(Received for publication, September 6, 1994; and in revised form, January 18, 1995 )
From the
We report here that, like nonheme iron, protein-bound
intracellular heme iron is also a target for destruction by
endogenously produced nitric oxide (NO). In isolated
rat hepatocytes
NO synthesis results in substantial
(approximately 60%) and comparable loss of catalase and cytochrome P450
as well as total microsomal heme, and decreased heme synthetic
(
-aminolevulinate synthetase and ferrochelatase) and increased
degradative (heme oxygenase) enzymatic activities. The effect is
reversible, and intact cytochrome P450 apoproteins are still present,
as judged by heme reconstitution of isolated microsomes. The effects on
-aminolevulinate synthetase and heme oxygenase are likely to be
secondary to heme liberation, while the effects on ferrochelatase
appear to be a direct effect of
NO, perhaps
destruction of its nonheme iron-sulfur center.
Nitric oxide (NO) is produced by specific
mammalian enzymes either in small quantities as a neural or vascular
messenger or in large quantities by certain cells of the immune system
as an effector where it is cytostatic/cytotoxic to many pathogenic
organisms and also to infected or transformed host
cells(1, 2) . Intracellular iron is a major target of
NO as an immune effector, and previous studies have
described loss of nonheme iron-containing enzyme
activities(1, 2) . Although heme iron is also a well
established target for exogenous
NO(3) , few
studies have demonstrated deleterious effects of endogenous
NO synthesis on intracellular heme-containing enzymes.
Recently it has been found that rat hepatic
NO
synthesis results in a decrease in both activity and levels of
cytochrome P450 (CYP)(
)(4, 5) .
Understanding the mechanism by which endogenous
NO
inhibits heme-containing proteins is likely to explain such important
phenomena as decreased drug tolerance in septic patients(6) .
We describe here one basis for this phenomenon, which involves
NO-induced intracellular heme loss and increased heme
degradation.
Catalase is a heme-containing enzyme that catalyzes the
dismutation of HO
to O
and
H
O. As shown in Fig. 1A, treatment of
hepatocytes with CME (which we have shown previously to induce
substantial
NO synthesis(9) ) results in a
decrease followed by gradual recovery of catalase activity. The minimum
activity (65% loss) occurred at approximately the same time as the
maximum rate of
NO synthesis, as we have shown
previously(20) . Inhibition of
NO synthesis by
NMMA results in oblation of catalase activity loss, demonstrating that
it is due to
NO production. This result raises the
possibility that inhibition of catalase may be a previously
unrecognized mechanism of
NO-induced potentiation of
oxidative injury(21) .
Figure 1:
Effects of endogenous and exogenous NO on catalase, CYP heme, and total microsomal heme
levels. Hepatocytes were isolated and treated and catalase, microsomal
protein, CYP heme, and total extractable heme were determined as
described under ``Experimental Procedures.'' A,
effects of endogenous
NO on catalase activity.
Hepatocytes were incubated in the absence (
) or presence
(
) of CME or of CME + NMMA (
), and at the indicated
times catalase activity was determined as described under
``Experimental Procedures.'' B, effects of
endogenous
NO on CYP and total microsomal heme.
Hepatocytes were incubated without or with CME, NMMA, or SNAP as
indicated (as described under ``Experimental Procedures'')
and assayed for total microsomal protein, CYP heme, and total
extractable heme. C, effects of
NO on CYP
heme in isolated microsomes after various times of exposure, as
described under ``Experimental Procedures'': I = 0; II = 20 min; III = 60
min. Spectrum IV is free heme (1 µM)-CO complex. D, heme reconstitution of
NO-induced loss of
CYP heme. Treatment of hepatocytes, preparation of microsomes, and
dialysis without (a) or with (b) heme were as
described under ``Experimental
Procedures.''
The CYPs are heme-containing enzymes
that catalyze the metabolism of a variety of endogenous and exogenous
compounds. We chose this class of enzymes for further studies on the
effects of NO because they represent the major heme
pool in hepatocytes(22) , and inactivation can be monitored by
the reduced versus reduced + CO difference spectrum of
the microsomal fraction(23) . Fig. 1B shows the
effects of endogenous and exogenous
NO on microsomal
CYP heme and total extractable microsomal heme (13) in cultured
rat hepatocytes. Exogenous
NO exposure was
accomplished by using SNAP, an
NO donor. Compared with
control cells there is an approximately 60% decrease in basal
(uninduced) CYP heme induced by either endogenous or exogenous
NO, as reported previously for inducible CYP
heme(4, 5) . CYP heme loss is prevented by inhibition
of
NO synthesis (NMMA). The parent compound of SNAP (N-acetylpenicillamine, not containing a nitrosyl group) had
no effect (data not shown); that was also true for all other studies
below with SNAP. In order to determine whether this effect is due to
prevention by
NO of CO binding to heme (thus masking
the appearance of the 450 nm peak(4) ) or instead loss of CYP
protein and/or heme, we assayed total extractable microsomal heme. Fig. 1B shows that both endogenous and exogenous
NO cause a loss of total microsomal heme that is
quantitatively indistinguishable from CYP heme loss.
