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Laboratories of Pulmonary Pathobiology (D.C.Z., J.E.B., C.R.M., S.W.), Experimental Pathology (J.F.), and Molecular Biophysics (K.B.T., C.P.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; the Department of Medicine, University of North Carolina (J.E.B.), Chapel Hill, North Carolina 27599; and the Department of Pathology, Duke University Medical Center (C.S.), Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Darryl C. Zeldin, Laboratory of Pulmonary Pathobiology, NIH, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: ZELDIN{at}NIEHS.NIH.GOV
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-, ß-,
-, and PP cells, respectively, showed that CYP2J protein expression
was abundantly present in all four cell types, but was highest in the
glucagon-producing -cells. Direct evidence for the epoxidation of
arachidonic acid by pancreatic cytochrome P450 was provided by
documenting, for the first time, the presence of epoxyeicosatrienoic
acids in vivo in human and rat pancreas by gas
chromatography/mass spectrometry. Importantly, the levels of
immunoreactive CYP2J2 in different human pancreatic tissues were highly
correlated with endogenous epoxyeicosatrienoic acid concentrations. We
conclude that human and rat pancreas contain an arachidonic acid
epoxygenase belonging to the CYP2J subfamily that is highly localized
to islet cells. These data together with previous work showing effects
of epoxyeicosatrienoic acids in stimulating insulin and glucagon
secretion from isolated rat pancreatic islets support the hypothesis
that epoxygenase products may be involved in stimulus-secretion
coupling in the pancreas. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Several studies have shown that cyclooxygenase, lipoxygenase, and P450 monooxygenase metabolites of arachidonic acid are produced in mammalian endocrine tissues and may be involved in the release of peptide hormones. For example, the rat anterior pituitary has been shown to metabolize arachidonic acid to 12-HETE, PGE2, and 5,6-EET (3, 4). Inhibitors of arachidonate 12-lipoxygenase block TRH-induced PRL secretion from anterior pituitary cells (5). Additionally, both PGE2 and 5,6-EET have been shown to evoke the release of gonadotropic hormones, GH, and PRL from the anterior pituitary (4, 6, 7, 8, 9). Microsomal fractions prepared from the rat neurohypophysis metabolize arachidonic acid to PGs and EETs, which stimulate the release of arginine vasopressin and oxytocin from this tissue (10). In the hypothalamus, PGE2 releases LHRH, and the EETs release somatostatin (11, 12). Adrenal glomerulosa cells primarily synthesize 12-HETE, inhibition of 12-lipoxygenase blocks angiotensin II-induced aldosterone secretion from these cells, and exogenous 12-hydroperoxyeicosatetraenoic acid restores aldosterone secretion in this system (13).
In the pancreas, glucose-induced insulin secretion is coupled to
activation of phospholipases and the release of arachidonic acid (14, 15). Isolated islets have been shown to biosynthesize 12-HETE,
thromboxane, PGE2, and PGF2 (15, 16).
Inhibition of PGH2 synthases with indomethacin completely
prevents islet synthesis of PGs, but does not influence glucose-induced
insulin secretion, suggesting that this pathway of arachidonic acid
metabolism is not involved in stimulus-secretion coupling in the
endocrine pancreas (17, 18). In contrast, glucose stimulates the
synthesis of 12-HETE, and inhibition of 12-HETE production suppresses
the glucose- and arginine-induced insulin release from isolated islets
(14, 18). However, 12-lipoxygenase products do not completely reverse
the effects of lipoxygenase inhibitors on insulin secretion from
isolated islets and have relatively weak insulin secretagogue effects
on cultured islet cells, suggesting that alternate pathways of
arachidonic acid metabolism may be involved in controlling insulin
release (17, 18). In this regard, Falck and co-workers have shown that
the EETs were potent mediators of insulin and glucagon release in
isolated rat pancreatic islets (19). These effects were highly
regioselective in that 5,6-EET stimulated insulin secretion but had no
effect on glucagon release, and 8,9-, 11,12-, and 14,15-EETs increased
glucagon release without affecting insulin secretion (19). Attempts at
detecting EETs derived from incubations of pancreatic microsomal
fractions with arachidonic acid have been largely unsuccessful (19, 20). Additionally, the documentation of EETs as endogenous constituents
of pancreatic tissue has not been reported. Furthermore, the identity
of the P450 isoform(s) responsible for the production of these
bioactive eicosanoids in the pancreas remains unknown.
