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Departments of Pathology and Laboratory Medicine (H.M.A.M.Q., M.A.E.-M., R.V.) and Biochemistry and Molecular Biology (R.V.), University of Louisville School of Medicine, Louisville, Kentucky 40292
Address all correspondence and requests for reprints to: Dr. Roland Valdes, Jr., Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292. E-mail: rvaldes{at}louisville.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of NKA,
stabilizing the phosphorylated intermediate, thus leading to the
inhibition of NKA activity and transport of sodium and potassium across
cell membranes (7, 8). Control of sodium pump activity is believed to
be an underlying mechanism in the pathophysiology of several diseases,
including cardiovascular, neurological, renal, hepatic, psychiatric,
and metabolic disorders (for reviews, see Refs. 9, 10, 11). A class of
compounds known as mammalian cardenolides is now believed to constitute
part of an hormonal axis-regulating activity of the sodium pump.
Concentrations of these factors in blood have been reported to be
increased in physiological and clinical conditions associated with
altered sodium pump activity (see reviews in Refs. 12, 13, 14). Most
findings to date suggest that two genre of mammalian-derived compounds,
digitalis-like factors (DLF or DLIF) and ouabain-like factors (OLF or
HIF), exist with properties similar to those of the plant-derived
cardenolides, digoxin and ouabain. There is considerable evidence to indicate that these mammalian-derived molecules are produced by endocrine-secreting tissues such as adrenals and hypothalamus (15, 16, 17) including recent reports indicating the presence of OLF in secretions from cultured bovine adrenocortical cells (18, 19, 20). Of significance is the recent discovery of several congeners of mammalian DLIF, including a series of deglycosylated species, DLIF-genin, -bis, and -mono components (21) as well as a dihydrodigoxin-like form, dihydro-DLIF (22). Of particular interest are recent data indicating a cytochrome p450-mediated metabolic conversion in vitro of a less active dihydro species (dihydrodigoxin) to a more biologically active species with digoxin-like immunoreactive properties (22). Although we recently reported the presence of a deglycosylated congener of OLF (OLF-genin) in humans (23), no reports have yet demonstrated the presence of a dihydro counterpart to OLF in vivo.
Dihydroouabain is a chemically reduced form of ouabain (Fig. 1
) that is used extensively to study the
biological activity of ouabain and its interaction with the sodium
pump. The reduced forms of both digoxin and ouabain show relatively
lower potencies than the oxidized counterparts for inhibition of sodium
pump activity. However, dihydroouabain has biophysical properties
(e.g. rapid washout from tissue), making the dihydro species
very attractive for potential fine regulation of sodium pump activity
if natural counterparts existed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Equipment and materials
We used a Polytron PT 3000 (Brinkmann Instruments, Inc., Westbury, NY) for homogenizing adrenocortical tissues.
Solid phase C18 cartridges (Sep-Pak) were
obtained from Waters Corp. (Milford, MA) so as the
C18 reverse phase µBondapak columns (3.9 x 300 mm,
10-µm particle size) connected to a Waters 600E system controller and
a Waters 966 photodiode array detector. Fractions collected with a
Waters fraction collector from Millipore Corp. (Milford,
MA) were evaporated with a Jouan Centrifugal Vacuum Concentrator RC
10.22 connected to a Jouan Refrigerated Trap RCT 60 (Winchester,
VA). Y-1 murine adrenocortical tumor cells were a gift from Dr. Bernard
Schimmer, University of Toronto (Toronto, Canada). These cells were
maintained and passaged in the laboratory of Dr. Barbara Clark
(University of Louisville, Louisville, KY).
For Western blots, we used monoclonal mouse antirabbit NKA isoform-specific antibodies purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat antimouse horseradish peroxidase-conjugate antibody was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA), and used as the second antibody in the detection of the sodium pump isoforms by Western analysis.
