RESUMO
Thyroid-stimulating hormone increased the cyclic 3',5'-adenosine monophosphate concentration in dog thyroid slices during a 1-minute incubation period and produced a maximum effect soon thereafter. The elevation persisted for at least 30 minutes. The concentrations of the cyclic 3',5'-adenosine monophosphate increased as the TSH concentration was increased from 0.125 to 50 milliunits per milliliter. Prostaglandin E(1), which increases glucose oxidation in dog thyroid slices, also increased the concentration of cyclic 3',5'-adenosine monophosphate. Although sodium fluoride stimulates thyroid adenyl cyclase, it did not increase concentration of cyclic 3',5'-adenosine monophosphate. Carbamylcholine and menadiol sodium diphosphate augment glucose oxidation in dog thyroid slices but do not change concentrations of cyclic 3',5'-adenosine monophosphate.
Assuntos
Nucleotídeos de Adenina/metabolismo , Prostaglandinas/farmacologia , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Adenilil Ciclases , Animais , Carbacol/farmacologia , AMP Cíclico/metabolismo , Cães , Fluoretos/farmacologia , Glucose/metabolismo , Técnicas In Vitro , Glândula Tireoide/efeitos dos fármacos , Vitamina K/farmacologiaRESUMO
Microtubules, microfilaments, and intermediate filaments were found to be associated with the cytoplasmic face of the plasma membrane and even localized on the cell surface following "perturbation" of the plasma membrane. Several hormones interacting with their surface receptors have an effect on the assembly, organization, and orientation of the cytoskeletal system thus inducing changes in cell morphology, motility and aggregation. The cytoskeletal system is probably responsible for the lateral and vertical mobility of plasma membrane receptors and for the efficient coupling of GTP-binding protein to the adenylate cyclase moiety. It is suggested that the cytoskeletal system may be involved in hormone-induced desensitization. The activity of cyclic nucleotide phosphodiesterase and protein kinase is modulated by Ca2+-calmodulin. These enzymes are associated with intermediate filaments and with microtubules which may control their activity and induce nuclear translocation of protein kinase. Stimulation of steroidogenesis by ACTH and LH, enhancement of H2O transport by vasopressin, elevation of the rate of amino acid and glucose transport by insulin, release of pancreatic insulin by glucose, and pituitary hormones by their respective hypothalamic releasing hormones, are only examples of a variety of hormonal responses that may be regulated by the cytoskeletal system. It is obvious that much more experimental study should be done to establish the role of the cytoskeletal system in hormonal action. I do hope this review will stimulate further ideas and experiments which might eventually lead to a better understanding of the role of the cytoskeletal system in the control of adenylate cyclase-cAMP system stimulated by hormones.
Assuntos
Adenilil Ciclases/fisiologia , Fenômenos Fisiológicos Celulares , AMP Cíclico/fisiologia , Hormônios/fisiologia , Animais , Fenômenos Biomecânicos , Membrana Celular/análise , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Células/enzimologia , Células/metabolismo , AMP Cíclico/biossíntese , Citoesqueleto/fisiologia , Ativação Enzimática , Proteínas de Membrana/análise , Microtúbulos/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologiaRESUMO
These studies evaluated the influence of an initial exposure of thyroid slices to thyroid-stimulating hormone (TSH) on the subsequent responsiveness to the hormone. Bovine thyroid slices were incubated with or without 50 mU/ml TSH for varying periods and then incubated in hormone-free medium for varying periods. Subsequently, slices were incubated for 20 min with 10 mM theophylline and with or without TSH. Cylic AMP was measured after the third incubation. Phosphodiesterase and adenylate cylase were assayed in homogenates prepared from slices after the second incubation. In some experiments prostaglandin E1, puromycin, thyroxine, and triiodothyronine and propylthiouracil were included in the media. In other experiments, low does of TSH (1 AND 10 mU/ml) were used instead of 50 mU/ml. Slices previously exposed to TSH have decreased responsiveness of the adenylate cyclase-cylic AMP system. Such refractoriness is hormone