RESUMEN
Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. OBJECTIVE: This study aimed to explore whether such effect is dependent on TLR9 signaling. MATERIAL AND METHODS: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. RESULTS: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. CONCLUSIONS: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.
Asunto(s)
Pérdida de Hueso Alveolar/tratamiento farmacológico , Ligando de CD40/farmacología , Citidina/farmacología , Nucleótidos de Guanina/farmacología , Oligodesoxirribonucleótidos/farmacología , Periodontitis/tratamiento farmacológico , Receptor Toll-Like 9/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos B/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Encía/efectos de los fármacos , Encía/patología , Interleucina-10/análisis , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Reproducibilidad de los Resultados , Factores de Tiempo , Receptor Toll-Like 9/análisisRESUMEN
Abstract Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. Objective: This study aimed to explore whether such effect is dependent on TLR9 signaling. Material and Methods: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. Results: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. Conclusions: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.
Asunto(s)
Animales , Oligodesoxirribonucleótidos/farmacología , Periodontitis/tratamiento farmacológico , Pérdida de Hueso Alveolar/tratamiento farmacológico , Ligando de CD40/farmacología , Citidina/farmacología , Receptor Toll-Like 9/efectos de los fármacos , Nucleótidos de Guanina/farmacología , Valores de Referencia , Factores de Tiempo , Ensayo de Inmunoadsorción Enzimática , Linfocitos B/efectos de los fármacos , Células Cultivadas , Adyuvantes Inmunológicos/farmacología , Reproducibilidad de los Resultados , Interleucina-10/análisis , Modelos Animales de Enfermedad , Receptor Toll-Like 9/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Citometría de Flujo , Encía/efectos de los fármacos , Encía/patología , Ratones Endogámicos C57BLRESUMEN
Guanine-based purines have been traditionally studied as modulators of intracellular processes, mainly G-protein activity. However, they also exert several extracellular effects not related to G proteins, including modulation of glutamatergic activity, trophic effects on neural cells, and behavioral effects. In this article, the putative roles of guanine-based purines on the nervous system are reviewed, and we propose a specific guanine-based purinergic system in addition to the well-characterized adenine-based purinergic system. Current evidence suggest that guanine-based purines modulate glutamatergic parameters, such as glutamate uptake by astrocytes and synaptic vesicles, seizures induced by glutamatergic agents, response to ischemia and excitotoxicity, and are able to affect learning, memory and anxiety. Additionally, guanine-based purines have important trophic functions affecting the development, structure, or maintenance of neural cells. Although studies addressing the mechanism of action (receptors and second messenger systems) of guanine-based purines are still insufficient, these findings point to the guanine-based purines (nucleotides and guanosine) as potential new targets for neuroprotection and neuromodulation.
Asunto(s)
Encéfalo/efectos de los fármacos , Nucleótidos de Guanina/farmacología , Guanosina/farmacología , Fármacos Neuroprotectores/farmacología , Adenosina Trifosfato/fisiología , Animales , Astrocitos/metabolismo , Conducta Animal/efectos de los fármacos , Ácido Glutámico/metabolismo , Guanosina Trifosfato/fisiología , Humanos , Receptores Purinérgicos P1/fisiología , Receptores Purinérgicos P2/fisiologíaRESUMEN
Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.
Asunto(s)
Encéfalo/efectos de los fármacos , Ácido Glutámico/metabolismo , Nucleótidos de Guanina/farmacología , Vesículas Sinápticas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Hidrólisis , Masculino , Ratas , Ratas WistarRESUMEN
Glutamate significantly increased levels of spontaneous chemiluminescence (CL) in rat hippocampal slices incubated under hypoxic conditions. Although it has been previously shown that guanine nucleotides (GN) displace glutamate from several of its receptors, in our study only GMP, as well as the glutamate antagonist MK-801, was able to reverse the increase in CL provoked by glutamate. On the other hand, not only GTP or Gpp(NH)p failed to reverse the action of glutamate, but they increased CL production like glutamate. This effect of GTP/Gpp(NH)p was also reversed by GMP. We concluded that, under neurotoxic conditions, GMP acted as an antagonist and GTP or Gpp(NH)p acted as agonists of glutamate. These results reinforced the evidence of the existence of extracellular site(s) for GN and indicated a possible role for GN in excitotoxicity.