Gel electrophoresis was performed on both a minigel and a vertical slab gel electrophoresis units (models SE245, SE260, and SE400) from Hoefer Scientific (San Francisco, CA). Acid gels were dried on a Slab Gel Dryer (SDG 4050) connected to a Savant gel pump (GP100; Farmingdale, NY). Densitometry measurements of the 32Pi-radiolabeled bands were performed on a laser densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). For cross-reactivity studies an ouabain EIA reagent kit was purchased from NEN Life Science Products.
Purification of OLF and dihydro-OLF
The major steps of the OLF and dihydro-OLF purification
procedure from adrenal cortex and human serum are essentially similar
to those described previously (23).
Tissue preparation
Bovine adrenal glands were obtained from Pel-Freeze Biologicals
(Rogers, AR) or were provided by a local abattoir. The cortexes were
separated from medulla, sliced, chopped, homogenized, and centrifuged
three times at 34,000 x g for 30 min at 4 C. The
proteins were precipitated by incubating the supernatants with 1%
5-sulfosalicylic acid at room temperature for 60 sec with continuous
stirring, followed immediately by adding an excess of
CaCO3 until a pH of 5.2 was reached. This extract
was then centrifuged at 80,000 x g for 10 min at 4 C,
followed by vacuum filtration using two layers of Whatman
no. 1 filter paper. Initial purification was performed by
C18 reverse phase, solid phase Sep-Pak extraction
cartridges (Vac 10 cc). The cartridges were primed with 1 vol
CH3CN followed by rinsing with 2 vol deionized
H2O. The supernatant was passed through the
cartridge twice at a rate of 1 ml/min. The cartridge was then washed
twice with 2–4 vol H2O (typically 20–30 ml)
before the compounds of interest were eluted with 20 ml 10%
CH3CN. To remove the CH3CN,
the eluates were evaporated to dryness in a vacuum desiccator,
reconstituted in H2O, and filtered through a
0.22-µm pore size filter from Whatman for removal of
particulates.
The Y-1 cells were grown in DMEM supplemented with 10% FBS (heat
inactivated) plus antibiotics. Four tissue culture flasks (75 mm) of
Y-1 cells at 2.5 x 106 cells/flask were
seeded and grown to 50–70% confluent density (at 37 C, 5%
CO2, 
48–56 h). The cells were then washed
twice with PBS, followed by adding fresh DMEM/Ham’s F-12 medium
without FBS to the cells, and the incubation was continued for
additional 48 h. It was estimated that Y-1 cells double in number
in approximately 30 h. At the end of the incubation, approximately
3 x 107 cells were centrifuged, and the
medium was collected and stored frozen at -80 C before analysis.
Before further use, the medium was extracted similarly to that of the
bovine adrenal homogenate using primed C18
reverse phase, solid phase Sep-Pak extraction cartridges. Further
chromatographic separation is described below.
Reverse phase HPLC
The HPLC separation was accomplished in two steps. In the first
step, the reconstituted eluates were fractionated using a linear
gradient of 20–80%CH3CN in
H2O, and 1 ml fraction/min was collected over 40
min, evaporated, and reconstituted. In the second chromatographic step,
fractions of interest eluting at 6 min (from the linear gradient) were
further chromatographed using an isocratic mode of 10% mobile phase
CH3CN in H2O for 40 min to
separate OLF, OLF-genin, and dihydro-OLF from each other. The purified
fractions (eluting, respectively, at 20, 24.5, 27.5, and 30 min) were
analyzed and measured by EIA for both ouabain and dihydroouabain
activities, and the absorbances at 220 and 196 nm were also
monitored.
Purification of human serum OLF and dihydro-OLF
Digoxin-free fresh-frozen human plasma obtained from the
American Red Cross (Louisville, KY) was treated as previously described
(23).
Molar absorptivity and concentration of OLF and dihydro-OLF
UV spectral properties, molar absorptivity, and concentrations
of these endogenous factors were calculated according to the method of
Qazzaz et al. (21). At their individual maximum absorbance
wavelengths, we assumed the molar absorptivities of OLF and dihydro-OLF
to be the same as those of ouabain and dihydroouabain (Dho-B),
respectively. Using the percent cross-reactivities of OLF and
dihydro-OLF obtained by EIA of ouabain or dihydroouabain, the apparent
molar immunoreactive concentrations of both molecules were determined.