Having
established that the effect of NO is due to decreased
holocytochrome P450 (and not prevention of CO binding), we next
determined whether this effect is due to a decrease in CYP protein
expression (as reported by Stadler et al.(5) for
inducible CYP) or to loss of enzyme-bound heme. We first determined if
exposure of microsomes from untreated hepatocytes to
NO induces loss of heme. As shown in Fig. 1C, exposure of microsomes from untreated
hepatocytes (I) to
NO for 20 min (II)
results in partial conversion of P450 into P420, indicative of sulfur
to nitrogen exchange of proximal axial heme ligand (23) . From
comparison with the spectrum of free heme (IV), after a further
40-min exposure (III) it can be concluded that a substantial
portion of CYP heme is fully detached from its normal binding in the
proteins, as judged by the appearance of the peak at 405
nm(24) . Therefore it appears that NO exposure induces P450 to
P420 conversion followed by release of heme from CYP.
In order to
determine whether a decrease in CYP is due to decreased CYP protein or
loss of heme, we determined whether CYP apoprotein is present after
loss of heme. We determined this by isolating microsomes from NO-synthesizing hepatocytes (or from hepatocytes
treated with SNAP), dialyzing the preparations versus heme,
and then measuring the reduced versus reduced plus CO spectrum
of the preparations to determine whether heme had reinserted into
apocytochrome P450 (Fig. 1D). This method has been used
successfully for reconstitution of several cytochrome P450s subsequent
to several methods of inducing heme loss/destruction, including
P450
(25) , P450
(26) , as
well as the system studied here, rat hepatic microsomal cytochrome
P450(27, 28) . Numerous studies have documented that
the characteristic peak at 450 nm (for which these enzymes are named)
is due to proximal cysteine ligation to the heme and that conversion to
nitrogenous proximal heme ligation results in a shift in the peak to
420 nm with consequent loss of activity(23) . As shown in
spectra I, dialysis versus heme of microsomes from
control hepatocytes did not result in appreciable effects on the total
CYP heme and resulted in no appreciable increase in either P420 or P450
heme absorption, as was also true for hepatocytes treated with the
cytokine mixture plus NMMA (III). As shown in II,
however, the substantial loss of CYP in microsomes from
NO-synthesizing hepatocytes was restored by dialysis versus heme. Spectra IV show an effect with
SNAP-treated cells that is similar to
NO-producing
cells, demonstrating that either endogenous or exogenous
NO induces comparable effects. These results
demonstrate that, in contrast to CYP induced by xenobiotic exposure (5) , the virtually exclusive effect of
NO on
basal CYP is loss of the heme prosthetic group with no appreciable
decrease in CYP protein. In addition, the CYP apoproteins are left in a
relatively native conformation since P450 (not P420) is reconstituted
by heme. We note that while observation of the feature at 450 nm
demonstrates that heme has indeed reinserted into the apoproteins (thus
showing that CYP apoproteins are present), we have not attempted to
determine whether dialysis versus heme results in
reconstitution of any specific P450 activity because different CYP
isoforms may be differently affected by
NO (as
described below).
We next determined whether intracellular heme
levels in NO-producing hepatocytes change as a result
of liberation from CYP, which is known to be the major heme pool in
these cells(22) . Previous studies have demonstrated
NO-induced loss of total cellular iron from cells
(including hepatocytes(29) ), although no differentiation of
heme versus nonheme iron has yet been
made(1, 2) . As shown in Fig. 2A,
there is a selective loss of whole cell total extractable heme iron
upon induction of
NO synthesis with CME, which is
prevented by NMMA. Similar loss occurs with SNAP-treated cells. No
significant change in total cellular nonheme iron occurs (Fig. 2B), although the total amount of nonheme iron is
much more than heme iron. Similar relative changes in heme also occur
in the microsomal and cytosolic subcellular fractions. Interestingly,
there is a relatively modest but detectable increase in nonheme iron in
the cytosolic fraction, which may represent increased iron storage as
ferritin(30) .
Figure 2: Heme and nonheme iron distribution in various cellular compartments in hepatocytes exposed to either endogenous or exogenous NO. Subcellular fractions were isolated from the hepatocytes cultured with CME for 24 h, CME plus NMMA for 24 h, and 1 mM SNAP for 9 h. The microsomes were resuspended in 0.3 ml of PBS prior to iron and protein analyses. Nonheme iron (B) is expressed as the difference between the total (14) and heme iron (A). The levels of significance were determined using the Student's t test: *, p < 0.005;**, p < 0.03;***, p > 0.1 versus control.