specific since initial exposure to prostaglandin E1 decreases the subsequent response to this substance but not to TSH. Refractoriness to TSH develops only when the first incubation is at least 30 min. It is not reversed by 5 h of incubation without hormone. Incubation of thyroid slices with puromycin does not eliminate refractoriness. The decreased response to TSH cannot be explained by release of thyroxine, triiodothyronine, or iodide from the slices. Phosphodiesterase activity is not increased during the refractory period. The decreased cyclic AMP response to TSH is associated with diminished response of adenylate cyclase activity to the hormone. Guanosine triphosphate (1 mM) increased adenylate cyclase activity in both control and TSH treated tissue, but the effect was significantly less in the latter. Although with guanosine triphosphate, TSH increased adenylate cyclase activity in TSH treated tissue, the enzyme activity was still less than that present in control tissue incubated with guanosine triphosphate and TSH. NaF caused an equivalent stimulation of adenylate cyclase in both control and TSH treated tissue. These results suggest that the refractoriness represents an alteration in hormone binding or the coupling of the bound hormone to the adenylate cyclase activity rather than any modification of the catalytic site of the enzyme.
Assuntos
AMP Cíclico/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Bovinos , Cães , Fluoretos/farmacologia , Guanosina Trifosfato/farmacologia , Humanos , Técnicas In Vitro , Cinética , Prostaglandinas E/farmacologia , Puromicina/farmacologia , Ovinos , Especificidade da Espécie , Glândula Tireoide/efeitos dos fármacos , Tiroxina/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
Vasopressin increased adenyl cyclase activity in homogenates of both inner and outer renal medulla of the rat. It also increased the concentration of cyclic 3',5'-adenosine monophosphate (AMP) in slices of both inner and outer medulla but not in renal cortex. In the inner medulla, a concentration of prostaglandin E(1) (PGE(1)), which was ineffective by itself significantly reduced the stimulation of adenyl cyclase activity and cyclic AMP concentration induced by vasopressin. These results are consistent with the hypothesis that PGE(1) can compete with vasopressin for adenyl cyclase-binding sites. However, the findings in the outer medulla suggest the situation is more complex. Although 10(-8) M PGE(1) had no effect by itself and inhibited the vasopressin-induced elevation of cyclic AMP, larger amounts of PGE(1) increased both adenyl cyclase activity and cyclic AMP levels. The maximum effect on the latter parameter was at least 6 times as great as that of maximum amounts of vasopressin.
Assuntos
Nucleotídeos de Adenina/metabolismo , Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Rim/metabolismo , Prostaglandinas/farmacologia , Vasopressinas/farmacologia , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/metabolismo , Adenilil Ciclases/análise , Animais , Técnicas de Cultura , AMP Cíclico/análise , Rim/análise , Rim/efeitos dos fármacos , Masculino , Prostaglandinas/metabolismo , Ratos , Ratos Endogâmicos , Estimulação Química , Fatores de Tempo , Vasopressinas/administração & dosagem , Vasopressinas/antagonistas & inibidores , Vasopressinas/metabolismoRESUMO
During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.
Assuntos
Cálcio/metabolismo , Queratinócitos/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Compostos de Anilina , Linhagem Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fluorescência , Humanos , Queratinócitos/efeitos dos fármacos , Medições Luminescentes , Lisofosfatidilcolinas/farmacologia , Oniocompostos/farmacologia , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Cianeto de Potássio/farmacologia , Temperatura , XantenosRESUMO
The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) by about 5-10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by an average of 50%. Maximal inhibition of hydrolysis of PIP2 and reduction in stimulation of IP3 formation was reached after 4 h of preincubation with 2.10(-6) M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2.10(-7) M) markedly suppressed antigen induced IP3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2.10(-6) M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.
