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Arginine Derived Nitric Oxide: Key to Healthy Skin


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Nitric oxide is generated in biologic tissues by specific nitric oxide synthases (NOS) that metabolize arginine and molecular oxygen to citrulline and nitric oxide. Arginine is an amino acid which is abundant in protamines and histones. Arginine, being a nonessential amino acid, can be manufactured by the human body and does not need to be obtained directly through the diet. l-Arginine-derived Nitric Oxide is a multifaceted molecular marvel that has been shown to provide a wide range of life-enhancing benefits, including repairing and preventing damage in blood vessels and stimulating regeneration in the skin as well as the heart, thymus gland, liver, kidneys, and other internal organs. l-Arginine-derived nitric oxide promotes the production of collagen. Nitric oxide is said to dilate the capillaries and increases healthy blood circulation to the skin. NO’s role in the progression of skin cancer is continually evolving. Arginine-derived NO is important when one is concerned with overall healthy skin.
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R.R. Watson and S. Zibadi (eds.), Bioactive Dietary Factors and Plant Extracts in Dermatology,
Nutrition and Health, DOI 10.1007/978-1-62703-167-7_8, © Springer Science+Business Media New York 2013
R. Saini
Molecular Biology Laboratory , Center of Hematology and Hemotherapy-HEMOCENTRO,
University of Campinas (UNICAMP) , Rua Carlos Chagas, 480 , Campinas , Sao Paulo , Brazil
S. L. Badole, M.Pharm., Ph.D. (Pharmacology) (*)
Department of Pharmacology , PE Society’s Modern College of Pharmacy ,
Sector 21, Yamuna Nagar , Nigadi, Pune , Maharashtra , India 411044
A.A. Zanwar
Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Medical college campus,
Off satara road, Dhankawadi, Pune 411043, Maharashtra, India
Key Points
Nitric oxide synthases (NOS) metabolize arginine and molecular oxygen to citrulline and nitric oxide.
Arginine is a nonessential a -amino acid.
Arginine is manufactured by the human body and does not need to be obtained directly through
the diet.
NO’s role in the progression of skin cancer is continually evolving.
Arginine derived NO is important when one is concerned with overall healthy skin.
Keywords Nitric oxide Arginine Skin care
The gaseous free radical nitric oxide (NO), once considered a noxious byproduct of combustion, has
now been shown to be an endogenous messenger that plays various physiological and pathophysio-
logical roles in nearly every organ system. The tremendous impact NO has had on research in biology
and medicine was re ected by the nomination of NO as the “molecule of the year 1992,” by the pres-
tigious journal “Science” [ 1 ] and in 1998, three American researchers received the Nobel Prize for
Medicine for their work with NO. Nitric oxide is generated in biologic tissues by speci c nitric oxide
synthases (NOS) that metabolize arginine and molecular oxygen to citrulline and nitric oxide. Besides
its function as a diffusible messenger in the vasculature and in neurons, nitric oxide also plays a key
role in innate immunity and in ammation. Recent progress has allowed the identi cation of the nitric
oxide pathway in several cell types that reside in the skin, including keratinocytes, melanocytes,
Chapter 8
Arginine Derived Nitric Oxide: Key to Healthy Skin
Rashmi Saini, Sachin L. Badole, and Anand A. Zanwar
74 R. Saini et al.
Langerhans cells, broblasts, and endothelial cells. Despite role of NO in various normal physiological
processes of skin, it is also involved in pathological conditions related to skin which include psoriasis
and other immune-mediated skin diseases as well as skin cancer. Arginine is the immediate precursor
of NO and Arginine-Derived Nitric Oxide (ADNO) has been shown to provide a wide range of life-
enhancing bene ts, including repairing and preventing damage in blood vessels and stimulating
regeneration in the skin. Arginine intake leads to a healthier, smoother, tighter, and wrinkle-free skin,
and thus it has been named as “magic anti-aging bullet.
l -Arginine: Semi-Essential Amino Acid
Arginine, an amino acid, which is abundant in protamines and histones (both proteins associated with
nucleic acids) was rst isolated from a lupin seedling extract in 1886 by the Swiss chemist Ernst
Schultze. Amino acids are generally classi ed as essential or nonessential. Essential amino acids are
those that the body cannot synthesize; a steady supply of amino acids must be provided through the diet.
The body can manufacture nonessential amino acids, so an exogenous supply of them in the diet is
unnecessary. Arginine is a unique amino acid, generally referred to as semi-essential. This noncommittal
label indicates that although the body can manufacture arginine, at times it does so in an amount that is
insuf cient to meet physiological needs and dietary supplementation may be required. This often occurs
during the periods of growth, illness, and metabolic stress. In other words, arginine is a nonessential
amino acid during the periods of maintenance, but is an essential amino acid during the periods of
growth and healing. In addition, newborns are not able to make their own supply of this substance, so
arginine is considered essential in the rst months of life.
