Hormonal interactions in mammalian collagenase regulation: Comparative studies in human skin and rat uterus

The production of collagenase by human skin explants in culture is prevented by 10^?8 M dexamethasone, 5·10^?4 M dibutyryl cyclic AMP, or 2.5· 10^?3 M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur.Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10^?7 M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen

Prostaglandin E1 and cholera toxin increased the intracellular levels of cyclic AMP of human lung fibroblasts. With prostaglandin E1, the increase in cyclic AMP occurred within 10 min followed by a decline to less than one-half of peak values in 6 h. With cholera toxin, the increase occurred within 60 min but the level of cyclic AMP remained increased for 6 h. Both agents caused a decrease in collagen production as expressed as the proportion of newly synthesized protein represented by collagen. The increase in cyclic AMP levels was accompanied by a marked increase in the proportion of newly synthesized collagen which was degraded intracellularly prior to secretion. Analysis of the degraded collagen showed it to be predominantly less than 1000 daltons in molecular mass, but still in peptide linkage. The data are consistent with the hypothesis that cyclic AMP levels in diploid fibroblasts regulate the amount of collagen produced by fibroblasts, at least in part, by modulating the level of intracellular collagen degradation.     

Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Paracrine regulation of epithelial progesterone receptor and lactoferrin by progesterone in the mouse uterus

The objective of this study was to determine whether uterine stromal and/or epithelial progesterone receptor (PR) is required for the antagonism by progesterone (P(4)) of estradiol-17beta (E(2)) action on expression of PR and lactoferrin in uterine epithelium. Uterine tissue recombinants were prepared with epithelium (E) and stroma (S) from wild-type (wt) and PR knockout (PRKO) mice: wt-S+wt-E and PRKO-S+wt-E. P(4) action on epithelial PR expression was studied in wt-S+wt-E and PRKO-S+wt-E tissue recombinants. E(2) down-regulated epithelial PR in both types of tissue recombinants, but P(4) blocked E(2)-induced down-regulation of epithelial PR only in wt-S+wt-E tissue recombinants. Thus, P(4) requires stromal PR to inhibit E(2)-induced down-regulation of epithelial PR. Epithelial PR is not sufficient in itself. The inhibitory effect of P(4) on lactoferrin expression was studied in 4 types of tissue recombinants (wt-S+wt-E, PRKO-S+wt-E, wt-S+PRKO-E, and PRKO-S+PRKO-E). E(2) induced lactoferrin in all 4 types of tissue recombinants. P(4) blocked E(2)-induced lactoferrin expression only in wt-S+wt-E tissue recombinants. In wt-S+PRKO-E tissue recombinants, P(4) inhibited lactoferrin expression only partially. P(4) failed to block E(2)-induced lactoferrin expression in PRKO-S+wt-E and PRKO-S+PRKO-E tissue recombinants. Thus, both epithelial and stromal PR are essential for full P(4) inhibition of E(2)-induced lactoferrin expression.     

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex.

1. Cyclic adenosine 3',5'-monophosphate and N-6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. N-6-2'-O-Dibutyryl adenosine 3'-5'-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.     

Inhibition of calmodulin-dependent cyclic AMP phosphodiesterase by phenoxazines

Phenoxazine derivatives were examined for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase, which is based on the hydrolysis of cAMP to AMP by phosphodiesterase in the presence or absence of inhibitor, followed by quantitative analysis by HPLC method. Anticalmodulin activity of phenoxazines with respect to substitution at C-2 position follows the order: 2-trifluoromethyl>2-chloro>unsubstituted phenoxazines. The interaction of phenoxazines with calmodulin using fluorescence spectroscopy has been performed. Binding study showed that calmodulin has two types of binding sites for phenoxazines. One is high affinity binding site (Kd value 0.07-0.46 microM) and the other, a low affinity binding site (Kd value 0.7-34.5 microM). The change in secondary structure of calmodulin upon binding to phenoxazines was studied by circular dichroism (CD) method, which showed that the percentage of helicity decreased with an extensive change in tertiary structure of calmodulin. Kinetic analysis of the phenoxazine-calmodulin interaction showed that phenoxazines competitively inhibited the activation of phosphodiesterase without affecting Vmax. Thus, these studies showed a good correlation between the ability of phenoxazines to block the activation of phosphodiesterase and their ability to bind to the activator.     

Inhibition by oestrogen of collagen breakdown in the involuting rat uterus

1. Both the post-partum involution of the rat uterus and the rapid breakdown of collagen that accompanies it are extensively inhibited by oestrogenic hormones. In the normal rat, 85% of the uterine collagen is degraded within 4 days after parturition; in rats treated with 100μg. of 17β-oestradiol/day, only 35% of uterine collagen is broken down in the same period. 2. Similar effects are produced by diethylstilboestrol if the dose is increased tenfold. 3. Collagen breakdown is inhibited to a greater extent than is the loss of wet weight by oestradiol but not by diethylstilboestrol. 4. The oestrogens appear to act by blocking the breakdown of collagen. There is a greatly decreased concentration of free hydroxyproline in the uterus of treated animals. 5. Acid hydrolase concentrations (β-glucuronidase, β-galactosidase, cathepsin D and acid phosphatase) in the uterus are decreased by oestrogen treatment compared with controls, but the total amounts of these enzymes in the uterus are somewhat elevated. Oestrogens do not appear to inhibit collagen breakdown by altering the concentration and total amount of acid hydrolases.     

