RESUMO
(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.
Assuntos
Proteoglicanas de Heparan Sulfato/análise , Células de Sertoli/metabolismo , Animais , Membrana Celular/química , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Glicosilfosfatidilinositóis/análise , Masculino , Ratos , Ratos Sprague-Dawley , Células de Sertoli/química , Tripsina , Fosfolipases Tipo CRESUMO
The influence of phorbol myristate acetate (PMA), dibutyryl cAMP and insulin-like growth factor (IGF-1) as well as cytoskeletal disrupting drugs on morphological changes has been studied in peritubular cells isolated from immature rat testis. Morphological studies were combined with immunofluorescence investigations of cytoskeletal elements and their rearrangements by various agents. The results were correlated with modulation of proteoglycan synthesis. Peritubular cells exposed to dibutyryl cAMP or cytochalasin D were transformed from flattened, fibroblast-like into neuronal-like morphology. In such cells, destruction of actin filaments was accompanied with a 50% decrease in cell-associated proteoglycan synthesis as well as with oversulfation of total proteoglycans. On the contrary, peritubular cell shape has been slightly altered after addition of PMA, IGF-1, vinblastine or colchicine. After these treatments, destruction or rearrangement of cytoskeletal elements was observed; cell-layer proteoglycan synthesis remained either unchanged or increased while total proteoglycans were always undersulfated. IGF-1, PMA and dibutyryl cAMP modified the peritubular cell morphology, cytoskeletal organization and proteoglycan production; the cytoskeleton disrupting drugs such as vinblastine, colchicine and cytochalasin D mimicked some of these effects. These observations suggest that alterations in proteoglycan biosynthesis, after activation of tyrosine kinase, protein kinase C and protein kinase A pathways might be mediated, at least in part, by the disorganization of the cytoskeleton structure.
Assuntos
Citoesqueleto/metabolismo , Proteoglicanas/biossíntese , Transdução de Sinais/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Animais , Bucladesina/farmacologia , Carcinógenos/farmacologia , Células Cultivadas , Citoesqueleto/ultraestrutura , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Microscopia Eletrônica de Varredura , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tubulina (Proteína)/análise , Vimentina/análiseRESUMO
In cultured peritubular cells (PT) from rat testis, protein kinase C (PKC) was activated by phorbol 12-myristate 13-acetate (PMA). PMA enhanced the synthesis of proteoglycans (PG) and to a lesser extent their catabolism; the stimulation of the synthesis appeared to be due to an increase in PG protein moiety production and, at the same time, to an increase in the glycanation process as revealed by the use of an exogenous acceptor, p-nitrophenyl-beta-d-xyloside. In the presence of PMA, the molecular weight of neosynthesized PG and the length of their constitutive glycosaminoglycan chains were not modified. Moreover, the distribution of proteochondroitin sulfate and proteoheparan sulfate in medium and in cell layer remained unchanged. However, PMA reduced the sulfation level of chondroitin sulfate and heparan sulfate chains, suggesting that PKC activation resulted in an independent modulation of the sugar chain formation and of the sulfate residue transfer. PMA effect on the synthesis of hyaluronan was also determined: PMA dramatically enhanced its production by PT cells.
Assuntos
Proteína Quinase C/metabolismo , Proteoglicanas/biossíntese , Testículo/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Glucosamina/análise , Glicosídeos , Ácido Hialurônico/biossíntese , Masculino , Proteoglicanas/química , Ratos , Ratos Sprague-Dawley , Sulfatos/análise , Radioisótopos de Enxofre , Testículo/enzimologia , TrítioRESUMO
The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [3H]-hexosamine incorporation in secreted PG and in cell-associated PG was reduced, whereas [35S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).
