 |
Introduction |
Prostaglandins, such as prostaglandin (PG)E2,1 are pivotal
modulators of tissue homeostatsis and their aberrant
regulation is known to cause serious pathophysiological
consequences (1). PGE2 biosynthesis is regulated by successive metabolic steps involving the phospholipase A2-
mediated release of arachidonic acid (AA) and its conversion to PGE2 by cyclooxygenase (COX), hydroperoxidase, and isomerase activities (1). Although cytosolic phospholipase A2 (cPLA2) is primarily responsible for agonist-induced
AA release from membrane phospholipids (5, 6), secretory
PLA2 may also be important in regulating AA availability
via a transcellular mechanism (7). Conversion of AA to
PGH2, the commited step in prostanoid biosynthesis, is
mediated by cyclooxygenases, COX-1 and -2, which are
encoded by two unique genes, located on different chromosomes (2). Generally, although COX-1 is constitutively expressed, the expression of COX-2 is highly inducible (2, 3). Based on their respective modes of expression, it is
thought that COX-1 is primarily involved in cellular homeostasis, whereas COX-2 plays a major role in inflammation and mitogenesis. The COX isoenzymes are thought to
be the primary target enzymes for nonsteroidal antiinflammatory drugs (NSAIDs), which act by inhibiting the COX
activity of COX-1 and -2, thereby blocking their ability to
convert AA to PGG2 (8, 9). In addition to the use of nonsteroidal antiinflammatory drugs as analgesics and for alleviation of acute and chronic inflammation, these agents have
proven effective in decreasing the frequency of heart attacks and strokes (8, 10, 11), and in reducing the incidence
of colon cancer (12, 13).
Since most cells invariably express both COX-1 and -2 under the appropriate conditions, it has been somewhat
problematic to determine the exact contribution of the two
COX isozymes towards basal and agonist-inducible PGE2
biosynthesis; the use of selective COX-1/COX-2 inhibitors to define relative contributions of the two isozymes has
also resulted in limited success. The purpose of this study
was to examine the effects of COX deficiency on the differential expression of the COX-1 and -2 isozymes, and compare the responses of wild-type, COX-1, and COX-2
knockout cells with respect to agonist-induced PGE2 biosynthesis. We demonstrate that the expression of COX,
cPLA2, and PGE2 production are significantly increased in
COX-deficient cells. Thus, COX deficiency, regardless of
whether it is COX-1 or -2, results in the enhanced basal
and inducible expression of the remaining COX isozyme as
well as the elevated expression of cPLA2. We interpret
these data to indicate that the elevated production of PGE2
in COX-1 or -2 isozyme-deficient cells is due to the compensatory expression of the remaining COX isozyme.
 |
Materials and Methods |
Isolation and Culture of COX-deficient Mouse Cells.
Lungs were
collected from wild-type C57BL/6J (B6), COX-1-deficient (14),
and COX-2-deficient (15) mice. The tissues were dissected into
small pieces and grown underneath coverslips in 10-cm plates
with MEM supplemented with PenStrep at a concentration of
300,000 U/liter penicillin G and 300 mg/liter streptomycin sulfate, nonessential amino acids (0.1 mM), Fungizone (1 mg/liter Amphotericin B), glutamine (292 mg/liter), ascorbic acid (50 mg/ liter), and 10% FCS in a humidified incubator with 5% CO2, and the media were changed three times per week. After 3 wk of culture under these conditions, only fibroblasts continued to grow.
The PenStrep was reduced to 100,000 U/liter penicillin G and
100 mg/liter streptomycin sulfate and the media were replaced
twice per week for another 3-5 wk. During subsequent passages,
cells were maintained in DMEM containing high glucose and
supplemented with PenStrep (100,000 U/liter penicillin G and
100 mg/liter streptomycin sulfate), nonessential amino acids (0.1 mM), Fungizone (1 mg/liter Amphotericin B), glutamine (292 mg/liter), ascorbic acid (50 mg/liter), and 10% FCS.
Transfection/Immortalization of COX-deficient Cells.
