Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.     

Staphylococcus aureus stimulates inducible nitric oxide synthase in articular cartilage.

OBJECTIVE: To determine if Staphylococcus aureus stimulates the L-arginine-nitric oxide (NO) synthase pathway in articular cartilage. METHODS: A heat-killed and sonicated (denatured) S. aureus preparation was added to cultures of bovine articular cartilage. NO production was measured as accumulated nitrite in the culture medium and by the NO synthase-dependent conversion of 3H-L-arginine to 3H-L-citrulline in cartilage homogenates. Inducible NO synthase (iNOS) messenger RNA (mRNA) expression was analyzed by Northern blot. Proteoglycan synthesis was measured by 35SO4 incorporation into glycosaminoglycan. RESULTS: Nitrite accumulation and 3H-L-citrulline formation in cartilage were elevated by denatured S. aureus (compared with unstimulated control cartilage) and inhibited by the NO synthase inhibitor N(G)-monomethyl-L-arginine. Northern blot analysis revealed increased iNOS mRNA expression in bovine chondrocytes in response to denatured S. aureus stimulation. Denatured S. aureus suppressed the accumulation of 35SO4-labeled macromolecules representing newly synthesized proteoglycans in bovine articular cartilage. The suppressed proteoglycan synthesis was due to the presence of NO. CONCLUSION: These findings support the hypothesis that a component of S. aureus can stimulate iNOS in articular cartilage, and that NO generated from this enzyme down-regulates cartilage matrix proteoglycan synthesis.     

Independence of the androgen-induced mRNA synthesis on exogenous glucose in the ventral prostate of the rat

Poly (A)-containing RNA species were isolated from prostatic polysomes after administration of testosterone to castrated rats. After exposure of prostatic tissue to radiolabelled uridine in vitro for 1 h the radioactivity of polysomal poly (A)-RNA increased at 6-12 h after testosterone treatment, which coincides with the hormonal activation of protein synthesis under the same experimental conditions. The increased radioactivity of polysomal mRNA was not due to changes in the uptake and phosphorylation or radiolabelled uridine or in the endogenous content of uridine nucleotides representing thus an increased mRNA content. Electrophoretic patterns of double-labelled polysomal mRNA failed to show detectable amounts of androgen-specific species. The larger part of increase in mRNA content thus consists of mRNAs common to prostatic cells of both testosterone-treated and non-treated castrated animals. The hormonal stimulation of polysomal poly (A)-RNA accumulation was not dependent upon exogenous glucose which is in contrast to protein synthesis. However, glucose was necessary for the hormonal activation of the phosphorylation of [3H]uridine leading ultimately to increased labelling of all RNA species. It is concluded that increased accumulation of polysomal mRNA may be necessary, but not sufficient for the hormonal activation of protein synthesis.     

Effects of limited exposure of rabbit chondrocyte cultures to parathyroid hormone and dibutyryl adenosine 3',5'-monophosphate on cartilage-characteristic proteoglycan synthesis

Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of [35S]sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either [35S]sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of [3H] serine into both proteoglycans and total protein. In all cultures described above, the stimulation of [3H]serine incorporation into proteoglycans exceeded that of [3H]serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of [35S]sulfate, the 3H radioactivity with glucosamine, and [3H]serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect [35S] sulfate incorporation into proteoglycans. These observations suggest that in addition to the transient rise of cAMP, other biochemical changes are required for elaboration of the effect of PTH on proteoglycan synthesis. Although cAMP analogs mimic some of the effects of PTH in chondrocytes, the nucleotides and PTH appear to stimulate proteoglycan synthesis by different mechanisms.     