Arginine (abbreviated as Arg or R) is a a -amino acid. The l -form is one of the 20 most common natural
amino acids. The amino acid side chain of arginine consists of a 3-carbon aliphatic straight chain, the
distal end of which is capped by a complex guanidinium group (Fig. 8.1 ). The guanidinium group is
positively charged in neutral, acidic, and even most basic environments, and thus imparts basic
chemical properties to arginine. Its IUPAC name is ( S )-2-Amino-5-guanidinopentanoic acid.
Biosynthesis of NO
NO is synthesized from the guanidino nitrogen in the l -arginine molecule, converting l -arginine to
NO and l -citrulline in a two-step reaction that requires cofactors including FAD, FMN, NADPH,
calmodulin (CaM), and tetrahydrobiopterin (BH
4 ) (Fig. 8.2 ) [ 2, 3 ] NOS isoforms are classi fi ed as
Fig. 8.1 l -Arginine
8 Arginine Derived Nitric Oxide: Key to Healthy Skin
constitutive neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric oxide synthase. Drs.
Marie-madeleine Cals-Grierson and Anthony Ormerod report in the June 2004 issue of “Nitric Oxide”
that skin cells contain three different types of nitric oxide synthase. Most cells in the skin contain
inducible nitric oxide synthase. The cells that maintain skin’s elasticity contain endothelial nitric
oxide synthase. Cells making up the majority of the epidermis release neuronal nitric oxide synthase.
Stem cells in the skin help maintain and repair damage to the skin’s outer layers, the epidermis and
the dermis. In the July 2010 issue of “Archives of Dermatological Research,” researchers at Polytechnic
University of Marche in Ancoma, Italy, reported the presence of three types of nitric oxide synthase
enzymes in skin stem cells (Fig. 8.2 )
Dietary Sources of Arginine
Arginine, being a nonessential amino acid, can be manufactured by the human body and does not need
to be obtained directly through the diet. The biosynthetic pathway, however, does not produce
suf cient arginine, and some must still be consumed through diet. Individuals who have poor nutri-
tion or certain physical conditions may be advised to increase their intake of foods containing argin-
ine. Arginine is found in a wide variety of foods, including
Animal sources: dairy products (e.g., cottage cheese, ricotta, milk, yogurt, whey protein drinks),
beef, pork (e.g., bacon, ham), gelatin, poultry (e.g. chicken and turkey light meat), wild game (e.g.,
pheasant, quail), seafood (e.g., halibut, lobster, salmon, shrimp, snails, tuna)
Plant sources: wheat germ and our, buckwheat, granola, oatmeal, peanuts, nuts (coconut, pecans,
cashews, walnuts, almonds, Brazil nuts, hazelnuts, pinenuts), seeds (pumpkin, sesame, sun ower),
chick peas, cooked soybeans, chocolate, Phalaris canariensis (canaryseed)
l -Arginine-Derived Nitric Oxide (ADNO) in Skin
The body needs arginine to produce nitric oxide, a chemical that causes blood vessel relaxation
(vasodilation). Preliminary studies indicate that arginine may be useful in the treatment of angina,
atherosclerosis, coronary artery disease, intermittent claudication, erectile dysfunction, female impo-
tence, migraine, and other conditions that are linked to reduced blood ow throughout the body.
ADNO ( l -Arginine derived Nitric Oxide) is a multifaceted molecular marvel that has been shown to
provide a wide range of life-enhancing bene ts, including repairing and preventing damage in blood
vessels and stimulating regeneration in the skin as well as the heart, thymus gland, liver, kidneys, and
Fig. 8.2 Biosynthesis of NO from l -Arginine. NO is synthesized from the guanidino nitrogen in the l -arginine molecule
by nitric oxide synthase (NOS) enzyme-converting l -arginine to NO and l -citrulline, requiring cofactors including FAD,
FMN, NADPH, Calmodulin, and Tetrahydrobiopterin (BH4)
76 R. Saini et al.
other internal organs. l -Arginine derived nitric oxide promotes the production of collagen. Nitric oxide
is said to dilate the capillaries and increases healthy blood circulation to the skin. The enhanced circula-
tion helps to bring a ood of nutrients saturating the malnourished skin with new life. It improves skin
texture, elasticity, thickness, stimulating cell regeneration, and restores moisture. It creates tighter,
smoother skin, reducing wrinkles as well as dark circles under the eyes. Arginine is used for speeding
up wound healing and increasing blood ow to cold hands and feet, especially in people with diabetes.
