RESUMO
PURPOSE: To investigate whether heat shock protein (Hsp) 72 is induced in retinal ganglion cells (RGCs) in experimental rat glaucoma and whether the induction of Hsp72 by heat stress or zinc (Zn(2+)) administration can increase survival of RGCs in the model. METHODS: Intraocular pressure (IOP) was elevated unilaterally in Wistar rats with argon laser irradiation of the trabecular meshwork 5 days after intracameral injection of india ink. Immunohistochemical staining for Hsp72 was performed. The rats with elevated IOP were treated with heat stress once a week (six rats) or intraperitoneal injection of zinc (10 mg/kg) every two weeks (six rats). Untreated rats with elevated IOP served as a control group (six rats). Quercetin, an inhibitor of Hsp expression was injected in the rats with heat stress (six rats) and zinc injection (seven rats). Subsequent to 4 weeks of IOP elevation, RGCs were counted. RESULTS: The IOP increase compared with the contralateral eyes was 48% +/- 4% throughout the study period. Hsp72 was detected only in the eyes with elevated IOP at 1 and 2 days and was weakly detected at 1 week of IOP elevation. A single administration of zinc strongly induced Hsp72 in RGCs of rats with elevated IOP for 2 weeks. Treatment with heat stress or zinc in rats with elevated IOP increased RGC survival after 4 weeks of IOP elevation, compared with the untreated control group (P = 0.004, n = 6). Quercetin reversed the positive effect of heat stress or zinc injection on RGC survival. CONCLUSIONS: These results demonstrate the possibility of a novel therapeutic approach to glaucoma through an enhanced induction of the endogenous heat shock response.
Assuntos
Citoproteção , Proteínas de Choque Térmico/biossíntese , Hipertensão Ocular/metabolismo , Células Ganglionares da Retina/citologia , Animais , Western Blotting , Contagem de Células , Sobrevivência Celular/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Choque Térmico HSP72 , Temperatura Alta , Pressão Intraocular , Masculino , Hipertensão Ocular/complicações , Hipertensão Ocular/patologia , Quercetina/farmacologia , Ratos , Ratos Wistar , Zinco/farmacologiaRESUMO
Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein gamma subunit (G gamma c, previously named G gamma b), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Ong et al., 1995, J. Biol. Chem. 270:8495-8500). We report here the characterization of human G gamma c and its gene structure. Human G gamma c subunit shares a high degree of sequence identity with the corresponding bovine G gamma c isoform (85%) and human rod G gamma 1 (63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-G gamma c antibodies. Nucleotide sequence analysis of the G gamma c gene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod G gamma 1 gene (GNGT1). By using fluorescence in situ hybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the G gamma c gene structure would facilitate the identification of genetic defects associated with cone degeneration.
Assuntos
Cromossomos Humanos Par 17/genética , Proteínas de Ligação ao GTP/genética , Células Fotorreceptoras Retinianas Cones/química , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação ao GTP/química , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The activation of cell-surface receptors usually produces a transient increase of the extracellular acidification rate in culture cells that can be detected with a biosensor-based instrument. We describe here the application of this method in monitoring hormonal response of Galphas-deficient fibroblasts from patients with pseudohypoparathyroidism type Ia (PHP-Ia). We found that following exposure to isoproterenol, the mean acidification response of fibroblasts from four PHP-Ia patients was only 18% of the response in normal cells. In contrast, these two groups of fibroblasts had similar levels of response to insulin and dibutyryl cAMP. This is the first reported experimental evidence correlating reduced Galphas activity with diminished cellular responsiveness to hormones in cultured living cells. Our results also indicate that this approach will be useful for rapid screening of other metabolic diseases caused by abnormal cellular signaling.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Sistemas Computacionais , Isoproterenol/farmacologia , Monitorização Fisiológica/métodos , Pseudo-Hipoparatireoidismo/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , OxirreduçãoRESUMO
The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.
Assuntos
Proteínas de Ligação ao GTP/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Complementar , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Soros Imunes , Imuno-Histoquímica , Dados de Sequência Molecular , Prenilação de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosAssuntos
Alquil e Aril Transferases , Cisteína/análogos & derivados , Diterpenos/síntese química , Proteínas de Ligação ao GTP/metabolismo , Prenilação de Proteína , Sequência de Aminoácidos , Animais , Cisteína/síntese química , Cisteína/metabolismo , Diterpenos/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Farnesiltranstransferase , Proteínas de Ligação ao GTP/isolamento & purificação , Substâncias Macromoleculares , Ácido Mevalônico/metabolismo , Dados de Sequência Molecular , Peso Molecular , Técnica de Diluição de Radioisótopos , Ratos , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transferases/metabolismo , TrítioRESUMO
A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Segmento Externo da Célula Bastonete/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Testes de Precipitina , Processamento de Proteína Pós-Traducional , Ratos , Ratos EndogâmicosRESUMO
Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyl]methionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the alpha subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory gamma subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the alpha cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the alpha-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.