Asunto(s)
Ácido Glutámico/farmacología , Nucleótidos de Guanina/farmacología , Hipocampo/metabolismo , Animales , AMP Cíclico/metabolismo , Maleato de Dizocilpina/farmacología , Femenino , Guanosina Trifosfato/farmacología , Guanilil Imidodifosfato/farmacología , Hipocampo/efectos de los fármacos , Hipoxia , Técnicas In Vitro , Mediciones Luminiscentes , Ratas , Ratas WistarRESUMEN
Metabotropic glutamate receptors (mGluRs) have been shown to modulate adenylate cyclase activity via G-proteins. In the present study we report similar results to the previously observed in the literature, showing that glutamate and the metabotropic agonists, 1S,3R-ACPD or quisqualate induced cAMP accumulation in hippocampal slices of young rats. Moreover, guanine nucleotides GTP, GDP or GMP, inhibited the glutamate-induced cAMP accumulation. By measuring LDH activity in the buffer surrounding the slices, we showed that the integrity of the slices was maintained, indicating that the effect of guanine nucleotides was extracellular. GMP, GDPbeta-S or Gpp(NH)p abolished quisqualate-induced cAMP accumulation. GDPbeta-S or Gpp(NH)p but not GMP inhibited 1S,3R-ACPD-induced cAMP accumulation. The response evoked by glutamate was also abolished by the mGluR antagonists: L-AP3 abolished glutamate-induced cAMP accumulation in a dose-dependent manner and MCPG was effective only at the 2 mM dose. DNQX was ineffective. We are reporting here, an inhibition induced by guanine nucleotides, via an extracellular site (s), similar to the observed with classical glutamate antagonists on a cellular response evoked by mGluR agonists.
Asunto(s)
AMP Cíclico/antagonistas & inhibidores , Nucleótidos de Guanina/farmacología , Receptores de Glutamato Metabotrópico/metabolismo , Animales , AMP Cíclico/metabolismo , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Ácido Glutámico/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Guanosina Monofosfato/farmacología , Guanosina Trifosfato/farmacología , Guanilil Imidodifosfato/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Neurotoxinas/farmacología , Ácido Quiscuálico/farmacología , Ratas , Ratas Wistar , Tionucleótidos/farmacologíaRESUMEN
Mammalian sperm must undergo an exocytotic event during fertilization, the acrosome reaction (AR). This process is specifically induced by egg-surface glycoproteins and it involves guanine nucleotide binding proteins (G-proteins). Neoglycoproteins (NGP) with mannose or N-acetylglucosamine residues has been demonstrated to induce the AR in human sperm. Activators of G-proteins, like GTP gamma S, GppNHp, mastoparan and AlF4- were capable of inducing the AR, while other nucleotides or analogues did not. When sperm were preincubated with these agents and then with NGPs, only the G-protein inhibitor GDP beta S decreased the AR rate. The preincubation of sperm with Pertussis toxin resulted in the inhibition of NGP-induced AR, while no effect was observed with cholera toxin. Results indicate that direct activation of G-proteins is sufficient to elicit the AR, and the induction of the AR in human sperm is mediated by N-acetylglucosaminyl and mannosyl binding sites involving PTx-sensitive G-proteins similar to the induction by zona pellucida glycoproteins.
Asunto(s)
Acrosoma/metabolismo , Exocitosis/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/farmacología , Espermatozoides/metabolismo , Acrosoma/efectos de los fármacos , Acrosoma/fisiología , Proteínas de Unión al GTP/antagonistas & inhibidores , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Nucleótidos de Guanina/metabolismo , Humanos , Masculino , Manosa/metabolismo , Fosforilasas/antagonistas & inhibidores , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiologíaRESUMEN
GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg2+, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [3H]glutamate binding by 30-40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [3H]glutamate binding occurred in the absence of Mg2+. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites.
Asunto(s)
Adenilil Ciclasas/metabolismo , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Nucleótidos de Guanina/farmacología , Animales , Encéfalo/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Guanosina Monofosfato/farmacología , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Magnesio/farmacología , Masculino , Ratas , Ratas Wistar , Sodio/farmacología , Tionucleótidos/farmacología , TritioRESUMEN
Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we have previously reported that, in the absence of externally added calcium, endosome fusion requires cytosol, ATP, and is sensitive to N-ethylmaleimide (NEM) and to anti-NEM-sensitive factor (NSF) antibody. This cytosol-dependent fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. Further studies have revealed, however, that in the presence of micromolar concentrations of free calcium, fusion is observed even in the absence of cytosol and ATP. At the electron microscope level, Ca(2+)-dependent endosome aggregation and fusion were similar to that observed for cytosol-dependent fusion. Calcium-dependent fusion was not affected by non-hydrolyzable analogs of GTP or GDP nor by NEM or anti-NSF antibody. However, Ca(2+)-dependent fusion was abrogated by trypsin treatment of the vesicles or by a membrane wash with 60 mM EDTA indicating that peripheral proteins are required. An anti-annexin II antibody and an annexin II peptide blocked Ca(2+)-dependent fusion by 50%. After the EDTA wash, Ca(2+)-dependent fusion was reconstituted by addition of purified annexin II and arachidonic acid. We conclude that endosomes can fuse by two mechanisms, one that has an absolute requirement for calcium and is probably mediated by annexins, and another that does not require calcium.
