Prospects for designing medicines based on carnosine (several new applications)]

The perspectives in application of carnosine, its analogs (histidine-containing dipeptides), and their derivatives as components of medicinal drugs are reviewed. These applications are based on antioxidative properties of carnosine and its analogs, their chelating activity towards transient valency metals as well as on their specific neurotransmitter functions in the brain. Combination of carnosine with other antioxidants and the use of copper or zinc complexes with histidine-containing dipeptides are considered as perspective trends in the design of new drugs.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

The effect of some drugs on the mitral cell odor-evoked responses in the gecko olfactory bulb

The activity of odor-evoked olfactory mitral cell response of the gecko was recorded extracellularly by glass microelectrodes. The activities of the mitral cell observed during the presentation of the odor (n-amyl acetate) could be described as excitation, suppression or zero. The present experiments were undertaken to study the neural activities of the mitral cell in the olfactory bulb by perfusion application of some drugs (cobalt chloride, carnosine, norepinephrine, GABA and D-L-homocysteate) on the olfactory bulb surface or iontophoretic application of some drugs (carnosine, norepinephrine, GABA and D-L-homocysteate) to the glomerulus and the external plexiform layer to change the physiological environment. The effect of the drugs suggested that the synaptic neurons on the mitral cell have different chemical characteristics.     

Carnosine stimulates vimentin expression in cultured rat fibroblasts.

Two-dimensional electrophoretic gel profiles were compared between rat 3Y1 fibroblasts cultured in the presence and absence of 30 mM L-carnosine (beta-alanyl-L-histidine) for one week without any replenishment of medium. While a number of cellular proteins changed their expression levels by the addition of carnosine, we identified one of the most prominently varied proteins as vimentin. Immunoblot analysis with anti-vimentin antibody demonstrated that the vimentin levels increased about 2-fold after one-week culture in the presence of carnosine. We also confirmed that the increase of vimentin expression was dependent on the concentration of carnosine added to the medium. Moreover, when cultured cells were stained with anti-vimentin antibody and observed by light microscopy, most cells grown in the presence of carnosine were found to have markedly developed vimentin filaments. The increase of vimentin expression was also observed by adding with carnosine related dipeptides, N-acetylcarnosine and anserine.     

A re-evaluation of the antioxidant activity of purified carnosine

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.     

Carnosine as a potential scavenger of oxidants generated by stimulated neutrophils

Luminol-dependent chemiluminescence of PMA-stimulated human neutrophils decrease more than by 50% in the presence of physiological concentrations of carnosine (20 mM). This inhibition is the result of carnosine ability to scavenge hypochlorite (OCl-), since carnosine exerts a similar effect on chemiluminescence produced by myeloperoxidase-H2O2-Cl- and OCl(-)-H2O2 systems. The previously undocumented property of this dipeptide to scavenge active oxygen species requires further experiments.     

Protective effects of carnosine on dehydroascorbate-induced structural alteration and opacity of lens crystallins: important implications of carnosine pleiotropic functions to combat cataractogenesis

The high level of dehydroascorbic acid (DHA) in the lenticular tissue is an important risk factor for the development of age-related cataracts. In this study, the effects of DHA on structure and function of lens crystallins were studied in the presence of carnosine using gel mobility shift assay, different spectroscopic techniques, and lens culture analysis. The DHA-induced unfolding and aggregation of lens proteins were largely prevented by this endogenous dipeptide. The ability of carnosine to preserve native protein structure upon exposure to DHA suggests the essential role of this dipeptide in prevention of the senile cataract development. Although the DHA-modified α-crystallin was characterized by altered chaperone activity, functionality of this protein was significantly restored in the presence of carnosine. The increased proteolytic instability of DHA-modified lens proteins was also attenuated in the presence of carnosine. Furthermore, the assessment of lens culture suggested that DHA can induce significant lens opacity which can be prevented by carnosine. These observations can be explained by the pleiotropic functions of this endogenous and pharmaceutical compound, notably by its anti-glycation and anti-aggregation properties. In summary, our study suggests that carnosine may have therapeutic potential in preventing senile cataracts linked with the increased lenticular DHA generation, particularly under pathological conditions associated with the oxidative stress.     

Carnosine Release from Olfactory Bulb Synaptosomes Is Calcium-Dependent and Depolarization-Stimulated

Abstract: The dipeptide carnosine (β-alanyl-L-histidine) has been proposed as a neurotransmitter in the mammalian olfactory pathway. Therefore, the efflux of in vivo -synthesized [¹⁴C]carnosine from mouse olfactory bulb synaptosomes was investigated. Carnosine was found to be released from the olfactory bulb synaptosomes by two mechanisms. The first is a slow spontaneous process that is independent of depolarization. The rate of this release was doubled in the presence of 1 m M external carnosine. Release by the second mechanism was markedly stimulated in the presence of calcium by depolarization with either 60 m M K⁺ or 300 μ M veratridine. Omission of calcium abolished the stimulatory effect of both of these agents. Further, blockage of the veratridine-induced depolarization by tetrodotoxin also inhibited carnosine release. These results are consistent with the hypothesis that carnosine acts as a neurotransmitter in the mouse olfactory pathway.     