From these data the percent cross-reactivities of OLF and congeners
with their respective antibodies were obtained.
Immunoreactive OLF and dihydro-OLF measurements
OLF was measured using ouabain EIA. This assay uses ouabain
covalently bound to the microtiter plate to compete with unbound
samples or standards for binding to a constant amount of antiouabain
antibody (24). Dihydroouabain-like immunoreactivity was measured by EIA
using a polyclonal dihydroouabain-specific antibody prepared according
to our specifications by HTI Bio-Product, Inc. (Ramona, CA).
Dihydroouabain was conjugated to keyhole limpet hemocyanin through the
rhamnose sugar ring, and the resulting conjugate was injected into
rabbits. The EIA was based on the competitive binding of bound with
free (unbound) dihydroouabain or sample to a constant amount of
antibody. Bound dihydroouabain-antibody complex was detected using a
secondary antibody enzyme conjugate (goat antirabbit horseradish
peroxidase conjugate). The breakdown of TMB substrate by the conjugated
enzyme resulted in a color change with an intensity inversely
proportional to the amount of dihydroouabain or dihydro-like factor in
the well. Color development was allowed for 30 min, after which the
reaction was stopped with TMB Stop buffer, and the plate was then read
at 450 nm. Our immunoasssays have a lower limit of quantitation of 250
pg/ml for OLF and 1000 pg/ml for dihydro-OLF.
Sodium pump catalytic activity inhibition assay
This assay was used to measure the effect of OLF and its
congeners on phosphate release in hydrolysis of ATP (25). The
inhibition assay was performed briefly by pipetting 20 µl sample
containing the desired concentration of glycoside (Tris buffer was used
for no inhibitor control) into a well of a microtiter plate placed in a
37 C water bath for 10 min. As a source of -subunit (containing
three -isoforms) of NKA (12), 20 µl of the porcine cerebral
cortical NKA solution diluted in Tris buffer, pH 7.8 (1 mg/ml), were
added, and a further 20-min incubation was performed. Twenty
microliters of ATP solution (10 mM in Tris buffer, pH 7.8)
were added and allowed to react for 15 additional min. After the
incubation period, we added 150 µl molybdate solution (per liter: 1.0
mmol molybdate, 11 mmol sulfuric acid, and 142 ml Tween-80/methanol
solution; 12:88, vol/vol). After 30 min of incubation, color
development was allowed to proceed for a maximum of 30 min, after which
the color intensity was measured at 340 nm. The color intensity is
proportional to the release of phosphate ions, which is a direct
indicator of ATP breakdown and therefore NKA activity. Duplicate
samples were corrected for background (assay buffer only), averaged,
and normalized to ouabain-sensitive NKA activity (100% inhibition at 1
mM ouabain). The percentage of NKA activity inhibition of
each compound represents the proportion of ouabain activity that is
inhibitable by that compound. The statistical analysis including a
best-fit by logit regression curves to determine the concentration of
inhibitor required for 50% inhibition (IC50) was
performed on SPSS for Windows, advanced statistical program version 7.5
(SPSS, Inc., Chicago, IL).