Assuntos
AMP Cíclico/metabolismo , Ácido Hialurônico/biossíntese , Proteoglicanas/biossíntese , Testículo/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Bucladesina/farmacologia , Células Cultivadas , Toxina da Cólera/farmacologia , Cromatografia em Gel , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA/análise , Glicosaminoglicanos/biossíntese , Glicosídeos/farmacologia , Meia-Vida , Masculino , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Testículo/citologiaRESUMO
The exposure of confluent peritubular (PT) cells from immature rat testis to insulin-like growth factor-1 (IGF-1) induced a time and dose-dependent increase of [35S]-sulfate and [3H]-D-glucosamine incorporations in newly synthesized proteoglycans (PG). This increased content of PG was the result of an enhancement of PG synthesis rather than a decreased rate of degradation. IGF-1 had no effect on the molecular weight of synthesized PG nor on the nature and distribution of the constitutive glycosaminoglycan chains, both in medium and in cell layer. The stimulation of PG synthesis by IGF-1 appeared to be due, at least partially, to an increase of glycosylation processes. IGF-1 effect was mediated by the classical tyrosine kinase signalling process, since IGF-1 action on PG synthesis was abolished by genistein and tyrphostin A9, two well known tyrosine kinase inhibitors. The increase of PG synthesis was accompanied with an undersulfation of constitutive glycosaminoglycan (GAG) chains (chondroitin sulfate and heparan sulfate chains) since the [35S]/[3H] ratio was reduced by about 20-25% in presence of IGF-1. Although the mechanism of hyaluronic acid synthesis was completely different from those of other GAG, IGF-1 also dramatically enhanced its production by PT cells.
Assuntos
Ácido Hialurônico/biossíntese , Fator de Crescimento Insulin-Like I/farmacologia , Proteoglicanas/biossíntese , Testículo/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Eletroforese em Gel de Campo Pulsado , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Glucosamina/metabolismo , Glicosídeos/farmacologia , Cinética , Masculino , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sulfatos/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
Proteoglycans present in follicular fluid are synthesized by granulosa cells under gonadotropin control. An inhibitor of proteoglycan synthesis, p-nitrophenyl-beta-D-xyloside (beta-D-xyloside) was used as a probe to study rabbit granulosa cell steroidogenesis and proliferation under abrogated proteoglycan synthesis. Granulosa cells isolated from rabbit preovulatory follicles were cultured 24 h in Minimum Essential Medium plus 2.5% fetal calf serum in the presence or absence of beta-D-xyloside and were then treated with FSH or dibutyryl cAMP (db-cAMP) alone or in combination with beta-D-xyloside for a further 24 h. The exposure for 48 h of granulosa cells to 1 mM beta-D-xyloside in the absence or presence of FSH inhibited proteoglycan synthesis and increased the amount of glycosaminoglycans (GAG). FSH-stimulated progesterone production was significantly correlated only with proteoglycan synthesis and not with GAG production. The addition of various concentrations of beta-D-xyloside (0.1-4 mM) for 48 h to granulosa cells induced a dose-dependent inhibition of FSH-stimulated progesterone secretion and [3H]thymidine incorporation into DNA. beta-D-Xyloside concentrations lower than 1 mM induced an inhibition of FSH-stimulated progesterone secretion but had no significant effect on FSH-induced proliferation. One millimolar beta-D-xyloside did not modify basal progesterone production, but in the presence of various doses (0.1-2.5 ng/ml) of FSH or hCG (0.1-1 IU/ml) it exerted a significant inhibitory effect on steroid secretion. Fifty percent inhibition was obtained for doses of FSH above 0.5 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Divisão Celular , Glicosídeos/farmacologia , Células da Granulosa/metabolismo , Progesterona/metabolismo , Proteoglicanas/antagonistas & inibidores , Animais , Bucladesina/farmacologia , Células Cultivadas , Gonadotropina Coriônica/farmacologia , AMP Cíclico/metabolismo , DNA/biossíntese , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Proteoglicanas/biossíntese , CoelhosRESUMO
Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG). Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (Kav = 0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column, they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.