Subconfluent
monolayers of COX-1
/
or COX-2
/
lung fibroblasts (passages 5-7) were cotransfected with pLE12S (containing adenovirus E1A gene; 8 µg/10-cm diam dish) and pREP4 (containing a hygromycin resistance gene; Invitrogen, Carlsbad, CA) plasmids (2 µg/10-cm diam dish) by the LipofectAMINE reagent (GIBCO
BRL, Gaithersburg, MD). Plasmid pLE12S was a gift of Dr. Margaret Quinlan (University of Tennessee, Memphis, TN). Cells were
maintained in the above media containing hygromycin (50 µg/ml)
for 1 wk; hygromycin in the culture media was increased 50 µg/ml
per week until the final concentration reached 250 µg/ml. Subsequent cell passage and subculture of cells used in all experiments
was done in media containing 250 µg/ml hygromycin.
Treatment of COX-1
/
and COX-2
/
Cells with Cytokines and
PMA.
Cells were seeded at 105 cells/ml in DMEM (high glucose) supplemented with PenStrep (100,000 U/liter penicillin G
and 100 mg/liter streptomycin sulfate), nonessential amino acids
(0.1 mM), Fungizone (1 mg/liter Amphotericin B), glutamine
(292 mg/liter), ascorbic acid (50 mg/liter), 10% FCS, and 250 µg/ml hygromycin in 24-well (0.9 ml/well) flat-bottomed tissue
culture plates (Costar, Cambridge, MA). Cells were incubated at
37°C in a humidified CO2 incubator (5% CO2) for 48 h until
confluent. The medium was then replaced with fresh DMEM
containing 0.5% FCS. Where indicated, cells were treated with
IL-1 (0.25 ng/ml), TNF (5 ng/ml), acidic fibroblast growth factor (FGF; 10 ng/ml), or PMA (12.5 ng/ml) along with the appropriate vehicle controls; at these concentrations, neither cytokines nor PMA affected cell morphology or viability. In Western blot experiments, cells were treated as above except they were seeded in 6-well culture plates (2.7 × 105 cells/well).
Western Blot Analysis.
The medium from COX-deficient
cells cultured in 6-well plates was aspirated and cell monolayers were
washed with cold PBS and lysed in the Laemmli sample buffer. The
samples were boiled for 3 min, and identical amounts of protein
applied to SDS-PAGE (7.5%) and later transblotted to an Optitran
membrane (Schleicher & Schuell, Keene, NH). The blot was blocked
with 5% nonfat dry milk before incubation with either a rabbit antibody against murine COX-2 (Cayman Chemical, Ann Arbor, MI),
a rabbit antibody against murine COX-1 (provided by Dr. D. DeWitt; reference 16), or mouse monoclonal anti-cPLA2 antiserum
(Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected
using either enhanced chemiluminescence or enhanced chemifluorescence kits from Amersham Corp. (Arlington Heights, IL).
PGE2 Measurement.
PGE2 in the media was measured by radioimmunoassay (RIA); this assay is based upon the competition
by PGE2 in the test sample with labeled PGE2 for anti-PGE2 antibody binding sites. A 2-100-µl aliquot of culture medium was
added to RIA assay buffer (0.1 mM phosphate buffer, pH 7.4, containing 0.9% sodium chloride, 0.1% sodium azide, and 0.1%
gelatin), and mixed with the appropriate amount of labeled tracer
and reconstituted antiserum. The mixture was incubated overnight at 4°C. Assay tubes were then placed in an ice bath, and 1 ml
of cold charcoal-dextran suspension was added. After a 15-min
incubation, the tubes were centrifuged at 2,200 g for 10 min at
4°C; the supernatants were decanted into scintillation vials, and
radioactivity was determined by scintillation spectrometry. Percent
binding was compared against a standard curve, and the amount
of PGE2 in the sample was calculated. In each case the amount of
PGE2 produced was normalized by cell number and all data is
presented as picograms of PGE2 per 103 cells. To determine the
potential effect of nonenzymatically produced, PGE2-immunoreactive products (e.g., isoprostanes) on RIA results, assays were
performed in the presence of an effective COX-1 inhibitor (indomethacin, 1 mM) and NS-398 (1 µM), a COX-2 inhibitor,
both of which block PGE2 biosynthesis, but should not block the
production of nonenzymatically generated AA metabolites; products were further analyzed by radio-thin-layer chromatography (17).
Statistical Analysis.
