Urate Synthesis in the Blood-sucking Insect Rhodnius
prolixus
STIMULATION BY HEMIN IS MEDIATED BY PROTEIN KINASE C*
Aurélio V.
Graça-Souza
,
Mário A. C.
Silva-Neto, and
Pedro L.
Oliveira
From the Departamento de Bioquímica Médica, Instituto
de Ciências Biomédicas, Centro de Ciências da
Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ,
Brasil, CEP 21910-590
 |
ABSTRACT |
Hemin is a catalyst of the formation of reactive
oxygen species. We proposed that hematophagous insects are exposed to
intense oxidative stress because of hemoglobin hydrolysis in their
midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella,
G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin
stimulates urate synthesis in the blood-sucking insect Rhodnius
prolixus (Graça-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released
by fat body cells, urate accumulates in the hemolymph, where this
radical scavenger constitutes an important defense against
blood-feeding derived oxidative stress.
Incubation of Rhodnius fat bodies with okadaic acid raises
the level of urate synthesis, suggesting that urate production can be
controlled by protein phosphorylation/dephosphorylation. Urate
synthesis is stimulated by dibutyryl cAMP and inhibited by
N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the
protein kinase C activator phorbol 12-myristate 13-acetate. In the
presence of hemin, however, inhibition of urate synthesis by H-89 does
not occur, suggesting that the hemin stimulatory effect is not mediated
by protein kinase A. Calphostin C completely inhibits the hemin-induced
urate production, suggesting that the triggering of urate antioxidant
response depends on protein kinase C activation. This conclusion is
reinforced by the observation that in fat bodies exposed to hemin, both
protein kinase C activity and phosphorylation of specific
endogenous polypeptides are significantly increased.
 |
INTRODUCTION |
Oxygen is toxic because of its ability to generate reactive
species that can damage cellular components such as nucleic acids, proteins and lipids (1-4). To survive in an oxygen-rich environment, aerobic organisms have developed an array of antioxidant mechanisms to
prevent or repair oxidative injury (5). Uric acid has been proposed to
be an important free radical scavenger (6-8). High levels of
allantoin, the product of urate oxidation, were found in patients under
oxidative stress (9). According to the free radical theory of aging,
high concentrations of urate in plasma may be correlated with
lengthening of the life span (10). Uric acid is the main end-product of
nitrogen metabolism in insects (11). It is synthesized in the fat body
cells (12) and secreted to the hemolymph for posterior absorption at
the Malpighian tubules during formation of insect urine (13, 14).
Increased susceptibility to oxidative stress was reported in mutants of
Drosophila melanogaster (15) that are not able to synthesize
uric acid, pointing to an antioxidative role of urate in insects.
Blood digestion by hematophagous insects creates an especially intense
source of oxidative stress, because hemin, iron, and hemoglobin itself
are promoters of free radical formation, mainly through Fenton-type
reactions (16-21). We have recently shown that in the blood-sucking
insect Rhodnius prolixus, urate is the most important low
molecular weight antioxidant present in the hemolymph (22). The
maintenance of high urate titer in its extracellular fluids (up to 5 mM) protects Rhodnius against oxidative damage caused by the intake of large amounts of hemin in a blood meal.
Reactive oxygen species have been shown to modulate the activity of
protein kinases and phosphatases (23). However, the triggering of a
signal transduction cascade by an oxidant challenge, resulting in the
activation of a low molecular weight antioxidant defense has not been
reported. Here we present evidence that the stimulation of urate
synthesis by hemin in R. prolixus is exerted through protein
kinase C activation.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Allopurinol, hemin, okadaic acid,
Bt2cAMP, PMA,1
soybean trypsin inhibitor, leupeptin, benzamidine, and
-mercaptoethanol were purchased from Sigma. Calphostin C, H-89, and
H-8 were purchased from Calbiochem. Other reagents were of analytical grade.
Insects--
R. prolixus were kept at 28 °C and
80% relative humidity. Experimental animals were adult, mated females,
fed directly on rabbits in their first cycle after the imaginal moult.
Organ Culture and Urate Determination--
Fat bodies were
dissected from adult females on the 4th or 5th day after a blood meal,
rinsed for 2 min in 50 ml of Rhodnius physiological saline
(24) and transferred to a 96-well microplate (3 fat bodies/well)
containing 200 µl of the same solution per well. After a 30 min
pre-incubation, the saline was discarded and fat bodies were incubated
in Rhodnius saline with the additions indicated in the
figure legends. Because Me2SO was used to solubilize most
compounds employed as effectors, all incubation media (including controls) contained 1% Me2SO. Urate secreted to the medium
was determined enzymatically (25) in a U-1100 Hitachi spectrophotometer using a medical diagnosis kit supplied by Doles (Goiânia, GO).