Recently, our laboratory has cloned a new human P450 complementary DNA (CYP2J2) and the corresponding rat homolog (CYP2J3),1 both of which were highly expressed in extrahepatic tissues (21). The recombinant human and rat proteins were active in the metabolism of arachidonic acid to EETs (21). In this report, we show that both CYP2J2 and CYP2J3 are abundantly expressed in the pancreas and that expression is highly localized to cells in the islets of Langerhans. We further document the presence of EETs in vivo in human and rat pancreas using gas chromatography/mass spectrometry (GC/MS), thus suggesting a role for CYP2J enzymes in the biosynthesis of EETs in the endocrine pancreas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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-bromo-2,3,4,5,6-pentafluorotoluene,
N,N-diisopropylethylamine,
N,N-dimethylformamide, and diazald were purchased
from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and
reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless
otherwise specified. Normal human pancreata were obtained through the
Cooperative Human Tissue Network (National Disease Research
Interchange, Philadelphia, PA) or from local tissue donors, snap-frozen
in liquid nitrogen immediately after collection, and stored at -80 C
until use or immediately fixed in formalin. Rat pancreata were obtained
from male Fisher 344 rats reared at the NIEHS, fed ad
libitum, and killed by lethal CO2 inhalation. All
animal studies described herein were conducted in accordance with
principles and procedures outlined in the NIH Guide for the Care and
Use of Laboratory Animals and approved by the NIEHS committee on animal
care and use. Approval for use of human tissues was obtained from the
Duke University and NIEHS institutional review boards.
Protein immunoblotting and immunohistochemistry
Microsomal fractions were prepared from frozen normal human and
rat pancreatic tissues by differential centrifugation at 4 C as
previously described (22). Polyclonal antihuman CYP2J2 IgG was raised
in New Zealand White rabbits against the purified recombinant human
CYP2J2 protein and affinity purified as previously described (21). For
immunoblotting, microsomal fractions or partially purified, recombinant
CYP2J2 were electrophoresed in SDS-10% (wt/vol) polyacrylamide gels
(80 x 80 x 1 mm), and the resolved proteins were
transferred electrophoretically onto nitrocellulose membranes.
Membranes were immunoblotted using rabbit antihuman CYP2J2 IgG (1:4000
dilution), goat antirabbit IgG conjugated to horseradish peroxidase
(Bio-Rad, Richmond, CA), and the ECL Western Blotting Detection System
(Amersham Life Sciences, Aylesbury, UK) as previously described (21).
Autoradiographs were scanned using an LKB Ultrascan XL Enhanced Laser
Densitometer (Pharmacia, Piscataway, NJ). The relative CYP2J2 content
of human pancreas samples was established by preparing standard curves
using the purified recombinant CYP2J2 protein. Neither preimmune IgG
nor rabbit nonimmune IgG (Biogenex Laboratories, San Ramon, CA)
significantly cross-reacted with microsomal fractions prepared from
human or rat pancreas. The anti-CYP2J2 IgG recognized a single protein
band corresponding to purified recombinant human CYP2J2 in microsomal
fractions prepared from several different human tissue samples and did
not cross-react with the following human P450s: CYP1A1, CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 (21). The
anti-CYP2J2 IgG also immunoreacted with a single protein band
corresponding to recombinant rat CYP2J3 in microsomal fractions
prepared from different rat tissues, but did not cross-react with the
following rat P450s: CYP1A1, CYP2A1, CYP2B1, CYP2B2, CYP2C11, CYP2C13,
CYP2C23, and CYP2E1 (see Footnote 1).
