(Received for publication, July 27, 1995; and in revised form, December 28, 1995)
From the
This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the
quantitatively major share of retinal for retinoic acid (RA) biogenesis
in rat tissues from the predominant substrate available
physiologically, holo-cellular retinol-binding protein, type I (CRBP).
With holo-CRBP as substrate in the absence of apo-CRBP microsomal
retinol dehydrogenases have the higher specific activity and capacity
to generate retinal used for RA synthesis by cytosolic retinal
dehydrogenases. In the presence of apo-CRBP, a potent inhibitor of
cytosolic retinol dehydrogenases (IC =
1
µM), liver microsomes provide 93% of the total retinal
synthesized in a combination of microsomes and cytosol. Cytosolic
retinol dehydrogenase(s) and the isozymes of alcohol dehydrogenase
expressed in rat liver had distinct enzymatic properties; yet ethanol
inhibited cytosolic retinol dehydrogenase(s) (IC
=
20 µM) while stimulating RA synthesis in a combination of
microsomes and cytosol. At least two discrete forms of cytosolic
retinol dehydrogenase were observed: NAD- and NADP-dependent forms.
Multiple retinal dehydrogenases also were observed and were inhibited
partially by apo-CRBP. These results provide new insights into pathways
of RA biogenesis and provide further evidence that they consist of
multiple enzymes that recognize both liganded and nonliganded states of
CRBP.
The metabolism of retinol (vitamin A) generates RA, ()a humoral factor critical to vertebrate
development(1, 2) . In the developing embryo, RA
transcriptionally regulates genes that specify body axis pattern and
may help program limb formation(3) . In mature vertebrates, RA
maintains epithelial tissues (prevents squamous cell metaplasia),
contributes to bone remodeling, and sustains reproductive processes,
including the estrus cycle, spermatogenesis, and placental growth. RA
aberrant in concentration, locus, or developmental stage causes
teratism and/or toxicity of the central nervous system and skeleton (4, 5, 6, 7) . RA acts through
ligand-activated receptors that comprise two distinct subfamilies of
the steroid hormone superfamily of receptors (8, 9, 10, 11) . These receptors
modify transcription as homodimers or by modifying the effects of other
receptors through heterodimerization. These pervasive and fundamental
effects of RA, as well as the consequences of its aberrant
distribution, imply that its biosynthesis must be regulated closely.
In many tissues, unesterified retinol occurs bound to CRBP. Thus, holo-CRBP may provide the most abundant substrate for RA biosynthesis. Direct transfer of retinol between holo-CRBP and enzymes that catalyze RA synthesis would circumvent uncontrolled diffusion of retinol through the aqueous phase and would participate in controlling the pathways of RA synthesis by protecting retinol from opportunistic reactions catalyzed by enzymes that do not recognize the high affinity, high specificity CRBP. Recent work has outlined a pathway of RA biosynthesis with the first step catalyzed by a NADP-dependent microsomal retinol DH, expressed in liver and in extrahepatic tissues, that recognizes holo-CRBP as substrate(12, 13, 14, 15) . Microsomes have low retinal DH activity and do not convert the retinal produced from holo-CRBP into RA at high rates. Rather, microsome-produced retinal undergoes conversion into RA by cytosolic retinal DHs, which can interact with CRBP-retinal complexes(16) .
During initial characterization of
microsomal retinol DH, we also observed cytosolic retinol DH activity
that was inhibited markedly by apo-CRBP and subsequently showed that
the inhibition might not result solely from sequestering free retinol,
because of ambiguities in the K value for
CRBP(12, 17) . Inhibition could result from apo-CRBP
interacting with a cytosolic enzyme that recognized holo-CRBP as
substrate. This suggested the presence of at least two pathways for
generating RA from holo-CRBP: one involving retinal synthesis in
microsomes and the other involving retinal synthesis in cytosol. In
either pathway, subsequent conversion of the retinal into RA occurs in
the cytosol. Ottonello and co-workers have analyzed cytosolic retinol
DH activity further and concluded that it catalyzes considerable RA
synthesis from holo-CRBP in the absence of apo-CRBP (18) . The
relative contributions, however, of microsomes and cytosol to overall
cellular RA synthesis remain uncertain, as well as the interactions
between these two pathways and/or their effects on each other.
