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Biomedical subjects

R Wen

Publications and source records attributed to R Wen.

At least 19 recordsLinked to original sources
PubMed2006

Biomarkers in transplantation: Prospective, blinded measurement of predictive value for the flow cytometry crossmatch after negative antiglobulin crossmatch in kidney transplantation.

This prospective, blinded observational study was conducted to measure the predictive value the of flow cytometric crossmatch for biopsy-proven acute rejection, graft loss, or death following kidney transplantation. Patients were selected for renal transplantation on the basis of a conventional antihuman globulin cytotoxic T-cell crossmatch. Flow crossmatch was performed simultaneously, but the results were not disclosed to the transplant team. A total of 257 kidney transplant recipients were enrolled in the study; 78 patients experienced biopsy-proven rejection in the first post-transplant year, and 41 patients lost their graft or died during the period of follow-up (mean: 2046 days). Kaplan-Meier estimates of rejection, graft loss, or patient death did not differ between subjects with a positive or negative flow crossmatch. Cox analyses showed no influence of the flow crossmatch on the risk of biopsy-proven acute rejection (P = 0.987). The sensitivity and specificity of the flow crossmatch for prediction of biopsy-proven rejection were 0.128 and 0.883, and the positive and negative post-test probabilities were 0.323 and 0.301, respectively. The magnitude of the channel shift did not influence the multivariate Cox regression model. The area under the receiver operating characteristic curve of the flow crossmatch was 0.483 (P = 0.71) and 0.572 (P = 0.38), respectively for the living and cadaver transplant recipients, indicating no discriminative value in this study population. Flow crossmatch appears to have no significant incremental value in predicting biopsy-proven acute rejection, graft loss, or death following kidney transplantation in patients who have a negative antihuman globulin cytotoxic T-cell crossmatch against their donor.

Adolescent↗
PubMed2001

Microtubule integrity regulates Pak leading to Ras-independent activation of Raf-1. insights into mechanisms of Raf-1 activation.

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser(338). Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser(338) to Ala or Tyr(340)-Tyr(341) to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4beta-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of (338)SSYY(341); 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser(338), an event crucial for Raf activation.

Amino Acid Substitution↗
PubMed2001

Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation.

The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.

Animals↗
PubMed2001

Variations in the human phospholipase Cgamma2 gene in patients with B-cell defects of unknown etiology.

Our recent studies using targeted gene disruption have shown that defects in phospholipase Cgamma2 (PLCgamma2) result in a B-cell abnormality that is very similar to that seen in Btk-deficient mice. Null mutations in either PLCG2 or BTK are associated with decreased numbers of mature B cells, failure to make antibodies to some T cell-independent antigens and the absence of CD5+ peritoneal B cells. Mutations in BTK in humans cause a more severe defect in B-cell development characterized by almost complete absence of B cells in the peripheral circulation, profound hypogammaglobulinemia and an inability to produce antibodies to any antigens. However, not all patients with severe defects in B-cell development have mutations in BTK or the components of the B-cell signal transduction complex. To explore the possibility that some patients with defects in B-cell development of unknown etiology might have mutations in PLCG2, we determined the genomic structure of this gene and established conditions to analyze the 32 exons of the gene and the flanking sequences by single-strand conformation polymorphism. Although 24 polymorphic variants of this gene were found in 35 patients, we did not identify any alterations that were likely to be the cause of disease.

Agammaglobulinemia↗
PubMed2001

Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein.

Cerebral ischemia resulting from a disruption of blood flow to the brain initiates a cascade of events that causes neuron death and leads to neurologic dysfunction. Reactive oxygen species are thought, at least in part, to mediate this disease process. The authors recently cloned and characterized an antioxidant protein, SAG (sensitive to apoptosis gene), that is redox inducible and protects cells from apoptosis induced by redox agents in a number of in vitro cell model systems. This study reports a neuroprotective role of SAG in ischemia/reperfusion-induced brain injury in an in vivo mouse model. SAG was expressed at a low level in brain tissue and was inducible after middle cerebral artery occlusion with peak expression at 6 to 12 hours. At the cellular level, SAG was mainly expressed in the cytoplasm of neurons and astrocytes, revealed by double immunofluorescence. An injection of recombinant adenoviral vector carrying human SAG into mouse brain produced an overexpression of SAG protein in the injected areas. Transduction of AdCMVSAG (wild-type), but not AdCMVmSAG (mutant), nor the AdCMVlacZ control, protected brain cells from ischemic brain injury, as evidenced by significant reduction of the infarct areas where SAG was highly expressed. The result suggests a rather specific protective role of SAG in the current in vivo model. Mechanistically, SAG overexpression decreased reactive oxygen species production and reduced the number of apoptotic cells in the ischemic areas. Thus, antioxidant SAG appears to protect against reactive oxygen species-induced brain damage in mice. Identification of SAG as a neuroprotective molecule could lead to potential stroke therapies.