Cyclic nucleotides and somatomedin action in cartilage

The role of cyclic nucleotides was evaluated in the stimulation of cartilage metabolism by somatomedin-C (Sm-C). The effects of cAMP and cyclic guanosine monophosphate (cGMP) analogs on matrix synthesis were evaluated. The effects of Sm-C on tissue concentrations of these cyclic nucleotides were investigated. Likewise, the direct effects of Sm-C on the activities of cartilage adenylate cyclase, guanylate cyclase, and phosphodiesterase were determined. We found that tissue concentrations of cAMP in cartilage declined rapidly during organ culture, despite the presence of serum or Sm-C, cGMP concentrations in cartilage declined rapidly during control incubations but were augmented significantly at 30 and 60 min of incubation with the addition of serum or Sm-C. Thereafter, cGMP concentrations declined toward the levels of incubated control cartilages. Sm-C had no effect on phosphodiesterase activity. N6-Monobutyryl cAMP stimulated sulfate uptake, but dibutyryl cGMP did not. Sm-C did not stimulate adenylate cyclase in purified plasma membranes from chondrocytes, whereas it stimulated both plasma membrane and cytosol guanylate cyclase at concentrations of Sm-C as low as 10(-12) M. These data would indicate that cAMP is not the intracellular second messenger for Sm-C in cartilage. The data for cGMP are provocative and suggest it as a candidate for a second messenger mediating a portion of Sm's stimulation of cartilage metabolism.     

Stimulation of uridine, leucine, and sulfate incorporation into rat cartilage macromolecules by adenosine 3',5'-monophosphate

Conflicting findings on the ability of cAMP analogs or phophodiesterase inhibitors to stimulate precursor incorporation into macromolecules of rat cartilage have been reported. Therefore, the effects of these compounds on the incorporation of uridine into RNA, leucine into proteins, and sulfate into proteoglycans have been reexamined in cartilage from normal and hypophysectomized rats. When cartilage was incubated for 24 h in a medium with the test agents and then pulsed for 2 h in the basal medium containing labeled precursors, both monobutyryl cAMP (BucAMP) and methylisobutylxanthine (MIX) enhanced the ability of the tissue to incorporate precursors into macromolecules. The effect of BucAMP was significant in most cases at a concentration of 30 microM, optimal at concentrations of 100-300 microM, and diminished at a concentration of 1000 microM. Similar stimulation was produced by dibutyrul cAMP [(Bu)2cAMP] or 8-dimethylamino cAMP, but monobutyryl cGMP was ineffective. MIX in a concentration of 20 microM increased precursor incorporation in most cases, and a concentration of 100 microM was optimal; at a concentration of 500 microM, MIX had no significant effect on leucine or sulfate incorporation. When cartilage from hypophysectomized rats was incubated in a medium with the test agents for 4-6 h and the labeled precursors were added for the last 2 h, BucAMP did not increase incorporation of any of the precursors. MIX was also ineffective, even though tissue cAMP levels were increased. However, precursor incorporation was increased by exposure to partially purified rat somatomedin for the same periods. The degree of stimulation of sulfate incorporation induced by either BucAMP or MIX was proportional to the time of exposure to these agents. Preincubation of cartilage in basal medium alone for 22 h or longer increased basal sulfate incorporation, but caused only a slight enhancement of the action of BucAMP. The addition of synthetic bovine PTH-(1-34) (1 microM) to the incubation medium increased sulfate incorporation into hypophysectomized rat cartilage by 24 h, and this effect was potentiated by MIX (10 microM). No stimulation was detectable by 6 h, even with MIX in the medium. PTH-(1-34) increased the cartilage cAMP level, and this effect was also potentiated by MIX. In the presence of MIX, PTH-(1-34) increased the level of cAMP within 30 min, while the rat somatomedin preparation had no effect on the cAMP level during 60 min of incubation. The level of cartilage cGMP was not raised by either PTH or somatomedin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Septic arthritis. Staphylococcal induction of chondrocyte proteolytic activity.

We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage after treatment with a purified staphylococcal culture medium or intraarticular infection with Staphylococcus aureus. Staphylococcal culture medium increased the release of gelatinolytic, collagenolytic, and caseinolytic activity into the medium of isolated chondrocytes or cartilage organ culture. The proteolytic activities were determined in assays using radiolabeled substrate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in staphylococcal septic arthritis is due to the production and activation of chondrocyte proteases.     