This work compares the contributions of microsomal and cytosolic retinol DH activities to RA synthesis and characterizes interactions that occur during RA biosynthesis between cytosol and microsomes. With holo-CRBP as substrate in the absence of apo-CRBP, microsomal retinol DH has the higher specific activity and capacity (60-83% of the total of cytosol plus microsomes, individually) to generate retinal for RA synthesis in the four tissues assayed. In the presence of apo-CRBP, the more likely condition in vivo, the microsomal contribution increased to 80-94% of the total retinal synthesized. In separating the activity of rat liver cytosolic retinol DH from that of the ADH isozymes expressed in rat liver, ADH-2 and ADH-3, we demonstrate further that ethanol inhibits cytosolic retinol DH activity. We also show that at least two forms of cytosolic retinol DH occur and that apo-CRBP inhibits not only cytosolic retinol DH activity but also conversion of retinal into RA. Thus, we establish here that multiple paths and complex interactions contribute to RA biosynthesis.
Figure 7:
Inhibition of cytosolic retinol DH.
IC values were determined for inhibition of cytosolic
retinol DH as described under ``Experimental Procedures'':
pentanol added in Me
SO (filled triangles); octanol
added in Me
SO (open circles); pentanol (open
triangles); and ethanol (filled circles). In the assays
with pentanol and octanol added in Me
SO, 1 µl of
Me
SO was added to each tube. Me
SO was used
because octanol was not miscible with water. Because Me
SO
itself causes some inhibition, pentanol was tested in the presence and
the absence of Me
SO so that the octanol data could be
related to the results with pentanol and ethanol. Each point is the
average of duplicate experiments.
Figure 1: Effects of apo-CRBP on RA synthesis in cytosol and the 10kS. Cytosol or the 10kS (300 and 500 µg of protein, respectively) were incubated with 5 µM holo-CRBP for 60 min with the indicated concentrations of apo-CRBP: RA synthesis by cytosol (filled circles) or 10kS (open circles). The inset shows the x axis expanded up to 1 µM apo-CRBP. In the absence of apo-CRBP, 105 and 97 pmol, respectively, of RA were generated by cytosol and the 10kS fraction.
In contrast to cytosol, 1 µM apo-CRBP did not inhibit RA synthesis by the 10kS, which includes cytosol and microsomes, and 50% inhibition of the 10kS required 5 µM apo-CRBP, a concentration equal to that of the holo-CRBP substrate. Lack of inhibition of RA synthesis in the 10kS by 1 µM apo-CRBP, a concentration that inhibited cytosolic RA synthesis >50%, suggests that cytosolic retinol DH activity was not contributing substantially to RA synthesis in the 10kS, i.e. in the presence of microsomes. The relatively apo-CRBP-resistant microsomal retinol DH, therefore, must have been providing retinal for RA synthesis by the cytosolic retinal DH component of the 10kS. Inhibition of RA synthesis in the 10kS by higher concentrations of apo-CRBP may result from a combination of inhibition of the microsomal retinol DH (12) and a decrease in the conversion of retinal into RA by cytosol (see below).
Figure 2: Time course of retinoid synthesis supported by holo-CRBP. Top panel, retinal synthesis by microsomes. Bottom panel, RA synthesis by cytosol (circles) or by a combination of cytosol and microsomes (diamonds). Experiments were done with 5 µM holo-CRBP in the absence (open symbols) or the presence (filled symbols) of 2 µM apo-CRBP. In each case 0.5 mg of cytosolic and/or 0.1 mg of microsomal protein were used.