In hepatocytes, free heme is known to
up-regulate its own degradation (by transcriptionally regulated
increase of HO) and down-regulate endogenous synthesis (by decrease of
mRNA stability and mitochondrial import of -aminolevulinate
synthetase(22) ). As shown in Fig. 3A, induction of
NO synthesis by hepatocytes results in a decrease in
-aminolevulinate synthetase and an increase in heme oxygenase
activities, consistent with intracellular heme liberation with
consequent effects on these two enzymes. This result also indicates
that the increase in heme oxygenase and decrease in
-aminolevulinate synthetase activities in rats injected with LPS (31, 32) are due to
NO-induced heme
liberation. It is worth noting that even in the presence of NMMA there
is still an approximately 2-fold increase in heme oxygenase activity,
which may indicate up-regulation by cytokine treatment (33, 34, 35) that is
NO-independent.
Figure 3:
Effects of NO on heme
metabolic enzymes. A, effects of endogenous
NO. Enzymatic activities were determined after the
indicated treatments for 24 h as described under ``Experimental
Procedures.'' B, time course of SNAP-induced effects on
heme metabolic enzymes. At zero time, hepatocytes were exposed to 1
mM SNAP and activities assayed at the indicated times as
described under ``Experimental Procedures.'' C, time
course of induction of nitrite + nitrate (NOx) and inducible
nitric oxide synthase and HO mRNA by endogenous and exogenous
NO. NOx, inducible nitric oxide synthase (iNOS), or HO mRNA were determined as described under
``Experimental Procedures'' at various times after induction
of endogenous
NO synthesis (CME) or SNAP or no further
treatment (CTRL). FC, ferrochelatase; ALAS,
-aminolevulinate
synthetase.
Ferrochelatase, recently discovered
to contain an FeS
nonheme iron-sulfur
center(36) , catalyzes the insertion of iron into porphyrin. As
shown in Fig. 3A,
NO synthesis also
decreases ferrochelatase activity, which represents a newly identified
nonheme iron enzymatic target for
NO. Fig. 3B presents a time course for the changes in the
activities of these three enzymes after exposure to SNAP. While a
decrease in
-aminolevulinate synthetase and an increase in heme
oxygenase activities require a time lag of 2-3 h (consistent with
previously described heme-induced regulatory mechanisms(22) ),
the decrease in ferrochelatase activity is virtually complete within 1
h. This indicates that
NO inhibits activity of this
enzyme by a direct effect, perhaps by destruction of its nonheme
iron-sulfur cluster(36) . This inhibition will contribute to a
decrease in intracellular heme levels in addition to a decrease in
-aminolevulinate synthetase and an increase in heme oxygenase
activities.
Finally, Fig. 3C demonstrates that NO synthesis results in increased heme oxygenase
transcription, as determined by Northern blot analysis using a cDNA
probe to human HO1(19) . The increase occurs slightly after
induction of
NO synthase and concomitant with
NO synthesis (appearance of NO
+ NO
in the medium, NOx). Heme
oxygenase mRNA is induced also by SNAP, without the lag required for
induction of
NO synthase by the cytokine mixture,
demonstrating that
NO alone (without cytokines) can
induce heme oxygenase. Although not shown, addition of 8-bromo-cyclic
GMP (20 µM) did not induce an increase in heme oxygenase
mRNA, indicating that the effect is not due to a cGMP signaling
mechanism.
With regard to the possible mechanism(s) of NO-induced heme loss, previous studies in oxygen-free
conditions have shown that
NO binds to both the
reduced and oxidized forms of CYP and that conversion to the inactive
P420 form can take place(37) . In the presence of oxygen, Wink et al.(38) have shown that
NO causes
both a transient, reversible inhibition of enzymatic activity and a
long lasting irreversible inhibition. The transient reversible
inhibition may be due to formation of nitrosyl-heme complexes; the
irreversible inhibition is substantially prevented by the addition of
albumin, suggesting the formation of a reactive nitrogen oxide species
from the reaction of
NO with O
(to form
nitrosonium ion equivalents [NO
]) or with
O
(to produce
peroxynitrite, ONOO
). Early work demonstrated the
formation of nitrosonium ion equivalents
([NO
]) in activated macrophages synthesizing
NO, and similar chemical reactivity is exhibited by
the reaction of
NO with O
in aqueous
solution(39, 40) . In addition, activated macrophages
synthesize peroxynitrite(41) . Previous studies have shown that
both heme and albumin are nucleophilic targets for nitrosation (42, 43) and that albumin is a target for
peroxynitrite(44) .
It is likely that all hemoproteins do
not respond identically to endogenous nitrogen oxides. For example, as
shown in Fig. 1B there appears to be a subpopulation of
CYP proteins that are resistant to 60-min exposure to NO, and mitochondrial cytochromes are also resistant
to enzymatic inhibition(1, 2) . In addition, at least
two enzymes (cyclooxygenase (45) and guanylyl
cyclase(46) ) are stimulated by
NO. In the
latter case, the results presented here may suggest that nitrosylation
of the heme in guanylate cyclase is a transient event, terminated by
dissociation of the NO-heme from the enzyme. This may explain the
transient nature of stimulation of cGMP synthesis by
NO.
These results also may have relevance to other
well known pathophysiological conditions. For example, formation of NO by the reticuloendothelial system under conditions
of immune activation may result in decreased erythropoiesis, perhaps
contributing to the well known anemia of inflammation and
infection(47) .