For immunohistochemistry, rat and human pancreatic tissues were fixed
in 10% neutral buffered formalin, processed routinely, and embedded in
paraffin. Localization of CYP2J2 and CYP2J3 protein expression was
investigated using the anti-CYP2J2 IgG (1:200 dilution). Rabbit
antiporcine glucagon IgG (1:3000 dilution), guinea pig antiporcine
insulin IgG (1:3000 dilution), rabbit antihuman somatostatin IgG
(1:3000 dilution), and rabbit antihuman pancreatic polypeptide (1:700
dilution; Dako Corp., Carpenteria, CA) were used to identify pancreatic
islet of Langerhans -, ß-, -, and PP cells, respectively. Each
antibody specifically immunoreacted with the corresponding rat and
human pancreatic polypeptides. Adjacent sections (5–6 µm) were
stained for either CYP2J2 (human) or CYP2J3 (rat), glucagon, insulin,
somatostatin, or pancreatic polypeptide. Slides were deparaffinized in
xylene and hydrated through a graded series of ethanol to 1x
automation buffer (25 mM Tris•HCl, pH 7.5, 150
mM NaCl, plus nonhazardous proprietary reagents) (Biomeda,
Burlingame, CA) washes. Endogenous peroxidase activity was blocked with
3% (vol/vol) hydrogen peroxide for 15 min. After rinsing in 1 x
automation buffer, slides were microwaved at high power (750 watts) in
sodium citrate buffer for 5 min, cooled for 15 min, and blocked with
normal goat serum. The primary antibodies were applied to the
respective slides for 30 min. Preimmune rabbit IgG was used as the
negative control in place of the CYP2J2 primary antibody. Normal guinea
pig serum (Vector Laboratories, Burlingame, CA) was used as the
negative control in place of the insulin primary antibody. Normal
rabbit serum (Vector Laboratories) was used as the negative control in
place of the primary antibodies to glucagon, somatostatin, and
pancreatic polypeptide. The bound primary antibodies were visualized by
avidin-biotin-peroxidase detection, using the Vectastain Rabbit Elite
Kit or the Vectastain Guinea Pig IgG Kit (Vector Laboratories)
according to the manufacturer’s instructions and using
3,3'-diaminobenzidine as the color-developing reagent. Slides were
counterstained with Harris hematoxylin, dehydrated through a graded
series of ethanol to xylene washes, and coverslipped with Permount
(Fisher, Springfield, NJ). Specific CYP2J2 and CYP2J3
immunohistochemical staining was confirmed by prestaining adsorption of
the anti-CYP2J2 IgG with a 300-fold molar excess of the purified
recombinant CYP2J2 antigen in 140 mM NaCl, 4 mM
KCl, and 10 mM sodium phosphate buffer (pH 7.4) at 4 C for
12 h.
Quantification of endogenous EETs in human and rat pancreas
The methods used to quantify endogenous EETs present in human
and rat pancreas were similar to those used to quantify endogenous EETs
in rat liver (23), rabbit lung (22), and human heart (21). Briefly,
freshly obtained tissues (0.5–1.5 g) were frozen in liquid nitrogen
and immediately homogenized in 10–15 ml 10 mM sodium
phosphate buffer (pH 7.4) containing 140 mM NaCl, 4
mM KCl, and 1 mg/ml tri-phenylphosphine, a
hydroperoxide reducing agent. The homogenate was extracted twice, under
acidic conditions, with 2 vol chloroform-methanol (2:1) and once more
with an equal volume of chloroform, and the combined organic phases
were evaporated in tubes containing mixtures of synthetic
[1-14C]8,9-, 11,12-, and 14,15-EET (55–57 µCi/µmol;
30–80 ng each) internal standards. Saponification to recover
phospholipid-bound EETs was followed by SiO2 column
purification. The eluent, containing a mixture of radiolabeled internal
standards and total endogenous EETs, was resolved into individual
regioisomers by HPLC as previously described (23). For chiral analysis,
rat pancreas EET regioisomers were derivatized to corresponding
EET-pentafluorobenzyl (EET-PFB) or EET-methyl esters, purified by
normal phase HPLC, and resolved into corresponding antipodes by chiral
phase HPLC as previously described (23, 24). For quantification,
aliquots of individual EET-PFBs were dissolved in dodecane and analyzed
by GC/MS on a VG TRIO-1 quadrupole mass spectrometer (Fisons/VG,
Altrincham, Manchester, UK) operating under negative ion chemical
ionization conditions (source temperature, 100 C; ionization potential,
75 eV; filament current, 500 µA) at unit mass resolution, and using
methane as a bath gas. Quantifications were made by selected ion
monitoring of m/z 319 (loss of PFB from endogenous EET-PFB) and m/z 321
(loss of PFB from [1-14C]EET-PFB internal standard). The
EET-PFB/[1-14C]EET-PFB ratios were calculated from the
integrated values of the corresponding ion current intensities.