Septic arthritis

We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage after treatment with a purified staphylococcal culture medium or intraarticular infection with Staphylococcus aureus. Staphylococcal culture medium increased the release of gelatinolytic, collagenolytic, and caseinolytic activity into the medium of isolated chondrocytes or cartilage organ culture. The proteolytic activities were determined in assays using radiolabeled substrate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in staphylococcal septic arthritis is due to the production and activation of chondrocyte proteases.     

Testosterone stimulation of a rapidly labeled, low-molecular-weight RNA fraction in human hepatic erythroid cells in culture.

The addition of erythropoietin to cell cultures of erythroid cells of human fetal liver resulted in an increased incorporation of thymidine, adenine, and uridine into trichloroacetic acid-insoluble cell fractions and in an increased uptake of adenine and uridine into the cell. Although the effects of testosterone and erythropoietin on heme synthesis in these cells are known to be very similar, there was no effect of testosterone on the total incorporation of radioactive precursors into DNA or RNA. The RNA synthesized after short pulses of radioactive uridine, when analyzed on sucrose gradients containing 1% sodium dodecyl sulfate, consisted of a homogeneous peak sedimenting at 10 plus or minus 2 S, which is quite different from the heterogeneous, high-molecular-weight RNA synthesized under identical conditions in primary cultures of human fetal lung, kidney, or liver parenchymal cells. Addition of testosterone to liver erythroid cells in cultures for 5 hr followed by a 1-hr uridine pulse resulted in a 3-fold increase of RNA species with an average sedimentation coefficient of 14 plus or minus 3 S. The similarity with the sedimentation coefficient of the globin mRNA described in other systems and the high degree of specialization of the erythroid cells suggest that this RNA may be a stable intermediate involved in the synthesis of hemoglobin.     

Biochemical study of normal and abnormal thyroid cells in tissue culture (author's transl)]

Thyrotropin induces the formation of a follicular structure of thyroid cells in tissue culture. It produces an increase in cyclic AMP content of thyrotropin treated cells. Thyrotropin induces on increase in iodine incorporation with the appearance of iodinated proteins which are mostly 19 S. thyroglobulin. In goiters, the level of cyclic AMP and the stimulation with thyrotropin differs from normal conditions indicating differences in cell metabolism and differences in the properties of the receptor. Thyrotropin also gives an increase in uridine incorporation in RNA and on increase not only in poly A but also in poly A-RNA associated with heavy polysomes. The level of poly A is higher in toxic adenoma and lower in microfollicular adenoma as compared to colloido nodular goiter which is in good agreement with our knowledge of thyroid cell metabolism.     

Hormonal stimulation of alpha-amylase synthesis in porcine pancreatic minces.

Minces of porcine pancreas maintained a linear rate or protein synthesis for more than 4 h with similar kinetics in the presence of absence of hormones. alpha-Amylase protein, quantitated by using glycogen percipitation, SDS polyacrylamide gel analysis, and radioimmunoassay, was found to represent 6-7% of the total protein content of the minces. The addition of prostaglandin E1 (10-7M), or a pancreozymin preparation (3-10 Crick-Harper-Raper units/ml) to the incubation media produced within 60 min an increase in alpha-amylase protein above that stored in the tissue 10- to 20-fold greater than that observed in control minces. The addiction of cAMP analogs (10(-3)M) to the medium produced a more variable stimulation of alpha-amylase content. Quantitation of the incorporation of radiolabeled amino acid into alpha-amylase protein with simultaneous determination of the specific activity of the precursor pool suggested that these agents produced at least 3- to 5-fold stimulation of de novo alpha-amylase synthesis. This stimulation was not the result of decreased rates of alpha-amylase catabolism. The addition of actinomycin D (20 mug/ml), a concentration inhibiting more than 93% of new RNA synthesis, failed to alter the stimulation of alpha-amylase synthesis by these agents. Apparent hormonal stimulation of non-alpha-amylase protein synthesis was also observed.     

