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Potential relationship between non-enzymatic antioxidants vitamin Cand oxytocin

Potential relationship between non-enzymatic antioxidants vitamin Cand oxytocin - Cell Biology and Cell Culture

Potential relationship between non-enzymatic antioxidants vitamin Cand oxytocin - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


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Old 09-16-2008, 08:52 PM
ian.jager@yahoo.com
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Default Potential relationship between non-enzymatic antioxidants vitamin Cand oxytocin



This is the first report regarding quantitation of enzymatic (eg.
superoxide dismutase, glutathione reductase & glucose-6-phosphate
dehydrogenase) and non-enzymatic (eg. glutathione) antioxidant levels
in isolated Leydig cells that I can find in the scientific
literature:

Mechanisms by which hypoxia augments Leydig cell viability and
differentiated cell function in vitro (PhD Dissertation, 1993)

by Mark A. Kukucka, MS, DVM, PhD

Department of Biomedical Sciences
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute & State University
Blacksburg, Virginia 24061-0442


ABSTRACT:

The 1980s heralded the discovery and identification of extra-pituitary
sources of the neurohypophysial hormone oxytocin in non-neural tissues
of several animal species. The presence, location and biosynthesis of
significant amounts of oxytocin in the ovarian corpus luteum was
followed by the immunocytochemical demonstration of an oxytocin-like
peptide in the testicular interstitial cells. Leydig cells, which
comprise up to 80% of the testicular intertubular cell population, are
known to synthesize testosterone in situ. Indirect evidence indicated
that an oxytocin-like peptide was also present in Leydig cells. The
question arose whether this peptide was synthesized de novo by Leydig
cells or was taken up and stored by the cells following biosynthesis
at some other intra- and/or extra-gonadal source(s). Since luteinizing
hormone (LH) and ascorbate are known to augment the production of
oxytocin in ovarian granulose cells, varying concentrations of these
two stimulants were used to monitor the biosynthesis of oxytocin from
isolated Leydig cells in culture.

Highly enriched populations of guinea pig Leydig cells were isolated
using a method that employed enzymatic dissociation and Percoll
gradient centrifugation. Since ambient oxygen tensions are toxic to
cultured Leydig cells leading to decreased steroidogenic capacity, the
antioxidant defense system of isolated Leydig cells was discerned.
Decreased levels of several antioxidants including superoxide
dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase
and total glutathione were measured. Using the dichlorofluorescin (DCF-
DA) assay, it was determined that isolated Leydig cells were capable
of accumulating hydrogen peroxide (H2O2). Leydig cells maintained in
an atmosphere composed of 19% oxygen produced H2O2 at a faster rate
than similar cells incubated at 3% oxygen.

Using a polyclonal antibody (Ab)-based immunoaffinity column, oxytocin
biosynthesis was monitored in Leydig cells incubated with a mildly
stimulating dose (0.1 ng/ml) of ovine LH for 24, 48 and 72 hours in
the presence of increasing concentrations of sodium ascorbate (1- 500
mM) under culture conditions of hypoxia and normoxia. Following solid
phase extraction and immunoaffinity purification, sample supernatants
were analyzed for both testosterone and oxytocin content as measured
by radioimmunoassay (RIA) and high performance liquid chromatography-
electrochemical detection (HPLC-ECD) respectively. Hypoxic culture
conditions and low (1-10 mM) concentrations of sodium ascorbate
augmented the production of oxytocin from Leydig cells in culture.
Higher (50-500 mM) levels of ascorbate and normoxic culture conditions
suppressed both testosterone and oxytocin production in isolated
Leydig cells. Because oxytocin synthesis was found to be cycloheximide-
sensitive, we conclude that Leydig cells possess the biosynthetic
machinery necessary to manufacture oxytocin. The isolated oxytocin
peptide was purified by HPLC with fraction collection followed by
polyclonal-Ab immunoaffinity column chromatography. Comparison of the
amino acid sequence of the isolated octapeptide with authentic
oxytocin provides unequivocal evidence that Leydig cells synthesize
oxytocin de novo. Considering the widespread use of vitamin C as a
dietary supplement, the research reported yields valuable mechanistic
information on the reproductive biologic role of vitamin C in gonadal
steroid and peptide hormone metabolism.

[Only registered users see links. ]


Vitamin C (aka ascorbate) is another non-enzymatic antioxidant found
in significant levels within the testes. As an antioxidant,
ascorbate's primary role is donate electrons to neutralize reactive
species of oxygen including superoxide (to H2O2) and hydroxyl free
radicals (to H2O). When ascorbate acts as a scavenger (by donating an
electron to a free radical), ascorbate is oxidized in the process to
the ascorbate free radical and dehydro-ascorbate. The ascorbate free
radical and the dehydro-ascorbate are reduced back to ascorbate either
by NADH catalyzed by semidehydroascorbate reductase (and forming NAD)
or reduced glutathione (GSH) catalyzed by dehydroascorbate reductase
(and forming oxidized glutathione (GSSG)).

n.b. Vitamin C also works along with glutathione peroxidase (a major
free radical-fighting enzyme) to revitalize vitamin E.

Interestingly, Kukucka et. al. reported finding significant levels of
oxytocin (a disulfide containing octapeptide) in isolated Leydig
cells. Kukucka theorized in the introduction of his PhD dissertation
(back in 1993) that open chain oxytoceine (the reduced form of
oxytocin) may also act as a scavenger (by donating an electron to a
free radical), oxytoceine may then be oxidized back to oxytocin. As
noted above, the ascorbate free radical and the dehydro-ascorbate are
reduced back to ascorbate either by NADH catalyzed by
semidehydroascorbate reductase (and forming NAD) or reduced
glutathione (GSH) catalyzed by dehydroascorbate reductase (and forming
oxidized glutathione (GSSG)).... why couldn't the ascorbate free
radical and dehydro-ascorbate be reduced back to ascorbate by reduced
oxytoceine (forming closed-ring oxytocin)?

Thus, the redox potential of oxytocin <---> oxytoceine may drive
ascorbate <---> dehydro-ascorbate or vice versa as part of the non-
enzymatic antioxidant defense system.


Other publications by Dr. Mark A. Kukucka
[Only registered users see links. ]
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