Ouabain-stimulated 32Pi phosphorylation of
NKA
Ouabain-stimulated phosphorylation of NKA by Pi was performed as
previously described (26). Acidic pH gel electrophoresis was used to
resolve the radioactively labeled protein species. A positive control
was preincubated with 20 µl of a 1 mM ouabain solution,
and a negative control was preincubated with 20 µl buffer alone. Ten
microliters of 32Pi (1 mCi in 100 µl) were
diluted with 250 µl phosphoric acid and purified through a 0.2-µm
pore size filter to remove polyphosphates. At the end of the
incubation, 20 µl of the 32Pi filtrate (8
µCi) were added to the mixture and allowed to incubate at room
temperature for an additional 15 min. The reaction was terminated by
8% HClO4, and the sample was then immediately
pelleted and resuspended in sample buffer containing (final
concentration) 0.5% (by volume) HClO4, 2.5% (by
weight) SDS, 10% (by volume) glycerol, and 0.1% (by weight) pyronin
Y, a bacterial ferric chloride complex stain. Samples were loaded onto
a 12.5% acid polyacrylamide gel and run at 4 C for 4–5 h at a
constant current of 30 mA. The gels were fixed in 40%
methanol-10% acetic acid, dried, autoradiographed, and quantitated
using a soft laser scanning densitometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that this condition separates
four well resolved chromatographic peaks corresponding to ouabain (at
30 min), ouabagenin (at 20 min) and two newly reported isomers of
dihydroouabain (Dho-A and Dho-B), which elute at 24.5 and 27.5 min,
respectively (27). Of significance is that the mammalian adrenocortical
OLF (ouabain immunoreactivity), which eluted at 6 min in the linear
gradient, when rechromatographed using 10% CH3CN
HPLC conditions further separates into three main congeners (see Fig. 2B): one previously identified as OLF, one as OLF-genin (20), and a new
species we now call dihydro-OLF, with properties almost identical to
one (Dho-B) of the two recently identified isomers of plant
dihydroouabain (see Fig. 2A) (28). This new dihydro-OLF species was
also evident in human serum (Fig. 2C). Interestingly, cultured mouse
adrenal Y-1 cells (Fig. 3) also produced
these HPLC fractions, indicating active in vivo synthesis of
these compounds (OLF and dihydro-OLF) in these cells. The cell pellet
was examined for the presence of digoxin and/or ouabain
immunoreactivity. Data showed clearly that Y-1 cells at 70% confluence
secreted most (90%) of their mammalian cardenolides and steroids
into the medium. Therefore, the medium only was collected for further
use. As control, neither the fresh medium (without incubating with Y-1
cells) nor the medium taken after 10 min of incubation with the 70%
confluent Y-1 cells showed detectable immunoreactivity to ouabain or
dihydroouabain. Note that the elution profiles are monitored by
immunoreactivity using antibodies to both ouabain and dihydroouabain
and also by absorbance at 220 and 196 nm for OLF and dihydro-OLF,
respectively.
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max at 196 nm) and
ouabain (max at 220 nm; Fig. 4, A and B). The 196 nm peak
absorbance observed for dihydro-OLF is consistent with the presence of
a chemically-reduced lactone ring (Fig. 1
), including the far UV shift
and lower absorptivity compared with the oxidized form. Both properties
are characteristic of the hydrogenated lactone ring. To estimate the
molar concentrations of the two species, OLF and dihydro-OLF, we
assumed identical molar absorptivities between OLF and dihydro-OLF and
those of the plant-related ouabain and dihydroouabain (Dho-B),
respectively. We also assumed similar extraction efficiencies of
dihydro-OLF and OLF. Table 1 shows the
amounts and relative concentrations of dihydro-OLF and OLF in bovine
adrenal cortical tissue and in human serum.
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), we used the molar
absorptivities to determine the concentrations needed to achieve a 50%
response in the assays.
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shows the relative
potencies for inhibition of sodium pump catalytic activity using
porcine cerebral cortex (containing three -isoforms) by ouabain and
dihydroouabain and by OLF and dihydro-OLF. The concentrations of OLF
and dihydro-OLF were determined assuming comparable molar absorptivity
between ouabain and OLF and between dihydroouabain (isomer Dho-B) and
dihydro-OLF. OLF is 10-fold more potent than ouabain, while dihydro-OLF
is 3-fold more potent than dihydroouabain (Dho-B). The order of
inhibitory activity also shows that OLF is 10-times more potent than
dihydro-OLF just as ouabain is more potent than dihydroouabain. Note
that the response curves of the mammalian-derived factors do not
parallel those of the plant-related counterparts, an indication of
possible differences in their binding to the individual
NKA-isoforms.
