. In female rats, cyclic changes in the level of both binding activities were evident during the estrous cycle. When considered in the light of evidence that sex steroids influence certain aspects of lung biochemistry, these data suggest that the demonstrated binding activities represent hormone receptor activities which may mediate a variety of biological events in the lung.

Triiodothyronine receptors in rat lung.

Specific triiodothyronine receptor activity has been detected in rat lung nuclei isolated by Triton X-100, which was sensitive to pH and exhibited temperature-dependent association kinetics. This activity represented a single order of high affinity binding sites, with an apparent dissociation constant for 3,3',5-triiodo-L-thyronine (L-T3) of 0.254 +/- 0.027 nM and an apparent capacity of 249.4 +/- 15.5 fmol 3,3',5-triiodo-L-thyronine/mg DNA. Its specificity was such that triiodothyronine derivatives were bound at least 10 times more avidly than were the tetraiodothyronines. The presence of specific triiodothyronine receptors in lung nuclei suggests that the effect of thyroid hormones on pulmonary surfactant metabolism may be mediated through these binding sites.

The presence in bovine trachealis muscle of cytosol receptors with high affinity for glucocorticoids.

Whereas receptors with high affinity for glucocorticoids have been reported present in both fetal and adult lungs, there has been no attempt to identify such receptors in airways smooth muscle. We have examined the binding characteristics of glucocorticoids for a cytosol fraction prepared from bovine trachealis muscle. The cytosol fraction, which was prepared in 10 mM Tris, 1.5 mM Na2 EDTA and 2 mM dithiothreitol, contained macromolecular high affinity receptors for 3H-dexamethasone (Kd = 5.6 nM) at a concentration of 0.20 pmol/mg protein. The comparative affinities of other steroids in descending order were: beclomethasone-17, 21-dipropionate, dexamethasone, beta-methasone, triamcinolone and triamcinolone acetonide. Progesterone and deoxycorticosterone only bound to a limited extent, whereas the binding with 5 alpha-dihydrotesterone and pregnenolone was negligible. We conclude that airways smooth muscle is a target tissue for glucocorticoids and postulate that the binding of glucocorticoids to these receptors may receptors may represent the initial step in the anti-asthmatic action of these drugs.

Pulmonary insulin responsivitiy: in vivo effects of insulin on the diabetic rat lung and specific insulin binding to lung receptors in normal rats.

Adult rats were rendered diabetic by a single iv injection of streptozotocin (70 or 75 mg/kg). In these rats, serum insulin fell to minimal levels during the 48 h following drug treatment, and this was roughly paralleled by a progressive decrease in the ability of the lung to oxidize glucose. The addition of insulin to diabetic rat lung slices in vitro had no restorative effect on the depressed glucose oxidative rate during a 2 h incubation period; however, two daily treatments of the rats with 1 unit of protamine, zinc insulin completely restored lung glucose oxidation rate to normal, without significantly reducing the hyperglycemic state of the rats. An examination of the temporal changes in glucose utilization by the rat lung after acute insulin treatment revealed that the diabetic lung responded directly to serum levels of insulin, whereas the normal lung appeared to be unaffected by serum insulin levels as hihg as 87 ng/ml. The reduced rate of glucose oxidation in the diabetic lung was apparent after perfusion of the lung with glucose-free medium, and was characterized by a significant reduction in Vmax without an alteration in Km. This was attended by a depressed ability of the lung to incorporate [3H]leucine into protein and an increased ability to produce lactate, but hexose monophosphate shunt activity was normal. Specific receptors for insulin have been identified and partially characterized in crude membrane preparations of normal rat lung. The interaction of insulin with these receptors was rapid, reversible, saturable, and was dependent upon time and temperature. The binding of labeled insulin was inhibited by low concentrations of unlabeled insulin and by high concentrations of proinsulin, whereas it was unaffected by the presence of glucagon, gastrin, prolactin, ACTH, or growth hormone in microgram amounts. These observations suggest that insulin regulates the transport and utilization of glucose in the rat lung, and that this tissue contains specific receptors for insulin.