When the body is not functioning at optimum, l -arginine production suffers. As it regulates blood
pressure, reduces plaque, lowers cholesterol, and prevents blood clots, it is not unusual for cardio-
vascular problems to follow. l -Arginine may help circulatory problems and the resultant dry skin.
Large concentrations of arginine are found in the skin, and this amino acid plays a key role in the
health of all the body’s connective tissues, particularly the muscles. Arginine helps the body process
both creatine, a natural substance that helps build muscle mass, and nitrogen, a chemical needed for
muscle metabolism. Laboratory research suggests that arginine may help reduce body fat and speed
up weight loss. Arginine has also been shown to help heal and repair damaged tissues, and thus may
be bene cial to both athletes and those suffering from arthritis.
NO and Skin Biology
Practically every cutaneous relevant cell type expresses some isoform of NOS and is therefore able
to generate and release NO for a broad spectrum of physiologic processes. Keratinocytes, the major
constituent of the epidermis, express both constitutive and inducible NOS and produce NO and hydro-
gen peroxide in response to in ammatory stimuli. This may act as one of the cardinal broad protective
mechanisms of the skin, as the epidermis is constantly exposed to foreign matter and organisms.
In addition, nely regulated responses are also exhibited by NOS species, such as in wound healing.
Fibroblasts, found in the dermis, regulate the structural framework of the skin by synthesizing extra-
cellular matrix, collagen, and brin, while orchestrating the complex steps of wound healing. Fibroblasts
demonstrate NOS expression, but this expression is inconsistent across different cells, possibly depen-
dent on cell maturation [ 4 ] . Additionally, eccrine glands express eNOS, melanocytes have shown eNOS
and iNOS expression [ 5 ] . Due to its widespread distribution, NO is able to participate in basic physi-
ological roles such as establishing and maintaining circulation, forming a protective barrier against
microorganisms, and UV-induced melanogenesis [ 6, 7 ] and development of erythema [ 8 ] .
It has become apparent that NO is also produced by other cell types residing in the skin. Expression
of both the constitutive and the inducible pathway has been demonstrated in dermal broblasts and
endothelial cells [ 9 ] . It will be of interest therefore to further elucidate the physiologic and/or
pathophysiologic roles of NO production in these cells with particular reference to cutaneous
in ammatory and immune responses. It has been discovered that NO is produced in Langerhans cells
of human skin [ 10 ] . From these studies it has been concluded that NO may affect Langerhans cell
functions such as microbicidal activity, antigen presentation, and cytotoxicity, and may also affect
adjacent keratinocytes and melanocytes. Dermal papilla cells play an important role in hair growth
and have been shown to produce NO after exposure to bacterial endotoxin [ 11 ] . Modulation of Ca 2 -
activated K channels by NO has been identi ed in these cells, although the biologic function of this
particular activity in human skin is not yet known.
NO is known to have role in normal physiological as well as pathophysiological processes of skin.
Biological role of NO in the regulation of vascular homeostasis [ 12, 13 ] is well known where the NO
produced from endothelial cells causes vascular smooth muscle relaxation. NO also has an important
role to play in immunological activities involved in skin which acts as a signi cant barrier against
many pathogens and thus contributing in rst line of defense [ 14 ] . Moreover, recent studies empha-
size the role of NO in ultraviolet-induced melanogenesis [ 15 ] apart from its signi cant role in wound
repairs and skin cancers.
8 Arginine Derived Nitric Oxide: Key to Healthy Skin
NO in Normal Physiological Processes of Skin
Vascular Homeostasis
Endothelial cells produce via eNOS activity small pulses of NO resulting in a basal level of vascular
smooth muscle relaxation. In addition to the regulation of systemic blood pressure, however, NO has
recently been reported to control the local blood ow to speci c vascular beds, in the brain, heart,
lung, gastrointestinal tract, and the skin [
16, 17 ] . A local de ciency of NO therefore could cause
vasospasm in selected organs. Nitric oxide helps control the blood ow to the skin thus restores some
of the youthful vibrancy to the skin. Reduced blood ow in the area under the eyes results in dark
circles. A study conducted by AGI Dermatics in New York published in “Nitric Oxide” in August
2006 shows increasing the release of nitric oxide in blood vessels under the eyes improves blood ow
in those vessels and decreases the appearance of dark circles under the eyes. The microvascular
endothelial cells have been shown to release NO in response to the vasodilatory neuropeptides calci-
tonin gene-related peptide and substance P, suggesting that NO provides a molecular link between the
nervous system and the skin [ 18 ] . Eczema are-ups often associated with dysfunction of the nervous
system; and, some hormonal imbalances (hypothyroidism being a very common example) are so
often associated with dry, itchy, in amed skin.
Immune System
The skin is a site of signi cant immunologic activity because of its constant exposure to environ-
mental challenges such as physical stress, trauma, chemical irritants, and infectious micro-organisms.