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-subunit is dependent on the binding of ouabain to its
binding site on the -subunits (7). This phosphorylation is enhanced
specifically by the cardenolides and incorporates Pi covalently into
the same aspartyl amino acid of the - subunit of NKA that is
phosphorylated by ATP during ATP hydrolysis (30). Figure 7 shows phosphorylation of the
-subunits induced by the mammalian-derived OLF and dihydro-OLF
isolated from both bovine adrenals as well as from human serum.
Moreover, the incorporation of 32Pi into the
-subunit, when incubated with OLF and dihydro-OLF, is dependent on
the concentrations of these factors (Fig. 8). In addition, the concentration of
dihydro-OLF required for phosphorylation is consistent with the
concentration range required for NKA catalytic inhibition (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we demonstrate the existence of a new molecular form of OLF in mammals that we term dihydro-OLF. Dihydro-OLF is unique in that it contains a chemically reduced lactone ring and is thus a hydrogenated counterpart to OLF, much like plant-derived dihydroouabain is to ouabain. The concentration of dihydro-OLF is considerably greater than that of OLF in bovine adrenal tissue, human blood, and cultured mouse adrenal Y-1 cells. The fact that both OLF and dihydro-OLF are produced by Y-1 cells indicates that these compounds can be synthesized de novo endogenously. The potency of mammalian dihydro-OLF to inhibit the catalytic activity of and to phosphorylate NKA is lower in specific activity compared with that of mammalian OLF.
The HPLC separation pattern of the mammalian endogenous pair, OLF and
dihydro-OLF, is similar to those of their respective plant-derived
counterparts, ouabain and dihydroouabain. A hydrogenated lactone ring
on dihydro-OLF was confirmed using absorbance spectra and antibody
reactivity. The max of ouabain and OLF are 220
nm, whereas the hydrogenated forms of these molecules (dihydroouabain
and dihydro-OLF) have maximum absorbance at 196 nm. The UV shift in
max observed for dihydro-OLF is consistent
with the double bond of the lactone ring being chemically reduced (32).
Two antibodies, one specific for ouabain and the other specific for
dihydroouabain and both sensitive to the lactone ring epitopes of these
molecules, indicate the presence of a reduced lactone ring on
dihydro-OLF. The ouabain antiserum has 100% cross-reactivity to
ouabain and less than 2% cross-reactivity to dihydroouabain.
Similarly, the dihydroouabain antiserum has 100% cross-reactivity to
dihydroouabain and less than 0.1% cross-reactivity to ouabain. The
HPLC elution pattern shows a separate immunoreactive peak that is not
ouabain-like, but is dihydroouabain-like by antibody reactivity and by
having an absorbance spectra characteristic of a cardenolide with a
reduced lactone ring. In addition, we detected both OLF and OLF-genin
(which has 60% cross-reactivity to the ouabain antiserum) as two
separate immunoreactive peaks eluting from the HPLC at 20 and 30 min,
respectively, on the same isocratic mode of 10%
CH3CN in dH2O for the
mobile phase. This finding is consistent with our previous report
identifying an OLF-genin species from mammalian tissues (23). It is of
interest to note that no detectable level (100 pg/ml) of
dihydroouabain-immunoreactive material comparable in HPLC elution
to the Dho-A fraction of dihydroouabain was observed in extracts from
any of the mammalian tissue sources used in the present study.
The molar concentrations of dihydro-OLF and OLF can be estimated by
assuming equivalent molar absorptivity at their respective absorbance
maxima between OLF and ouabain (220 nm) and between dihydro-OLF and
dihydroouabain (196 nm). This assumption allows estimation of the molar
immunoreactivity, the tissue extraction efficiency, the molar
concentrations needed to inhibit the catalytic activity (potency) and
enhance phosphorylation of the -subunit of the sodium pump, and the
molar ratio of dihydro-OLF to OLF in different mammalian tissues. The
molar immunoreactivities of OLF and dihydro-OLF are almost identical to
those of ouabain and dihydroouabain. These particular specific
antibodies may have practical use for various clinical studies in
humans.
