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1.  Anti-Stress Effects of Carnosine on Restraint-Evoked Immunocompromise in Mice through Spleen Lymphocyte Number Maintenance 
PLoS ONE  2012;7(4):e33190.
Carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, has been characterized as a putative neurotransmitter and serves as a reservoir for brain histamine, which could act on histaminergic neurons system to relieve stress-induced damages. However, understanding of the role of carnosine in stress-evoked immunocompromise is limited. In this study, results showed that when mice were subjected to restraint stress, spleen index and the number of spleen lymphocytes including Natural Killer (NK) cells were obviously decreased. Results also demonstrated that restraint stress decreased the cytotoxic activity of NK cells per spleen (LU10/spleen) while the activity of a single NK cell (LU10/106 cells) was not changed. However, oral administration of carnosine (150 and 300 mg/kg) increased spleen index and number of spleen lymphocytes (including NK cells), and elevated the cytotoxic activity of NK cells per spleen in restraint-stressed mice. These results indicated that carnosine ameliorated stress-evoked immunocompromise through spleen lymphocyte number maintenance. Carnosine was further found to reduce stress-induced elevation of plasma corticosterone level. On the other hand, results showed that carnosine and RU486 (a glucocorticoids receptor antagonist) treatment prevented the reduction in mitochondrion membrane potential and the release of mitochondrial cytochrome c into cytoplasm, increased Bcl-2/Bax mRNA ratio, as well as decreased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in spleen lymphocytes of stressed mice. The results above suggested that the maintenance of spleen lymphocyte number by carnosine was related with the inhibition of lymphocytes apoptosis caused by glucocorticoids overflow. The stimulation of lymphocyte proliferation by carnosine also contributed to the maintenance of spleen lymphocyte number in stressed mice. In view of the elevated histamine level, the anti-stress effects of carnosine on restraint-evoked immunocompromise might be via carnosine-histamine metabolic pathway. Taken together, carnosine maintained spleen lymphocyte number by inhibiting lymphocyte apoptosis and stimulating lymphocyte proliferation, thus prevented immunocompromise in restraint-stressed mice.
PMCID: PMC3325237  PMID: 22511917
2.  Carnosine Inhibits the Proliferation of Human Gastric Cancer SGC-7901 Cells through Both of the Mitochondrial Respiration and Glycolysis Pathways 
PLoS ONE  2014;9(8):e104632.
Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.
PMCID: PMC4130552  PMID: 25115854
3.  Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine. 
British Journal of Cancer  1996;73(8):966-971.
Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco's modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha-ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate.
PMCID: PMC2075811  PMID: 8611433
4.  Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts 
BMC Cancer  2014;14:358.
Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function.
The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.
Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.
Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.
PMCID: PMC4061103  PMID: 24886661
Carbonic anhydrase IX; Hypoxia; Carnosine; pH regulation
5.  Carnosine: can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential? 
The dipeptide carnosine (β-alanyl-L-histidine) has contrasting but beneficial effects on cellular activity. It delays cellular senescence and rejuvenates cultured senescent mammalian cells. However, it also inhibits the growth of cultured tumour cells. Based on studies in several organisms, we speculate that carnosine exerts these apparently opposing actions by affecting energy metabolism and/or protein homeostasis (proteostasis). Specific effects on energy metabolism include the dipeptide’s influence on cellular ATP concentrations. Carnosine’s ability to reduce the formation of altered proteins (typically adducts of methylglyoxal) and enhance proteolysis of aberrant polypeptides is indicative of its influence on proteostasis. Furthermore these dual actions might provide a rationale for the use of carnosine in the treatment or prevention of diverse age-related conditions where energy metabolism or proteostasis are compromised. These include cancer, Alzheimer's disease, Parkinson's disease and the complications of type-2 diabetes (nephropathy, cataracts, stroke and pain), which might all benefit from knowledge of carnosine’s mode of action on human cells.
PMCID: PMC3602167  PMID: 23442334
Carnosine; Energy metabolism; Reactive oxygen species (ROS); Methylglyoxal; Proteolysis; Alzheimer’s disease; Parkinson’s disease; Diabetes; Cancer; Yeast
6.  Safety and Efficacy Evaluation of Carnosine, An Endogenous Neuroprotective Agent for Ischemic Stroke 
Background and Purpose
An urgent need exists to develop therapies for stroke which have high efficacy, long therapeutic time windows and acceptable toxicity. We undertook preclinical investigations of a novel therapeutic approach involving supplementation with carnosine, an endogenous pleiotropic dipeptide.