In consequence, a complex set of immune reactions can be observed in the skin, providing an appro-
priate defense under a variety of circumstances. The rst indication that NO might be an integral part
of the immune response in human skin came from in vitro experiments showing that in ammatory
stimuli induce keratinocytes to produce NO as well as hydrogen peroxide [ 19 ] . Numerous studies
have demonstrated that NO synthesis is a necessary component of nonspeci c defense mechanisms for
several pathogens, including bacteria, viruses, parasites, and fungi [ 20 ] . In particular, NO synthesized
at high concentrations eliminates intracellular pathogens, such as Mycobacterium tuberculosis and
Mycobacterium leprae , Leishmania species , Trypanosoma cruzi , and Plasmodium falciparum , and is
thought to block viral replication. Initially, this type of NO-mediated cytotoxicity was presumed to be
restricted to macrophages; however, it has now become apparent that other cell types that express
iNOS, such as keratinocytes and endothelial cells, may also contribute to this innate immunity.
Speci cally, the skin, acting as immunologic barrier, appears to be well equipped for this rst line of
defense. NO is able to regulate skin ora through this non-NOS-dependent synthetic pathway. Nitrite
is a known constituent of both blood and sweat, and in the case of sweat, the acidity of the skin surface
allows for the reduction of nitrite to NO [
21 ] . The acidi ed nitrite functions as a moat, so to speak,
of protective antimicrobial NO, preventing pathogen access to the body.
Melanogenesis: Reponses to Ultraviolet Irradiation
Melanin pigments produced in human melanocytes are classi ed into two categories; black coloured
eumelanin and reddish-yellow pheomelanin. Nitric oxide (NO) is melanogenesis-stimulating factor.
The ratio of eumelanin and pheomelanin increased signi cantly with the addition of NO and thus
contribute to UV-induced hyperpigmentation by enhancing eumelanogenesis [
22 ] . Within the epidermal-
melanin unit, melanocytes synthesize and transfer melanin to the surrounding keratinocytes.
Keratinocytes produce paracrine factors that affect melanocyte proliferation, dendricity, and melanin
78 R. Saini et al.
synthesis. It has been demonstrated that normal human keratinocytes secrete nitric oxide (NO) in
response to UVA and UVB radiation and this involves constitutive isoform of keratinocyte NO
synthase [ 23 ] . Melanogenic effect of NO by keratinocytes in response to UV radiation was investi-
gated using melanocyte and keratinocyte cocultures. Conditioned media from UV-exposed keratino-
cytes stimulate tyrosinase activity of melanocytes. This effect is reversed by NO scavengers, suggesting
an important role for NO in UV-induced melanogenesis. These observations suggest that NO plays an
important role in the paracrine mediation of UV-induced melanogenesis [ 24 ] .
Several melanogenic factors released from keratinocytes and other cells surrounding melanocytes
in the skin following UV radiation are reported to up-regulate tyrosinase gene expression through a
different pathway, but most regulate tyrosinase via microphthalmia-associated transcription factor
(MITF). NO donors increase tyrosinase activity and melanin synthesis in human melanocytes. This
effect is positively correlated with an increased amount of both tyrosinase and tyrosinase-related pro-
tein 1 (TRP-1), two enzymes involved in melanogenesis [ 23, 24 ] . Recent research provide exciting
new evidence that NO can enhance melanogenesis in alpaca skin melanocytes by stimulating MITF
phosphorylation [ 25 ] .
Ultraviolet irradiation is one of the major assailants to the skin and it is constantly exposed to this
stressor capable of inducing oxidative cellular damage. As one of the skin’s primary defense mecha-
nisms, keratinocytes produce sustained concentrations of NO upon exposure to both UVA and UVB
irradiation, which only declines after 3 days and coincides with the time course of sun-induced ery-
thema. In this regard, NO may quench free radical damage that can result from UV radiation exposure
if generated photoproducts are allowed to propagate unhindered. This proposed role of NO is sup-
ported by reports that endothelial cells are protected from UVA-induced apoptosis by NO [ 21 ] and
that UV-induced lesions in cutaneous lupus erythematosus demonstrate reduced expression of iNOS.
More recently, a non-enzymatic pathway of NO production was elucidated, where NO is derived from
biologically relevant NO-related products in the human epidermis, super cial vascular dermis and
sweat. In the setting of acute UVA exposure, these products are quickly mobilized within 30 min to
generate NO, resulting in keratinocyte cytoprotection from ultraviolet radiation-induced apoptosis.