Assuntos
Membrana Celular/química , Proteoglicanas/isolamento & purificação , Testículo/química , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteoglicanas de Heparan Sulfato , Heparina , Heparitina Sulfato/isolamento & purificação , Heparitina Sulfato/metabolismo , Masculino , Octoxinol , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Testículo/citologiaRESUMO
In rat Sertoli cells, linoleate addition modified cell membrane fatty acid composition and changes depended on linoleate concentrations. In presence of the lowest 18:2 n-6 concentrations (2.5 and 7.5 microM), decrease in proteoglycan synthesis paralleled increase in n-6 linoleate-derived metabolites. At high concentration (21 microM), linoleate accumulated in membranes and level of n-6 linoleate-derived metabolites returned to basal value, without change in proteoglycan synthesis. Linoleate modified proteoglycan distribution in Sertoli cells by an increase in peripheral proteoglycans and a concomitant decrease in medium proteoglycans. Vitamin E (100 microM) did not alter fatty acid composition in control and linoleate-treated cells, but enhanced proteoglycan production. Furthermore, this agent counteracted linoleate-induced modifications in proteoglycan cell distribution.
Assuntos
Ácidos Linoleicos/farmacologia , Proteoglicanas/biossíntese , Células de Sertoli/metabolismo , Vitamina E/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ácidos Graxos/metabolismo , Ácidos Graxos Ômega-6 , Ácidos Graxos Insaturados/metabolismo , Ácido Linoleico , Masculino , Proteoglicanas/análise , Ratos , Células de Sertoli/efeitos dos fármacosRESUMO
Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.
Assuntos
Polietilenoglicóis , Proteoglicanas/análise , Células de Sertoli/química , Testículo/química , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Detergentes , Masculino , Octoxinol , Ratos , Ratos Endogâmicos , SolubilidadeRESUMO
Rabbit articular chondrocytes in confluent monolayer cultures were treated with D-Penicillamine (D-Pen) during 3 or 5 days. The [35S]-sulfate incorporation in neosynthesized proteoglycans was not modified by D-Pen doses ranging from 50 to 800 micrograms/ml. After treatment during 5 days with D-Pen concentrations of 50 or 400 micrograms/ml, the chemical characteristics of proteoglycans from medium and cell-layer were determined. The aggregation capacity of proteoglycans from medium, the monomer molecular size, the glycosaminoglycan chain length and the relative rates of the different glycosaminoglycans (chondroitins, chondroitin 6-sulfate, chondroitin 4-sulfate, hyaluronic acid) remained unchanged. These results suggest that D-Pen does not alter some of the cartilage mechanical properties due to the presence of proteoglycans.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Penicilamina/farmacologia , Proteoglicanas/biossíntese , Animais , Cartilagem Articular/metabolismo , Células Cultivadas , Cromatografia em Gel , Matriz Extracelular/metabolismo , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/isolamento & purificação , Conformação Molecular , Peso Molecular , Proteoglicanas/isolamento & purificação , CoelhosRESUMO
In rabbit articular chondrocytes, phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DG) and calcium ionophore (A23187), reduced the proteoglycan synthesis, in a dose-dependent manner. The combined treatment by PMA and A23187 resulted in an enhanced inhibition of proteoglycan production, indicating a synergistic effect. In presence of PMA or A23187, the release of prostaglandin E2 (PGE2) was dramatically increased. The addition of indomethacin and BW755c to chondrocytes stimulated by PMA or A23187, suppressed the liberation of PGE2, but did not stop the decrease of proteoglycan synthesis.