Adenoviridae↗
PubMed2001

Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development.

Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

Animals↗
PubMed2001

Extended photoreceptor viability by light stress in the RCS rats but not in the opsin P23H mutant rats.

PURPOSE: To determine the effect of light stress on retinal function and long-term photoreceptor viability in Royal College of Surgeons (RCS) rats and the applicability of the light treatment to the opsin P23H mutant rats. METHODS: RCS rats at postnatal day (P)23 were illuminated with 120 foot-candles (fc) white light for 10 hours. Photoreceptor survival and basic fibroblast growth factor (bFGF) expression were measured at P60 and P83. Retinal function was evaluated by electroretinography. Opsin P23H transgenic rats were treated with light at P28 and analyzed at P70 for photoreceptor viability, ultrastructure, and bFGF expression. RESULTS: Light-treated RCS rats at P60 had four to five rows of nuclei versus one to two rows in untreated littermates. The average amplitude of the ERG b-wave was 28 microV in treated rats, compared with 6 microV in untreated littermates. By P83 there was still significant preservation of the ONL in treated rats. Immunoblot analysis showed a high expression of bFGF in the treated retinas even 2 months after treatment. Illumination of P23H rats at P28 with 120 fc white light for 10 hours caused substantial photoreceptor cell death, although bFGF expression was upregulated. Lowered illumination dosages continued to cause photoreceptor damage until levels were reached that neither caused damage nor enhanced survival. CONCLUSIONS: Although light stress promotes photoreceptor survival and function in the RCS rat, it elicits death signals in the P23H rats that may not be overcome by survival-promoting factors. Therefore, use of light stress to promote photoreceptor survival should be considered with regard to sensitivity of the mutation to light damage.

Animals↗
PubMed2001

[Synthesis and biological activities of new 5-HT2A selective ligands--N-substituted-piperidinyl-4-phenylthioether and sulfone derivatives].

AIM: A series of 4-piperidinylthioether and sulfone derivatives of 4-[1-hydroxy-1-(2,3-dimethoxyphenyl) methyl]-N-2-(4-fluorophenylethyl) piperidine (MDL 100907) were synthesized in order to find new 5-HT2A selective ligands. METHODS: Title compounds 2a-2c were synthesized from 2, 3-dimethoxythiophenol and tested for their affinities to 5-HT2A, 5-HT2C, 5-HT6 and 5-HT7 receptors and some other nervous transmitter receptors in vitro. RESULTS: Compounds 2a-2c are new compounds. The results of the binding assay demonstrated that they have relatively high selectivity for 5-HT2A receptor in vitro. CONCLUSION: Some sulfur containing analogues of MDL 100907 showed selective affinity to 5-HT2A receptor and are worth further study.

Animals↗
PubMed2000

Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis.

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explanted DMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19(ARF), Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARF and functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null and ARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1(+/+) animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

3T3 Cells↗
PubMed2000

Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Activation of caspase-3 in the retina of transgenic rats with the rhodopsin mutation s334ter during photoreceptor degeneration.

The role of caspase-3 in photoreceptor degeneration was examined in a line of transgenic rats that carry a rhodopsin mutation S334ter. Photoreceptor degeneration in these animals is rapid. It is detected as early as postnatal day (PD) 8, and by PD 20, only one of the original 12 rows of nuclei remain in the outer nuclear layer. At PD 11 and 12, the number of photoreceptors dying per day reaches a peak of approximately 30% of the total photoreceptors in the retina. Coincident with this rapid degeneration is an increase in caspase-3-like activity as assessed by the cleavage of a fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and an increase in activated caspase-3 as determined by Western blot analysis for its 12 kDa subunit. Intraocular injection of an irreversible caspase-3 inhibitor N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyk etone partially protected photoreceptors from degeneration. These findings indicate that a caspase-3-dependent mechanism is operative in photoreceptor death in the transgenic rats under investigation.