Hormonal evaluation of the intrauterine progesterone contraceptive system.

Nine women were studied for one menstrual cycle prior to the insertion of an intrauterine progesterone contraceptive system (IPCS) delivering 65 microng progesterone/day into the uterus and again at 1 month after its insertion. Eight of these women were again studied between 6-8 months after the insertion of the IPCS. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin and relaxin were measured in each plasma sample. The data from each study were combined according to the day of the LH peak. Ovulation occurred in all the cycles studied in spite of an elevation in plasma estradiol-17beta and a depression of prolactin and relaxin immunoactivities at the 6-8 month follow up. Menstruations noted at the 6-8 month of use occurred while levels of estradiol-17beta and progesterone were elevated.

PIP: Plasma levels of luteinizing hormone, follicle stimulating hormone, estradiol-17beta, progesterone, prolactin, and relaxin were studied in 9 women before and after insertion of an Alza, progesterone-releasing, IUD. The IUD releases approximately 65 mcg/day of progesterone into the uterus. The lengths of the preovulatory and postovulatory phases of the menstrual cycle were not affected by the device. Ovulation occurred in all the cycles, even though plasma levels of estradiol-17beta were significantly (p less than .05) increased and plasma levels of prolactin and relaxin were significantly (p less than .05) decreased as late as 6-8 months after insertion. During menstruation at 6-8 months after insertion, plasma levels of estradiol-17beta and progesterone were high. The results are discussed.

The binding of phenol red to rabbit renal cortex.

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 - 10(3) M-1 and a binding capacity of 2.41 mumol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 - 10(3) M-1 and a maximal binding capacity of 55.06 mumol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive to the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./l, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.

Venous outflow of the hormones secreted by the rat pituitary autotransplanted beneath the kidney capsule.

In adult female or male rats subjected to hypophysectomy and autotransplantation of the pituitary beneath the renal capsule, the levels of prolactin in the renal vein of the pituitary-bearing kidney were not significantly different from those found either in the renal vein of the contralateral kidney or in the jugular veing. LH levels were slightly but highly significantly higher than those found in hypophysectomized rats; the levels found in the two renal veins and the jugular vein were also not sigificantly different from one another.

Radioimmunologic assessment of the level of circulating LH antibodies after passive immunization of the rat: relation to the level of LH secretion.

A double antibody radioimmunologic technique was used to estimate circulating levels of "free" LH antibodies (Ab) (i.e., Ab available for binding to radioiodinated rat LH in vitro) after a single injection of an equine antiserum to bovine LH (LH-AS) into cyclic (diestrus-1), early pregnant (d 6 or d 8), short-term (1 d) or long-term (12 d) ovariectomized, or 8-d hypophysectomized female rats. In both cyclic and pregnant rats given 0.5 ml LH-AS sc the blood Ab levles peaked 1 to 2 days after injection and then decreased exponentially (half-time: 14.6-17.0 h) and equally: vaginal estrus also appeared in the cyclic and the aborting pregnant rats on the 6th day, and ovulation on the 7th day after LH-AS treatment, when the blood levels had fallen to very low values. The rate of exponential decrease in blood Ab levels was not affected by other routes of administration (iv or ip) of LH-AS, or by sc doses of 1.0 or 0.25 ml, although the peak levels and their duration were related to dose and route of administration. Ovariectomy significantly increased the rate of exponential Ab decrease (half-time: 9.8 h in rats tested 12 days after operation) and hypophysectomy markedly diminished it (half-time: 63.0 h in rats tested 8 days after operation). Daily treatment of hypophysectomized rats with 20 mu/day of ovine LH from the time of LH-AS injection increased the rate of exponential Ab decrease to one approaching that of the intact rats (half-time: 20.5 h). Free LH antibodies thus seem to disappear from the circulation at a rate proportional to the amount of LH in the circulation.