Paired t test was used to determine the differences in the PGE2 levels between control samples of wild-type,
COX-1
/
and COX-2
/
cells, and between control samples
and samples from cytokine-treated cells. Differences were considered significant if P <0.05.
 |
Results and Discussion |
We examined the effects of IL-1 on PGE2 production in
cells containing both COX isozymes (wild type) compared
with cells that had only COX-1 (COX-2
/
) or COX-2
(COX-1
/
), respectively. As shown in Fig. 1 A both
COX-1
/
or COX-2
/
cells synthesized 6-8-fold higher
amounts of PGE2 compared with their wild-type counterparts. Interestingly, basal PGE2 production was higher in
both COX-2
/
(66.71 ± 3.54 pg/103 cells; n = 6) and
COX-1
/
(90.23 ± 3.29 pg/103 cells; n = 8) cells compared with wild-type (11.07 ± 0.62 pg/103 cells; n = 8).
(All values are mean ± SE.) IL-1 treatment of wild-type
and COX-1
/
cells further enhanced their PGE2 output.
In contrast, IL-1 treatment of COX-2
/
cells did not significantly enhance PGE2 biosynthesis (Fig. 1 A).

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Fig. 1.
The effect of IL-1
on PGE2 biosynthesis, COX-1,
and -2 expression. (A) Wild-type, COX-2 / , and COX-1 /
cells were treated with vehicle
(Control) or with IL-1 (0.25 ng/
ml) as described in Materials and
Methods. After 24 h of culture, media was collected and analyzed for PGE2 by RIA. Data are
means ± SE of at least six separate determinations (wells). Values that are significantly different from the wild-type value in the control group by paired t
test (P <0.05). * Values that are
significantly different from the
control values of each respective cell type by paired t test. (B)
Western blot analysis of COX-2 protein levels in IL-1-stimulated
cells. Wild-type, COX-2 / ,
and COX-1 / cells were
treated with vehicle (Control) or
with IL-1 (0.25 ng/ml) as described in Materials and Methods. After 24 h of culture, media
was removed and Western blot
analysis was carried out as described in Materials and Methods.
(C) Western blot analysis of
COX-1 protein levels in IL-1-stimulated cells. Wild-type, COX-2 / ,
and COX-1 / cells were treated with vehicle (Control) or with IL-1
(0.25 ng/ml) as described in Materials and Methods. After 24 h of culture, media was removed and Western blot analysis was carried out as described in Materials and Methods.
|
|
These dramatic differences in basal PGE2 biosynthesis
between wild-type and COX-deficient cells, prompted us
to compare the expression of genes encoding three key enzymes (COX-1, COX-2, and cPLA2) regulating PGE2 biosynthesis in untreated and IL-1-treated wild-type, COX-1
/
,
and COX-2
/
cells. A comparison of basal and IL-1-
stimulated levels of COX-1 and COX-2 protein by immunoblot assay in wild-type and COX-1
/
cells is shown in
Fig. 1, B and C. Consistent with numerous previous observations, the basal expression of COX-2 protein in wild-type cells was barely detectable (Fig. 1 B). Constitutive levels of COX-2 proteins were also significantly increased
(2.4-fold) in untreated COX-1
/
cells (Fig. 1 B). The elevated level of COX-2 protein correlates well with the
higher basal PGE2 levels in COX-1
/
cells compared with
those in wild-type cells. When treated with IL-1, COX-2
protein levels increased moderately in wild-type cells (Fig.
1 B), but the increase in COX-2 protein was much more
dramatic in COX-1
/
cells (41-fold). The overall pattern
of COX-2 protein expression in wild-type and COX-1
/
cells correlated with increased PGE2 production seen in
cells with unique COX phenotypes (see Fig. 1 A).
Next, we examined the basal and IL-1-stimulated levels of
COX-1 protein in identically treated wild-type, COX-2
/
,
and COX-1
/
cells, respectively (Fig. 1 C). In wild-type
cell extracts, the level of COX-1 protein was barely detectable and IL-1 treatment was apparently inconsequential.