Early Stimulation of RNA Synthesis by Erythropoietin in Cultures of Erythroid Precursor Cells

The effect of erythropoietin on cultured erythroid precursor cells from 13-day mouse-fetal livers was examined. Within 1 hr, erythropoietin causes a 2- to 3-fold stimulation of uridine incorporation into RNA by these cells. The types of RNA preferentially stimulated by erythropoietin during the first hour of exposure of the cells to the hormone include ribosomal RNAs and their precursors, as well as 4-5S RNA. No unique RNA species, not present in control cells, could be detected by sucrose gradient sedimentation or gel electrophoresis. Inhibition of protein synthesis for up to 1 hr does not abolish the stimulatory effect of erythropoietin on RNA synthesis, suggesting that the effect of the hormone on RNA synthesis is not mediated by a newly synthesized protein.     

Effects of cyclic nucleotides on deoxyribonucleic acid synthesis in hypophysectomized rat cartilage: stimulation of thymidine incorporation and potentiation of the action of somatomedin by analogs of adenosine 3',5'-monophosphate or a cyclic nucleotide phosphodiesterase inhibitor

The actions of cyclic nucleotides on basal and somatomedin-stimulated thymidine incorporation into DNA by costal cartilage from hypophysectomized rats were investigated. Three analogs of cAMP (dibutyryl, 8-bromo, and 8-dimethylamino derivatives, which are alternate activators of cAMP-dependent protein kinase and resistant to degradation by cAMP phosphodiesterase but represent a wide difference in potency as phosphodiesterase inhibitors) in range of concentrations from about 10(-5) to 3 X 10(-4) M enhanced basal and somatomedin-stimulated thymidine incorporation. Each cAMP analog at optimal concentration produced combined effects with a suboptimal concentration of somatomedin which were additive or greater. cAMP itself, 5'-AMP, adenosine, 8-Br-5'-AMP, 8-Br-AMPT, and cGMP at concentrations from 10(-7)--10(-3) M or dibutyryl cGMP at concentrations from 10(-10)--10(-3) M did not reproduce the effects of the cAMP analogs. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) at concentrations of 100 or 500 microM also potentiated the effects of somatomedin. At 100- or 500-microM concentrations, the phosphodiesterase inhibitor increased cartilage levels of cAMP and cGMP. These results suggest a role for cAMP in DNA synthesis in rat cartilage. However, they fail to support the hypothesis that all effects of somatomedin on that process are mediated by cAMP, since stimulation of thymidine incorporation by the hormone can be demonstrated in cartilage maximally stimulated by analogs of cAMP.     

Insulin stimulates the incorporation of 32Pi into ribonucleic acid in cultured sertoli cells

Chronic treatment of cultured Sertoli cells with insulin, FSH, or testosterone does not affect the number of attached cells or the amount of total RNA or poly(A)+ RNA per culture dish. However, Sertoli cells cultured in the presence of insulin have a nearly 2-fold stimulated incorporation of 32Pi into poly(A)+ RNA. The other hormone treatments did not induce any stimulation over control values. The t1/2 of the total poly(A)+ RNA was determined in pulse-chase experiments. None of the hormone treatments affected the t1/2 (16 h) of the total poly(A)+ RNA. The incorporation of [3H]uridine and 32Pi into the total nucleotide pools and into some individual nucleotides was determined by high pressure liquid chromatography analysis of acid-soluble extracts. Insulin-treated cells had approximately a 1.5-fold increased specific activity of [3H]uridine or 32Pi into the total nucleotide pool and in ATP, GTP, and UTP pools. Insulin apparently stimulates the uptake of phosphorylation (or both) of free nucleosides.     

Isolation of cells from rat seminal vesicles and epididymis and their use in studying androgen action

Cells isolated enzymically from seminal vesicles and epididymides of normal and castrated rats were shown by electron microscopy to be intact and representative of the tissue. The cells synthesize and secrete tissue-specific proteins. Short-term incorporation of [3H]uridine and [35S]methionine was measured to determine the effects of castration on RNA and protein synthesis. Epididymal cells and tissue incorporated uridine at similar rates which were unaltered by castration. Similarly castration failed to diminish uridine incorporation by seminal vesicle cells and tissue. Therefore, androgens may principally control RNA degradation. A similar situation pertained to methionine incorporation by epididymal cells and tissue so here too control may be via protein degradation. In contrast, castration greatly decreased methionine incorporation by seminal vesicle tissue but not by isolated cells. Isolated cells were more active than in tissue, particularly those from castrated rats, and may be released from stromal-epithelial interactions and controls.     