The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum]

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.     

Retardation of the Senescence of Cultured Human Diploid Fibroblasts by Carnosine

We have examined the effects of the naturally occurring dipeptide carnosine (β-alanyl-L-histidine) on the growth, morphology, and lifespan of cultured human diploid fibroblasts. With human foreskin cells, HFF-1, and fetal lung cells, MRC-5, we have shown that carnosine at high concentrations (20-50 mM) in standard medium retards senescence and rejuvenates senescent cultures. These late-passage cultures preserve a nonsenescent morphology in the presence of carnosine, in comparison to the senescent morphology first described by Hayflick and Moorhead. Transfer of these late-passage cells in medium containing carnosine to unsupplemented normal medium results in the appearance of the senescent phenotype. The serial subculture of cells in the presence of carnosine does not prevent the Hayflick limit to growth, although the lifespan in population doublings as well as chronological age is often increased. This effect is obscured by the normal variability of human fibroblast lifespans, which we have confirmed. Transfer of cells approaching senescence in normal medium to medium supplemented with carnosine rejuvenates the cells but the extension in lifespan is variable. Neither D -carnosine, (β-alanyl-D-histidine), homocarnosine, anserine, nor β-alanine had the same effects as carnosine on human fibroblasts. Carnosine is an antioxidant, but it is more likely that it preserves cellular integrity by its effects on protein metabolism.     

Vascular smooth muscle actions of carnosine as its zinc complex are mediated by histamine H(1) and H(2) receptors

The endogenous dipeptide carnosine (beta-alanyl-L-histidine), at 0.1-10 mM, can provoke sustained contractures n rabbit saphenous vein rings with greater efficacy than noradrenaline. The effects are specific; anserine and homocarnosine are ineffective, as are carnosine's constituent amino acids histidine and beta-alanine. Zinc ions enhance the maximum carnosine-induced tension (to 127 +/- 13% of control at 10 microM Zn(total)) and muscle sensitivity is potentiated (mean K(0.5) reduced from 1.23 mM to 17 microM carnosine with 15 microM Zn(total)). The dipeptide acts as a Zn-carnosine complex (Zn. Carn). The effects of carnosine at 1 microM-10 mM (total) in the presence of 1-100 microM Zn(2+) (total) can be described as a unique function of [Zn.Carn] with an apparent K(0.5) for the complex of [7.4)(10(-8)] M. Contractures are reduced at low [Ca(2+)], unaffected by adrenoceptor antagonists, but can be blocked by antagonists to several receptor types. The most specific effect is by mepyramine, the H(1) receptor antagonist. With Zn present, carnosine can inhibit the H(1)-specific binding of [(3)H]mepyramine to isolated Guinea pig cerebella membranes. This effect of carnosine can be described as a function of the concentration of Zn.Carn with an apparent IC(50) of 2.45 microM. Like histamine, carnosine evoked an H2-mediated (cimetidine-sensitive) relaxation in the presence of mepyramine, but was less potent (10.8 +/- 3.1% of initial tension remaining at 10 mM carnosine compared with 13.4 +/- 7.5% remaining at 0.1 mM histamine). Preliminary studies with a Zn-selective fluorescent probe indicate that functionally significant levels of Zn can be released from adventitial mast cells that could modulate actions of carnosine in the extravascular space as well as those of histamine itself. We conclude that carnosine can act at the smooth muscle H(1)-receptor to provoke vasoconstriction and that it also has the potential to act at H(1)-receptors in the central nervous system. Carnosine's mode of action is virtually unique: a vascular muscle receptor apparently transduces the action of a dipeptide in the form of a metal chelate. The functional relationship of carnosine with histamine and the possible physiological relevance of Zn ions for the activity of both agents have not previously been reported.     

Low plasma carnosinase activity promotes carnosinemia after carnosine ingestion in humans

A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon carnosine ingestion, 8 of the 25 subjects (responders) displayed a measurable increase in plasma carnosine up to 1 h after supplementation. Subjects with no measurable increment in plasma carnosine (nonresponders) had ～2-fold higher plasma carnosinase protein content and ～1.5-fold higher activity compared with responders. Urinary carnosine recovery was 2.6-fold higher in responders versus nonresponders and was negatively dependent on both the activity and protein content of the plasma carnosinase enzyme. In conclusion, low plasma carnosinase activity promotes the presence of circulating carnosine upon an oral challenge. These data may further clarify the link among CNDP1 genotype, carnosinase, and diabetic nephropathy.     