This result was not unexpected since COX-1 expression is
not known to be inducible under many conditions. We
observed that basal expression of COX-1 protein in untreated COX-2
/
cells (Fig. 1 C) was much greater (14-fold) than that in wild-type cells. This overexpression of
COX-1 protein corresponds with greater basal PGE2 levels
in COX-2
/
cells, compared with the basal levels in wild-type cells. IL-1 had no stimulatory effect on COX-1 protein levels in COX-2
/
cells (Fig. 1 C) and as expected,
COX-1
/
cells did not express detectable COX-1 protein. Another important enzyme in the prostaglandin biosynthetic pathway is PGE2 synthase, the isomerase that
converts PGH2 to PGE2. Although PGE2 synthase has neither been sequenced nor cloned, making it difficult to study, available evidence does seem to indicate that this enzyme is not a rate-limiting reaction in PGE2 biosynthesis.
However, based upon our findings, we cannot rule out the
possibility that PGE2 synthase expression may also be altered in COX null cells.
To examine the possibility that iso-PGE2 or other isoprostanes (18) may be generated nonenzymatically from a
buildup of endoperoxide intermediate that cross-reacts
with the anti-PGE2 used in our RIA leading to erroneously
high estimations of COX and/or PGE2 synthase activity,
we performed two experiments. First, we treated wild-type
and COX
/
cells with either indomethacin or NS-398,
COX-1, and COX-2 selective inhibitors, respectively since
these COX inhibitors should block PGE2 synthesis without
affecting iso-PGE2 formation. We found that either indomethacin or NS-398 completely blocked both the basal
and cytokine-induced formation of immunoreactive PGE2
in wild-type and COX
/
cells (data not shown). Second,
radio-thin-layer chromatography was used to confirm that
PGE2 was by far the predominate prostanoid product generated by wild-type and COX
/
cells and that no other
AA metabolites in addition to PGE2 were generated in
COX
/
cells (data not shown).
To compare the effects of IL-1 (see Fig. 1 A) to other inducers of PGE2 biosynthesis, we tested the effects of TNF,
acidic FGF, and PMA on PGE2 production in wild-type,
COX-2
/
, and COX-1
/
cells (Fig. 2). Compared to
stimulated wild-type cells, there was significantly more
PGE2 produced in either COX-1
/
or COX-2
/
cells,
with the possible exception of TNF that induced comparable PGE2 biosynthesis in each cell type. In response to
FGF, the amount of PGE2 that was produced by COX-2
/
cells was elevated and in COX-1
/
cells, PGE2 was even
more dramatically elevated compared to wild type. Both
COX-2
/
and COX-1
/
cells treated with PMA also
produced much more PGE2 than wild type. Thus, in general, COX-isozyme deficiency results in increased PGE2
biosynthesis, but the relative contributions of COX-1 and COX-2 are clearly dependent upon the specific agonists
involved.

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Fig. 2.
The effect of cytokines and PMA on PGE2 biosynthesis.
Wild-type (A), COX-2 / (B), and COX-1 / (C) mouse cells were
treated with IL-1 (0.25 ng/ml), TNF (5 ng/ml), FGF (10 ng/ml), or
PMA (12.5 ng/ml) as described in Materials and Methods. After 24 h of
culture, media was collected and analyzed for PGE2 by RIA. Data are
means ± SE of at least six separate determinations (wells). * Values that
are significantly different from the control values of each respective cell
type by paired t test (P <0.05).
|
|
Since constitutive COX-2 protein expression and PGE2
production in COX-1
/
cells was significantly enhanced,
we were also curious about the status of cPLA2 gene expression in COX-deficient cells. We reasoned that cPLA2
activity could be involved in regulating levels of free AA
for conversion to PGE2 and thereby could play a critical role in compensating for COX-isozyme deficiency. We were
somewhat surprised to find that basal levels of cPLA2 protein in either COX-2
/
(Fig. 3 B) or COX-1
/
(Fig. 3 C)
cells were significantly higher than levels of cPLA2 in wild-type cells (Fig. 3 A). It is conceivable, therefore, that enhanced expression of cPLA2 could directly contribute to
higher PGE2 levels in both of the COX-deficient cells by
generating greater AA substrate for PGE2 biosynthesis.
