The Prostaglandin Biosynthetic
|To date, we have found four
distinct pathways by which arachnid acid (AA) and prostaglandins (PG) can
be produced in the macrophage P388D1 cells. The following is a brief description
on each pathway.
|go to Pathway
1, Pathway 2, Pathway 3, Pathway
1: Immediate AA release and PG production
|The most straightforward pathway
for AA mobilization and prostaglandin production takes place immediately
after the stimulus has interacted with its receptor. For the P388D1 macrophages
this occurs when the cells are stimulated with zymosan. For PG production
this involves the release of AA from phospholipids via a PLA2 and its conversion
to PGs via one of the two known cyclooxygenases (COX-1 or COX-2). The process
itself may last from minutes up to a few hours, and its distinguishing characteristic
is that pre-existing enzymes mediate it. The phospholipases and cyclooxygenases
involved are already present in the cells and are not synthesized de novo.
The immediate pathway for AA mobilization and PG production operates in
almost all cell types and is mediated by the early activation of the GIV
PLA2. This PLA2 appears to be responsible for the release of the entire
AA produced. There is no apparent involvement of the sPLA2 in this pathway.
Since the GIV PLA2 requires an initial burst of Ca2+ to interact with its
membrane substrate, this pathway is typically activated by short-term stimuli
that induce transient increases in the intracellular Ca2+ concentration.
|In most cases, the GIV PLA2
is coupled to the COX-1 for the production of prostaglandins. There are
some cases, however, where COX-2 is also constitutively present in the cell.
When this is the case, COX-2 also participates in the process.
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2: Primed immediate AA release and PG production
|The primed immediate pathway
of AA mobilization and prostaglandin production involves the sequential
exposure of the cells to two different stimuli. The first one does not activate
the cells itself but leaves them prepared to maximally respond to a second
stimulation. This kind of response is typical of macrophages exposed to
LPS. Scheme 1 represents our current understanding
of the mechanism of the primed immediate pathway during inflammatory stimulation
of the P388D1 cells.
|LPS primes the cells thus preparing
them for activation by inflammatory mediators such as PAF. The LPS priming
step can be prevented by inhibitors of RNA transcription and protein translation,
indicating that de novo protein synthesis is a required event. Thus, this
pathway involves the participation of both constitutive and inducible enzymes.
By stimulating protein synthesis, LPS increases the levels of PLA2 and COX
activity in the cells, and this allows the cells to subsequently produce
a larger response.
|In the second phase of the primed
immediate pathway, the primed cells are activated by PAF. Acting through
a specific receptor, PAF produces at least two kinds of signal which, acting
in concert, lead to the immediate activation of the GIV PLA2 in an intracellular
compartment. It is now clear that one of these signals is
the liberation of sequestered intracellular Ca2+. The second signal remains undefined. However, we have found that PIP2 activates GIV
PLA2 in vitro thus opening the possibility that PIP2 production constitutes
the second signal.
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|The PAF signals accomplish two
tasks. They initiate the rapid secretion of an sPLA2, which, once secreted, re-associates with the cellular
surface. They also lead to the activation of the GIV PLA2 that produces
a transient elevation of intracellular free AA levels. Elevated free AA
during the initial stages of P388D1 cell activation may serve important
metabolic and biochemical functions, one of which appears to be to help
activate the GV PLA2 which is now found on the outside of the cell surface.
The molecular mechanism involved in the activation of the outer surface
GV PLA2 by AA derived from the GIV PLA2 is unknown. AA containing phospholipid
must be transported to the external leaflet of the plasma membrane for the
GV PLA2 to have access to it. Therefore membrane asymmetry and phospholipid
transport across the membrane may play a role in activating this GV PLA2. It is not clear whether PAF in any way directly
activates the GV PLA2 or its expression in the cells.
|Once the GV PLA2 is activated
at the outer surface, it hydrolyzes phospholipids and releases AA.