Radioisotopes--
Carrier-free 32Pi was
purchased from Comissão Nacional de Energia Nuclear (São
Paulo, SP), purified by ion-exchange chromatography on a Dowex 1X-10
column (26), and used in metabolic labeling experiments and in the
enzymatic synthesis of [
-32P]ATP (27).
PKC Assay--
Typically 30-40 fat bodies were dissected and
homogenized in a Potter-Elvehjem tissue grinder in the presence of 150 mM NaCl, 250 mM sucrose, 2.5 mM
MgCl2, 2.5 mM EGTA, 50 mM
-mercaptoethanol, 0.05 mg/ml soybean trypsin inhibitor, 0.05 mg/ml
leupeptin, and 1 mM benzamidine in 10 mM
Tris-Cl, pH 7.4. After centrifugation at 4,000 × g for
10 min to remove tissue debris, the supernatant was centrifuged at
100,000 × g for 1 h at 4 °C. The pellet was discarded, and the supernatant was used as a crude extract to measure
PKC activity.
PKC activity against endogenous substrates was determined as described
by Kikkawa et al. (28). Briefly, reaction medium (0.2 ml)
contained 10 mM Tris-Cl, pH 7.4, 2.5 mM
MgCl2, 1 mM CaCl2, and 100 µg of
crude fat body extract. Reactions were started by addition of
[
-32P]ATP to a final concentration of 10 µM (1,000 cpm/pmol). After 10 min of incubation at
37 °C, 25-µl aliquots of the reaction medium were transferred to a
phosphocellulose sheet and washed three times for 15 min each with 10 ml of 25% ice-cold trichloroacetic acid. Incorporated radioactivity
was determined by liquid scintillation. Phosphatidylserine, PMA,
calphostin C, and hemin were added as described in the figure legends.
Protein concentration was estimated accordingly Lowry et al.
(29).
Protein Phosphorylation in Intact Cells--
Fat bodies from
adult females on the 5th day after a blood meal were dissected and
incubated under a Zeiss stereomicroscope, rinsed for 2 min in 50 ml of
phosphate-free Rhodnius saline, and transferred to a 96-well
microplate (3 fat bodies/well) containing 200 µl of
Rhodnius saline in the presence of 100 µCi
32Pi. After 30 min of pre-incubation to allow
endogenous formation of [
-32P]ATP, the medium was
discarded and replaced by Rhodnius saline with the additions
indicated in the figure legends. All incubations were carried out at
28 °C. 1 h later, organs were homogenized in SDS sample buffer,
immediately heated at 100 °C for 3 min, and samples were separated
by 10% SDS-polyacrylamide gel electrophoresis (30). After Coomassie
Blue staining, gels were dried and exposed to a Kodak X-Omat AR-5 film
for 3 days at -70 °C. Densitometry of the autoradiographs was
carried out using a computer scanner (4,800 dpi) and the gel analysis
software QuantiScan (Biosoft, Cambridge, UK). Molecular masses were
determined using the following protein standards: myosin (205 kDa),
-galactosidase (116 kDa), phosphorylase b (98 kDa),
albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and
cytochrome c (12.3 kDa).
 |
RESULTS AND DISCUSSION |
In a previous report we observed that hemin injection or exposure
to 70% O2, two conditions that cause oxidative stress,
lead to increased urate levels in R. prolixus hemolymph
(22). Brief (20 min) exposure of isolated fat bodies to hemin were
enough to induce higher rates of urate synthesis (data not shown),
suggesting the existence of short-term regulatory mechanisms. Here we
show that treatment of Rhodnius fat bodies with okadaic
acid, an inhibitor of protein phosphatases (31, 32), greatly enhances
uric acid release (Fig. 1). This result
suggests that purine metabolism depends on protein phosphorylation in
the fat body cell. The effect of okadaic acid was caused by increased
de novo synthesis of uric acid, and not merely increased
uric acid secretion by the organ, because addition of allopurinol, a
specific inhibitor of xanthine dehydrogenase, prevented the okadaic
acid stimulation of urate production (Fig. 1).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Urate synthesis is regulated by protein
phosphorylation in R. prolixus fat body. Fat
bodies were dissected and pre-incubated in a 96-multiwell microplate
with Rhodnius physiological saline during 30 min. After this
time the medium was removed and replaced by physiological saline alone
(control), saline + 400 µM allopurinol
(allopurinol), 0.1 or 1 µM okadaic acid, or
400 µM allopurinol + 1 µM okadaic acid.