Efficacy and safety of carnosine treatment was evaluated in rat models of permanent or transient middle cerebral artery occlusion. Mechanistic studies used primary neuronal/astrocytic cultures and ex vivo brain homogenates.
Intravenous treatment with carnosine exhibited robust cerebroprotection in a dose-dependent manner, with long clinically-relevant therapeutic time windows of 6 h and 9 h in transient and permanent models, respectively. Histological outcomes and functional improvements including motor and sensory deficits were sustained at 14 d post-stroke onset. In safety and tolerability assessments, carnosine did not exhibit any evidence of adverse effects or toxicity. Moreover, histological evaluation of organs, complete blood count, coagulation tests and the serum chemistry did not reveal any abnormalities. In primary neuronal cell cultures and ex vivo brain homogenates, carnosine exhibited robust anti-excitotoxic, antioxidant, and mitochondria protecting activity.
In both permanent and transient ischemic models, carnosine treatment exhibited significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. Carnosine was well tolerated and exhibited no toxicity. Mechanistic data show that it influences multiple deleterious processes. Taken together, our data suggest that this endogenous pleiotropic dipeptide is a strong candidate for further development as a stroke treatment.
PMCID: PMC3678096  PMID: 23250994
carnosine; neuroprotection; ischemic stroke; efficacy; safety
7.  Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model 
Molecular Cancer  2010;9:2.
It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy.
A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 μl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo.
As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.
PMCID: PMC2818694  PMID: 20053283
8.  Impairment of jejunal absorption rate of carnosine by glycylglycine in man in vivo. 
Gut  1976;17(4):252-257.
Using a double-lumen tube jejunal perfusion system in vivo, the mutual effects of carnosine (beta-alanyl-L-histidine) and glycylglycine on their respective absorption rates have been studied in six Zambian African adults. Data on the effect of the constituent amino-acids of carnosine on glycylglycine absorption rate have similarly been obtained. The solutions infused in each subject contained (A) carnosine (50 mmol l.-1), (B) carnosine (50 mmol l.-1) and glycylglycine (50 mmol l.-1), (C) glycylglycine (50 mmol l.-1), and (D) glycylglycine (50 mmol l.-1), L-histidine (50 mmol l.-1) and beta-alanine (50 mmol l.-1). Glycylglycine produced a significant impairment in the mean rate of histidine absorption from carnosine (P less than 0-01). However, carnosine did not have a significant effect on the mean rate of glycine absorption from glycylglycine. Mean rate of histidine absorption from solution D was significantly higher than that from solution A (P less than 0-01). Mean rate of glycine absorption from glycylglycine was not significantly different during infusion of solutions B, C, and D. The results are consistent with the concept that carnosine on glycylglycine is probably because the affinity of mechanism; the lack of influence of carnosine on glycylglycine is probably because the affinity of carnosine for the dipeptide uptake mechanism is relatively low. A gross difference has been shown between mean absorption rate of histidine from free L-histidine (solution D) (25-8 mmol h-1) and when it is given in the form of carnosine in the presence of another dipeptide (solution B) (8-7 mmol h-1); that emphasizes the complexity of amino acid and peptide interaction during absorption, which must be important in nutrition.
PMCID: PMC1411104  PMID: 773786
9.  Ergogenic Effects of β-Alanine and Carnosine: Proposed Future Research to Quantify Their Efficacy 
Nutrients  2012;4(7):585-601.
β-alanine is an amino acid that, when combined with histidine, forms the dipeptide carnosine within skeletal muscle. Carnosine and β-alanine each have multiple purposes within the human body; this review focuses on their roles as ergogenic aids to exercise performance and suggests how to best quantify the former’s merits as a buffer. Carnosine normally makes a small contribution to a cell’s total buffer capacity; yet β-alanine supplementation raises intracellular carnosine concentrations that in turn improve a muscle’s ability to buffer protons. Numerous studies assessed the impact of oral β-alanine intake on muscle carnosine levels and exercise performance. β-alanine may best act as an ergogenic aid when metabolic acidosis is the primary factor for compromised exercise performance. Blood lactate kinetics, whereby the concentration of the metabolite is measured as it enters and leaves the vasculature over time, affords the best opportunity to assess the merits of β-alanine supplementation’s ergogenic effect. Optimal β-alanine dosages have not been determined for persons of different ages, genders and nutritional/health conditions. Doses as high as 6.4 g day−1, for ten weeks have been administered to healthy subjects. Paraesthesia is to date the only side effect from oral β-alanine ingestion. The severity and duration of paraesthesia episodes are dose-dependent. It may be unwise for persons with a history of paraesthesia to ingest β-alanine. As for any supplement, caution should be exercised with β-alanine supplementation.