Assuntos
Glucocorticoides/farmacologia , Fosfatos de Inositol/metabolismo , Leucemia Basofílica Aguda/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Antígenos/metabolismo , Cicloeximida/metabolismo , Dexametasona/farmacologia , Dinitrofenóis/metabolismo , Hidrocortisona/farmacologia , Inositol/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Leucemia Basofílica Aguda/patologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfatidilinositóis/metabolismo , Fosfoinositídeo Fosfolipase C , Ratos , Soroalbumina Bovina/metabolismo , Células Tumorais CultivadasRESUMO
Rat basophilic leukemia (RBL-2H3) cells serve as a model to examine the role of elevated internal Ca2+ concentration ([Ca2+]i), following antigen (DNP10BSA)-induced stimulation of leukotriene C4 (LTC4) formation. A novel action of hydrocortisone (HC), to reduce increased [Ca2+]i and consequently inhibit LTC4 formation is assessed. Half-maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3-fold increase) was reached within 2-3 min. This high [Ca2+]i level waned gradually by 27% during 20 min of incubation. For induction of LTC4 formation, however, there was a refractory period of about 2 min, and half-maximal elevation was at 11 min. Following pretreatment with HC, the antigen-stimulated increase in [Ca2+]i was stunted by 41% at 2-3 min and by 73% at 20 min. LTC4 formation was almost abolished. There was a lag period of at least 2 h to observe any inhibition in both parameters, and the maximal inhibition was about 4 h. Cycloheximide, and receptor antagonist to glucocorticosteroid (RU486) completely prevented the inhibitory effects of HC on elevated [Ca2+]i and LTC4 formation. Estradiol and aldosterone (each at 2.10(-6) M) were virtually inactive, while another glucocorticosteroid, dexamethasone (2.10(-7) M) markedly suppressed antigen induction in both parameters. It is proposed that the inhibitory effect of HC on the formation of LTC4 could be attributed mainly to its ability to reduce elevated [Ca2+]i.
Assuntos
Cálcio/metabolismo , Hidrocortisona/farmacologia , Leucemia Experimental/metabolismo , SRS-A/biossíntese , Animais , Citosol/metabolismo , Dinitrofenóis/farmacologia , Antagonismo de Drogas , Mifepristona/farmacologia , Ratos , Soroalbumina Bovina/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Previously we have shown that reactive oxygen species (ROS) formation induced by phorbol ester in association with vanadate is essential for protein tyrosine phosphorylation and phospholipase A2 (PLA2) activation. Here we show that the interaction of beta-glucan particles (glucanp) or zymosan with complement receptor type 3 (CR3) leads, when associated with vanadate, to a cascade of reactions culminating in PLA2 activation. Vanadate + zymosan (or glucanp) markedly enhance protein tyrosine phosphorylation in bone marrow derived macrophages (BMMs), whereas neither of the agents alone has any effect. The enhancement was due to both sustained activation of protein tyrosine kinase (PTK) and inactivation of protein tyrosine phosphatase (PTP) as assessed in lysates of treated cells. Zymosan elevates membranal PKC, an effect that is potentiated by vanadate. Activation of both PTK and PKC leads to the activation of NADPH oxidase and to ROS formation. The formed ROS together with vanadate are potent inactivators of PTP leading to amplification of tyrosine phosphorylation and myelin basic protein kinase (MBP-K) activation. The activation of the cascade of protein kinases eventually leads to activation of PLA2. All the activation steps, i.e., activation of PTK, NADPH oxidase, MBP-K,PLA2 and the inactivation of PTP are sensitive to the NADPH oxidase inhibitor diphenyleneiodonium (DPI), to antioxidants and to PKC inhibitors. Thus, ROS formation (in the presence of vanadate) is critical for protein phosphorylation processes constituting the regulatory pathway of PLA2 activation by ligand-CR3 interaction.