After incubation for 10-90 min, the media were collected, and the
amount of urate produced was determined as described under
"Experimental Procedures." Data are presented as mean ± S.D.
for four determinations.
|
|
When the specific protein kinase A inhibitors H-8 (33) and H-89 (34),
were added to the incubation medium, a significant (p < 0.05) inhibition (30 and 38%, respectively) was observed in both
cases (Fig. 2). Incubation of fat bodies
with Bt2cAMP resulted in a two-fold stimulation of urate
synthesis (Fig. 2), an effect that was counteracted by H-8 and H-89,
confirming that the stimulatory effect of Bt2cAMP should be
attributed to protein kinase A activation. An analogous experiment was
performed to test the role of PKC in the control of urate synthesis.
The phorbol ester analogue PMA (35, 36) was a very effective stimulator of urate synthesis (Fig. 3), and the
activation of urate production by this compound was blocked by
calphostin C (37) and sphingosine (38) at the concentration used (2.5 and 50 µM, respectively). When we incubated the fat
bodies with calphostin C and sphingosine in the absence of PMA (Fig.
3), we did not observe inhibition of urate formation, indicating that
this signaling cascade was not actively modulating the urate formation
pathway.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Protein kinase A and urate synthesis.
Fat bodies were dissected and pre-incubated as described in the legend
to Fig. 1, and then incubated for 90 min with the following additions:
Rhodnius saline (ctrl), 50 µM H-8,
1 µM H-89, 20 µM Bt2cAMP
(cAMP), 50 µM H-8 + 20 µM
Bt2cAMP, 1 µM H-89 + 20 µM
Bt2cAMP, 400 µM allopurinol
(allo), and 400 µM allopurinol + 20 µM Bt2cAMP. After incubation, the media were
collected and the amount of urate produced was determined as described
under Experimental Procedures. Data are presented as mean ± S.D.
for four determinations.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Protein kinase C and urate synthesis.
Fat bodies were dissected and pre-incubated as described in the legend
to Fig. 1 and then incubated for 90 min with the following additions:
Rhodnius saline (ctrl), 20 µM
sphingosine (sphg), 2.5 µM calphostin C
(cph C), 50 µM PMA (pma), 20 µM sphingosine + 50 µM PMA, 2.5 µM calphostin C + 50 µM PMA, 400 µM allopurinol (allo), and 400 µM allopurinol + 50 µM PMA. After
incubation, the media were collected, and the amount of urate produced
was determined as described under "Experimental Procedures." Data
are presented as mean ± S.D. for four determinations.
|
|
When hemin was added to the incubation medium, high rates of urate
synthesis were observed (Fig. 4). This
augmented urate production seems to involve PKC, because calphostin C
reduced urate synthesis to levels close to those of the control without hemin. On the other hand, addition of H-89 together with hemin had no
effect on urate secretion by the fat body, indicating that protein
kinase A does not participate in the stimulation of urate production by
hemin. However, the urate overproduction induced by hemin seems to be a
PKC-dependent phenomenon, a conclusion that led us to
investigate the effect of hemin on protein phosphorylation. When
32Pi-labeled fat bodies were incubated with
hemin, phosphorylation of specific polypeptides (151 and 73 kDa)
increased by 190 and 100%, respectively (Fig.
5, lanes 2 and 3)
when compared with control (Fig. 5, lane 1). This
phosphorylation was blocked by calphostin C (Fig. 5, lane
4), suggesting once more that the action of hemin on fat body
cells involves PKC activation. Although regulation of PKC activity by
superoxide (39) and other pro-oxidant species (40, 41) has already been
reported, this is the first study providing evidence that the synthesis
of a low molecular weight antioxidant can be regulated by an oxidant
agent through PKC activation.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Hemin stimulation of urate synthesis is
mediated by protein kinase C. Fat bodies were dissected and
pre-incubated as described in Fig. 1 and then incubated for 90 min with
the following additions: Rhodnius saline alone (none), 500 µM hemin (ctrl), 500 µM hemin + 400 µM allopurinol (allo), 500 µM hemin + 20 µM sphingosine
(sphg), 500 µM hemin + 2.5 µM
calphostin C (cph C), or 500 µM hemin + 1 µM H-89. After incubation, the media were collected and
urate levels determined (see "Experimental Procedures"). Data are
presented as mean ± S.D. for four determinations.
