The place of carnosine among physiologically active peptides]

The results of analysis of EROP-Moscow data base concerning structural and functional peculiarities of endogenous regulatory oligopeptides are reviewed in relation to carnosine, the first endogenous peptide bioregulator. The dipeptide fragment ala-his is widely distributed in natural systems, in particular in various representatives of living organisms. The main structural peculiarity of carnosine is its elongated "filamentous" structure with a positively charged N-terminus and a cyclic radical characteristic of large physiologically active oligopeptides. The relatedness of carnosine to other oligopeptides also becomes apparent during the analysis of its role in various regulatory systems of the body.     

Use of carnosine as a natural anti-senescence drug for human beings

Carnosine is an endogenous free-radical scavenger. The latest research has indicated that apart from the function of protecting cells from oxidation-induced stress damage, carnosine appears to be able to extend the lifespan of cultured cells, rejuvenate senescent cells, inhibit the toxic effects of amyloid peptide (A beta), malondialdehyde, and hypochlorite to cells, inhibit glycosylation of proteins and protein-DNA and protein-protein cross-linking, and maintain cellular homeostasis. Also, carnosine seems to delay the impairment of eyesight with aging, effectively preventing and treating senile cataract and other age-related diseases. Therefore, carnosine may be applied to human being as a drug against aging.     

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropteridine reductase and dihydrofolate reductase; and tetrahydrobiopterin content in mammalian ocular tissues and in human senile cataracts

The enzymatic activities of GTP cyclohydrolase, sepiapterin reductase, dihydropterin reductase and dihydrofolate reductase were determined in the ocular tissues of rat, rabbit, calf and human. The enzymatic activities of the pteridine biosynthesis and the content of tetrahydropteridine (BH4) were higher in retina and ciliary body-iris as compared with lens tissue in all mammalian species tested. The activities of the pteridine synthesizing enzymes and BH4 content were decreased in human senile cataracts as compared with age-matched clear human lenses. The loss of BH4 may result in lenticular proteins more susceptible to oxidation and contribute to high molecular weight protein formation in cataracts.     

Characteristics of chloramine complexes of carnosine with hypochlorite anion]

Carnosine interaction with CIO results in the formation of a stable chloramine complex. The binding of the whole bulk of hypochlorite to carnosine is completed within one minute of incubation. During subsequent 2-hour incubation no more than 15% of the chloramine complex is destroyed; this property of carnosine makes it similar to taurine. Unlike histidine and beta-alanine, glutathione rapidly interacts with hypochlorite. However, in contrast with these compounds and carnosine, glutathione does not form stable chloramine complexes with CIO. The putative role of myeloperoxidase in the development of senile human lens opacities is discussed.     

Anti-crosslinking properties of carnosine: significance of histidine

Carnosine, a histidine-containing dipeptide, is a potential treatment for Alzheimer's disease. There is evidence that carnosine prevents oxidation and glycation, both of which contribute to the crosslinking of proteins; and protein crosslinking promotes beta-amyloid plaque formation. It was previously shown that carnosine has anti-crosslinking activity, but it is not known which of the chemical constituents are responsible. We tested the individual amino acids in carnosine (beta-alanine, histidine) as well as modified forms of histidine (alpha-acetyl-histidine, 1-methyl-histidine) and methylated carnosine (anserine) using glycation-induced crosslinking of cytosolic aspartate aminotransferase as our model. beta-Alanine showed anti-crosslinking activity but less than that of carnosine, suggesting that the beta-amino group is required in preventing protein crosslinking. Interestingly, histidine, which has both alpha-amino and imidazolium groups, was more effective than carnosine. Acetylation of histidine's alpha-amino group or methylation of its imidazolium group abolished anti-crosslinking activity. Furthermore, methylation of carnosine's imidazolium group decreased its anti-crosslinking activity. The results suggest that histidine is the representative structure for an anti-crosslinking agent, containing the necessary functional groups for optimal protection against crosslinking agents. We propose that the imidazolium group of histidine or carnosine may stabilize adducts formed at the primary amino group.     