Part of this AA is released to the extracellular media while the
rest is re-captured by the cells and made accessible to COX-2 for the generation
of prostaglandins during cellular activation. We have evidence that the
COX-1, the constitutively expressed enzyme that seems to be responsible
for the production of the PG needed for maintenance functions, does not
see this AA. It apparently obtains
its AA from another source, perhaps from the GVI Ca2+-independent PLA2.
|Enhanced mobilization of AA
is followed by its enhanced conversion to PGs by either COX-1 or, if present,
by COX-2. When both COX isoenzymes are present, whether one of them is utilized
preferentially over the other appears to depend on cell type and stimulation
conditions. In the P388D1 macrophage cell line exposed to LPS/PAF, most
of the PG produced arises from COX-2, not COX-1, but this might not constitute
the general mechanism.
|An interesting feature of the
primed immediate pathway is that the sPLA2 releases most of the AA under
these conditions, not the GIV PLA2. However, the activity of the sPLA2 appears
to be strikingly controlled by the GIV PLA2. This means, that, even though
the GIV PLA2 is no longer the major provider of AA, it is still the key
enzyme in the release due to its upstream position in the signaling cascade.
How this happens is uncertain at present, but it appears that the GIV PLA2
directly regulates the mechanism of activation of the sPLA2 . Even though
the sPLA2 contributes the majority of the AA acid, the GIV PLA2 still liberates
AA that is converted to PGs. Therefore, the sPLA2 may be acting more as
an enhancer rather than as an absolutely required component of the pathway.
|Subsequent to our studies establishing
the model depicted in Scheme 1, other research groups have recognized it
as an applicable model for immediate PG production in activated mammalian
cells. Our discovery that GV PLA2 is a novel and important effector for
AA release, was confirmed by the groups of Herschman
and Arm in murine mast cells. Moreover, the key discovery that there are
two different PLA2s involved in the AA release, acting in different roles,
but in an inter-related manner, has also been confirmed in a variety of
other cell types and has decisively influenced further research in the field. Scheme 1 is now widely accepted as the model
of PG production in cells of the immune system and may be applicable to
other types as well.
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3: Delayed AA release and PG production
proceeds gradually for several hours and requires de novo synthesis
of particular enzymes. This is the response elicited by LPS and other
pro-inflammatory stimuli when incubated for long periods of time. PAF
activation is not required for this process. AA release occurs only
after a time lag of about 2-3 h, followed by an enhanced PG production.
Delayed AA release has been correlated to the LPS-induced expression
of sPLA2 (either GIIA or GV, depending on cell type), and PG production
has been correlated with the induction of COX-2. Thus, increased synthesis
of sPLA2 by the cells eventually results in large increases in free
AA release. Part of this AA will be metabolized to prostaglandins by
COX-2. As in the primed immediate pathway, in the delayed pathway the
major provider of free AA is not the GIV PLA2 but the sPLA2. Yet, the
sPLA2 activity is still controlled by the GIV PLA2. Thus, GIV PLA2 activation
is again an important signaling step. We and others have provided strong evidence that GIV PLA2
activation is required for the cells to synthesize new sPLA2 protein.
This finding suggests that the GIV PLA2 can affect gene expression in
addition to its direct role in AA release. Enhanced COX-2 expression
at the mRNA level is another striking biochemical marker of the delayed
phase, and also appears to be downstream of GIV PLA2 activation. It
is important to note that in the delayed phase, COX-2 is always present
and is solely responsible for the delayed synthesis of prostaglandins.
COX-1, albeit present, does not participate in this process. Why this
is so is unknown at present, but this appears to be the general mechanism.
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Pathway 4: Ca2+-independent
Immediate AA release and PG production
|In our studies of the WISH cells,
we have found a PMA stimulated pathway that is similar to the immediate
release pathway described above. It does not require an
sPLA2, LPS, or PAF, and it occurs in a short time period. It differed in
one important way; it did not require a Ca2+ spike. This may represent a
fourth pathway. We have found that a similar pathway is also present in
the P388D1 cells.
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