Treatment of COX-1
/
or COX-2
/
cells with IL-1 resulted in a modest increase in the amount of cPLA2 protein
(4-fold in COX-1
/
and 1.4-fold in COX-2
/
). This
was in contrast to wild-type cells, which showed no change in the levels of cPLA2 protein after treatment with IL-1. As
an important control, we examined the quantitative parameters of PGE2 production, and COX-1, COX-2, and cPLA2
gene expression in wild-type, COX-2
/
, and COX-1
/
cells from primary cell cultures and found essentially the
same patterns in primary cells as those observed in the immortalized cells (data not shown). Therefore, the characteristic pattern of expression of COX-1, COX-2, and cPLA2
proteins in COX-2
/
and COX-1
/
cells is not elicited
as a result of immortalization caused by the E1A adenovirus
gene. Taken together these data indicate that COX-1
/
cells express enhanced levels of both basal and cytokine-stimulated COX-2 protein, and increased basal expression
of cPLA2 protein. We postulate that the significantly increased levels of COX-2 and cPLA2 in COX-1
/
cells are
likely to account for the increased rates of PGE2 biosynthesis; these data also implicate the existence of compensatory
mechanisms for PGE2 production in COX-isozyme-deficient cells.

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Fig. 3.
Expression of cPLA2
in IL-1-stimulated cells. Wild-type, COX-2 / , and COX-1 /
mouse cells were treated with
vehicle (Control) or with IL-1 (0.25 ng/ml) as described in Materials and Methods. After 24 h of culture, media was removed
and Western blot analysis was
carried out as described in Materials and Methods. (A) Proteins
from wild-type cells. (B) Proteins from COX-2 / cells. (C) Proteins
from COX-1 / cells. A, B, and C are from the same blot, which is a
representative of three replications.
|
|
To distinguish between preferences of COX-1 and -2 for endogenous and/or exogenous AA for conversion to
PGE2, and to verify that COX-1 was indeed expressed in
COX-2
/
cells as judged by its ability to synthesize PGE2,
we added exogenous AA to wild-type, COX-2
/
, or
COX-1
/
cells that were either untreated or treated
with IL-1, TNF, FGF, or PMA. The results shown in Fig. 4
demonstrate that cells expressing only COX-1 (COX-2
/
)
synthesized similar amounts of PGE2 as wild-type or
COX-1
/
cells supplemented with exogenous AA.
Therefore, COX-1 is expressed and enzymatically active in
COX-2
/
cells, but cytokines neither enhance COX-1
protein biosynthesis nor PGE2 biosynthesis. Thus, agonists
that induce PGE2 biosynthesis in COX-2
/
cells in the
absence of exogenous AA, do so by increasing endogenous substrate availability. Based on these data, we conclude that in COX-2
/
cells, substrate is likely to be limiting for
constitutively expressed COX-1-mediated PGE2 biosynthesis. Fig. 4 also shows that COX-1
/
cells are able to
use both exogenous and endogenous substrates (also see
Fig. 1 A). However, IL-1, TNF, and FGF significantly enhanced the ability of COX-1
/
cells to produce PGE2,
most likely by enhancing COX-2 expression as shown in
Fig. 2. In addition, COX-1
/
cells treated with PMA did
not produce elevated levels of PGE2, even when exogenous AA was provided. This indicates that PMA likely increased PGE2 production by increasing the availability of
endogenous AA in COX-1
/
cells, whereas IL-1, TNF,
and FGF likely affect AA mobilization and COX-2 expression. PMA affected the wild-type cells similarly. These results clearly raise the possibility that in COX-1 or -2 null
cells, there is a coordinate upregulation of the expression and/or activities of COX-1, COX-2, and cPLA2, leading
to increased PGE2 biosynthesis. These data also demonstrate that both COX-1
/
and COX-2
/
cells can effectively use AAs from either endogenous or exogenous sources.

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Fig. 4.
The effect of exogenous arachidonic acid on cytokines or
PMA induced PGE2 production. Wild-type (A), COX-2 / (B), and
COX-1 / (C) mouse cells were treated with IL-1 (0.25 ng/ml), TNF
(5 ng/ml), FGF (10 ng/ml), or PMA (12.5 ng/ml) as described in Materials and Methods. After 24 h of culture, media was removed, the cells were
washed to remove any accumulated PGE2, and fresh serum-free media
containing AA (50 µM) was added. After a 15-min incubation in the
presence of added AA, media was collected and PGE2 measured by RIA.