In the presence of 2 µM apo-CRBP (Fig. 2, top panel, filled symbols), the
amounts of retinal produced by microsomes alone were comparable
quantitatively throughout the incubation to the retinal produced by
microsomes in the absence of apo-CRBP. RA production by cytosol,
however, was below detection limits before 30 min, when it reached a
plateau, which on the average was only 13% of the amount of RA observed
in the absence of apo-CRBP (Fig. 2, bottom panel). In
the combination of microsomes and cytosol, retinal concentrations
reached a plateau of 30 pmol and declined after 60 min (data not
shown). RA production by the combination increased throughout the
incubation and at all times exceeded RA production by cytosol alone,
even though the microsomal protein added to the combination was 5-fold
less than that of the cytosolic protein present.
The specific
activities of microsomal and cytosolic retinol DHs were also compared
in the absence (open symbols) and the presence (filled
symbols) of apo-CRBP (Fig. 3). Microsomal retinol DH was
linear to 0.1 mg of protein and generated retinal from 5 µM holo-CRBP at rates of 23 pmol/min/mg protein in the absence
of apo-CRBP and
15 pmol/min/mg protein in the presence of 2
µM apo-CRBP. In the absence of apo-CRBP, cytosolic retinol
DH activity was linear to 0.25 mg of protein and functioned at a rate
of
4 pmol/min/mg of protein, i.e.
6-fold less than
the rate due to microsomal retinol DH. In the presence of 2 µM apo-CRBP, cytosolic retinol DH activity was not observed below
0.25 mg of protein but was linear between 0.25 and 1 mg of protein. The
rate was
0.33 pmol/min/mg of protein or
50-fold less than
that of microsomal retinol DH in the presence of apo-CRBP.
Figure 3: Retinol metabolism supported by holo-CRBP versus protein concentration. Top panel, retinal production by microsomes. Bottom panel, RA production by cytosol. Incubations were done for 30 min with 5 µM holo-CRBP in the absence (open symbols) or the presence (filled symbols) of 2 µM apo-CRBP.
Figure 4: Effect on RA and retinal syntheses of titrating microsomes into cytosol. Top panel, RA synthesis; bottom panel, retinal synthesis. Incubations were done with 5 µM holo-CRBP in the absence (open symbols) or the presence of 2 µM apo-CRBP (filled symbols) for 30 min with 0.35 mg of cytosolic protein and the indicated amount of microsomal protein.
To test the possibility that apo-CRBP inhibits the conversion of retinal into RA, cytosolic RA production was monitored from retinal bound to CRBP in the presence of increasing concentrations of apo-CRBP (Fig. 5). An apo-CRBP concentration 50% of the CRBP/retinal concentration inhibited <20% (filled circles). An apo-CRBP concentration equal to the CRBP/retinal concentration of 0.5 µM caused the maximum inhibition of 50%. Apo-CRBP up to 9-fold greater than CRBP/retinal resulted in no additional inhibition. These data indicate that CRBP/retinal acted as substrate for RA synthesis, because the concentrations of unbound retinal and the rate of RA synthesis did not coincide, i.e. the rate of retinal synthesis was not dependent on the concentration of unbound retinal. The decrease in RA synthesis with increasing apo-CRBP, therefore, was caused by inhibition of retinal dehydrogenation by apo-CRBP. This phenomenon probably contributes to the inhibition of RA synthesis in the 10kS by the higher concentrations of apo-CRBP (see above).
Figure 5:
Effect of apo-CRBP on the conversion of
retinal into RA by cytosol. Assays were done with 0.5 mg of cytosolic
protein, 0.5 µM CRBP/retinal, and the indicated
concentrations of apo-CRBP for 30 min: RA measured (filled
circles); retinal concentrations calculated (open
circles). Retinal concentrations were calculated using a K of 100 nM for the affinity
between retinal and CRBP(25) . 50 pmol of RA were generated in
the absence of apo-CRBP.