Acute metabolic effects of nitrogen mustard and thiotepa on rabbit articular cartilage and synovium.

Metabolic alterations in immature rabbit joint tissue were examined following in vitro and in vivo exposure to the alkylating agents Thiotepa and nitrogen mustard. Brief exposure in vitro to either agent resulted in marked suppression of incorporation of radiolabeled precursors of protein, RNA, and glycosaminoglycan synthesis in articular cartilage, which was partially reversible after Thiotepa exposure. In vivo, nitrogen mustard has little effect on synovium and transient inhibitory effects on cartilage vital processes, whereas Thiotepa caused a prolonged inhibition of synovial metabolism with little effect on cartilage. Autoradiographic localization of labeled agents indicated that synovial tissue and cartilage were readily penetrated by nitrogen mustard, but only a few synovial lining cells and superficial chondrocytes were labeled with 35S-Thiotepa. Furthermore, trypsin significantly reduced labeling of cartilage with 14C-nitrogen mustard. These data suggest that alkylating agents differentially affect metabolic processes in joint tissues in vivo and that with Thiotepa, this interference occurs primarily in the synovium. The degree of interference is apparently dependent upon the time of exposure to the agents and the relative DNA-RNA synthetic activity of the joint tissue.     

Cellular and molecular correlates of the decline in responsiveness of cartilage and thymus to growth hormone in aged animals

The responses in vitro of cartilage to somatomedin and of thymocytes to growth hormone have been studied in tissues derived from rats of various ages. The basal and somatomedin stimulated incorporation of radioactive proline into proteins and of sulphate into mucopolysaccharides diminishes markedly with age. Chondrocytes per unit area in cartilage of old rats are about 4- to 5-fold reduced in number as compared to those in cartilage from 1-day old rats. The organ size and yield of thymocytes are reduced in aged rats. Besides the fall in number of cells, the average metabolic activity of cells as measured by uridine incorporation into RNA in vitro is also diminished. There is a progressive decline in stimulation by growth hormone of uridine incorporation in isolated thymocytes with age. Immunocytochemical studies reveal the location of the hormone along the membrane. The amount of the hormone bound by thymocytes, estimated by immunoenzymatic methods is 3- to 9-fold lower in thymocytes from 14-months old rats as compared to similar preparations from 4-week old rats. Thymocytes from both young and old rats are composed of subpopulations, one of which binds growth hormone. The proportion of the hormone binding cells is higher in thymocytes from young rats as compared to those from aged animals.     

Acute Metabolic Effects of Nitrogen Mustard and Thiotepa on Rabbit Articular Cartilage and Synovium

Metabolic alterations in immature rabbit joint tissues were examined following in vitro and in vivo exposure to the alkylating agents Thiotepa and nitrogen mustard. Brief exposure in vitro to either agent resulted in marked suppression of incorporation of radiolabeled precursors of protein, RNA, and glycosaminoglycan synthesis in articular cartilage, which was partially reversible after Thiotepa exposure. In vivo, nitrogen mustard had little effect on synovium and transient inhibitory effects on cartilage vital processes, whereas Thiotepa caused a prolonged inhibition of synovial metabolism with little effect on cartilage. Autoradiographic localization of labeled agents indicated that synovial tissue and cartilage were readily penetrated by nitrogen mustard, but only a few synovial lining cells and superficial chondrocytes were labeled with ³⁵S-Thiotepa. Furthermore, trypsin significantly reduced labeling of cartilage with ¹⁴C-nitrogen mustard. These data suggest that alkylating agents differentially affect metabolic processes in joint tissues in vivo and that with Thiotepa, this interference occurs primarily in the synovium. The degree of interference is apparently dependent upon time of exposure to the agents and the relative DNA-RNA synthetic activity of the joint tissue.