Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Pharmacokinetics of doxycycline hyclate in pigs with a new feed premix formulation.

This study aimed to evaluate the administration of doxycycline hyclate in a long-acting pharmaceutical preparation in pigs when administered either ad libitum as a feed medication or an oral bolus dose. In all instances, the studied dose was 20 mg/kg b.w. A total of 48 healthy crossbred, castrated male pigs (Landrace-Yorkshire) weighing 23 ± 4.3 kg were included in this trial. They were randomly assigned to six groups as follows: two groups for the experimental prototype 1 of doxycycline hyclate administering it ad libitum (Fad-lib) or as forced bolus (Fbolus); two groups for the experimental prototype 2 of doxycycline hyclate as for the former groups (FCad-lib and FCbolus), and two control groups receiving the same dose of doxycycline hyclate, but of a commercial premix, also as previously explained (Cbolus and Cad-lib). Statistical analysis of the mean pharmacokinetic values was carried out with Kruskal-Wallis and Dunn's tests. The relative bioavailability (Fr) of the best prototype, when administered ad libitum (FCad-lib), was five times larger than the reference group (Cadlib). These results allow the proposal that the referred differences achieved in the presented prototypes can mark a notable clinical difference, particularly in pathogens with some resistance.

Changes in glutamate and glutamine distributions in the retinas of cystine/glutamate antiporter knockout mice.

Purpose: The cystine/glutamate antiporter is involved in the export of intracellular glutamate in exchange for extracellular cystine. Glutamate is the main neurotransmitter in the retina and plays a key metabolic role as a major anaplerotic substrate in the tricarboxylic acid cycle to generate adenosine triphosphate (ATP). In addition, glutamate is also involved in the outer plexiform glutamate-glutamine cycle, which links photoreceptors and supporting Müller cells and assists in maintaining photoreceptor neurotransmitter supply. In this study, we investigated the role of xCT, the light chain subunit responsible for antiporter function, in glutamate pathways in the mouse retina using an xCT knockout mouse. As xCT is a glutamate exporter, we hypothesized that loss of xCT function may influence the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate. Methods: Retinas of C57BL/6J wild-type (WT) and xCT knockout (KO) mice of either sex were analyzed from 6 weeks to 12 months of age. Biochemical assays were used to determine the effect of loss of xCT on glycolysis and energy metabolism by measuring lactate dehydrogenase activity and ATP levels. Next, biochemical assays were used to measure whole-tissue glutamate and glutamine levels, while silver-intensified immunogold labeling was performed on 6-week and 9-month-old retinas to visualize and quantify the distribution of glutamate, glutamine, and related neurochemical substrates gamma-aminobutyric acid (GABA) and glycine in the different layers of the retina. Results: Biochemical analysis revealed that loss of xCT function did not alter the lactate dehydrogenase activity, ATP levels, or glutamate and glutamine contents in whole retinas in any age group. However, at 6 weeks of age, the xCT KO retinas revealed altered glutamate distribution compared with the age-matched WT retinas, with accumulation of glutamate in the photoreceptors and outer plexiform layer. In addition, at 6 weeks and 9 months of age, the xCT KO retinas also showed altered glutamine distribution compared with the WT retinas, with glutamine labeling significantly decreased in Müller cell bodies. No significant difference in GABA or glycine distribution were found between the WT and xCT KO retinas at 6 weeks or 9 months of age. Conclusion: Loss of xCT function results in glutamate metabolic disruption through the accumulation of glutamate in photoreceptors and a reduced uptake of glutamate by Müller cells, which in turn decreases glutamine production. These findings support the idea that xCT plays a role in the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate and derived neurotransmitters in the retina.

Hyper-reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy.

The aim of this study is to correlate small dot hyper-reflective foci (HRF) observed in spectral domain optical coherence tomography (SD-OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD-OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba-1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba-1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia-mediated inflammatory component of progressive diseases showing HRF.

Early onset of age-related changes in the retina of cystine/glutamate antiporter knockout mice.