|
|

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
Hemin stimulates the phosphorylation of 151- and 73-kDa bands through PKC activation. Fat bodies were dissected
and pre-incubated with 100 µCi 32Pi for 30 min. After this period, the medium was discarded and replaced by
Rhodnius physiological saline alone or with the additions
described below. After 1 h of incubation, the medium was
discarded, and the organs were homogenized in SDS-polyacrylamide gel
electrophoresis sample buffer and analyzed by 10% SDS-polyacrylamide
gel electrophoresis. Panel A, autoradiogram of the gel: 1, control; 2, 0.1 mM hemin; 3, 0.5 mM hemin; 4, 0.5 mM hemin + 2.5 µM calphostin C. Panel B, densitometric analysis of the 151- and 73-kDa
bands. Further conditions as described under "Experimental
Procedures."
|
|
Because of the amphiphilic nature of the hemin molecule, its partition
into cellular membranes is expected, and therefore these membranes
could be the site where hemin exerts its stimulatory effect. However,
PKC activation induced by hemin may not be dependent on the presence of
the plasma membrane. Incubation of fat body cytosolic (100,000 × g) fraction with different concentrations of hemin resulted
in a 120% increase in PMA-stimulated/calphostin-inhibited phosphorylation of endogenous substrates (Fig.
6). This could be explained either by
activation of some other upstream soluble component of the cascade or
by a direct action of hemin (or hemin-derived reactive species) on PKC
itself.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 6.
Hemin stimulates PKC activity in R. prolixus fat body cytosolic fraction. Fat bodies were
homogenized and PKC activity was assayed in a cytosolic fraction
obtained as described under "Experimental Procedure." Incubation
media were as follows: Rhodnius saline (control);
20 µM PMA + 20 µg PS/tube; 20 µM PMA + 20 µg PS/tube in the presence of 50 nM calphostin C; 0.01, 0.1, or 0.5 mM hemin or the same concentrations of hemin in
the presence of 50 nM calphostin C.
|
|
PKC has been demonstrated to have its activity directly stimulated by
superoxide anion, redox cycling quinones and micromolar levels of
periodate (42, 43). Several mechanisms have been proposed to explain
PKC modulation by these effectors, including oxidation of regulatory
and binding domains of the kinase itself (44). Another possible
mechanism for PKC activity modulation is a thiol-dependent
inactivation of protein phosphatases 1 and 2A (45). Threonine/tyrosine
phosphorylation catalyzed by mitogen-activated protein kinase has been
shown to be stimulated by H2O2, ionizing radiations, and phorbol esters in NIH-3T3 cells, but a PKC-independent pathway was involved (46). Nevertheless, the hemin-stimulated urate
synthesis reported here could involve a cross-talk between mitogen-activated protein kinase and PKC, because it has been demonstrated that mitogen-activated protein kinase can be activated by
PKC (47, 48).
As pointed out before, hemin is produced in large amounts in the
digestive tract of Rhodnius, and it constitutes an intense physiological source of oxidative stress for this animal (49, 50). The
regulation of urate production according to hemin availability seems to
be an important adaptation of this insect to blood feeding. Urate has
been identified as an important low molecular weight antioxidant in
extracellular fluids of vertebrates (9). It would be interesting to
test whether the control of urate formation by oxidative stress also
occurs in mammalian systems.
 |
ACKNOWLEDGEMENTS |
We express our gratitude to Dr. Martha M. Sorenson for a critical reading of the manuscript, to Rosane
O. M. M. da Costa, Heloísa S. L. Coelho,
Lílian S. C. Gomes, José de Souza L. Júnior,
and José F. de Souza Neto for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação de Coordenação de
Aperfeiçoamento do Pessol de Nível Superior (CAPES),
Financiadora de Estudos e Projetos (Finep), Programa de Núcleos
de Excelência (PRONEX), and Programa de Apoio ao Desenvolvimento
Científico e Tecnológico (PADCT).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 55-21-270-8647;
E-mail: avsouza{at}server.bioqmed.ufrj.br.
 |
ABBREVIATIONS |
The abbreviations used are:
PMA, phorbol
12-myristate 13-acetate;
H-89, N-(2-((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide;
H-8, N(-2-(methylamino)ethyl)-5-isoquinolinesulfonamide;
Me2SO, dimethyl sulfoxide;
PKC, protein kinase C;
PS, phosphatidylserine.
 |
REFERENCES |
-
Ames, B. N.,
Shigenaga, M. K.,
and Hogen, J. M.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
90,
7915-7922[Abstract/Free Full Text]
-
Richter, C.,
Park, J. W.,
and Ames, B. N
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
6465-6467[Abstract]
-
Chance, B.,
Sies, H.,
and Boveris, A.