These studies suggests that intake of external sources of NO precursors, such as, arginine may
in uence the innate and acute cutaneous response to ultraviolet radiation [ 21 ]
NO in Pathophysiological Processes of Skin
Skin Cancer
Our understanding of NO’s role in the progression of skin cancer is continually evolving. Over the
past two decades, its precise role in tumor pathophysiology has been a matter of great debate. There
is extensive evidence that tumor expressed NOS and subsequent NO production can be both pro- and
anti-carcinogenic, depending on the concentration of NO produced. NO may function in an anti-
carcinogenic role via the induced apoptosis of mutated cells or through modulation of growth
responses and gene expression patterns. Dong et al. [ 25 ] demonstrated the potential anti-carcinogenic
features seen with increased expression of NOS2. In a comparative study between several nonmetastatic
and highly metastatic melanoma clones, it was demonstrated that the nonmetastatic clones expressed
much higher levels of endogenous NO. Since then, a multitude of projects designed to generate high intra-
tumoral levels of NO have been pursued, including the use of NO donor drugs and the transfection of a
functional NOS2 gene [ 26 ] . Additionally, NO is able to further exert its anti-carcinogenic effects through
the regulation of MMP levels [ 27 ] and by increasing the release of cytochrome c, which activates caspases
and induces the release of massive quantities of cellular calcium both of which result in apoptosis.
It is clear that NO is involved in a multitude of signaling pathways that vary based on cell type and
the level of NO produced. Therefore, it is not surprising that a myriad of effects have been observed
8 Arginine Derived Nitric Oxide: Key to Healthy Skin
following the modi cation of NO levels in different tumor cell lines. It appears that at modest
concentrations, the effects of NO could be characterized as pro-malignant, whereas, at highly elevated
concentrations, NO acts as a potent anticancer agent, promoting apoptosis and inhibiting metastasis.
Wound Repair
Wound healing of the skin represents a highly ordered process of important tissue movements that
aims for a rapid closure of the wound site and a subsequent regeneration of the injured tissue. The
factors ensuring the intercellular communication during repair are only known in part. However,
although protein-type mediators are well-established players in this process, it has become evident
that the diffusible, gaseous molecule nitric oxide (NO) participates in the orchestration of wound heal-
ing. NO also accelerates wound healing which is greatly needed in the case of major burns or injuries.
The role of wound-derived NO critically in uences macrophage, broblast, and keratinocyte behav-
iour within the intercellular communication network during repair [ 28, 29 ] . NO synthesis in human
broblasts has been shown to attenuate the pathophysiologic sequelae in wound healing early during
the in ammatory stages and later during stages of proliferation and tissue remodeling [ 30 ] . Additionally,
reduced NO synthesis has been demonstrated in broblasts of hypertrophic scar tissue [ 31 ] . These
ndings suggest that changes in NO levels can lead to signi cant alterations of cellular responses to
wounding. Several cytokines and growth factors that are known regulators of iNOS expression con-
trol critical aspects of wound healing, suggesting a complex network of autocrine and paracrine cel-
lular responses. The inducible isoform (iNOS) is synthesized in the early phase of wound healing by
in ammatory cells, mainly macrophages. However many cells participate in NO synthesis during the
proliferative phase after wounding. NO released through iNOS regulates collagen formation, cell
proliferation, and wound contraction in distinct ways [ 32 ] .
Mode of Action of NO in Skin
In multicellular organisms tissue homeostasis is maintained through a delicate balance between cell
proliferation and cell death. Arrest of cell division is a prerequisite for cells to enter a program of
terminal differentiation. Mitogenesis and cytostasis can be induced by diverse intrinsic and extrinsic
stimuli, and convincing evidence suggests that alterations in this process contribute to the pathogen-
esis of several human skin diseases, including psoriasis and other hyperproliferative diseases [ 18 ] . NO
could mediate cessation of growth and synchronize commitment for differentiation in epidermal kera-
tinocytes. In human skin, NO has important role to play in normal development as well as host
responses to infection and tissue injury, which is orchestrated through an intricate and ordered series
of interactions between cells resident to the skin as well as cytokines, growth factors, and extracellular
matrix proteins. Pertubations in these cell–cell and cell–matrix interactions have been shown to result
in a loss of skin integrity, as characterized, for example, during wound healing processes. Reduced
blood ow causes dead and dry skin, NO dilates the blood vessels, increases the blood ow, and
results in rejuvenated healthier skin [ 16, 17 ] .