Animals↗
PubMed1999

Light treatment enhances photoreceptor survival in dystrophic retinas of Royal College of Surgeons rats.

PURPOSE: To determine whether treatment with bright light elicits a protective response that enhances photoreceptor survival in Royal College of Surgeons (RCS) rats with inherited retinal degeneration. METHODS: RCS rats were illuminated for 10 to 12 hours with 130 foot-candles (fc) of white or green light. Untreated littermates that were kept under low cyclic light levels were used as control subjects. Photoreceptor survival was determined by quantitative analysis of photoreceptor nuclei and ultrastructural assessment of cellular organization. Basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) gene expression were determined at the mRNA and protein levels. RESULTS: Treatments of RCS rats with a single dose of bright light on postnatal day 23 (P23) greatly enhanced photoreceptor survival. Ultrasturctural analysis revealed intact inner segments in light-treated retinas, whereas in untreated retinas only remnants of inner segments were observed. By P42, numerous viable nuclei were counted in the posterior retina of light-treated rats, whereas most of the remaining nuclei in untreated RCS rat retinas were highly pyknotic. At 2.5 days after treatment with a single dose of bright light, bFGF gene expression was significantly higher than in untreated RCS rat retinas. By P42, bFGF protein levels were still significantly higher in the treated retinas. CONCLUSIONS: Exogenous bFGF has been shown to promote photoreceptor survival in the RCS rat retina. Thus, the increased bFGF expression that was measured in the light-treated RCS rat retinas may be a protective response to light stress, which supports the observed rescue of photoreceptors in light-treated RCS rat retinas.

Animals↗
PubMed1998

Preconditioning with bright light evokes a protective response against light damage in the rat retina.

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12-48 hr preexposure, followed by a 48 hr "rest phase" of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.

Animals↗
PubMed1998

Continuous exposure to bright light upregulates bFGF and CNTF expression in the rat retina.

PURPOSE: To examine mRNA expression of neurotrophic factors in the retina after exposure to bright light. METHODS: Male adult Sprague-Dawley rats were exposed to light of 115-130 ft-c. Retinas were collected after 1, 2, 4 or 7 days of exposure. Northern blot analysis was performed to determine mRNA levels for the following factors and their receptors: basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), acidic fibroblast growth factor (aFGF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1) and glial fibrillary acidic protein (GFAP). Expression of bFGF, CNTF and GFAP was localized by in situ hybridization. RESULTS: Exposure to light of 115-130 ft-c resulted in a substantial increase in bFGF and CNTF expression that persisted during the entire 7-day period of exposure. The peak expression of bFGF was almost 9-fold at day 2. The increase in CNTF mRNA reached a maximum of 6-fold at day 4. A small increase (50%) in IGF-1 mRNA was also seen at day 4. Among the receptors, an elevation of 3-fold in FGF receptor 1 (FGFR-1) was observed at day 2. There was also a small increase (70%) in IGF-1 receptor (IGF-1R) at day 2. In addition, the expression of GFAP showed a rapid elevation of about 8-fold by day 1 and 9-fold by day 2, and 18-fold by day 4. There was, however, no significant alteration in the expression of aFGF and BDNF. In situ hybridizations showed that the elevation of bFGF, CNTF and GFAP occurred across the entire retina with especially prominent increases over specific layers for each gene. CONCLUSIONS: Continuous exposure to bright light upregulates bFGF, CNTF, FGFR-1 and GFAP expression in the rat retina. The pattern of induced expression closely resembles that induced by mechanical injury, implying a common underlying mechanism.

Animals↗
PubMed1998

[Mechanism of preventing the recurrence of bladder carcinoma by intravesical instillation of BCG plus IL-2].

OBJECTIVE: To study the mechanism of preventing the recurrence of bladder carcinoma by intravesical instillation of BCG plus IL-2. METHOD: Intravesical instillation of BCG or BCG plus IL-2 were carried out in the bladder transitional cell carcinoma patients undergoing surgical operation (16, 19 cases respectively). RESULT: They were followed up for 14-22 months. The recurrence rates were 31.25% and 21.05% respectively. At 6 weeks after the instillation of IL-2 plus BCG, the peripheral blood NKCF activity was increased significantly and there was a significant positive correlation between the activities of NKCF and IL-2. CONCLUSION: The intravesical instillation of IL-2 plus BCG is clearly superior to only BCG in preventing recurrence of bladder carcinoma. There may be immuno-enhancement and immuno-synergism.