To determine the role of the cystine/glutamate antiporter on retinal structure and function, retinas of C57Bl/6J wild-type and xCT knockout mice, lacking the xCT subunit of the cystine/glutamate antiporter were examined from 6 weeks to 12 months of age. Fundoscopy, optical coherence tomography (OCT), and whole mount retinal autofluorescence imaging were used to visualise age-related retinal spots. Glial fibrillary acidic protein (GFAP) immunolabelling was used to assess retinal stress. Retinal function was evaluated using full-field and focal electroretinograms. Examinations revealed retinal spots in both wild-type and xCT knockout mice with the number of spots greater at 9 months in the knockout compared to wild-type. OCT confirmed these discrete spots were located at the retinal pigment epithelium (RPE)-photoreceptor junction and did not label with drusen markers. Whole mount lambda scans of the 9 month xCT knockout retinas revealed that the photoreceptor autofluorescence matched the spots, suggesting these spots were retinal debris. GFAP labelling was increased in knockout retinas compared to wild-type indicative of retinal stress, and the discrete spots were associated with migration of microglia/macrophages to the RPE-retina intersection. OCT revealed that the superior retina was thinner at 9 months in knockout compared to wild-type mice due to changes to the outer nuclear and photoreceptor layers. While global retinal function was not affected by loss of xCT, focal changes in retinal function were detected in areas where spots were present. Tother these results suggest that the xCT KO mice exhibit features of accelerated ageing and suggests that this mouse model may be useful for studying the underlying cellular pathways in retinal ageing.

Orally Delivered Connexin43 Hemichannel Blocker, Tonabersat, Inhibits Vascular Breakdown and Inflammasome Activation in a Mouse Model of Diabetic Retinopathy.

Diabetic retinopathy (DR), a microvascular complication of diabetes, is associated with pronounced inflammation arising from the activation of a nucleotide-binding and oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome. Cell culture models have shown that a connexin43 hemichannel blocker can prevent inflammasome activation in DR. The aim of this study was to evaluate the ocular safety and efficacy of tonabersat, an orally bioavailable connexin43 hemichannel blocker, to protect against DR signs in an inflammatory non-obese diabetic (NOD) DR mouse model. For retina safety studies, tonabersat was applied to retinal pigment epithelial (ARPE-19) cells or given orally to control NOD mice in the absence of any other stimuli. For efficacy studies, either tonabersat or a vehicle was given orally to the inflammatory NOD mouse model two hours before an intravitreal injection of pro-inflammatory cytokines, interleukin-1 beta, and tumour necrosis factor-alpha. Fundus and optical coherence tomography images were acquired at the baseline as well as at 2- and 7-day timepoints to assess microvascular abnormalities and sub-retinal fluid accumulation. Retinal inflammation and inflammasome activation were also assessed using immunohistochemistry. Tonabersat did not have any effect on ARPE-19 cells or control NOD mouse retinas in the absence of other stimuli. However, the tonabersat treatment in the inflammatory NOD mice significantly reduced macrovascular abnormalities, hyperreflective foci, sub-retinal fluid accumulation, vascular leak, inflammation, and inflammasome activation. These findings suggest that tonabersat may be a safe and effective treatment for DR.

Effect of hypobaric hypoxia on hematological parameters related to oxygen transport, blood volume and oxygen consumption in adolescent endurance-training athletes.

Objective: To analyze the effect of altitude on hematological and cardiorespiratory variables in adolescent athletes participating in aerobic disciplines. Methods: 21 females and 89 males participated in the study. All were adolescent elite athletes engaged in endurance sports (skating, running and cycling) belonging to two groups: permanent residents in either low altitude (LA, 966 m) or moderate altitude (MA, 2640 m). Hematocrit (Hct), hemoglobin concentration ([Hb]), total hemoglobin mass (Hbt), blood, plasma and erythrocyte volumes (BV, PV and EV), VO2peak and other cardiorespiratory parameters were evaluated. Results: Sex differences were evident both in LA and HA skating practitioners, the males having higher significant values than the females in oxygen transport-related hematological parameters and VO2peak. The effect of altitude residence was also observed in Hct, [Hb], Hbt and EV with increased (14%-18%) values in the hematological parameters and higher EV (5%-24%). These results matched the significantly higher values of VO2peak measured in MA residents. However, BV and PV did not show differences between LA and MA residents in any case. Sports discipline influenced neither the hematological variables nor most of the cardiorespiratory parameters. Conclusions: LA and MA adolescent skaters showed sex differences in hematological variables. Endurance-trained male adolescent residents at MA had an increased erythropoietic response and a higher VO2peak compared to their counterparts residing and training at LA. These responses are similar in the three aerobic sports studied, indicating that the variables described are highly sensitive to hypoxia irrespective of the sports discipline.

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1ß and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Differential Action of Connexin Hemichannel and Pannexin Channel Therapeutics for Potential Treatment of Retinal Diseases.