Assuntos
Glucanos/farmacologia , Macrófagos/efeitos dos fármacos , Fosfolipases A/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zimosan/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Quinase 3 da Glicogênio Sintase , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A2 , Proteína Quinase C/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Vanadatos/farmacologiaRESUMO
Parathyroid hormone (PTH), which increases cAMP levels, also induces an increase in the activity of the brain isozyme of creatine kinase and in DNA synthesis in osteoblast-enriched bone cell cultures by a cAMP-independent mechanism. The following results lead us to the conclusion that PTH induction of brain isozyme of creatine kinase activity and DNA synthesis occurs by activation of membranal phospholipid metabolism leading to increased protein kinase C activity and Ca2+ mobilization, a mechanism demonstrated for several growth factors and other hormones. (1) Binding of membranal phospholipids by agents such as gentamycin or antiphospholipid antibodies abolishes the stimulation by PTH of creatine kinase activity and DNA synthesis but not of cAMP production. (2) Treatment of cell cultures with exogenous phospholipase C increases brain isozyme of creatine kinase activity and DNA synthesis, but not cAMP production; these stimulations are also blocked by serum containing anti-phospholipid antibodies. PTH has no additional effect on stimulation of creatine kinase activity by phospholipase C (and only a slight effect on DNA synthesis). (3) A synthetic diacylglycerol (1-oleyl-2-acetyl glycerol) or phorbol ester (phorbol 12-myristate 13-acetate) or Ca2+ ionophore, A23187 induces creatine kinase activity and DNA synthesis in the cultures. However, this effect is not blocked by antiphospholipid sera and PTH has no additional effect. (4) Inhibition of protein kinase C activity by drugs reported to inhibit the enzyme (retinoic acid, quercetin) abolishes the stimulation of brain isozyme of creatine kinase activity and of DNA synthesis by PTH.
Assuntos
Osso e Ossos/metabolismo , Creatina Quinase/biossíntese , Replicação do DNA/efeitos dos fármacos , Diglicerídeos/farmacologia , Glicerídeos/farmacologia , Hormônio Paratireóideo/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Calcitonina/farmacologia , Células Cultivadas , AMP Cíclico/metabolismo , Embrião de Mamíferos , Indução Enzimática , Quercetina/farmacologia , RatosRESUMO
Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.
Assuntos
Adenilil Ciclases/metabolismo , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Folículo Ovariano/enzimologia , Prostaglandinas E/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Folículo Ovariano/efeitos dos fármacos , RatosRESUMO
Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.
Assuntos
Membrana Celular/ultraestrutura , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/ultraestrutura , Fluidez de Membrana/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Cinética , Lipídeos de Membrana/análise , Ácidos Oleicos/farmacologia , Ratos , Espectrometria de Fluorescência , ViscosidadeRESUMO
The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.
Assuntos
Antígenos/farmacologia , Ácidos Araquidônicos/metabolismo , Calcimicina/farmacologia , Dexametasona/farmacologia , Ionomicina/farmacologia , Macrófagos/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Alcaloides/farmacologia , Animais , Carbazóis/farmacologia , Linhagem Celular , Membrana Celular/enzimologia , Células Cultivadas , Imunoglobulina E , Alcaloides Indólicos , Cinética , Leucemia Basofílica Aguda , Leucemia Experimental , Macrófagos/efeitos dos fármacos , Dibutirato de 12,13-Forbol/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Proteína Quinase C/antagonistas & inibidores , Ratos , Soroalbumina Bovina/imunologia , Soroalbumina Bovina/farmacologia , EstaurosporinaRESUMO