Other methods
EETs were prepared by total chemical synthesis according to
previously described procedures (25, 26). [1-14C]EET
internal standards were synthesized from
[1-14C]arachidonic acid (55–57 µCi/µmol) by
nonselective epoxidation as previously described (27). Synthetic EETs
and [1-14C]EET were purified by reverse phase HPLC as
previously described (22, 23). Methylations were performed using an
ethereal solution of diazomethane (22). PFB esters were formed by
reaction with pentafluorobenzyl bromide as previously described (22).
Protein determinations were performed according to the method of
Bradford (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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, anti-CYP2J2 IgG immunoreacted
with an electrophoretically distinct band at approximately 56 kDa in
microsomal fractions prepared from rat and human pancreas. In rats,
there was relatively low interanimal variability in the expression of
pancreatic CYP2J3 (Fig. 1). In contrast, the human interindividual
variability in pancreatic CYP2J2 expression was significantly greater;
thus, samples HP2 and HP3 contained significantly more immunoreactive
CYP2J2 than samples HP1 and HP4 (densitometric values: HP1, 0.26; HP2,
0.91; HP3, 1.62; HP4, 0.51; Fig. 1). Based on these data, we conclude
that 1) CYP2J2 protein is expressed in human pancreas; 2) CYP2J3
protein is expressed in rat pancreas; and 3) there is some
interindividual differences in expression of CYP2J2 protein in human
pancreas.
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, CYP2J3 immunostaining was highly localized to islets
of Langerhans cells in rat pancreas. Positive staining was present
throughout the islets, but the most intense staining was observed along
a narrow rim at the periphery of the islets (Fig. 2, A and C). Positive
staining, albeit much less intense, was also noted in pancreatic acinar
cells, vascular smooth muscle cells, and epithelial cells lining the
intralobular ducts, whereas interlobular connective tissue did not
stain (Fig. 2, A and C). Both preimmune IgG (Fig. 2, B and D) and
rabbit nonimmune IgG (data not shown) produced negative staining
throughout the entire rat pancreas. Furthermore, prestaining adsorption
of anti-CYP2J2 IgG with excess purified recombinant CYP2J2 abolished
the positive reaction, thus demonstrating the specificity of the
immunostaining for the CYP2J3 protein (Fig. 2E). An identical pattern
was observed in five different rat pancreatic specimens immunostained
with the anti-CYP2J2 IgG.
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, A, C, and E). As in rat pancreas,
positive staining was present throughout the islets; however, the
proportion of intensely stained cells in the human islets was greater
than that in the rat. Furthermore, the intensely stained cells were not
limited to the periphery of the human islets (Fig. 3, A, C, and E). As
with the rat, both preimmune IgG (Fig. 3, B, D, and F) and rabbit
nonimmune IgG (data not shown) produced negative staining throughout
the human pancreas. In addition, prestaining adsorption of anti-CYP2J2
IgG with excess purified, recombinant CYP2J2 abolished the positive
staining in human pancreas, thus demonstrating the specificity of the
immunostaining for the CYP2J2 protein (Fig. 3G). An identical pattern
was observed in five different human pancreatic specimens immunostained
with the anti-CYP2J2 IgG.
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-, ß-, -, and PP cells, respectively. Glucagon-producing
-cells at the periphery of the rat islets (Fig. 4D)
expressed the highest levels of CYP2J3 immunoreactive protein (Fig. 4B). The expression of CYP2J3 protein in insulin-producing ß-cells
(Fig. 4C), somatostatin-producing -cells (Fig. 4E), and pancreatic
polypeptide-producing PP cells (Fig. 4F) was substantial, but appeared
to be lower than that in -cells (Fig. 4D). Therefore, in rat
pancreas, although all four islet cell types expressed abundant CYP2J3
protein, the highest levels of expression were present in -cells. In
human pancreatic islets, the difference in CYP2J2 expression among
-, ß-, -, and PP cells was not as obvious as that in rat
islets, and it appeared that there was substantial expression in all
four cell types (Fig. 5). These immunohistochemical data
together with a previous report that EETs stimulate insulin and
glucagon secretion in pancreatic islets (19) support the hypothesis
that these enzymes and their bioactive eicosanoid products may be
involved in the control of stimulus-secretion coupling in the
pancreas.