Adjuvants, Immunologic↗
PubMed1998

Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Müller cells.

PURPOSE: To investigate the induction of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene expression by prostaglandin E2 (PGE2) in cultured rat Müller cells and to study the mechanism of the induction. METHODS: Müller cells were obtained from neonatal Sprague-Dawley rat retinas and cultured in essential modified Eagle's medium supplemented with 10% fetal calf serum for up to four passages. Cells were treated with PGE2, protein kinase A (PKA) inhibitors H-89 or SQ 22536, protein kinase C (PKC) inhibitors calphostin C or GF 109203X, PKC activator phorbol 12-myristate 13-acetate (PMA), or the PKA activator forskolin. Northern blot analysis was performed to determine the levels of VEGF and bFGF mRNA. RESULTS: PGE2 induced VEGF and bFGF mRNA expression in a dose- and time-dependent manner. VEGF and bFGF mRNA reached peaks of 2- and 3.5-fold at 10 microM PGE2. No further increases were observed at 100 microM PGE2. When treated with 10 microM PGE2, the increases in VEGF and bFGF mRNA reached maximum by 2 hours, then slowly declined toward the control level within 24 hours of PGE2 treatment. The inductions of VEGF and bFGF mRNA expression by PGE2 were blocked by the specific PKA inhibitors H-89 (30 microM) or SQ 22536 (500 microM, 1000 microM). Forskolin (10 microM), a cyclic adenosine monophosphate activator, also stimulated VEGF and bFGF mRNA expression. However, the effects of forskolin and PGE2 on VEGF gene expression were not additive, whereas forskolin enhanced the effect of PGE2 on bFGF mRNA expression. The specific PKC inhibitors, GF 109203X (2 microM) and calphostin C (1 microM), did not inhibit PGE2-induced VEGF gene expression, whereas PGE2-induced bFGF expression was blocked by the PKC inhibitor GF 109203X. In addition, downregulation of PKC by PMA (0.8 microM) treatment did not block the induction of VEGF gene expression, whereas it did inhibit the induction of bFGF mRNA expression. CONCLUSIONS: These results indicate that PGE2 stimulates VEGF and bFGF mRNA expression in cultured rat Müller cells. The induction of VEGF seems to occur through activation of the PKA pathway, whereas that of bFGF occurs through PKA and PKC activation. These findings raise the possibility that endogenous PGE2 stimulates VEGF and bFGF mRNA expression in Müller cells in vivo under conditions in which PGE2 production is increased, such as in injury.

Animals↗
PubMed1998

Alpha2-adrenergic agonists selectively activate extracellular signal-regulated kinases in Müller cells in vivo.

PURPOSE: Alpha2-adrenergic agonists have specific and selective effects on the retina to induce expression of basic fibroblast growth factor and to protect photoreceptors. This work explores the signaling pathway that mediates these effects. METHODS: Alpha2-adrenergic agonists xylazine and clonidine were administered systemically to male adult Sprague-Dawley rats. The activation state of extracellular signal-regulated kinases (ERKs) in the retina was assessed by immunoblot analysis, using antibodies that specifically recognize the dually phosphorylated forms of p44/p42 ERKs. Localization of phosphorylated ERKs was determined by immunocytochemistry. RESULTS: Intramuscular injection of 6 mg/kg xylazine induced an increase in ERK phosphorylation in the retina within 30 minutes that lasted 3 hours. Xylazine induced ERK phosphorylation at 1 mg/kg and reached a maximum at 10 mg/kg. Injection of clonidine also induced ERK phosphorylation in the retina. Yohimbine, a specific alpha2-adrenergic antagonist, completely prevented the induction of ERK phosphorylation. Immunocytochemical studies showed that the increase in ERK phosphorylation occurred mainly in Müller cells. In the brain, xylazine injection resulted in a decrease in ERK phosphorylation. CONCLUSIONS: Our results indicate that systemically administered alpha2-adrenergic agonists selectively activate ERKs in retinal Müller cells. The induced activation of ERKs in Müller cells is probably one of the early events that result in photoreceptor protection. These results also indicate that Müller cells are unique in response to alpha2-adrenergic agonists and imply a role for Müller cells in alpha2-adrenergic agonist-induced photoreceptor protection.

Adrenergic alpha-2 Receptor Agonists↗