PMCID: PMC3407982  PMID: 22852051
carnosine; dietary supplement; amino acid; paraesthesia
10.  Dietary Carnosine Prevents Early Atherosclerotic Lesion Formation in ApoE-null Mice 
Arteriosclerosis, thrombosis, and vascular biology  2013;33(6):10.1161/ATVBAHA.112.300572.
Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions.
Approach and Results
We examined the efficacy of carnosine, a naturally occurring β-alanyl-histidine dipeptide in preventing aldehyde toxicity and atherogenesis in apoE-null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes - HNE and malonaldialdehyde in Cu2+-oxidized LDL. Preloading bone marrow-derived macrophages with cell-permeable carnosine analogs reduced HNE-induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apoE-null mice and attenuated the accumulation of protein-acrolein, protein-HHE and protein-HNE adducts in atherosclerotic lesions, while urinary excretion of aldehydes as carnosine conjugates was increased.
The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.
PMCID: PMC3869200  PMID: 23559625
Atherosclerosis; carnosine; aldehydes; oxidized LDL
11.  Analysis of an H1 receptor-mediated, zinc-potentiated vasoconstrictor action of the histidyl dipeptide carnosine in rabbit saphenous vein 
British Journal of Pharmacology  1998;125(6):1272-1280.
The contractile action of the dipeptide carnosine (β-alanyl-L-histidine), active as a Zn·carnosine complex (Zn·Carn), was investigated in isolated rings of rabbit saphenous vein (RSV) and was found to be antagonized by the H1 antagonist mepyramine.Mepyramine-sensitive, histamine-induced contractures in RSV, were smaller (73±0.1%) and less well sustained than carnosine-induced contractures.Schild plot values for mepyramine antagonism were, for carnosine-induced contractures; pA2=7.97±0.12, slope=1.33±0.06 (r=0.793) and for histamine-induced contractures; pA2=8.48±0.07, slope=0.63±0.05, r=0.957).Serotonergic antagonists methiothepin and ketanserin, antagonize both carnosine- and histamine-induced contractures in RSV, probably reflecting coincidental inhibition at the H1-receptor.Carnosine, with Zn present, can inhibit the H1-specific binding of [3H]-mepyramine to isolated guinea-pig cerebellar membranes (log IC50s−2.78±0.02, −3.93±0.03 and −4.64±0.03 at 10, 30 and 80 μM Zn respectively; values corrected for the Zn-specific inhibition which has a logIC50 of −4.20).In the radioligand binding assay, the effect of carnosine can be described as a function of Zn·Carn concentration with an apparent logIC50 of −5.61. This value is consistent with that obtained from the functional studies on RSV.Histamine-induced contractures have an indomethacine-sensitive component (27.2±8.3% of control response), not apparent with carnosine-induced contractures.Like histamine, carnosine evoked an H2-mediated (cimetidine-sensitive) relaxation in the presence of mepyramine, but was less potent (10.8±3.1% residual tension at 10 mM carnosine compared with 13.4±7.5% at 0.1 mM histamine).Carnosine, like mepyramine, can 'reveal' the H2-mediated relaxation of histamine providing further evidence that carnosine binds at the H1 receptor.We conclude that carnosine can act at the smooth muscle H1-receptor to provoke vasoconstriction and that it also has the potential to act at H1-receptors in CNS.
PMCID: PMC1565695  PMID: 9863657
Carnosine; histamine; H1 receptor; H2 receptor; mepyramine; cimetidine; rabbit saphenous vein; guinea-pig cerebellum; vasoconstrictor
12.  Central administration of dipeptides, beta-alanyl-BCAAs, induces hyperactivity in chicks 
BMC Neuroscience  2007;8:37.