Moderate caloric restriction delays cataract formation in the Emory mouse

Eye lens senile cataract is a major cause of blindness, affecting the elderly in particular. The etiology of the disorder has been elusive, and attempts to delay the onset of senile cataracts have been unsuccessful. The need for more information is underscored by epidemiologists who estimate that the ability to delay cataract formation in humans by only 10 years would eliminate the need for 50% of the cataract extractions performed annually in the United States. The Emory mouse provides the best model for human senile cataracts. Feeding Emory mice a diet that was restricted in calories by approximately 21% delayed the onset of cataracts. This is the first study that demonstrates in vivo the delay of senile-type cataracts. In these animals, aging and cataracts are associated with diverse changes in the proportion of various proteins (particularly 21, 22, 31-34 kDa) and with transformation of proteins from a soluble to an insoluble state. In advanced cataracts, there is a loss of total protein. Within a cataract grade, there is no difference between restricted and nonrestricted animals in relative proportion of specific lens proteins or in amounts of total or soluble proteins. The transition from a clear to cataractous lens appears when the soluble-to-total protein ratio falls below about 0.58. The exclusive use of gamma-crystallin as an indicator of lens viability is questioned. To the extent that cataract formation is due to lens protein oxidation and/or an inability to proteolytically remove damaged protein, it would appear that caloric restriction results in enhanced protection against lens oxidative stress or in prolonged proteolytic function.     

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.     

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: synthesis and characterization of their copper(II) complexes

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of several animal species. Many functions have been proposed for this compound, such as antioxidant and metal ion-chelator properties. However, the main limitation on therapeutic use of carnosine on pathologies related to increased oxidative stress and/or metal ion dishomeostasis is associated with the hydrolysis by the specific dipeptidase carnosinase. Several attempts have been made to overcome this limitation. On this basis, we functionalized carnosine and its amide derivative with small sugars such as glucose and lactose. The resistance of these derivatives to the carnosinase hydrolysis was tested and compared with that of carnosine. We found that the glycoconjugation protects the dipeptide moiety from carnosinase hydrolysis, thus potentially improving the availability of carnosine. The copper(II) binding properties of all the new synthesized compounds were investigated by spectroscopic (UV-Visible and circular dichroism) and ESI-MS studies. Particularly, the new family of amide derivatives that are not significantly hydrolyzed by carnosinase is a very promising class of carnosine derivatives. The sugar moiety can act as a recognition element. These new derivatives are potentially able to act as chelating agents in the development of clinical approaches for the regulation of metal homeostasis in the field of medicinal inorganic chemistry.     

Neuroprotective features of carnosine in oxidative driven diseases

Carnosine (β-alanyl-L-histidine) is a natural dipeptide widely and abundantly distributed in excitable tissues of several animal tissues. Although its physiological role has not been completely understood yet, many beneficial actions have been attributed to carnosine, such as being an antioxidant, antiglycating and ion-chelating agent, a wound healing promoter and a free-radical scavenger. The role of carnosine in the neuroprotection of oxidative stress-driven disorders has been reviewed. The effects of carnosine have been extensively studied both in vivo and in vitro models of cerebral damages, such as neurodegenerative disorders and hypoxia-ischemia injuries. Beside the classical sacrificial agent, carnosine has been reevaluated as a molecular chaperon and an inducer of antioxidant systems in oxidative stress conditions. Thus, beneficial effects on most of the common biochemical events that characterize neurological disorders make carnosine a very promising molecule among all the endogenous compounds in the treatment and/or prevention of oxidative driven diseases.     