Dysregulation of retinal function in the early stages of light-induced retinal degeneration involves pannexins and connexins. These two types of proteins may contribute to channels that release ATP, leading to activation of the inflammasome pathway, spread of inflammation and retinal dysfunction. However, the effect of pannexin channel block alone or block of both pannexin channels and connexin hemichannels in parallel on retinal activity in vivo is unknown. In this study, the pannexin channel blocker probenecid and the connexin hemichannel blocker tonabersat were used in the light-damaged rat retina. Retinal function was evaluated using electroretinography (ERG), retinal structure was analyzed using optical coherence tomography (OCT) imaging and the tissue response to light-induced injury was assessed immunohistochemically with antibodies against glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1 (Iba-1) and Connexin43 (Cx43). Probenecid did not further enhance the therapeutic effect of connexin hemichannel block in this model, but on its own improved activity of certain inner retina neurons. The therapeutic benefit of blocking connexin hemichannels was further evaluated by comparing these data against results from our previously published studies that also used the light-damaged rat retina model. The analysis showed that treatment with tonabersat alone was better than probenecid alone at restoring retinal function in the light-damaged retina model. The results assist in the interpretation of the differential action of connexin hemichannel and pannexin channel therapeutics for potential treatment of retinal diseases.

Proinflammatory cytokines trigger biochemical and neurochemical changes in mouse retinal explants exposed to hyperglycemic conditions.

Purpose: Diabetic retinopathy (DR) is one of the most frequent complications of diabetes affecting the retina and eventually causing vision impairment. Emerging evidence suggests that inflammation plays a vital role in DR progression. In this study, we evaluated the early biochemical and neurochemical changes in mouse retinal explants to understand the contribution of proinflammatory cytokines to disease progression. Methods: DR was modeled in vitro by incubating mouse retinal explants in a physiological buffer supplemented with high glucose and the proinflammatory cytokines TNF-α and IL-1ß. Key metabolites of retinal energy metabolism, including glucose, lactate, ATP, glutamate, glutamine, and enzymes supporting retinal ATP levels were assessed 40 min after the application of high glucose and proinflammatory cytokines. As retinal energy metabolism is tightly coupled to retinal neurochemistry, we also determined the short-term effect on the amino acid distribution of glutamate, gamma aminobutyric acid (GABA), glutamine, and glycine. Results: The results indicated that the combined application of high glucose and proinflammatory cytokines increased retinal glucose, lactate, and ATP levels, and decreased retinal glutamate, without affecting glutamine levels or the enzymes supporting ATP levels. Moreover, we observed a statistically significant increase in ATP and glutamate release. Correspondingly, statistically significant alterations in amino acid distribution were observed in retinal explants coexposed to high glucose and proinflammatory cytokines. Conclusions: These data suggest that short-term exposure to proinflammatory cytokines contributes to the early biochemical and neurochemical changes caused by hyperglycemia, by affecting retinal energy metabolism and amino acid distribution. These data are consistent with the idea that early intervention to prevent inflammation-triggered loss of metabolic homeostasis in patients with diabetes is necessary to prevent DR progression.

Adherence to UK dietary guidelines is associated with higher dietary intake of total and specific polyphenols compared with a traditional UK diet: further analysis of data from the Cardiovascular risk REduction Study: Supported by an Integrated Dietary Approach (CRESSIDA) randomised controlled trial.

Adherence to dietary guidelines (DG) may result in higher intake of polyphenols via increased consumption of fruits, vegetables and whole grains. We compared polyphenol dietary intake and urinary excretion between two intervention groups in the Cardiovascular risk REduction Study: Supported by an Integrated Dietary Approach study: a 12-week parallel-arm, randomised controlled trial (n 161; sixty-four males, ninety-seven females; aged 40-70 years). One group adhered to UK DG, whereas the other group consumed a representative UK diet (control). We estimated polyphenol dietary intake, using a 4-d food diary (4-DFD) and FFQ, and analysed 24-h polyphenol urinary excretion by liquid chromatography-tandem MS on a subset of participants (n 46 control; n 45 DG). A polyphenol food composition database for 4-DFD analysis was generated using Phenol-Explorer and USDA databases. Total polyphenol intake by 4-DFD at endpoint (geometric means with 95 % CI, adjusted for baseline and sex) was significantly higher in the DG group (1279 mg/d per 10 MJ; 1158, 1412) compared with the control group (1084 mg/d per 10 MJ; 980, 1197). The greater total polyphenol intake in the DG group was attributed to higher intake of anthocyanins, proanthocyanidins and hydroxycinnamic acids, with the primary food sources being fruits, cereal products, nuts and seeds. FFQ estimates of flavonoid intake also detected greater intake in DG compared with the control group. 24-h urinary excretion showed consistency with 4-DFD in their ability to discriminate between dietary intervention groups for six out of ten selected, individual polyphenols. In conclusion, following UK DG increased total polyphenol intake by approximately 20 %, but not all polyphenol subclasses corresponded with this finding.