Carnosine (β-alanyl-L-histidine) is a putative neurotransmitter and has a possible role in neuron-glia cell interactions. Previously, we reported that carnosine induced hyperactivity in chicks when intracerebroventricularly (i.c.v.) administered. In the present study, we focused on other β-alanyl dipeptides to determine if they have novel functions.
In Experiment 1, i.c.v. injection of β-alanyl-L-leucine, but not β-alanyl-glycine, induced hyperactivity behavior as observed with carnosine. Both carnosine and β-alanyl-L-leucine stimulated corticosterone release. Thus, dipeptides of β-alanyl-branched chain amino acids were compared in Experiment 2. The i.c.v. injection of β-alanyl-L-isoleucine caused a similar response as β-alanyl-L-leucine, but β-alanyl-L-valine was somewhat less effective than the other two dipeptides. β-Alanyl-L-leucine strongly stimulated, and the other two dipeptides tended to stimulate, corticosterone release.
These results suggest that central β-alanyl-branched chain amino acid stimulates activity in chicks through the hypothalamus-pituitary-adrenal axis. We named β-alanyl-L-leucine, β-alanyl-L-isoleucine and β-alanyl-L-valine as Excitin-1, Excitin-2 and Excitin-3, respectively.
PMCID: PMC1892563  PMID: 17537271
13.  Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme 
PLoS ONE  2013;8(12):e81982.
Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.
PMCID: PMC3859581  PMID: 24349167
14.  Distribution of carnosine-like peptides in the nervous system of developing and adult zebrafish (Danio rerio) and embryonic effects of chronic carnosine exposure 
Cell and tissue research  2009;337(1):45-61.
Carnosine-like peptides (carnosine-LP) are a family of histidine derivatives that are present in the nervous system of various species and that exhibit antioxidant, anti-matrix-metalloproteinase, anti-excitotoxic, and free-radical scavenging properties. They are also neuroprotective in animal models of cerebral ischemia. Although the function of carnosine-LP is largely unknown, the hypothesis has been advanced that they play a role in the developing nervous system. Since the zebrafish is an excellent vertebrate model for studying development and disease, we have examined the distribution pattern of carnosine-LP in the adult and developing zebrafish. In the adult, immunoreactivity for carnosine-LP is specifically concentrated in sensory neurons and non-sensory cells of the olfactory epithelium, the olfactory nerve, and the olfactory bulb. Robust staining has also been observed in the retinal outer nuclear layer and the corneal epithelium. Developmental studies have revealed immunostaining for carnosine-LP as early as 18 h, 24 h, and 7 days post-fertilization in, respectively, the olfactory, corneal, and retinal primordia. These data suggest that carnosine-LP are involved in olfactory and visual function. We have also investigated the effects of chronic (7 days) exposure to carnosine on embryonic development and show that 0.01 μM to 10 mM concentrations of carnosine do not elicit significant deleterious effects. Conversely, treatment with 100 mM carnosine results in developmental delay and compromised larval survival. These results indicate that, at lower concentrations, exogenously administered carnosine can be used to explore the role of carnosine in development and developmental disorders of the nervous system.
PMCID: PMC3725833  PMID: 19440736
Carnosine-like peptides; Nervous system; Development; Embryo toxicity; Zebrafish; Danio rerio (Teleostei)
15.  Differential Neuroprotective Effects of Carnosine, Anserine, and N-Acetyl Carnosine against Permanent Focal Ischemia 
Journal of neuroscience research  2008;86(13):2984-2991.
Carnosine (β-alanyl-L-histidine) has been shown to exhibit neuroprotection in rodent models of cerebral ischemia. In the present study, we further characterized the effects of carnosine treatment in a mouse model of permanent focal cerebral ischemia and compared them with its related peptides anserine and N-acetylated carnosine. We also evaluated the efficacy of bestatin, a carnosinase inhibitor, in ameliorating ischemic brain damage. Permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery (pMCAO). Mice were subsequently randomly assigned to receive an intraperitoneal injection of vehicle (0.9% saline), carnosine, N-acetyl carnosine, anserine, bestatin alone, or bestatin with carnosine. Infarct size was examined using 2,3,5-triphenyltetrazolium chloride staining 1, 3, and 7 days following pMCAO, and neurological function was evaluated using an 18-point-based scale. Brain levels of carnosine were measured in treated mice using high-performance liquid chromatography 1 day following pMCAO. We demonstrated that treatment with carnosine, but not its analogues, was able to significantly reduce infarct volume and improve neurological function compared with those in vehicle-treated mice. These beneficial effects were maintained for 7 days post-pMCAO. In contrast, compared with the vehicle-treated group, bestatin-treated mice displayed an increase in the severity of ischemic lesion, which was prevented by the addition of carnosine. These new data further characterize the neuroprotective effects of carnosine and suggest that carnosine may be an attractive candidate for testing as a stroke therapy.