(1979)
Physiol. Rev.
59,
527-605[Free Full Text]
-
Carney, J. M.,
Starke-Reed, P. E.,
Oliver, C. N.,
Landum, R. W.,
Cheng, M. S.,
Wu, J. F.,
and Floyd, R. A.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3633-3636[Abstract]
-
Halliwell, B.,
and Gutteridge, J. M. C.
(1990)
Arch. Biochem. Biophys.
280,
1-8[Medline]
[Order article via Infotrieve]
-
Davies, K. J. A.,
Sevanian, A.,
Muakkassah-Kelly, S. F.,
and Hochstein, P.
(1986)
Biochem. J.
235,
747-754[Medline]
[Order article via Infotrieve]
-
Matsushita, S.,
Ibuki, F.,
and Aoki, A.
(1963)
Arch. Biochem. Biophys.
102,
446-451[Medline]
[Order article via Infotrieve]
-
Meadows, J.,
and Smith, R. C.
(1986)
Arch. Biochem. Biophys.
246,
838-845[Medline]
[Order article via Infotrieve]
-
Ames, B. N.,
Cathcart, R.,
Schwiers, E.,
and Hochstein, P
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
6858-6862[Abstract]
-
Cochran, D. G.
(1985)
Annu. Rev. Entomol.
30,
29-49[CrossRef]
-
Bursell, E
(1965)
Adv. Insect Physiol.
9,
33-67
-
Cochran, D. G.
(1975)
in
Insect Biochemistry and Function (Candy, D. J., and Kilby, B. A., eds), pp. 177-281, Chapman & Hall, London
-
Wigglesworth, V. B.
(1931)
J. Exp. Biol.
8,
448-451
-
O'Donnel, M. J.,
Maddrell, S. H. P.,
and Gardiner, B. O. C.
(1983)
J. Exp. Biol.
103,
169-184
-
Hilliker, A. J.,
Duyf, B.,
Evans, D.,
and Phillips, J. P.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
4343-4347[Abstract]
-
Tappel, A. L.
(1955)
J. Biol. Chem.
217,
721-733[Free Full Text]
-
Gutteridge, J. M. C.,
and Smith, A.
(1988)
Biochem. J.
256,
861-865[Medline]
[Order article via Infotrieve]
-
Sadrzadeh, S. M.,
Graf, E.,
Panter, S. S.,
Hallaway, P. E.,
and Eaton, J. W.
(1984)
J. Biol. Chem.
259,
14354-14356[Abstract/Free Full Text]
-
Halliwell, B.,
and Gutteridge, J. M. C.
(1986)
Arch. Biochem. Biophys.
256,
501-514
-
Vincent, S. H.,
Grady, R. W.,
Shaklai, N.,
Snider, J. M.,
and Muller-Eberhard, U.
(1988)
Arch. Biochem. Biophys.
265,
539-550[Medline]
[Order article via Infotrieve]
-
Aft, R. L.,
and Mueller, G. C
(1983)
J. Biol. Chem.
258,
12069-12072[Abstract/Free Full Text]
-
Graça-Souza, A. V.,
Petretsky, J. H.,
Demasi, M.,
Bechara, E. J. H.,
and Oliveira, P. L.
(1997)
Free Radical Biol. Med.
22,
209-214[CrossRef][Medline]
[Order article via Infotrieve]
-
Suzuki, Y. J.,
Forman, H. J.,
and Sevanian, A.
(1997)
Free Radical Biol. Med.
22,
269-285[CrossRef][Medline]
[Order article via Infotrieve]
-
Maddrell, S. H. P.
(1969)
J. Exp. Biol.
51,
71-79
-
Domagk, G. F.,
and Schlicke, H. H.
(1963)
Anal. Biochem
22,
219-224[CrossRef]
-
de Meis, L.,
and Masuda, H.
(1974)
Biochemistry
13,
2057-2062[Medline]
[Order article via Infotrieve]
-
Maia, J. C. C.,
Gomes, S. L.,
and Juliani, M. H.
(1983)
in
Genes and Antigenes of Parasites: A Laboratory Manual Proceedings (Morel, C. M., ed), pp. 144-157, Fiocruz, RJ, Brazil
-
Kikkawa, U.,
Minakuchi, R.,
Takai, Y.,
and Nishizuka, Y.