The amounts of dihydro-OLF extractable from tissues are 0.36 ± 0.34 x 10-10 mol/g adrenal cortical tissue and 50 ± 0.46 x 10-10 mol/liter human serum and are greater than those of OLF by 22- and 13-fold, respectively. These data indicate that adrenocortical tissues contain higher amounts of both of the chemically reduced mammalian cardenolides, dihydro-DLIF (22) and dihydro-OLF (this report), than their respective oxidized species DLIF and OLF. This is of particular interest because if the reduced lactone ring species (e.g. dihydro-OLF and dihydro-DLIF) are acting as precursor substrates for biotransformation to OLF and DLIF, respectively, in adrenals, then one might expect higher amounts of the precursor substrate to accommodate enzymatic regulation of secretion of OLF and DLIF into the blood. Understanding control of the molar ratios of these compounds and their respective oxidized/reduced ratios in blood may be important in view of recent reports showing selective reactivity of mammalian cardenolides with specific individual isoforms of the sodium pump (33). Also, recent observations of specific binding proteins in plasma as carriers of these compounds (34, 35, 36) suggest other potential mechanisms for control of plasma concentrations of these factors. Thus, the relative amounts of these compounds in blood (unbound vs. bound to proteins, etc.) may play a role in their physiological function.
Goto and Yamada (1), in a recent review, documented a wide range of concentrations in human plasma for the ouabain-like factors measured by different immunoassays (e.g. RIA, EIA, and RRA). The reported concentrations of OLF in human plasma within each study ranged widely, for example, 25 pM (37), 34–95 pM (38), 50–750 pM (39), 55–168 pM (40), and 204 pM (41). Our EIA measurements of OLF are very consistent with OLF concentrations reported by others. In any event, the concentration of dihydro-OLF we measured by EIA is higher than any of the OLF concentrations reported to date, which suggests that dihydro-OLF may potentially be a richer source of material for further structural and functional characterization.
It has been difficult to characterize the structural features of these mammalian cardenolides (e.g. OLF and DLIF) because of their low concentrations in human tissues. For example, it is thought that OLF is indistinguishable from ouabain using mass spectroscopy and other related techniques (18, 42). However, more recent studies using an exciton-coupled circular dichroism technique showed that OLF and HIF (an isomer of ouabain from bovine hypothalamus) are structurally different from ouabain (43). In the case of DLIF, there is clear evidence of chromatographic, spectral, and molar immunoreactivity differences that suggests that DLIF is more structurally distinct from digoxin than OLF is from ouabain (21). Nonetheless, sufficient quantities of these compounds are still required to fully permit the identification of fine structure.
We assessed the relative biological activities of OLF and dihydro-OLF
by their abilities to inhibit the catalytic activity and to
phosphorylate the -subunit of NKA. OLF and dihydro-OLF are 10 and 3
times more potent than plant-derived ouabain and dihydroouabain
(Dho-B), respectively (28). Other studies have shown that a chemically
reduced lactone ring on cardenolides decreases their ability to inhibit
sodium pump activity compared with the oxidized state (44). Feng and
Lingrel (44) have shown that the dihydro species of cardiac glycosides
in general have distinctly different inhibitory potencies for
individual NKA -subunits. It is interesting to note that the
concentration-dependent inhibitory responses of the mammalian
cardenolides do not parallel those of the plant-derived compounds. One
explanation for this may be the presence of three distinct isoforms of
the -subunit of NKA in porcine cerebral cortex and the likelihood of
a difference in -subunit isoform-specific interaction with the
cardenolides. Differences in affinity for NKA between the mammalian and
plant cardenolides as well as their oxidized and reduced species are
particularly intriguing because of its implication in targeted
selective regulation of individual NKA isoforms by these compounds as
reported for the plant-derived counterparts (8, 45, 46).