PMCID: PMC2805719  PMID: 18543335
carnosine; anserine; N-acetyl carnosine; bestatin; permanent cerebral focal ischemia
16.  Control of ATP homeostasis during the respiro-fermentative transition in yeast 
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition.
Yeast cells are exposed to strongly fluctuating nutrient concentrations in their natural environment, which requires rapid and efficient adaptation through rearrangements on all levels of their metabolism. The quantitative understanding of these adaptation processes represents the basis for a directed optimization of the microorganism to suit the needs of biochemical production processes that often impose non-uniform or harsh cultivation conditions (Lara et al, 2006), or require a redirection of metabolic fluxes to improve productivity (Bailey, 1991). In this context, controlled perturbation experiments represent a valuable tool, as they allow studying the transition from one defined physiological state to another under well-characterized conditions. Measurements of metabolite pool dynamics and enzymatic activities in response to different perturbations enable the quantitative mathematical analysis of glycolytic dynamics, which is ultimately meant to discriminate the impact of allosteric regulation and changes in the enzymatic make-up of the cell on the overall metabolic response (Rizzi et al, 1997; Teusink et al, 2000; Daran-Lapujade et al, 2007; van den Brink et al, 2008).
A long-standing problem in the context of these studies was the apparent loss of adenine nucleotides, which immediately follows the relief from glucose limitation (Theobald et al, 1997; Kresnowati et al, 2006). In this study, we showed that the transient accumulation of the purine salvage pathway (PSP) metabolites, IMP and inosine, account for the loss of AXP nucleotides. The pathway for inosine formation and recycling was identified, and the interplay between different pathways during the respiro-fermentative transition is schematically summarized in Figure 10. The presence of a quickly metabolizable sugar causes accumulation of the phosphorylated metabolites F1,6P, T6P and G3P, which results in a transient imbalance of ATP-consuming and ATP-regenerating reactions, and provokes a drop in both ATP and intracellular phosphate (Pi) concentrations. The joint action of fast ATP consumption and the Adk1 reaction results in the net-production of AMP. The accumulation of AMP, however, is prevented by Amd1 and Isn1, which readily convert AMP via IMP into inosine. Recycling of inosine into IMP is facilitated by Pnp1 and the concomitant action of Hpt1 and Xpt1, with Hpt1 having the predominant role. We suggest that inosine formation serves to prevent unscheduled AMP accumulation, and to store AXP nucleotides in a metabolically ‘neutral' form until re-equilibration of glycolysis eventually allows recovery of ATP levels. Recycling of inosine via IMP represents an energy-saving way to replenish the AXP pool, as de novo synthesis of IMP starting from PRPP requires 4 ATP molecules, whereas IMP production from inosine and PRPP is an ATP-neutral process (Figure 10).
In contrast to the behavior observed in the presence of fermentable carbon sources, neither IMP nor inosine were formed in response to perturbations of the cellular energy balance under respiratory conditions. Instead, the drop in ATP level caused a concomitant increase in AMP concentration. Therefore, we conclude that the conversion of AXP nucleotides into inosine represents a specific short-term metabolic response to the perturbation of cellular energy homeostasis, which is controlled by the presence of a fermentable carbon source (or a sugar derivative that can undergo rapid phosphorylation). The discrimination between AMP accumulation and AMP-to-inosine conversion seems to be controlled by Amd1 activity. At the present stage of investigation, we cannot conclude whether regulation of Amd1 is brought about by allosteric control or by posttranslational modification. However, given that inorganic phosphate is a potent inhibitor of Amd1 (Merkler et al, 1989) and intracellular phosphate concentration transiently drops when cells become exposed to fermentable carbon sources (our data, and e.g. Hohmann et al, 1996), phosphate availability is likely to have a pivotal role in the regulation of AXP pool size and inosine formation.