Data are means ± SE of at least six separate determinations (wells). * Values that are significantly different from the control values of each respective cell type by paired t test (P <0.05).
|
|
Our data are consistent with the hypothesis that the
long-term of COX-isozyme deficiency results in the altered expression of the remaining two enzymes that regulate mobilization and conversion of arachidonic acid to prostaglandins. Fig. 5 summarizes the patterns of COX and
cPLA2 expression in COX null cells compared with normal cells in response to IL-1. The scheme shows the compensatory expression of the alternative COX isozyme and cPLA2
when one of the COX isozymes is absent. In cells lacking
the housekeeping isozyme COX-1, overcompensation results in the overexpression of COX-2 and cPLA2, and in
turn elevated PG biosynthesis. Similarly, cells lacking the
inducible isozyme, COX-2, elicit the enhanced expression
of COX-1 and cPLA2. Although we are uanble to comment as to the precise status of PGE2 synthase expression in
wild-type, COX-1
/
or COX-2
/
cells, we have depicted its expression in each cell type; since PGE2 is the predominate prostanoid product, its expression would not appear
to be rate-limiting given the great potential for PGE2 biosynthesis in the presence of exogenous AAs (see Fig. 4).
Thus, our data clearly show that COX-deficient cells have
the potential to overcome the lack of expression of one or
the other COX isoenzymes by overexpressing the alternate
COX isoform and increased cPLA2 expression. Such a potential mechanism for producing PGE2 by cells in vitro is
not surprising since neither COX-1
/
(14) nor COX-2
/
mice (15) showed severe developmental arrest in utero or
immediate postnatal mortality. However, in contrast to the
results shown here using lung fibroblasts, Langenbach et al.
(14) did not report any compensatory COX-2-mediated
PGE2 production in glandular stomachs of COX-1-deficient mice, suggesting that tissue specificity may also be an
important factor for further investigation. Together, these
findings underscore the importance of elucidating the potential long-term effects of COX-1 or COX-2 inhibition
with respect to alterations in the quantitiative and/or qualitative patterns of AA metabolism.

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Fig. 5.
The coordinate regulation of COX-1, COX-2, and
cPLA2 expression in COX null
cells by IL-1. In wild-type cells,
inducible COX-2 is responsible
for the increase in IL-1-induced
PGE2 production as represented
by proportionally larger characters and lines throughout the diagram. Although the constitutive
level of COX-1 expression is
low, exogenous AA (exo. AA) is
effectively converted to PGE2. In
COX-2 / or COX-1 / cells,
the overexpression of cPLA2 may
play a role in increased basal
PGE2 biosynthesis compared to
wild-type cells by increasing the availability of endogenous AA (endo. AA). IL-1 greatly induces COX-2 accumulation in COX-1 / cells resulting in enhanced PGE2 biosynthesis. In COX-2 / cells, overexpression of COX-1 and cPLA2 lead to an increase in basal PGE2 biosynthesis compared to wild
type. However, IL-1 does not enhance PGE2 biosynthesis in COX-2 / due to the lack of increased COX-1 expression. As in wild-type cells, exogenous AA is effectively used by COX-1 in COX-2 / cells as indicated by a high level of PGE2 accumulation. At present, the effects of COX-isozyme
deficiency on the expression of PGE2 synthase are not known, but this enzyme does not appear to be rate limiting.
|
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Received for publication Received for publication 17 September 1997 and in revised form 18 November 1997..
This work was supported by research funds from the Department of Veterans Affairs (DVA), The Arthritis
Foundation, and grants
1.
|
Goetzl, E.J.,
S. An, and
W.L. Smith.
1995.
Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases.
FASEB J.
9:
1051-1058
[Abstract/Free Full Text].
|
2.
|
Smith, W.L.,
R.M. Garavito, and
D.L. DeWitt.
1996.
Prostaglandin endoperoxide H synthase (cyclooxygenases)-1 and -2.
J. Biol. Chem.
271:
33157-33160
[Free Full Text].
|
3.
|
Herschman, H..
1996.
Prostaglandin synthase 2.
Biochim. Biophys. Acta.