Differences exist in the rates at which hormones are inactivated by, or dissociate from, their target tissues. The present studies examined the binding of biologically active TSH to thyroid slices and compared its characteristics to those of PGE. Canine thyroid slices were initally incubated with 5 mU/ML OF BOVINE TSH (TSH-Inital) for 15 min, washed and incubated in media free of hormone for 3 hr. At the conclusion of this second incubation period all slices were again washed. Some were then transferred to media containing 10-2M theophylline for a final 10 min incubation and subsequent measurement of cAMP and protein kinase, while others were transferred to media containing (l-14C)glucose without theophylline for a final 45 min incubation to assess glucose oxidation. Identically treated slices never exposed to TSH served as controls, while others were exposed to TSH only during the final 10 or 45 min incubation periods (TSH-Final). cAMP content determined after significantly increased in TSH-Initial (mean 2.98 plus or minus 0.36 (se) pmol/mg wet wt) compared to control (0.35 plus or minus 0.04), but was less than that in TSH-Final (5.76 plus or minus 0.51). This phenomenon was not unique to canine thyroid, since comparable results were noted in studies of human, bovine or porcine thyroid slices. The protein kinase activity ratio (-cAMP/+cAMP) and glucose oxidation of TSH-Initial were also significantly increased above control following the final 10 min or 45 min incubations respectively. Addition of trypsin to the 3 h incubation abolished the subsequent increase in cAMP in TSH-Initial, while addition of TSH antiserum appreciably reduced this increase. These results are consistent with the persistent binding of biologically active TSH to thyroid. By contrast, evidence of similar persistent binding of PGE1 to thyroid, glucagon to liver, or parathyroid hormone to renal cortex was lacking when assessed by an identical experimental procedure. Differences between the duration of interaction of TSH and PGE1 with thyroid may be dependent or a more gradual dissociation to tissue bound TSH, a more rapid inactivation of bound-PGE1, or both.
Assuntos
Receptores de Superfície Celular , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Animais , Reações Antígeno-Anticorpo , Sítios de Ligação , Bovinos , AMP Cíclico/metabolismo , Cães , Glucose/metabolismo , Humanos , Soros Imunes , Técnicas In Vitro , Córtex Renal/metabolismo , Cinética , Fígado/metabolismo , Especificidade de Órgãos , Especificidade da Espécie , Suínos , Teofilina/farmacologia , Fatores de Tempo , TripsinaRESUMO
Deglycosylated LH ( DGLH ) abolished the stimulatory effect of LH on cAMP production by mature granulosa cells. DGLH , however, had no such inhibitory effect on the stimulatory activity of FSH, prostaglandin E2, isoproterenol, or choleragen. We have examined whether DGLH could prevent the desensitization of the cAMP response induced by continuous exposure to LH, or if it would induce receptor down-regulation. Cells were cultured with DGLH and LH concomitantly for a period of 3 h or 20 h and afterwards washed in acidic medium to dissociate bound ligands from their receptors. The cells were then challenged with fresh LH or with labeled 125I-human CG. At 20 h of culture with LH the cAMP response to the LH challenge was only 10% of the control, whereas after a 20-h culture with DGLH there was a 55% response to challenge with LH. DGLH , however, prevented LH from inducing desensitization in a dose-dependent manner (0.2-1.0 microgram/ml). Both LH and DGLH (1.0 microgram/ml each) markedly reduced (85%) the extent of binding of 125I-human CG to membrane receptors. At 3 h of culture, the cAMP response to challenge with LH was 25% of the control, whereas after 3-h culture with DGLH there was a 65% response to challenge with LH. DGLH (1.0 micrograms/ml) again prevented LH-induced desensitization, although only a moderate reduction in receptor-binding capacity (33%) by LH or DGLH was noted. The results suggest that occupancy of LH receptors alone for a long period is essential, but not sufficient to induce hormone desensitization. DGLH on its own is as active as LH in inducing a loss of membrane receptors, but causes only a moderate desensitization. The data support previous evidence that desensitization may result from postreceptor events, such as uncoupling of the receptors from the adenylate cyclase moiety, rather than receptor disappearance.