Arginine–Skin Connection: Liver, Hormones, and Blood Sugar Levels
Arginine is known to improve insulin sensitivity. One of the main physiological problems in type 2
diabetes is that the body’s cells become increasingly resistant to the action of insulin. This is the hor-
mone that helps cells take in glucose (the “fuel” the body needs to stay alive) from the blood. If insulin
80 R. Saini et al.
resistance develops, glucose is not transported into the cells as ef ciently as it should be, and it builds
up in the blood. That is why people with diabetes are often said to have high blood sugar—and it must
be controlled. Research study showed that arginine supplementation may help people with type 2
diabetes utilize glucose more ef ciently by improving their insulin sensitivity [ 33 ] . Regulation of
blood sugar levels is essential for healthy skin. Both high and low blood sugar levels dry the skin, and
high blood sugar can also lead to the cracked, split skin sometimes seen in diabetics. People with
arginine de ciency show fat build ups in their liver. Arginine helps eliminate toxins. Poor liver
function, results in a buildup of toxins which then get excreted through the skin causing skin dry-
ness and in ammation. When liver function is poor, metabolism slows down which causes dry skin,
but as we gain weight from improper metabolism of fats our circulation slows down and the skin
dries even further.
l -Arginine De fi ciency
Arginine is a nonessential amino acid during the periods of maintenance, but is an essential amino
acid during the periods of growth and healing. l -Arginine helps the body get rid of waste and synthesize
proteins. When the body is not functioning at optimum, l -arginine production suffers and arginine
de ciency occurs in those ghting infection, severe burns, undergoing dialysis, experiencing rapid
growth, or those with trouble processing urea. Certain conditions such as protein de ciencies and
malnutrition also affect ability to produce l -arginine. People with l -arginine de ciency may have fat
build ups in their liver. Its de ciency is accompanied by symptoms such as alopecia (hair loss), skin
rashes, poor wound healing, and other skin problems.
l -Arginine Supplements: Side Effects?
l -arginine supplements can cause some side effects such as abdominal pain, bloating, diarrhea, gout,
blood abnormalities, allergies, airway in ammation, worsening of asthma, and low blood pressure.
l -arginine should not be taken by pregnant or nursing women as it stimulates growth hormone in
young children. Persons having herpes should also avoid taking arginine as it can stimulate the herpes
infection. People taking medication for high blood pressure, impotence, migraines, or any other
problem, or having a history of kidney or liver disease, should check with their doctors before taking
l -arginine supplements. Long-term use of arginine supplements is not recommended, as it may result in
thickening and coarsening of the skin and/or nitrogen imbalance in the body. Arginine has also been
shown to increase or decrease the effects of certain medications, including lysine, NSAIDS (non-steroi-
dal anti-in ammatories), ACE inhibitors, or potassium sparing diuretics. People with herpes or schizo-
phrenia should avoid arginine supplementation altogether as it may aggravate these conditions.
NO responses known from other biologic systems, such as vasodilation, neurotransmission, as well as
cytotoxicity and immunoregulation, are also of signi cant importance in human skin. Characterization
of the role of NO in cutaneous disease will not only provide an important addition to our understand-
ing of cutaneous biology but also is likely to be the foundation for the development of new therapeutic
approaches that can modify, arrest, or reverse the course of human skin disease. The demonstrable and
8 Arginine Derived Nitric Oxide: Key to Healthy Skin
potential roles of nitric oxide in skin disease pathogenesis and treatment has led to the use of arginine
as a source of NO in diet. More recent modalities that have been evaluated and developed include
continuous horizontal- ow delivery of gaseous NO, and its local skin effects like quick healing
wounds [ 34 ] but arginine-derived NO is important when one is concerned with overall healthy skin.
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... This EAA also termed as 'conditionally EAA'. This EAA found in G. changii was important to certain conditions such as during growth, illness, and metabolic stress, as well as during the first month of a new born (Saini, Badole, & Zanwar, 2013). The second highest EAA presence was leucine; representing 18.42% of the total EAAs. ...
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Nitric oxide (NO) synthase (iNOS) is required for the resolution of acute cutaneous leishmaniasis in resistant C57BL/6 mice. As is the case in several other infections, the clinically cured host organism still harbors small amounts of live Leishmania major parasites. Here, we demonstrate lifelong expression of iNOS at the site of the original skin lesion and in the draining lymph node of long-term-infected C57BL/6 mice. iNOS activity in the lymph node was dependent on CD4+, but not on the CD8+ T cells. By double labeling techniques, iNOS and L. major were each found in macrophages (F4/80+, BM-8+, and/or MOMA-2+) and dendritic cells (NLDC-145+), but not in granulocytes or endothelial cells. In situ triple labeling of lymph node sections revealed that approximately 30-40% of the L. major foci were associated with iNOS-positive macrophages or dendritic cells. The majority of the L. major foci (60-70%), however, was located in areas that were negative for both iNOS and the macrophage and dendritic cell markers. In L. major-infected C57BL/6 mice, which had cured their cutaneous lesions, administration of L-N6-iminoethyl-lysine (L-NIL), a potent inhibitor of iNOS, led to a 10(4)-10(5)-fold increase of the parasite burden in the cutaneous and lymphoid tissue and caused clinical recrudescence of the disease. Persistent expression of iNOS and resumption of parasite replication after application of L-NIL was also observed in resistant C3H/HeN and CBA/J mice. We conclude that iNOS activity is crucial for the control of Leishmania persisting in immunocompetent hosts after resolution of the primary infection. Failure to maintain iNOS activity might be the mechanism underlying endogenous reactivation of latent infections with NO-sensitive microbes during phases of immunosuppression.