Vinpocetine protects inner retinal neurons with functional NMDA glutamate receptors against retinal ischemia.

Retinal ischemia is involved in the pathogenesis of many major vision threatening diseases. Vinpocetine is a natural drug, which has a range of neuroprotective actions against retinal ischemia including modulating cation flow, improving metabolic activity and preventing apoptosis. The exact mechanism behind these actions remains unknown but may involve glutamate receptors, major components of the ischemic cascade. This study examined the effects of vinpocetine in association with specific ionotropic glutamate receptor agonists: N-methyl-D-aspartate (NMDA) and kainate. Vinpocetine's actions to improve cation channel permeability and cell marker immunoreactivity following ischemia appeared to be limited to NMDA activation with no changes observed following kainate stimulation. Vinpocetine's actions were lost in the presence of an NMDA receptor inhibitor further suggesting they may be secondary to NMDA receptor activation. NMDA receptor function was also necessary for vinpocetine's actions on glucose availability during ischemia but not lactate dehydrogenase (LDH) activity in the ischemic retina suggesting not all of vinpocetine's actions are linked to NMDA receptor function. These results may explain vinpocetine's effectiveness as a neuroprotective agent as the NMDA receptor is implicated in the pathogenesis of ischemia in a range of tissues of the central nervous system.

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release.

BACKGROUND: Connexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease. METHODS: Retinal pigment epithelial cells were exposed to HG, 10ng/mL pro-inflammatory cytokines IL-1ß and TNF-α, or a combination of both. Quantitative Cytometric Bead Array analysis was used to measure the release of inflammatory cytokines IL-6, IL-8, MCP-1, and sICAM-1, as well as VEGF and ATP. To determine the role of connexin43 hemichannels in the disease process, changes in cytokine and ATP release were evaluated following treatment with Peptide5, a connexin43 hemichannel blocker. Immunohistochemistry was used to compare NLRP3 inflammasome assembly under control and treatment conditions. RESULTS: Co-application of HG and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF and ATP, to significantly higher levels compared to cytokines alone. Peptide5 prevented cytokine release and prevented the increase in ATP release following co-application of HG and cytokines. Adding exogenous ATP negated Peptide5-mediated protection against inflammatory cytokine release in injury conditions. CONCLUSIONS: Our findings show that connexin43 hemichannels play an important role in the amplification and perpetuation of inflammation by mediating an ATP autocrine feedback loop in the inflammasome/inflammation cycle. GENERAL SIGNIFICANCE: Targeting connexin43 hemichannels offers a potential therapeutic strategy to break the inflammatory cycle in diseases such as DR, but also other chronic inflammatory indications.

Pre-treatment with vinpocetine protects against retinal ischemia.

Vinpocetine has been shown to have beneficial effects for tissues of the central nervous system subjected to ischemia and other related metabolic insults. We recently showed vinpocetine promotes glucose availability, prevents unregulated cation channel permeability and regulates glial reactivity when present during retinal ischemia. Less is known however about the ability of vinpocetine to protect against future ischemic insults. This study explores the effect of vinpocetine when used as a pre-treatment in an ex vivo model for retinal ischemia using cation channel permeability of agmatine (AGB) combined with immunohistochemistry as a measure for cell functionality. We found that vinpocetine pre-treatment reduced cation channel permeability and apoptotic marker immunoreactivity in the GCL and increased parvalbumin immunoreactivity of inner retinal neurons in the inner nuclear layer following ischemic insult. Vinpocetine pre-treatment also reduced Müller cell reactivity following ischemic insults of up to 120 min compared to untreated controls. Many of vinpocetine's effects however were transient in nature suggesting the drug can protect retinal neurons against future ischemic damage but may have limited long-term applications.

Immunohistochemical Characterization of Connexin43 Expression in a Mouse Model of Diabetic Retinopathy and in Human Donor Retinas.

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1ß and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression.

Macromolecular markers in normal human retina and applications to human retinal disease.

Macromolecular cell markers are essential for the classification and characterization of the highly complex and cellularly diverse vertebrate retina. Although a plethora of markers are described in the current literature, the immunoreactivity of these markers in normal human tissue has not been fully determined. This is problematic as they are quintessential to the characterization of morphological changes associated with human retinal disease. This review provides an overview of the macromolecular markers currently available to assess human retinal cell types. We draw on immunohistochemical studies conducted in our laboratories to describe marker immunoreactivity in human retina alongside comparative descriptions in non-human tissues. Considering the growing number of eye banks services offering healthy and diseased human retinal tissue, this review provides a point of reference for future human retina studies and highlights key species specific disease applications of some macromolecular markers.