The impact of defective AXP cycling on the global metabolic response to glucose addition was tested under conditions in which respiration was inhibited by antimycin A. The amd1 mutant, in which adenine nucleotide cycling was completely blocked, showed the strongest deviations from the wild-type behavior. In this mutant, adenylic energy charge exhibited a strong drop after glucose addition, and recovered much slower than in wild-type cells. Furthermore, deletion of AMD1 resulted in strong accumulation of AMP and pronounced changes in the dynamics of trehalose-6-phosphate, glycerol-3-phosphate, and PRPP after glucose addition (Figure 6).
Despite pronounced changes in metabolite pool dynamics caused by the deletion of AMD1, no alterations in the production of the fermentative end products, ethanol and glycerol, nor in the consumption of glucose were observed (Figure 9B). However, the amd1 and isn1 mutant strains showed delayed growth acceleration after glucose addition (Figure 9C). The observation of unaltered fermentation capacity but concomitant delay in growth acceleration in the amd and isn1 mutants points to the possibility that regulation of glycolysis is not the major target of AMP. Indeed, our study revealed a delayed increase of PRPP concentration in the amd1 strain after glucose addition. The PRPP protein has a central role as a precursor for purine nucleotide de novo synthesis and the synthesis of amino acids (Vavassori et al, 2005). Hence, limitation of this important precursor may negatively affect growth. In addition, AMP accumulation may have an important signaling function acting, for example, through the cAMP/PKA pathway (Thevelein et al, 2005; Zaman et al, 2009) and/or via Snf1 (Celenza and Carlson, 1984). However, experimental evidence to support this potential link is missing. Thus, the assumption of a signaling role of AMP remains speculative, although intriguing, when asking for the actual function of AXP-to-inosine conversion during the respiro-fermentative transition.
On the basis of this study, we put forward the implication of adenine nucleotide cycling through the PSP in energy homeostasis in yeast. Answering the question whether this pathway is also active in humans could potentially contribute to a better understanding of metabolic processes that control the metabolic transition from respiratory to respiro-fermentative energy supply in muscle upon heavy exercise. In particular, it may help to understand conflicting results on the impaired physical performance of individuals carrying an AMP deaminase dysfunction under these conditions (Tarnopolsky et al, 2001; Fischer et al, 2007).
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5′-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions.
PMCID: PMC2824524  PMID: 20087341
ATP homeostasis; metabolic regulation; purine nucleotide metabolism; respiro-fermentative transition; yeast
17.  Carnosine Inhibits the Proliferation of Human Gastric Carcinoma Cells by Retarding Akt/mTOR/p70S6K Signaling 
Journal of Cancer  2014;5(5):382-389.
Carnosine (β-alanyl-L-histidine), described as an enigmatic peptide for its antioxidant, anti-aging and especially antiproliferation properties, has been demonstrated to play an anti-tumorigenic role in certain types of cancer. However, its function in human gastric carcinoma remains unclear. In this study, the effect of carnosine on cell proliferation and its underlying mechanisms were investigated in the cultured human gastric carcinoma cells. The mTOR signaling axis molecules were analyzed in carnosine treated cells. The results showed that treatment with carnosine led to proliferation inhibition, cell cycle arrest in the G0/G1 phase, apoptosis increase, and inhibition of mTOR signaling activation by decreasing the phosphorylation of Akt, mTOR and p70S6K, suggesting that proliferation inhibition of carnosine in human gastric carcinoma was through the inhibition of Akt/mTOR/p70S6K pathway, and carnosine would be a mimic of rapamycin.
PMCID: PMC4007526  PMID: 24799956
Carnosine; rapamycin; proliferation; gastric carcinoma cell; mTOR signalling pathway
18.  The Anti-Proliferative Effect of L-Carnosine Correlates with a Decreased Expression of Hypoxia Inducible Factor 1 alpha in Human Colon Cancer Cells 
PLoS ONE  2014;9(5):e96755.
In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS) production. In the present study we identified the Hypoxia-Inducible Factor 1α (HIF-1α) as a possible target of L-carnosine in HCT-116 cell line. HIF-1α protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1α induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1α. In the current study we found that L-carnosine reduces the HIF-1α protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1α degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively). Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1α expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1α activity has not been yet elucidated, this ability may be promising in treating hypoxia-related diseases.