Assuntos
Calcimicina/farmacologia , Cartilagem Articular/metabolismo , Prostaglandinas E/metabolismo , Proteoglicanas/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina , Animais , Ácidos Araquidônicos/fisiologia , Diglicerídeos/farmacologia , Dinoprostona , Sinergismo Farmacológico , Indometacina/farmacologia , Pirazóis/farmacologia , CoelhosRESUMO
Arthritic-rendered rabbits were treated in vivo with 50 mg/kg D-penicillamine (D-Pen) daily during 4 months. Glycosaminoglycan (GAG) synthesis by synovial fibroblast cultures from D-Pen treated and untreated normal or arthritic animals was studied. Cells from arthritic-rendered animals synthesized hyaluronic acid (HA) at the same rate as cells isolated from control rabbits. When D-Pen was administered to arthritic-rendered rabbits, it significantly inhibited GAG production by fibroblasts. The hyaluronate synthetase activity determined on synovial fibroblast homogenates, however, was not modified whatever the treatment undergone by the rabbits. Moreover, synovial fibroblasts from arthritic rabbits treated or not with D-Pen generally synthesized HA with a high molecular weight similar to that produced by D-Pen treated or untreated control animals.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite/tratamento farmacológico , Glicosaminoglicanos/biossíntese , Glicosiltransferases , Proteínas de Membrana , Penicilamina/uso terapêutico , Membrana Sinovial/efeitos dos fármacos , Transferases , Proteínas de Xenopus , Animais , Artrite Experimental/metabolismo , Células Cultivadas , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucuronosiltransferase/análise , Hialuronan Sintases , Masculino , Peso Molecular , Pronase/metabolismo , Coelhos , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
Cultured human synovial cells treated with an interleukin-1-like mononuclear cell factor incorporated more 35S in proteoglycans than control cultures, but the radioactivity distribution between medium and cell layer was not modified. Proteoglycans were synthesized essentially in monomeric form and the mononuclear cell factor increased the molecular weight of these monomers. The [3H]hexosamine/[14C]serine ratio in purified proteoglycans on the one hand, and the study of [35S]glycosaminoglycan molecular weight, on the other hand, indicated that this increase is not modulated through enhanced synthesis of core protein but by an increase in the glycosaminoglycan chain length. After enzyme hydrolysis, dermatan sulfate (62% of the total glycosaminoglycans) and chondroitin 4/6-sulfate (30%) were found to be the major glycosaminoglycans synthesized by cultured synovial cells, and the existence of 8% heparan sulfate was evidenced by nitrous acid treatment. In the presence of the mononuclear cell factor, the dermatan sulfate synthesis was decreased (47%), with a concomitant increase of chondroitin sulfate synthesis (45%).
Assuntos
Proteínas/farmacologia , Proteoglicanas/biossíntese , Membrana Sinovial/metabolismo , Idoso , Células Cultivadas , Sulfatos de Condroitina/biossíntese , Feminino , Glicosaminoglicanos/biossíntese , Hexosaminas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Monocinas , Serina/metabolismo , Membrana Sinovial/efeitos dos fármacosRESUMO
A partially purified monocyte factor, with Interleukin-1 properties (MCF/IL-1), enhances the proteoglycan synthesis of human neonatal articular chondrocytes in culture, and changes the repartition of these macromolecules between medium and cell layer. The size of the proteoglycan monomers, the length of the glycosaminoglycan chains and the respective levels of chondroitin-6-sulfate, chondroitin-4-sulfate and non sulfated chondroitin remain unchanged under MCF/IL-1 exposures. The addition of indomethacin reduces the stimulation effect by 60-70% only, suggesting that the MCF/IL-1 action is partially dependent on prostaglandins but seems also related to mechanisms distinct from the cyclooxygenase pathway.
Assuntos
Cartilagem Articular/metabolismo , Interleucina-1/farmacologia , Monócitos/fisiologia , Proteoglicanas/biossíntese , Cartilagem Articular/citologia , Células Cultivadas , Glicosaminoglicanos/análise , Humanos , Indometacina/farmacologia , Peso Molecular , Monócitos/análise , Monocinas , Prostaglandinas/fisiologia , Proteínas/farmacologia , Proteoglicanas/análiseRESUMO
Treatment of cultured human synovial cells with a mononuclear cell factor (MCF) enhanced their ability to synthesize glycosaminoglycans (GAG), but GAG repartition between extracellular, pericellular and intracellular compartments was found to be the same as in control. Hyaluronic acid (HA) production, which represents 80-90% of all secreted GAG, was stimulated 2 1/2-3-fold, but the HA molecular weight was not modified. The MCF increased the hyaluronate synthetase activity of synovial cells in similar proportions. Actinomycin D inhibited the increase in hyaluronate synthetase activity produced by MCF, indicating that this increase involves new synthesis of mRNA. Stimulation of both HA synthesis and hyaluronate synthetase activity by MCF was suppressed by 10(-4)-10(-5) M indomethacin (an inhibitor of cyclo-oxygenase), suggesting that MCF effect is prostaglandin-dependent.