1299:
125-140
[Medline].
|
4.
|
Herschman, H.R.,
R.S. Gilbert,
W. Xie,
S. Luner, and
S.T. Reddy.
1995.
The regulation and role of TIS10 prostaglandin
synthase-2.
Adv. Prostaglandin Thromboxane Leukotreine Res.
23:
23-28
[Medline].
|
5.
|
Lin, L.L.,
A.Y. Lin, and
J.L. Knopf.
1992.
Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.
Proc. Natl. Acad. Sci. USA.
89:
6147-6151
[Abstract].
|
6.
|
Clark, J.D.,
L.L. Lin,
R.W. Kriz,
C.S. Ramesha,
L.A. Sultzman,
A.Y. Lin,
N. Milona, and
J.L. Knopf.
1991.
A novel
arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and
GAP.
Cell.
65:
1043-1051
[Medline].
|
7.
|
Reddy, S.T., and
H.R. Herschman.
1996.
Transcellular prostaglandin production following mast cell activation is mediated by proximal secretory phospholipase A2 and distal prostaglandin synthase 1.
J. Biol. Chem.
271:
186-191
[Abstract/Free Full Text].
|
8.
|
Vane, J.R., and
R.M. Botting.
1995.
A better understanding
of anti-inflammatory drugs based on isoforms of cyclooxygenase (COX-1 and COX-2).
Adv. Prostaglandin Thromboxane
Leukotreine Res.
23:
41-48
[Medline].
|
9.
|
DeWitt, D.L.,
E.A. Meade, and
W.L. Smith.
1993.
PGH synthase isozyme selectivity: potential safer nonsteroidal antiinflammatory drugs.
Am. J. Med.
95:
40s-44s
[Medline].
|
10.
|
Vane, J.R., and
R.M. Botting.
1994.
Regulatory mechanisms
of the vascular endothelium: an update.
Pol. J. Pharmacol.
46:
499-421
[Medline].
|
11.
|
Vane, J.R., and
R.M. Botting.
1995.
New insights into the
mode of action of anti-inflammatory drugs.
Inflamm. Res.
44:
1-10
[Medline].
|
12.
|
Kargman, S.L.,
G.P. O'Neill,
P.J. Vickers,
J.F. Evans,
J.A. Mancini, and
S. Jothy.
1995.
Expression of prostaglandin G/H
synthase-1 and -2 protein in human colon cancer.
Cancer Res.
55:
2556-2559
[Abstract].
|
13.
|
Rigas, B.,
I.S. Goldman, and
L. Levine.
1993.
Altered eicosanoid levels in human colon cancer.
J. Lab. Clin. Med.
122:
518-523
[Medline].
|
14.
|
Langenbach, R.,
S.G. Morham,
H.F. Tiano,
C.D. Loftin,
B.I. Ghanayem,
P.C. Chulada,
J.F. Mahler,
C.A. Lee,
E.H. Goulding,
K.D. Kluckman, and
O. Smithies.
1995.
Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced
gastric ulceration.
Cell.
83:
483-492
[Medline].
|
15.
|
Morham, S.G.,
R. Langenbach,
C.D. Loftin,
H.F. Tiano,
N. Vouloumanos,
J.C. Jennette,
J.F. Mahler,
K.D. Kluckman,
A. Ledford,
C.A. Lee, and
O. Smithies.
1995.
Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.
Cell.
83:
473-482
[Medline].
|
16.
|
Morita, I.,
M. Schindler,
M.K. Regier,
J.C. Otto,
T. Hori,
D.L. De Witt, and
W.L. Smith.
1995.
Different intracellular
locations for prostaglandin endoperoxide H synthase-1 and -2.
J.
Biol. Chem.
270:
10902-10908
[Abstract/Free Full Text].
|
17.
|
Baily, J.M.,
B. Muza,
T. Hla, and
K. Salata.
1985.
Restoration of prostacyclin synthase in vascular smooth muscle cells
after aspirin treatment: regulation by epidermal growth factor.
J. Lipid Res.
26:
54-61
[Abstract].
|
18.
|
Roberts, L.J., and
J.D. Morrow.
1997.
The generation and
actions of isoprostanes.
Biochim. Biophys. Acta.
1345:
121-135
[Medline].
|