Phosphorylation of the -subunit of NKA is a characteristic feature
of the specificity of interaction between the cardenolides and sodium
pump as receptors. It is well documented that ouabain-stimulated
32Pi phosphorylation of the NKA -subunit is
dependent on the binding of ouabain to its binding site on the
-subunits (7). This interaction is specific to ouabain (and other
related cardenolides), in that the cardenolides do not enhance
phosphorylation of other ATPases, such as the
Ca2+-ATPases. This specific phosphorylation
incorporates Pi covalently into the same aspartyl amino acid of the
-subunit of NKA that is phosphorylated by ATP during ATP hydrolysis
(30). Our studies indicate that phosphorylation of the -subunit by
both dihydro-OLF and OLF is specific and concentration dependent. The
molar concentration of the newly discovered dihydro-OLF needed to
enhance phosphorylation is in a range consistent with the concentration
required for inhibition of NKA catalytic.
Our previous studies using a cytochrome P-450 and NADPH-dependent reductase model isolated from cortical cells demonstrated conversion of dihydrodigoxin to an oxidized species, DLIF. This suggests a metabolic route for endogenous conversion of lactone ring-related oxidation-reduction of these compounds by cortical cells. Those findings taken altogether with our present study marking the discovery of a dihydro species of OLF now provides support for an hypothesis that the oxidized (DLIF and OLF) and reduced (Dh-DLIF and dihydro-OLF) species of these mammalian factors are probably metabolically linked.
Interestingly, extensive review of the literature fails to reveal evidence that the cardiac glycoside dihydroouabain occurs naturally in plants. It is known that the dihydro species of the cardenolides are produced in vitro through hydrogenation of the cyclobutenolide ring of the plant-derived oxidized species using either platinum oxide (room temperature) or palladium-carbon (50 C) as catalysts (47). This chemical modification of plant-derived ouabain yields two isomers of dihydroouabain (Dho-A and Dho-B), each with distinct, but similar, properties. However, as reported previously, Dho-B (IC50 = 1.7 µM) has greater potency than Dho-A (IC50 = 7.4 µM) (25). The dihydro-OLF we have now discovered has properties consistent with one of these two isomers (Dho-B). The production and the secretion of these factors in vitro by cultured Y-1 cells suggest that dihydro-OLF is very likely produced de novo in mammals.
This study demonstrates the existence of a previously unrecognized,
naturally occurring dihydro-OLF in adrenal tissues and serum. The
importance of this discovery lies in its suggestion of a metabolic link
with OLF in the adrenal glands or other tissues and provides a possible
metabolic route for the formation and secretion of OLF into the
circulation. The possibility of in vivo conversion of
mammalian OLF to dihydro-OLF or vice versa seems plausible.
In addition to the enzymatic P-450 metabolic routes discussed above,
there is another route for potential interconversion of the lactone
ring in mammals. This, as depicted in Fig. 9, is modeled from previously reported
data on the conversion of digoxin to dihydrodigoxin by
Eubacterium lentum, a common anaerobic bacteria of the human
gastrointestinal tract microflora (48).
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Acknowledgments |
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Footnotes |
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Received March 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit:
site-directed mutagenesis of glutamine-111 to arginine and
asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.
Biochemistry 27:8400–8408[CrossRef][Medline]
subunit identifies threonine 797 as a determinant of
ouabain sensitivity. Biochemistry 33:4218–4224[CrossRef][Medline]
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 R. I. Dmitrieva and P. A. Doris Cardiotonic Steroids: Potential Endogenous Sodium Pump Ligands with Diverse Function Experimental Biology and Medicine, September 1, 2002; 227(8): 561 - 569. [Abstract] [Full Text] [PDF]
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 P. Manunta, B. P. Hamilton, and J. M. Hamlyn Structure-Activity Relationships for the Hypertensinogenic Activity of Ouabain : Role of the Sugar and Lactone Ring Hypertension, February 1, 2001; 37(2): 472 - 477. [Abstract] [Full Text] [PDF]
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