Hormonal regulation of progesterone receptor mRNA expression in the hypothalamus of whiptail lizards: regional and species differences

The effects of gonadal steroid hormones on steroid receptor mRNA expression vary across nuclei within the brain, between the sexes, and between species. We report that exogenous estrogen increases progesterone receptor (PR) mRNA levels in the periventricular preoptic area in an ancestor and descendant species pair of whiptail lizards, and also that this effect of estrogen is significantly stronger in females of the descendant species. Second, while progesterone strongly decreases PR mRNA in the ventromedial hypothalamus of whiptail lizards and rodents, we find that there is no discernible effect of progesterone on PR mRNA levels in the periventricular preoptic area in females of the ancestral member of this species pair. These findings are a further demonstration of the variability of steroid effects on steroid receptor mRNA levels across brain nuclei. This variability may be important both in behavioral transitions over the course of the ovarian cycle in this ancestor-descendant species pair of lizards and in the evolution of pseudosexual behavior in the descendant parthenogen species.     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum.

When amoebae of Dictyostelium discoideum, suspended in buffer, were treated with 100 nM pulses of cAMP, the extracellular cAMP phosphodiesterase (ePD) activity increased dramatically and the synthesis of the phosphodiesterase inhibitor (PDI) was repressed. In addition, the time of appearance on the cell surface of contact sites A, membrane-bound cAMP phosphodiesterase, and cAMP binding sites was accelerated by 3–4 hr and the concentration of intracellular cAMP increased ?20-fold. When the concentration of the cAMP pulse was reduced to 1 nM, the effect of the pulses on membrane differentiation and on the cAMP pool was virtually the same, while the effect on the ePD-PDI system was reduced. When cAMP was added to the suspension continuously, the nucleotide had no effect on membrane differentiation and failed to stimulate the intracellular cAMP pool, however, the ePD-PDI system was regulated normally. When the developmental mutant, HC112, was treated with cAMP pulses, membrane differentiation and the level of the cAMP pool were unaffected, while the ePD-PDI system responded to the exogenous cAMP. In another mutant, HC53, membrane differentiation was stimulated by cAMP pulses and this response was accompanied by a sharp increase in the concentration of the cAMP pool. These results suggest that the ePD-PDI system and membrane differentiation are regulated independently by exogenous cAMP and that regulation of the ePD-PDI system does not require activation of the adenylyl cyclase.     

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.     

Inhibition by prolactin of gonadotrophin-induced cyclic AMP accumulation in the preovulatory ovary

We investigated whether prolactin acts at the ovarian level by interfering with the accumulation of gonadotrophin-induced ovarian cyclic AMP. Mouse ovaries were incubated with hCG and varying doses of prolactin. At the end of the incubation, the cyclic AMP which accumulated in the tissue + medium was measured. In ovaries devoid of corpora lutea, a significant inverse correlation (r = -0.93, P less than 0.05) was obtained between the doses of prolactin (0.1-25.6 micrograms ovine prolactin) and hCG-induced accumulation of ovarian cyclic AMP. In the presence of the phosphodiesterase inhibitor IBMX, however, the same doses of prolactin failed to exhibit any restricting influence on the accumulation of cyclic AMP. In luteinized ovaries, the same doses of prolactin in the absence of IBMX did not inhibit the hCG-induced cyclic AMP accumulation.     

The regulation of cell proliferation by calcium and cyclic AMP

Calcium, in partnership with cyclic AMP, controls the proliferation of non-tumorigenic cells in vitro and in vivo. While it does not seem to be involved in the proliferative activation of cells such as hepatocytes (in vivo) or small lymphocytes (in vitro), it does control two later stages of prereplicative (G1) development. It must be one of the very many regulatory and permissive factors affecting early prereplicative development, because severe calcium deprivation reversibly arrests some types of cell early in the G1 phase of their growth-division cycle in vitro. However, calcium more specifically and much more often regulates a later (mid or late G1) stage of prereplicative development. Thus, regardless of its severity or the type of cell, calcium deprivation in vitro or in vivo reversibly stops proliferative development at that part of the G1 phase in which the cellular cyclic AMP content transiently rises and the synthesis of the four deoxyribonucleotides begins. The evidence points to calcium and the cyclic AMP surge being co-generators of the signal committing the cell to DNA synthesis. The evidence is best explained so far by the cyclic AMP surge causing a surge of calcium ions which combine with molecules of the multi-purpose, calcium-dependent, regulator protein calmodulin (CDR) somewhere between the cell surface and the cytosol. The resulting Ca-calmodulin complexes then stimulate many different (and possibly membrane-associated) enzymes such as protein kinases, one of which produces the DNA-synthetic initiator. Calcium has little or no influence on the proliferation of tumor cells. Some possible explanations of this very important loss of control are considered.