Energy metabolism, proteotoxic stress and age-related dysfunction - protection by carnosine

This review will discuss the relationship between energy metabolism, protein dysfunction and the causation and modulation of age-related proteotoxicity and disease. It is proposed that excessive glycolysis, rather than aerobic (mitochondrial) activity, could be causal to proteotoxic stress and age-related pathology, due to the generation of endogenous glycating metabolites: the deleterious role of methylglyoxal (MG) is emphasized. It is suggested that TOR inhibition, exercise, fasting and increased mitochondrial activity suppress formation of MG (and other deleterious low molecular weight carbonyl compounds) which could control onset and progression of proteostatic dysfunction. Possible mechanisms by which the endogenous dipeptide, carnosine, which, by way of its putative aldehyde-scavenging activity, may control age-related proteotoxicity, cellular dysfunction and pathology, including cancer, are also considered. Whether carnosine could be regarded as a rapamycin mimic is briefly discussed.     

Carnosine protects against the inactivation of esterase induced by glycation and a steroid

Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes. Esterase activity in the lens is decreased in senile cataract and diabetes. Previously, we reported that glycation and a steroid inactivate esterase. Here we tested the inactivation of esterase with fructose, fructose 6-phosphate (F6P) and ribose as model glycation reactions and prednisolone-21-hemisuccinate (P-21-H) as a model steroid and investigated the ability of carnosine to protect esterase against inactivation. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. The modified esterase was examined electrophoretically. The esterase was progressively inactivated by F6P, fructose, ribose and P-21-H. P-21-H was more effective than the sugars. Carnosine significantly inhibited the inactivation of esterase induced by all four compounds. Carnosine decreased the extent of the cross-linking. These results provide further evidence for carnosine's role as an anti-glycation compound. It is also proposed that carnosine may be an anti-steroid agent.     

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.     

Inhibition of tumour cell growth by carnosine: some possible mechanisms

The naturally occurring dipeptide carnosine (β-alanyl-l-histidine) has been shown to inhibit, selectively, growth of transformed cells mediated, at least in part, by depleting glycolytic ATP levels. The mechanism(s) responsible has/have yet to be determined. Here, we discuss a number of probable and/or possible processes which could, theoretically, suppress glycolytic activity which would decrease ATP supply and generation of metabolic intermediates required for continued cell reproduction. Possibilities include effects on (i) glycolytic enzymes, (ii) metabolic regulatory activities, (iii) redox biology, (iv) protein glycation, (v) glyoxalase activity, (vi) apoptosis, (vii) gene expression and (viii) metastasis. It is possible, by acting at various sites that this pluripotent dipeptide may be an example of an endogenous “smart drug”.     

Role of the non-enzymatic glycosylation of protein in the formation of human cataracts

We have measured the free epsilon amino groups in soluble and insoluble proteins of clear human lenses and diabetic and non-diabetic senile cataractous lenses. The free epsilon amino groups content of soluble and insoluble proteins was significantly lower in diabetic cataracts than in clear lenses and non diabetic senile cataracts. Our results seem to demonstrate that non-enzymatic glycosylation of lens protein could play a role in the pathogenesis of cataract in diabetes.     

Prevention of lens protein glycation by taurine

Modifications in lens protein structure and function due to nonenzymic glycosylation and oxidation have been suggested to play a significant role in the pathogenesis of sugar and senile cataracts. The glycation reaction involves an initial Schiff base formation between the protein NH₂ groups and the carbonyl group of a reducing sugar. The Schiff base then undergoes several structural modifications, via some oxidative reactions involving oxygen free radicals. Hence certain endogenous tissue components that may inhibit the formation of protein-sugar adduct formation may have a sparing effect against the cataractogenic effects of sugars and reactive oxygen. The eye lens is endowed with significant concentration of taurine, a sulfonated amino acid, and its precursor hypotaurine. It is hypothesized that taurine and hypotaurine may have this purported function of protecting the lens proteins against glycation and subsequent denaturation, in addition to their other functions. The results presented herein suggest that these compounds are indeed capable of protecting glycation competitively by forming Schiff bases with sugar carbonyls, and thereby preventing the glycation of lens proteins per se. In addition, they appear to prevent oxidative damage by scavenging hydroxyl radicals. This was apparent by their preventive effect against the formation of the thiobarbituric acid reactive material generated from deoxy-ribose, when the later was exposed to hydroxyl radicals generated by the action of xanthine oxidase on hypoxanthine in presence of iron.     

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.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

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.