Assuntos
Gonadotropina Coriônica/metabolismo , AMP Cíclico/biossíntese , Células da Granulosa/metabolismo , Hormônio Luteinizante/análogos & derivados , Hormônio Luteinizante/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Células Cultivadas , Feminino , Gonadotropinas Equinas/farmacologia , Células da Granulosa/efeitos dos fármacos , Cinética , Hormônio Luteinizante/farmacologia , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores do LHRESUMO
Administration of antiserum to synthetic thyrotropin-releasing hormone (TRH) to male and female rats cause a 50% and a 70% suppression in serum levels of prolactin and thyrotropin, respectively, as compared with controls injected with normal rabbit serum. The degree of suppression was similar in diestrous and proestrous female rats and in male rats. These findings support the view that, in addition to its original designation, TRH also has a physiological role in regulating release of pituitary prolactin.
Assuntos
Prolactina/sangue , Hormônio Liberador de Tireotropina/imunologia , Tireotropina/sangue , Animais , Diestro , Feminino , Soros Imunes/farmacologia , Masculino , Gravidez , Proestro , RatosRESUMO
Reference preparations of ovine follicle-stimulating hormone (NIH-FSH-S8 and S9; 10-50 mug/ml) induced ovum maturation and stimulated cyclic AMP formation, as well as progesterone and 17beta-estradiol secretion, by rat Graafian follicles in vitro. These actions of NIH-FSH were retained after immunoabsorption of any contaminating luteinizing hormone (LH) present in the preparations, by treatment with an antiserum to the beta-subunit of purified ovine LH (anti-betaLH). In contrast, the corresponding biological actions of NIH-LH-S18 (0.5-10 mug/ml) were abolished by treatment with this anti-betaLH serum. A highly purified FSH preparation (64-96 CD, 0.25 mug/ml) also triggered oocytic meiosis and increased follicular progesterone secretion in vitro. Intraperitoneal (ip) administration of anti-betaLH-treated NIH-FSH-S9 (50 mug/rat at 1430 h) consistently induced ovulation in proestrous rats in which the endogenous gonadotropin surge had been blocked by ip injection of either Nembutal (1345 h) or antiserum to the LH-releasing hormone (1200 h). Injection (ip) of anti-betaLH serum on its own into proestrous rats at 1200 h prevented ovum maturation and follicular rupture. We conclude that currently available reference preparations of ovine FSH possess the capacity to stimulate follicular adenylate cyclase, steroidogenesis, and ovum maturation in vitro, as well as ovulation in vivo, in the rat, and that this capacity cannot be attributed to contamination with material immunochemically identical with LH. However, it is inferred that the physiological triggering of ovulation and related events in this species depends principally on LH.
Assuntos
AMP Cíclico/metabolismo , Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Óvulo/crescimento & desenvolvimento , Animais , Estradiol/biossíntese , Feminino , Hormônio Foliculoestimulante/imunologia , Hormônio Foliculoestimulante/isolamento & purificação , Hormônio Liberador de Gonadotropina/imunologia , Soros Imunes , Hormônio Luteinizante/fisiologia , Folículo Ovariano/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Pentobarbital/farmacologia , Progesterona/biossíntese , Ratos , Estimulação QuímicaRESUMO
Twenty-six patients with migraine attacks were treated for 3 to 16 months with flufenamic acid (125 mg four to six times per attack), an inhibitor of prostaglandin synthesis and action. In 25 patients the drug afforded symptomatic relief in 195 of 200 treated attacks. Side effects observed were mild dyspepsia (eight patients) and severe upper gastrointestinal symptoms (two patients). None of the eight patients treated with placebo reported any relief (20 attacks). The "common" antimigraine drugs afforded symptomatic relief in 12 of the patients, partial relief in seven, and no relief in seven. Treatment with flufenamic acid was based on the hypothesis that prostaglandins are involved in migraine attack and that the drug relieves migraine by inhibition of the vasoactivity of prostaglandins.