Because ethanol inhibits cytosolic
retinol DH but not microsomal retinol DH (see below), it was used to
provide further insight into the contribution of retinal generated by
cytosol to RA synthesis (Fig. 6). Ethanol (hatched
bars) inhibited by 90% cytosolic RA synthesis supported by
holo-CRBP (compare bars 1 and 2) but stimulated
40% retinal synthesis in microsomes (bars 3 and 4) and in a combination of microsomes and cytosol (bars 5 and 6). These data indicate that ethanol inhibits the
cytosolic retinol DH, but neither ethanol nor its metabolite
acetaldehyde inhibits the cytosolic retinal DHs and also that cytosolic
retinal DHs do not discriminate against cytosolic or microsomally
produced retinal. Most importantly, these data show that in the
combination of cytosol and microsomes, inhibition of cytosolic retinol
DH does not diminish RA synthesis.
Figure 6: Effect of ethanol on retinol metabolism in cytosol, microsomes, and a combination of cytosol and microsomes. Cytosol (bars 1 and 2, 0.2 mg of protein) or a combination of microsomes and cytosol (bars 5 and 6, 0.1 and 0.2 mg of protein, respectively) were assayed for RA synthesis or microsomes were assayed for retinal synthesis (bars 3 and 4, 0.1 mg of protein) from 5 µM holo-CRBP in the absence of ethanol (open bars) or in the presence of 250 mM ethanol (2.5% v/v; striped bars) for 30 min. The error bars represent S.D. of 4 replicates. *, all of the ethanol-added groups were significantly different from the control without ethanol (p < 0.001).
The interactions of
these compounds with ADH-2 and ADH-3, the two isozymes of the medium
chain alcohol dehydrogenases expressed in rat liver cytosol, were
compared with their effects on microsomal and cytosolic retinol DH
activities (Table 2). ADH-3 is far more sensitive to
4-methylpyrazole than is either retinol DH. Even though the
sensitivities of cytosolic retinol DH and ADH-2 to 4-methylpyrazole are
similar, carbenoxolone had no impact on ADH-2, thereby distinguishing
cytosolic retinol DH and ADH-2. Carbenoxolone did not inhibit ADH-3
either. Unlike the case with retinol DHs, apo-CRBP also did not depress
ADH-2 or ADH-3 activity in concentrations up to 10 µM. The
differences in the K values of ethanol, pentanol,
and octanol for ADHs and their IC
values for cytosolic
retinol DH further indicate that the structure-function properties of
cytosolic retinol DH and ADH are readily distinguishable.
The generation of RA in cytosol from holo-CRBP with NADP as the sole cofactor indicated that NADP alone supports cytosolic retinal synthesis. Consistent with this, cytosolic preparations without the NADP-supported cytosolic retinol DH activity, as well as cytosol with the NADP-supported cytosolic retinol DH activity, converted retinal into RA with NADP as sole cofactor (Table 4). Ethanol had a modest or no effect on the rates of RA synthesis from CRBP/retinal.
This work demonstrates the primary contribution
quantitatively of microsomal retinol DH to RA biosynthesis relative to
cytosolic retinol DH activity. Not only was microsomal retinol DH
severalfold greater in specific activity than cytosolic retinol DH
activity in a comparison of subcellular fractions from four rat
tissues, it had a greater capacity to generate retinal (the majority of
enzyme units) and was relatively resistant to inhibition by apo-CRBP.
Most revealing were the data showing that cytosolic retinol DH could be
inhibited in a mixture of cytosolic retinol DH and microsomal retinol
DH without decreasing the production of RA ( Fig. 1and Fig. 6). For example, 1 µM apo-CRBP inhibited
cytosolic retinol DH 50% but had no effect on the amount of RA produced
by the 10kS fraction, whereas ethanol caused 90% inhibition of
cytosolic retinol DH but enhanced RA production in a combination of
cytosol and microsomes, similar to its effect on retinal synthesis in
microsomes. These results are consistent with either cytosolic retinol
DH not functioning in the presence of microsomal retinol DH or
microsomal retinol DH compensating for the quantitatively minor
production of retinal by cytosolic retinol DH in cases of cytosolic
retinol DH inhibition. Notably, under conditions likely to prevail
physiologically, i.e. a mixture of holo-CRBP and apo-CRBP with
concentrations in the range of 5 and
2 µM,
respectively, microsomal retinol DH accounted for >90% of the
retinal-generating capacity in three of the four tissues screened.