Vinpocetine modulates metabolic activity and function during retinal ischemia.

Vinpocetine protects against a range of degenerative conditions and insults of the central nervous system via multiple modes of action. Little is known, however, of its effects on metabolism. This may be highly relevant, as vinpocetine is highly protective against ischemia, a process that inhibits normal metabolic function. This study uses the ischemic retina as a model to characterize vinpocetine's effects on metabolism. Vinpocetine reduced the metabolic demand of the retina following ex vivo hypoxia and ischemia to normal levels based on lactate dehydrogenase activity. Vinpocetine delivered similar effects in an in vivo model of retinal ischemia-reperfusion, possibly through increasing glucose availability. Vinpocetine's effects on glucose also appeared to improve glutamate homeostasis in ischemic Müller cells. Other actions of vinpocetine following ischemia-reperfusion, such as reduced cell death and improved retinal function, were possibly a combination of the drug's actions on metabolism and other retinal pathways. Vinpocetine's metabolic effects appeared independent of its other known actions in ischemia, as it recovered retinal function in a separate metabolic model where the glutamate-to-glutamine metabolic pathway was inhibited in Müller cells. The results of this study indicate that vinpocetine mediates ischemic damage partly through altered metabolism and has potential beneficial effects as a treatment for ischemia of neuronal tissues.

Gap junction proteins in the light-damaged albino rat.

PURPOSE: Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. METHODS: Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. RESULTS: Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. CONCLUSIONS: The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.

The origin and diversification of the merozoite surface protein 3 (msp3) multi-gene family in Plasmodium vivax and related parasites.

The genus Plasmodium is a diversified group of parasites with more than 200 known species that includes those causing malaria in humans. These parasites use numerous proteins in a complex process that allows them to invade the red blood cells of their vertebrate hosts. Many of those proteins are part of multi-gene families; one of which is the merozoite surface protein-3 (msp3) family. The msp3 multi-gene family is considered important in the two main human parasites, Plasmodium vivax and Plasmodium falciparum, as its paralogs are simultaneously expressed in the blood stage (merozoite) and are immunogenic. There are large differences among Plasmodium species in the number of paralogs in this family. Such differences have been previously explained, in part, as adaptations that allow the different Plasmodium species to invade their hosts. To investigate this, we characterized the array containing msp3 genes among several Plasmodium species, including P. falciparum and P. vivax. We first found no evidence indicating that the msp3 family of P. falciparum was homologous to that of P. vivax. Subsequently, by focusing on the diverse clade of nonhuman primate parasites to which P. vivax is closely related, where homology was evident, we found no evidence indicating that the interspecies variation in the number of paralogs was an adaptation related to changes in host range or host switches. Overall, we hypothesize that the evolution of the msp3 family in P. vivax is consistent with a model of multi-allelic diversifying selection where the paralogs may have functionally redundant roles in terms of increasing antigenic diversity. Thus, we suggest that the expressed MSP3 proteins could serve as "decoys", via antigenic diversity, during the critical process of invading the host red blood cells.

Sildenafil alters retinal function in mouse carriers of retinitis pigmentosa.

Sildenafil, the active ingredient in Viagra, has been reported to cause transient visual disturbance from inhibition of phosphodiesterase 6 (PDE6), a key enzyme in the visual phototransduction pathway. This study investigated the effects of sildenafil on the rd1(+/-) mouse, a model for carriers of Retinitis Pigmentosa which exhibit normal vision but may have a lower threshold for cellular stress caused by sildenafil due to a heterozygous mutation in PDE6. Sildenafil caused a dose-dependent decrease in electroretinogram (ERG) responses of normal mice which mostly recovered two days post administration. In contrast, rd1(+/-) mice exhibited a significantly reduced photoreceptor and a supernormal bipolar cell response to sildenafil within 1 h of treatment. Carrier mice retinae took two weeks to return to baseline levels suggesting sildenafil has direct effects on both the inner and outer retina and these effects differ significantly between normal and carrier mice. Anatomically, an increase in expression of the early apoptotic marker, cytochrome C in rd1(+/-) mice indicated that the effects of sildenafil on visual function may lead to degeneration. The results of this study are significant considering approximately 1 in 50 people are likely to be carriers of recessive traits leading to retinal degeneration.