Assuntos
Ácido Flufenâmico/uso terapêutico , Transtornos de Enxaqueca/tratamento farmacológico , Prostaglandinas/biossíntese , Adolescente , Adulto , Criança , Relação Dose-Resposta a Droga , Dispepsia/induzido quimicamente , Feminino , Ácido Flufenâmico/administração & dosagem , Ácido Flufenâmico/efeitos adversos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We studied the effects on survival of allogeneic skin grafts after treatment with hydrocortisone and/or inhibitors of prostaglandin synthesis: indomethacin and flufenamate. We found a marked synergistic effect of combined treatment with hydrocortisone and indomethacin or flufenamate. Neither hydrocortisone nor indomethacin or flufenamate, when given alone in relatively small doses, caused delayed graft rejection. However, when small doses of hydrocortisone were used in combination with flufenamate or indomethacin, the median survival time (MST) of allogeneic grafts was prolonged from 11.4 days to 20.9 and 23.8 days, respectively. Moreover, the increase in graft survival was comparable to that obtained by treatment with relatively high doses of azathioprine alone or combined with hydrocortisone. The finding of synergism between low doses of prostaglandin synthesis inhibitors and glucocorticoids in delaying graft rejection suggests that such treatment might provide a relatively safer means of achieving clinical immunosuppression than the high doses of steroids and azathioprine currently in use.
Assuntos
Hidrocortisona/farmacologia , Prostaglandinas/biossíntese , Transplante de Pele , Animais , Ácido Flufenâmico/farmacologia , Sobrevivência de Enxerto , Indometacina/farmacologia , Masculino , Camundongos , Fatores de Tempo , Transplante HomólogoRESUMO
Prostaglandin E (PGE) concentration the aqueous humor of an intact rabbit eye was less than 0.1 ng. per milliliter and increased to 19 +/- 3 ng. per milliliter 60 minutes following paracentesis. The rise in PGE level was associated with clinical signs of ocular inflammation. Pretreatment with triamcinolone reduced both the accumulation of PGE in the aqueous humor and the inflammatory response following paracentesis. intravitreal injection of E. coli endotoxin into rabbit eyes increases PGE level in the anterior chamber to 72 +/- 17 ng. per milliliter and induced acute uveitis. slices of iris and ciliary body (ICB) derived from either rabbit eyes with endotoxin-induced uveitis or normal eyes were incubated for 60 to 240 minutes and the rate of PGE release into the medium was measured by radioimmunoassay. after a 4 hour incubation, the PGE release from inflamed ICB was threefold higher than that of normal ICB. incubation of inflamed ICB with hydrocortisone, or Millicorten (100 mug per milliliter) for 4 hours reduced PGE accumulation in the medium by 50 and 81 per cent, respectively. Aldosterone had no effect on the rate of PGE release from inflamed ICB throughout the incubation period. Hydrocortisone or Millicorten also reduced PGE tissue content of inflamed ICB by about 74 per cent during the period of incubation. Indomethacin (100 mug per milliliter) abolished PGE accumulation. The suppressive action of hydrocortisone on PGE release into the incubation medium was prevented by the addition of arachidonic acid (2 mug per milliliter), a substrate for prostaglandin synthesis. By contrast , the inhibitory action of indomethacin was not affected by provision of arachidonic acid. We suggest that glucocorticosteroids reduce PGE accumulation by limiting the availability of the substrate for prostaglandin biosynthesis and thus suppress the inflammatory response.
Assuntos
Glucocorticoides/farmacologia , Prostaglandinas E/biossíntese , Uveíte/metabolismo , Aldosterona/farmacologia , Animais , Humor Aquoso/efeitos dos fármacos , Ácidos Araquidônicos/farmacologia , Endotoxinas/farmacologia , Escherichia coli , Glucocorticoides/uso terapêutico , Hidrocortisona/farmacologia , Indometacina/farmacologia , Prostaglandinas E/análise , Coelhos , Uveíte/induzido quimicamente , Uveíte/tratamento farmacológicoRESUMO
The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 10(5) cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the beta-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.