| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 85 === Part I: In previous studies, we have shown that mouse RAW
264.7 macrophagespossess pyrimidinoceptors coupled to
phosphoinositide-specific phospholipase C,with higher
specificity for UTP than for ATP.In the present study,we
exploredthe mechanism involved in the UTP-induced intracellular
acidification seen inthis cell line. UTP(30 mM) caused
reversible decrease in pHi of 0.16 unit. The response to 30 mM
UTP was unaffected by removal of extracellular Cl- or Na+ions or
by pretreatment with EIPA (10 mM),NPPB(100 mM),staurosporine (1
mM) or Ro 31-8220 (1 mM); however , removalof extracellular
Ca2+ abolished the pHiresponse to 30 mM UTP,1 mM thapsigargin or
1 mM ionomycin,all of which induced similar extent and time-
dependency of acidification, but in a nonadditivemanner.To
further investigate the Ca2+-dependent mechanism, the
involvement ofarachidonic acid (AA) and eicosanoid metabolites
were studied. Addition of AA(10 mM), but not arachidic acid
(100 mM), produced a reduction in pHi. UTP,thapsigargin and
ionomycin can induce Ca2+-dependent arachidonic acid releasein
RAW264.7 cells.Furthermore,4-bromo-phenacyl bromide (30 mM,a
PLA2 inhibitor), NDGA (50 mM, a lipoxygenaseinhibitor) and
MK-886 (10 mM, a FLAP inhibitor)abolished the responses to UTP
and ionomycin , while indomethacin (30 mM,a cyclooxygenase
inhibitor ) andbaicale in (10 mM, a selective
12-lipoxygenaseinhibitor)had no effects. Incontrast, the pHi
response to AA was unaffected by4-bromo-phenacyl bromide
andremoval of extracellular Ca2+ ions , but wasabolished
by addition of NDGA . Exogenous 5-HPETE (2 mM) also
producedmarked acidification, and both UTP and ionomycin
could induce peptideleukotriene formation. In conclusion, this
is the first report indicating thatlipoxygenase metabolites act
as mediators of the Ca2+-dependent acidificationseen in
macrophages in response to UTP andionomycin via activation of
cPLA2and AA release. Part II: 1. Lysophosphatidi acid
(LPA) has been widelyexamined as a naturally occurring and
multifunctional phospholipid messengerin diverse tissue and cell
types.The G protein-mediated inhibition of adenylylcyclase (AC)
has been identified in the action of LPA in 3T3 fibroblasts
andPC12 cells. In this study, the effects of LPA on the
intracellular cAMP levelswere examined in mouse RAW 264.7
macrophages and rat C6 glioma cells.2. In RAW264.7 cells, we for
the first time found that LPA at 3-50 mM can increase thecAMP
formation in a concentration-dependent manner, which exhibits an
additiveeffect with forskolin or cholera toxin, while a
synergistic effect with PGE1or isoproterenol. On the
contrary, LPA causes an inhibition on the cAMPresponse to
isoproterenol or forskolin in C6 glioma cells.3. The
pretreatmentwith pertussis toxin has no effect on LPA-induced
cAMP stimulation in RAW264.7cells, but abolishes the inhibitory
effect of LPA in C6 cells.4. The effectsof LPA on the
cytoskeletal structure were compared in RAW 264.7 and C6
cells. A rapid formation of stress fiber is induced by LPA in
quiescent C6 cells,but not in quiescent RAW264.7 cells.5.The
cAMP effects of LPA is also comparedin both cell types cultured
in normal medium containing 10% serum (controlcells) or in
serum-deprived medium (quiescent cells). The cAMP
potentiationseffect of LPA in RAW 264.7 macrophages is
unaffected by the removal of serum,
while its inhibitory effect in C6 glioma cells disappears in
quiescent cells.6. In RAW 264.7 cells, both colchicine (
a microtubule disrupter ) andcytochalasin B (an actin filament
disrupter) can potentiate cAMP response toPGE1, and further
increase the synergistic effect of LPA and PGE1. 7. Althougha
concentration-dependent increase in [Ca2+]i, resulting from
the influx ofextracellular Ca2+, is induced by LPA (1-30 mM) in
RAW 264.7 macrophages, thecAMP potentiation effect of LPA is
unaffected by the removal of extracellularCa2+. By contrast,
LPA has no effect on [Ca2+]i in C6 glioma cells.8. In RAW
264.7 cells, pretreatment with genistein, PD 98059, herbimycin A
or H-89 has no effect on the cAMP response caused by LPA.9. PMA-
induced PKC activation results in the potentiation of PGE1-
elicited cAMP response in RAW 264.7 cells,and this
potentiation is non-additive to that elicited by LPA. PKC
inhibitors(staurosporine, calphostin and Ro 31-8220, but not
Go6976) can significantlyinhibit the LPA-induced cAMP
potentiation.10.Seven PKC isoforms (a, BI, BII, d, m, l and z)
are expressed in RAW 264.7 macrophages, ad a, BI, BII,and d
isoforms can be down-regulated by long-term treatment (24hr)
with PMA.11.In RAW 264.7 cells, LPA treatment within 7 min can
cause a slight cytosolic translocation of PKC a and bI,
whereas a significant membrane translocation of PKC m and l.
Although LPA cannot induce PKCz translocation, a marked
increase in PKCz activity is observed after LPA treatment.12.
Pharmacological manipulations also exclude the involvement of
PI-PLC, PC-PLC, PLD and PI3-kinase activation in the cAMP
potentiation effect of LPA in RAW 264.7 cells. 13. In RAW
264.7 macrophages, the b-adrenoceptor-induced
homologousdesensitization of cAMP response is unaffected by LPA.
14. In RAW 264.7 cells,the potentiation effect of LPA depends on
its preincubation time. A maximalincrease is observed within
5 min preincubation followed by a time-dependentdecrease
between 10-60 min. 15. Scatchard plot analysis in RAW 264.7
cellsindicates the existence of specific binding sites for LPA,
with Bmax value of15.7 pmol/mg.16.Taken together, different
effects of LPA on cAMP formation aredemonstrated in mouse RAW
264.7 macrophages and rat C6 glioma cells. In RAW264.7 cells,
LPA can increase AC activity possibly by the activation of
PKCm,l and z, while in C6 cells, LPA decreases AC activity via a
pertussis toxin-senstitive Gi protein.
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