Because there is a RA response element in the CRBP gene (28) and RA induces CRBP expression in vivo(29) , Ottonello et al. have proposed that generation of CRBP by RA may provide apo-CRBP as a signal in a feedback loop to inhibit RA synthesis from cytosolic retinol DH(18) . This hypothesis has limitations. Firstly, CRBP may affect the amount of retinol sequestered by cells; as CRBP increases, the concentration of holo-CRBP could increase as long as plasma retinol were available(30) . Generation of CRBP, therefore, does not necessarily result in elevated concentrations of apo-CRBP. It seems more reasonable that RA acts as an on/off signal inducing constitutive expression of CRBP rather than acutely regulating relatively modest changes in the concentration of CRBP. The ``feedback'' loop hypothesis encounters another problem during vitamin A depletion. During times of less than optimal blood and cell retinol concentrations, RA synthesis would need to continue efficiently to generate the active humoral agent, even though substrate concentrations were diminishing. Potent inhibition of cytosolic retinol DH during vitamin A depletion, when apo-CRBP concentrations would increase, appears to be counterproductive to RA generation at a time when RA generation would be needed.
Lack of an obvious role for cytosolic
retinol DH in RA generation, because it would be inhibited under normal
conditions by the apo-CRBP present, doesn't imply cytosolic
retinol DH has no role; perhaps cytosolic retinol DH reduces retinal
generated by -carotene metabolism into retinol. Such a function
would make sense with respect to the sensitivity of cytosolic retinol
DH to inhibition by apo-CRBP. If cytosolic retinol DH were a reductase,
inhibiting it during vitamin A depletion would provide increased
retinal from
-carotene for conversion into RA. Additional work
will ultimately address these issues.
This and previous works have
demonstrated unequivocally that microsomal retinol DH does not belong
to the medium chain ADH
family(12, 13, 14, 15) . This work
has also distinguished between the two known rat liver ADH isozymes and
cytosolic retinol DH. Differences in affinities for apo-CRBP,
carbenoxolone, and 4-methylpyrazole distinguish cytosolic retinol DH
and ADH-3, whereas differences in affinities for apo-CRBP,
carbenoxolone, and the short chain alcohols (ethanol, pentanol, and
octanol) distinguish cytosolic retinol DH and ADH-2. These results are
consistent with our previous results obtained using 4-methylpyrazole to
study RA synthesis from retinol not bound with CRBP in cytosols
prepared from rat tissues and from the tissues of ADH and ADH
deermice(31, 32, 33) . 4-Methylpyrazole
inhibited potently (>94%) the conversion of unbound retinol into RA
catalyzed by liver cytosol from rat or ADH
deermouse.
In cytosol from the ADH
deermouse, however, which
showed only 13% of the retinol DH activity of the ADH
deermouse, 4-methylpyrazole was a much less effective inhibitor.
Thus, these previous data had demonstrated the presence of a
quantitatively minor retinol DH activity in liver cytosol that was not
affected markedly by 4-methylpyrazole. The present data suggest that
the cytosolic retinol DH activity that recognizes holo-CRBP as
substrate substantially accounts for this 4-methylpyrazole-insensitive
DH. The earlier report by Ottonello et al.(18) had
concluded that cytosolic retinol DH was distinct from ADH, in part
because 1 mM pyrazole did not inhibit cytosolic retinol DH;
however, 1 mM pyrazole does not inhibit ADH-2(24) . On
the other hand, carbenoxolone inhibition characterizes several short
chain dehydrogenases/reductases but not the medium chain
ADHs(26, 27) . Whether cytosolic retinol DH, like
microsomal retinol DH, belongs to the short chain superfamily remains
to be established.