Asunto(s)
Calcio/metabolismo , Endosomas/metabolismo , Fusión de Membrana , Animales , Anexina A1/inmunología , Anexina A2/inmunología , Células Cultivadas , Endocitosis , Nucleótidos de Guanina/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Fosfolipasas A/antagonistas & inhibidoresRESUMEN
In G protein-coupled receptors, neurotransmitter-induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor-G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP-PNP, a non-hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP-binding sites are pre-saturated with GMP-PNP. Experiments carried out with these membranes show that GMP-PNP, GDP-S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP-PNP activates adenylate cyclase activity by binding to G proteins.
Asunto(s)
Adenilil Ciclasas/metabolismo , Nucleótidos de Guanina/farmacología , Ácido Kaínico/metabolismo , Colículos Superiores/efectos de los fármacos , Colículos Superiores/metabolismo , Animales , Sitios de Unión , Pollos , Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Guanilil Imidodifosfato/metabolismo , Guanilil Imidodifosfato/farmacología , Técnicas In VitroRESUMEN
Adenylate cyclase activity and binding of neurotransmitters to some receptors can be modulated simultaneously by guanine nucleotides. Furthermore it has been shown, in different neurotransmitter systems, that the ability of GTP to inhibit agonist binding is related to the capacity of the transmitter to modulate adenylate cyclase activity. In the present report we show that in chick optic tectum and cerebellum the effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity can be dissociated. In lysed membrane preparations, GTP, GDP, and GMP, or their analogs, displace binding of kainic acid with the same efficiency, whereas only GTP stimulates adenylate cyclase. In vesicle preparations, all three nucleotides inhibit binding of kainic acid without modifying adenylate cyclase activity. The present results suggest that, if adenylate cyclase is indeed coupled to this particular type of excitatory amino acid receptor, the coupling mechanism would be probably different from those operating in other neurotransmitter systems and also that the displacement of kainic acid by GDP and GMP (and even perhaps by GTP) is not likely to depend on the interaction between the receptor and a Gs-protein-mediated effector system.
Asunto(s)
Adenilil Ciclasas/metabolismo , Cerebelo/metabolismo , Nucleótidos de Guanina/farmacología , Ácido Kaínico/metabolismo , Receptores de Neurotransmisores/metabolismo , Colículos Superiores/metabolismo , Animales , Membrana Celular/metabolismo , Cerebelo/efectos de los fármacos , Pollos , Cinética , Receptores de Ácido Kaínico , Receptores de Neurotransmisores/efectos de los fármacos , Colículos Superiores/efectos de los fármacosRESUMEN
These studies examine the regulation of adenylate cyclase in renal cortical membranes from phosphate-deprived and phosphate-deprived acidotic dogs. Enzyme stimulation by parathyroid hormone (PTH) was decreased in phosphate deprivation [Vmax 1,578 +/- 169 vs. 2,581 +/- 219 pmol adenosine 3',5'-cyclic monophosphate (cAMP).mg protein-1 x 30 min-1 in controls, P less than 0.01]. Metabolic acidosis further decreased PTH-stimulated activity. Membranes from phosphate-deprived dogs showed a decrease in Gs alpha-content by cholera toxin-dependent ADP-ribosylation (174 +/- 18 arbitrary units vs. 266.4 +/- 13.6 in controls, P less than 0.01). Metabolic acidosis further decreased Gs alpha-content, P less than 0.01. Gi content by pertussis-dependent ADP-ribosylation was also lower in phosphate-deprived and phosphate-deprived acidotic animals. Gs function was examined by its property to protect the catalytic unit from inactivation by N-ethylmaleimide when preincubated with GTP gamma S. In controls, protection of inactivation was 80% of the maximal activity, whereas in phosphate deprivation protection was less than 50%. In conclusion, metabolic acidosis enhances adenylate cyclase resistance to PTH in phosphate deprivation. These alterations are associated with a decrease in the content and function of Gs alpha, suggesting a role of Gs in the renal adaptation to phosphate depletion and acidosis.