PMCID: PMC4013086  PMID: 24804733
19.  Pharmacological influence on processes of adjuvant arthritis: Effect of the combination of an antioxidant active substance with methotrexate 
Interdisciplinary Toxicology  2012;5(2):84-91.
Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis. A certain correlation was observed between oxidative stress, arthritis and the immune system. Reactive oxygen species produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative burst, exceed the physiological buffering capacity and result in oxidative stress. The excessive production of ROS can damage protein, lipids, nucleic acids, and matrix components. Patients with rheumatoid arthritis have an altered antioxidant defense capacity barrier. In the present study the effect of substances with antioxidative properties, i.e. pinosylvin and carnosine, was determined in monotherapy for the treatment of adjuvant arthritis (AA). Moreover carnosine was evaluated in combination therapy with methotrexate. Rats with AA were administered first pinosylvin (30 mg/kg body mass daily per os), second carnosine (150 mg/kg body mass daily per os) in monotherapy for a period of 28 days. Further, rats with AA were administered methotrexate (0.3 mg/kg body mass 2-times weekly per os), and a combination of methotrexate+carnosine, with the carnosine dose being the same as in the previous experiment. Parameters, i.e. changes in hind paw volume and arthritic score were determined in rats as indicators of destructive arthritis-associated clinical changes. Plasmatic levels of TBARS and lag time of Fe2+-induced lipid peroxidation (tau-FeLP) in plasma and brain were specified as markers of oxidation. Plasmatic level of CRP and activity of γ-glutamyltransferase (GGT) in spleen and joint were used as inflammation markers. In comparison to pinosylvin, administration of carnosine monotherapy led to a significant decrease in the majority of the parameters studied. In the combination treatment with methotrexate+carnosine most parameters monitored were improved more remarkably than by methotrexate alone. Carnosine can increase the disease-modifying effect of methotrexate treatment in rat AA.
PMCID: PMC3485659  PMID: 23118593
arthritis; oxidative stress; pinosylvin; carnosine; methotrexate; combination therapy
20.  Mechanical Stress Promotes Cisplatin-Induced Hepatocellular Carcinoma Cell Death 
BioMed Research International  2015;2015:430569.
Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death).
PMCID: PMC4317602  PMID: 25685789
21.  Switching the mode of sucrose utilization by Saccharomyces cerevisiae 
Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.
We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source.
Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.
PMCID: PMC2268662  PMID: 18304329
22.  Detoxification of aldehydes by histidine-containing dipeptides: from chemistry to clinical implications 
Chemico-biological interactions  2013;202(0):288-297.
Aldehydes are generated by oxidized lipids and carbohydrates at increased levels under conditions of metabolic imbalance and oxidative stress during atherosclerosis, myocardial and cerebral ischemia, diabetes, neurodegenerative diseases and trauma. In most tissues, aldehydes are detoxified by oxidoreductases that catalyze the oxidation or the reduction of aldehydes or enzymatic and nonenzymatic conjugation with low molecular weight thiols and amines, such as glutathione and histidine dipeptides. Histidine dipeptides are present in micromolar to millimolar range in the tissues of vertebrates, where they are involved in a variety of physiological functions such as pH buffering, metal chelation, oxidant and aldehyde scavenging. Histidine dipeptides such as carnosine form Michael adducts with lipid-derived unsaturated aldehydes, and react with carbohydrate-derived oxo- and hydroxy- aldehydes forming products of unknown structure. Although these peptides react with electrophilic molecules at lower rate than glutathione, they can protect glutathione from modification by oxidant and they may be important for aldehyde quenching in glutathione-depleted cells or extracellular space where glutathione is scarce. Consistent with in vitro findings, treatment with carnosine has been shown to diminish ischemic injury, improve glucose control, ameliorate the development of complications in animal models of diabetes and obesity, promote wound healing and decrease atherosclerosis. The protective effects of carnosine have been linked to its anti-oxidant properties, it ability to promote glycolysis, detoxify reactive aldehydes and enhance histamine levels. Thus, treatment with carnosine and related histidine dipeptides may be a promising strategy for the prevention and treatment of diseases associated with high carbonyl load.
PMCID: PMC4418549  PMID: 23313711
Carnosine; aldehyde; histidine dipeptides; oxidative stress
23.  Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A 
BMC Systems Biology  2008;2:60.
The yeast Saccharomyces cerevisiae is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce.