(1983)
Methods Enzymol.
99,
288-298[Medline]
[Order article via Infotrieve]
-
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. R.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275[Free Full Text]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Bialojan, C.,
and Takai, A.
(1988)
Biochem. J.
256,
283-290[Medline]
[Order article via Infotrieve]
-
Cohen, P.,
Holmes, C. F. B.,
and Tsukitani, Y.
(1990)
Trends Biochem. Sci.
15,
98-102[CrossRef][Medline]
[Order article via Infotrieve]
-
Hidaka, H.,
Inagaki, M.,
Kawamoto, S.,
and Sasaki, Y.
(1984)
Biochemistry
23,
5036-5041[Medline]
[Order article via Infotrieve]
-
Chijiwa, T.,
Mishima, A.,
Hagiwara, M.,
Sano, M.,
Hayashi, K.,
Inoue, T.,
Naito, K.,
Toshioka, T.,
and Hidaka, H.
(1990)
J. Biol. Chem.
265,
5267-5272[Abstract/Free Full Text]
-
Nishizuka, Y.
(1988)
Nature
334,
661-665[CrossRef][Medline]
[Order article via Infotrieve]
-
Feener, E. P.,
Shiba, T.,
Hu, K. Q.,
Hilden, P. A.,
White, M. F.,
and King, G. L.
(1994)
Biochem. J.
303,
43-50[Medline]
[Order article via Infotrieve]
-
Kobayashi, E.,
Nakano, H.,
Morimoto, M.,
and Tamaoki, T.
(1989)
Biochem. Biophys. Res. Commun.
159,
548-553[Medline]
[Order article via Infotrieve]
-
Hannun, Y. A.,
Loomis, C. R.,
Merril, A. H., Jr.,
and Bell, R. M.
(1986)
J. Biol. Chem.
261,
12604-12609[Abstract/Free Full Text]
-
Klan, E.,
Roberson, E. D.,
Knapp, L. T.,
and Sweat, J. D.
(1998)
J. Biol. Chem.
273,
4516-4522[Abstract/Free Full Text]
-
Brawn, M. K.,
Chiou, W. J.,
and Leach, K. L.
(1995)
Free Radical Res.
22,
23-37[Medline]
[Order article via Infotrieve]
-
Kuo, M. L.,
Lee, K. C.,
Lin, J. K.,
and Huang, T. S.
(1995)
Biochim. Biophys. Acta
1268,
229-236[Medline]
[Order article via Infotrieve]
-
Kass, G. E. N.,
Duddy, S. K.,
and Orrenius, S.
(1989)
Biochem. J.
260,
499-507[Medline]
[Order article via Infotrieve]
-
Gopalakrishna, R.,
and Anderson, W. B.
(1991)
Arch. Biochem. Biophys.
285,
382-387[CrossRef][Medline]
[Order article via Infotrieve]
-
Gopalakrishna, R.,
and Anderson, W. B.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
6758-6762[Abstract]
-
Nemani, R.,
and Lee, E. Y. C.
(1993)
Arch. Biochem. Biophys.
300,
24-29[CrossRef][Medline]
[Order article via Infotrieve]
-
Liu, Y.,
Guyton, K. Z.,
Gorospe, M.,
Xu, Q.,
Lee, J. C.,
and Holbrook, N. J.
(1996)
Free Radical Biol. Med.
21,
771-781[CrossRef][Medline]
[Order article via Infotrieve]
-
Qiu, Z.,
and Leslie, C. C.
(1994)
J. Biol. Chem.
269,
19480-19487[Abstract/Free Full Text]
-
Yamaguchi, K.,
Ogita, K.,
Nakamura, S.,
and Nishizuka, Y.
(1995)
Biochem. Biophys. Res. Commun.
210,
639-647[CrossRef][Medline]
[Order article via Infotrieve]
-
Oliveira, P. L.,
Kawooya, J. K.,
Ribeiro, J. M. C.,
Meyer, T.,
Poorman, R.,
Alves, E. W.,
Walker, F.,
Machado, E. A.,
Nussenzveig, R. H.,
Padovan, G. J.,
and Masuda, H.
(1995)
J. Biol. Chem.
270,
10897-10901[Abstract/Free Full Text]
-
Petretsky, M. D.,
Ribeiro, J. M. C.,
Atella, G. C.,
Masuda, H.,
and Oliveira, P. L.
(1995)
J. Biol. Chem.
270,
10893-10896[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.