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Ultraviolet (UV) B radiation can cause skin-tanning via the synthesis of melanin which is synthesized by specific tyrosinase and tyrosinase-related enzymes expressed in melanocytes. It is reported that several melanogenic factors are released from keratinocytes and other cells surrounding melanocytes in the skin following UV radiation. Some of them are reported to up-regulate tyrosinase gene expression through a different pathway, but most regulate tyrosinase via microphthalmia-associated transcription factor (MITF). It is unknown whether an NO-induced pathway regulates melanogenesis via MITF in vitro. In this study, we investigated this problem because it is important for our understanding of how to enhance the coat color of alpaca. We set up three groups for experiments using alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures were also allowed 5 days of growth like the control group, but were then irradiated once everyday with 312 mJ/cm(2) of UVB; the UV + L-NAME group was the same as the UV group, but with the addition of 300 μM L-NAME every 6 h. To determine the NO inhibition effect, NO product was measured. To determine the effect of NO on MITF, the expression levels of the MITF gene and protein were measured by immunofluorescence, quantitative real-time PCR and western immunoblotting. To determine the influence of NO on MITF phosphorylation, phosphorylated MITF protein (p-MITF) was measured by western immunoblotting. To determine the effect of NO on melanogenesis, the melanin content was measured. The results provide exciting new evidence that NO can enhance melanogenesis in alpaca skin melanocytes by stimulating MITF phosphorylation.
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In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.
The pathways regulating rat and mouse embryonic and lung fibroblast nitric oxide production were analyzed in an attempt to evaluate the potential role of these cells in nonspecific host defense and inflammation. Interleukin-1β (IL-1β) was found to be the strongest single activator in all types of fibroblasts examined. In addition, lipopolysaccharide (LPS) was synergistic with IL-1β or tumor necrosis factor-α (TNF-α) in induction of nitric oxide synthesis. These patterns of responsiveness are not observed in macrophages and may be significant in initiation of early host defense processes, before specific interferon-γ (IFN-γ)–mediated immune responses have become operative. Rat and mouse fibroblasts were also found to produce nitric oxide when primed with IFN-γ and simultaneously treated with IL-1, TNF-α, or LPS. The doses of IFN-γ effective in priming fibroblasts for nitric oxide production were as low as 1-10 U/ml. Furthermore, effective triggering doses of LPS, TNF-α, and IL-1 were 10 ng/ml, 100 U/ml, and 0.2 ng/ml, respectively. These results demonstrate that fibroblasts are activated more readily to produce nitric oxide than interstitial macrophages and may be the major source of this mediator in tissues. Immunohistochemical studies demonstrated that fibroblasts are heterogeneous with respect to inducible nitric oxide synthase expression with the majority of cells not involved in the response. Fibroblasts were also found to be distinct from macrophages in their sensitivity to the suppressive effects of Transforming growth factor-β, which in fibroblasts inhibited both IFN-γ plus LPS–and IFN-γ plus TNF-α-induced nitric oxide production. At the stage of growth crisis, a dramatic increase in nitric oxide production was observed in rat fibroblasts in response to IFN-γ or TNF-α that may be directly correlated with cellular senescence. Taken together, our data suggest that mouse and rat fibroblasts are potential effectors in both IFN-γ-dependent and -independent nitric oxide-mediated processes and that the patterns regulating nitric oxide metabolism in these cells are distinct from those of macrophages.
Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
Harald H. H. W. Schmidt and Ulrich Walter Medizinische Universitatsklinik Wiirzburg Klinische Biochemie und Pathobiochemie Versbacher Strasse 5 D-97078 Wiirzburg Federal Republic of Germany Nitroglycerine has been used for over a century to treat coronary heart disease, and it has long been suggested that humans synthesize oxides of nitrogen (Mitchell et al., 1916). These observations have recently been brought into focus by the demonstration that endogenous nitric oxide (NO) regulates mammalian blood vessels and other systems (Moncada and Higgs, 1991) such that virtually every mammalian cell is under the influence of NO. The three “classics” of NO-mediated functions-endothelium- dependent relaxation (Furchgott and Zawadzki, 1980) neurotransmission (Garthwaite et al., 1988; Gillespie et al., 1989) and cell-mediated immune response (Nathan and Hibbs, 1991)-have suggested principles for the mode of action of NO and for its functions. General Principles Networks In many systems, NO derives from two or more different cellular sources, forming networks of paracrine communi- cation (Figure 1). For example, we now know that vascular and bronchial NO originates not only from endothelial cells, where it iscalledendotheliumderived relaxing factor (EDRF), but also from adventitial nerves and epithelial cells (Schmidt et al., 1992a; Wilcox et al., 1992), where it mediates endothelium-independent smooth muscle relax- ation. Neurons use NO to regulate transmitter release of adjacent neurons (Meffert et al., 1994) and also to match cerebral blood flow with neuronal activity; similarly, bron- chial epithelial and endothelial cells use NO to match venti- lation and perfusion (Gaston et al., 1994). Macula densa renal tubular epithelial cells release NO to dilate the neigh- boring afferent artery and increase glomerular filtration (Wilcox et al., 1992). NO Toxicity NO is a double-edged sword (Table l), beneficial as a messenger or modulator and for immunologic self-defense, but potentially toxic. In several different scenarios (Figure 2) with factors such as oxidative stress, generation of reactive oxygen intermediates (ROls), and deficient anti- oxidant systems, NO switches from friend to foe. A pre- dominant mechanism by which this occurs is through the diffusion-limited reaction of NO with superoxide to gener- ate peroxynitrite (Beckman et al., 1990) which may modu- late signaling functions of NO (Gaston et al., 1994; Moro et al., 1994) and is directly cytotoxic (Beckman, 1991). e.g., by causing extensive protein tyrosine nitration (Beck- man et al., 1994).
The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly; only a few survive to produce metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Experimental studies concluded that metastatic cells survive in the circulation whereas nonmetastatic cells do not. In part, this difference is due to an inverse correlation between expression of endogenous inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) and metastatic potential. Direct evidence for the role of iNOS in metastasis has been provided by our data on transfection of highly metastatic murine K-1735 clone 4 (C4.P) cells which express low levels of iNOS, with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM of the specific iNOS inhibitor NG-L-methyl arginine (NMA). C4.P, C4.Neo, and C4.S2 cells were highly metastatic, whereas C4.L8 cells were not. Moreover, C4.L8 cells produced slow-growing subcutaneous tumors in nude mice, whereas the other 3 cell lines produced fast-growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with either cytostasis or cytolysis via apoptosis, depending upon NO output. The tumor cells producing high levels of NO underwent autocytolysis and produced cytolysis of bystander cells under both in vitro and in vivo conditions. Multiple i.v. injections of liposomes containing a synthetic lipopeptide upregulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Transfection of interferon-beta suppressed tumor formation and eradicated metastases, which was apparently linked to iNOS expression and NO production in host cells such as macrophage. Besides mediating cell death, NO produced tumor suppression by regulating expression of genes related to metastasis, e.g., survival, invasion, and angiogenesis. Suppression of metastasis can be achieved through use of immunomodulators that induce iNOS expression in tumor lesions or by the direct delivery of the iNOS gene to tumor cells or host cells through liposome and/or viral vectors.
NO (nitric oxide) molecule is produced by various mammalian cell types and plays a significant role in inflammation, infection and wound healing processes. Recently, gNO (gaseous nitric oxide) therapy has been utilized for its potential clinical application as an antimicrobial agent, with special focus on skin infection. In a previous study, we demonstrated that 200 ppm gNO, 8 h/day for three consecutive days significantly reduced the number of bacteria in dermal wounds without compromising the viability and function of skin cells. To increase the feasibility and ease of its clinical use, we propose that different doses of gNO (5 to 10 K ppm) for 8 h and as short as 10 min be used, respectively. To achieve this, we set up in vitro experiments and asked whether (i) different doses of gNO have any toxic effect on immune cells and (ii) gNO has any modulating effect on key ECM (extracellular matrix) components in fibroblasts. To further investigate the effect of gNO, expression of more than 100 key ECM genes have been examined using gene array in human fibroblasts. As immune cells play an important role in wound healing, the effect of gNO on proliferation and viability of human and mouse lymphocytes was also examined. The findings showed that, the 5, 25, 75 and 200 ppm of gNO for 8 h slightly increased the expression of Col 5A3 (collagen type V alpha 3), and gNO at 5 ppm decreased the expression of MMP-1 (matrix metalloproteinase 1), while exposure of fibroblast to 10 K ppm of gNO for 10 min does not show any significant changes in ECM genes. Exposure to gNO resulted in inhibition of lymphocyte proliferation without affecting the cell viability. Taken together, our findings show that skin could be treated with gNO without compromising the role of ECM and immune cells in low concentrations with long time exposure or high concentrations for a shorter exposure time.