Metabolic flux distributions of S. cerevisiae CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O2 in the inlet gas were quantified by 13C-MFA. Metabolic flux ratios from fractional [U-13C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of S. cerevisiae.
While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO2. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.
13C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.
PMCID: PMC2507709  PMID: 18613954
24.  Possible ameliorative effects of antioxidants on propionic acid / clindamycin - induced neurotoxicity in Syrian hamsters 
Gut Pathogens  2013;5:32.
Propionic acid (PA) found in some foods and formed as a metabolic product of gut bacteria has been reported to mimic/mediate the effects of autism. The present study was undertaken to compare the effect of orally administered PA with that of clindamycin-induced PA-microbial producers in inducing persistent biochemical autistic features in hamsters. The neuroprotective potency of carnosine and carnitine supplements against PA toxicity was also investigated.
The following groups were studied. 1. Control group, which received phosphate buffered saline orally, 2. Propionic acid treated group which were given PA at a dose of 250 mg/kg body weight/day for 3 days orally, 3. Clindamycin treated group which received a single dose of the antibiotic orogastrically at a dose of 30 mg/kg on the day of the experiment, 4. Carnosine-treated group which were given carnosine at a dose of 10 mg/kg body weight/day orally for one week, 5. Carnitine treated group given 50 mg/kg body weight/day carnitine orally daily for one week. Group 6. Carnosine followed by PA, Group 7. Carnitine followed by PA. Dopamine, adrenaline and noradrenaline, serotonin and Gamma amino-butyric acid (GABA) were measured in the cortex and medulla of the nine studied groups.
PA administration caused significant decrease in the neurotransmitters in the brains of treated hamsters while clindamycin caused a significant decrease only in dopamine in hamster brains (cortex and medulla) and GABA in the cerebral cortex of the treated hamsters. Administration of carnosine and carnitine which are known antioxidants caused no significant changes in the levels of neurotransmitters when administered alone to hamsters. However when administered with PA both carnosine and carnitine restored the altered neurotransmitters to near normal levels.
Carnosine and carnitine may be used as supplements to protect against PA neurotoxicity.
PMCID: PMC3828401  PMID: 24188374
Autism; Clindamycin; Propionic acid; Carnosine; Carnitine; Cortex; Medulla
25.  Intravitreal injection of forskolin, homotaurine, and L-carnosine affords neuroprotection to retinal ganglion cells following retinal ischemic injury 
Molecular Vision  2015;21:718-729.
Retinal ganglion cell (RGC) death is the final event leading to visual impairment in glaucoma; therefore, identification of neuroprotective strategies able to slow down or prevent the process is one of the main challenges for glaucoma research. The purpose of this study was to evaluate the neuroprotective potential of RGC death induced by the in vivo transient increase in intraocular pressure (IOP) of a combined treatment with forskolin, homotaurine, and L-carnosine. Forskolin (7beta-acetoxy-8, 13-epoxy-1a, 6β, 9a-trihydroxy-labd-14-en-11-one) is an activator of adenylate cyclase that decreases IOP by reducing aqueous humor production and functions as a neuroprotector due to its neurotrophin-stimulating activity. Homotaurine is a natural aminosulfonate compound endowed with neuromodulatory effects, while the dipeptide L-carnosine is known for its antioxidant properties.
Retinal ischemia was induced in the right eye of adult male Wistar rats by acutely increasing the IOP. Forskolin, homotaurine, and L-carnosine were intravitreally injected and RGC survival evaluated following retrograde labeling with FluoroGold. Total and phosphorylated Akt and glycogen synthase kinase-3β (GSK-3β) protein levels, as well as calpain activity, were analyzed with western blot. Protein kinase A (PKA) was inhibited by intravitreal injection of H89.
A synergic neuroprotective effect on RGC survival was observed following the combined treatment with forskolin, homotaurine, and L-carnosine compared to forskolin alone. The observed neuroprotection was associated with reduced calpain activity, upregulation of phosphoinositide 3-kinase (PI3K)/Akt pathway, and inhibition of GSK-3β but was independent from PKA activation and distinct from the hypotensive effects of forskolin.
A multidrug/multitarget approach, by interfering with several pathways involved in RGC degeneration, may be promising to achieve glaucoma neuroprotection.
PMCID: PMC4483367  PMID: 26167113

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