There has been some speculation that an ADH
isozyme may contribute to RA biosynthesis, in addition to metabolizing
various xenobiotic and endogenous long and short chain alcohols. The
proposed isozyme has been revised, however, as evidence has eliminated
candidates. There are convincing arguments against this hypothesis,
however, that include the requirements for control and specificity in
RA biogenesis; the occurrence of holo-CRBP as the predominant substrate
available in vivo; the ability of cytosol of the
ADH deermouse mutant to convert retinol into
RA(32, 33) , and the expression of distinct enzymes
(microsomal and cytosolic retinol DHs) with extraordinary built in
specificity, i.e. they recognize holo-CRBP as
substrate(12, 13, 14, 15) .
Ethanol inhibits cytosolic retinol DH potently despite differences
between cytosolic retinol DH and the known liver ADH isozymes. Thus,
this is the first demonstration of ethanol inhibition of a retinol DH,
identified by its affinity for holo-CRBP (0.4 µMK, rat liver cytosolic retinol DH, this work;
0.8 µMK
, calf liver cytosolic
retinol DH, (18) ). The impact, if any, however, of ethanol
consumption on the retinoid humoral system cannot be predicted from
this observation, especially because ethanol stimulates RA biogenesis
in microsomes and in a combination of microsomes and cytosol. Certainly
chronic excessive ethanol consumption decreases hepatic vitamin A
stores(34, 35) , and alcoholism leads to testicular
atrophy and loss of testes function (36, 37, 38) , similar to vitamin A
deficiency(39, 40) . Because of these observations,
the possibility has been proposed that the medium chain ADHs catalyze
retinol metabolism in vivo and thereby ethanol may
competitively inhibit retinol metabolism(34, 35) . For
the reasons discussed above, the potential involvement physiologically
of an ADH isozyme in retinol metabolism remains in question. But our
data suggest an alternative model, namely, that a cytosolic retinol DH,
which is not a known ADH, is present and is inhibited by low
concentrations of ethanol.
Another significant result was the variability among different batches of rat liver cytosol in the NADP-supported cytosolic retinol DH-catalyzed reaction in contrast to the constancy of the NAD-supported reaction. This suggests two or more cytosolic retinol DHs occur, an NAD-dependent enzyme and a NADP-dependent enzyme. More potent ethanol inhibition of the NAD than the NADP-dependent activity (80 versus 30% inhibition, respectively, with 2% ethanol) supports the occurrence of two distinct enzymes.
Our data also show that either NAD or NADP can support the
conversion of retinal into RA. Three isozymes with retinal
dehydrogenase activity have been separated by anion exchange
chromatography of cytosol from rat liver. The major one, representing
67% of the total liver cytosolic NAD-dependent units, has been
purified and does not use NADP as cofactor(16) . The other
isozymes, and additional isozymes resolved chromatographically from
kidney and testis, have not been tested with NADP and may not differ
from the NADP-dependent activity observed here. Nevertheless, this
result, along with the observation of cytosolic retinol DH
multiplicity, clearly broadens the scope of enzymes and/or pathways
involved in RA biogenesis.
In summary, several new insights have resulted from this work. The quantitatively dominant contribution has been demonstrated of microsomal versus cytosolic retinol DH to RA biogenesis, including the ability of microsomal retinol DH to supplant retinal produced by cytosolic retinol DH. At least two cytosolic retinol DHs have been demonstrated: a NAD-dependent DH and a NADP-dependent DH. Intriguingly, ethanol in micromolar concentrations inhibits cytosolic retinol DH-catalyzed RA synthesis, despite the differences between cytosolic retinol DH and the known ADHs, and stimulates RA synthesis by microsomes or a combination of microsomes and cytosol. Finally, the complex nature of retinal DHs has been confirmed and extended. It is becoming more apparent that RA biogenesis involves intricate metabolic pathways that involve multiple enzymes that recognize both liganded and nonliganded forms of CRBP.