Carnosine exhibits significant antiviral activity against Dengue and Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito‐borne diseases whereas ZIKV infection occasionally re‐emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β‐alanyl‐l ‐histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell‐based assays were performed to validate the computational results. Mode‐of‐inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC₅₀ values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode‐of‐inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Carnosine reacts with a glycated protein

Oxidation and glycation induce formation of carbonyl (CO) groups in proteins, a characteristic of cellular aging. The dipeptide carnosine (beta-alanyl-L-histidine) is often found in long-lived mammalian tissues at relatively high concentrations (up to 20 mM). Previous studies show that carnosine reacts with low-molecular-weight aldehydes and ketones. We examine here the ability of carnosine to react with ovalbumin CO groups generated by treatment of the protein with methylglyoxal (MG). Incubation of MG-treated protein with carnosine accelerated a slow decline in CO groups as measured by dinitrophenylhydrazine reactivity. Incubation of [(14)C]-carnosine with MG-treated ovalbumin resulted in a radiolabeled precipitate on addition of trichloroacetic acid (TCA); this was not observed with control, untreated protein. The presence of lysine or N-(alpha)-acetylglycyl-lysine methyl ester caused a decrease in the TCA-precipitable radiolabel. Carnosine also inhibited cross-linking of the MG-treated ovalbumin to lysine and normal, untreated alpha-crystallin. We conclude that carnosine can react with protein CO groups (termed "carnosinylation") and thereby modulate their deleterious interaction with other polypeptides. It is proposed that, should similar reactions occur intracellularly, then carnosine's known "anti-aging" actions might, at least partially, be explained by the dipeptide facilitating the inactivation/removal of deleterious proteins bearing carbonyl groups.     

Carnosine Protects Against Aβ42-induced Neurotoxicity in Differentiated Rat PC12 Cells

(1) The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Aβ42-induced impairment in differentiated PC12 cells. (2) PC12 cells were exposed to Aβ42 (5 μM) for 24 h after carnosine (5 mM) applied for 18 h. Histamine receptor antagonists (diphenhydramine, zolantidine, thioperamide, clobenpropit) or histidine decarboxylase inhibitor (α-fluoromethylhistidine) were added 15 min before carnosine. Cell viability, glutamate release or cell surface expression of NMDA receptor was examined. (3) Aβ42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with carnosine ameliorated this impairment. This amelioration was reversed by the H₃ receptor antagonists thioperamide and clobenpropit, but not by either the H₁ receptor antagonist diphenhydramine or the H₂ receptor antagonist zolantidine. Further, α-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, also had no effect. In the presence of Aβ42, carnosine significantly decreased glutamate release and carnosine increased the surface expression of NMDA receptor. (4) These results indicate that the mechanism by which carnosine attenuates Aβ42-induced neurotoxicity is independent of the carnosine–histidine–histamine pathway, but may act through regulation of glutamate release and NMDA receptor trafficking.     

Superoxide-scavenging activity of carnosine in the presence of copper and zinc ions

In the presence of Cu2+ and Zn2+ carnosine (beta-alanyl-L-histidine) possesses a superoxide-scavenging activity. The efficiency of scavenging as measured by the inhibition of tetrazolium nitroblue reduction in superoxide anion generation systems (phenazine methasulfate/NADH and xanthine/xanthine oxidase) is concentration-dependent and shows a maximum in the presence of millimolar concentrations of carnosine and equimolar concentrations of Cu2+ and Zn2+. In the presence of Cu2+ and Zn2+ histidine also exhibits a superoxide-scavenging activity. The feasible role of the superoxide-scavenging activity of histidine-containing dipeptide complexes with bivalent metal ions in the realization of physiological function of these dipeptides in skeletal muscles is discussed.     

Neuroprotective effect of carnosine on primary culture of rat cerebellar cells under oxidative stress

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatographymass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.     

In vitro demonstration of histamine biosynthesis from carnosine by kidneys of pregnant mice

Kidneys of pregnant mice synthesize histamine when incubated in the presence of carnosine, manganese, and pyridoxal phosphate. Intensity of biosynthesis increases linearly with the amount of enzyme and the incubation time. The reaction can only be catalysed by two enzymes that are located in kidneys and act in succession: carnosinase, which hydrolyzes carnosine into its two moieties, and histidine decarboxylase, which transforms histidine, a product of carnosine degradation, into histamine. The biosynthesis of histamine from carnosine seems to increase with the progress of pregnancy. In nonpregnant mice, kidneys do not effect this biosynthesis. The above results directly demonstrate that carnosine may be used for histamine synthesis when the activity of histidine decarboxylase is high, as in pregnant mouse kidney. Vertebrate carnosine, its role still enigmatic, might thus be mainly a potential histidine reservoir that would be mobilized any time there is a significant requirement for histidine, such as for histamine biosynthesis.