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, rat
pancreas contained about 280 ng total EET/g tissue. The major EET
regioisomer present in rat pancreas was 8,9-EET (47% of the total),
followed by lower amounts of 14,15- and 11,12-EETs (32% and 21% of
the total, respectively; Table 1). The labile 5,6-EET suffered
extensive decomposition during the extraction and purification process
used and, therefore, could not be quantified. Chiral analysis of rat
pancreatic EETs revealed that the 14(R),15(S)-
and 11(S),12(R)-EETs were the predominant
antipodes (optical purity, 59% and 66%, respectively; Table 1). In
contrast, 8,9-EET was recovered in nearly racemic mixtures (Table 1).
To our knowledge, this is the first demonstration of EETs as endogenous
constituents of pancreatic tissue.
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).
Despite variability in the total amount of EETs in human pancreas, the
regiochemical distribution remained relatively consistent from specimen
to specimen. Thus, in each case, the predominant EET regioisomer
present was 8,9-EET (45–49% of the total), followed by lower amounts
of 14,15-EET (28–41% of the total) and 11,12-EET (14–23% of the
total; Table 2). As before, the labile 5,6-EET suffered extensive
decomposition during the extraction and purification process used and,
therefore, could not be quantified. Interestingly, the amount of
immunoreactive CYP2J2 present in a given human pancreatic specimen, as
determined by scanning densitometry, was highly correlated with the
amount of total EETs recovered from the specimen (Pearson’s
correlation coefficient, r = 0.98; P = 0.007; Fig. 1
and Table 2). This suggests that CYP2J2 is one of the primary
epoxygenases present in human pancreas. The limited availability of
fresh normal human pancreatic tissue precluded stereochemical analysis
of human pancreatic EETs. Attempts to document de novo
synthesis of EETs in human or rat pancreas by incubating pancreatic
microsomal fractions with arachidonic acid in the presence of NADPH and
an NADPH-regenerating system were unsuccessful.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Previous studies have shown constitutive expression of a number of P450
monooxygenases in rat and human pancreas, including members of the
CYP1A, CYP2B, and CYP3A subfamilies (31, 32). Without exception, in
both rats and humans, immunohistochemical studies have demonstrated
that P450 enzyme expression is present only in acinar cells of the
exocrine pancreas, without significant P450 expression in the islets of
Langerhans (31, 32). In contrast, CYP2J2 and CYP2J3 expression is
highly localized to pancreatic islet cells, with substantially less
CYP2J expression in acinar tissue. Furthermore, CYP2J proteins are
present in all four cell types within pancreatic islets and at
exceptionally high levels in the glucagon-producing -cells.
Interestingly, Hall and co-workers (33) recently showed that NADPH-P450
reductase, a key enzyme that provides reducing equivalents from NADPH
to P450, is also expressed, albeit at low levels, in pancreatic islet
cells.
Several researchers have reported that the lipoxygenase inhibitors nordihydroguaiaretic acid and eicosa-5,8,11,14-tetraynoic acid inhibit glucose- and arginine-induced insulin release, suggesting that lipoxygenase products may participate in insulin secretion (14, 17, 18). However, lipoxygenase products do not completely reverse the effects of lipoxygenase inhibitors on insulin secretion from isolated islets and have relatively weak insulin secretagogue effects on cultured islet cells (17, 18). Furthermore, these inhibitors possess other actions, including interference with arachidonate oxygenation by the P450 enzyme system (34). These results suggest that P450-derived eicosanoids might also play a role in the control of pancreatic hormone secretion. In this regard, Falck and co-workers have shown that the EETs stimulate the release of insulin and glucagon from isolated rat pancreatic islets at concentrations as low as 10 nM (19). Currently, the mechanism by which the EETs control peptide hormone secretion in the pancreas is unknown. However, the pivotal role of intracellular Ca2+ in coupling glucose metabolism to insulin secretion (35, 36, 37, 38) together with the known effects of the EETs in mobilizing intracellular Ca2+ in the pituitary and other tissues (8, 9, 39, 40) suggest that the insulin and glucagon secretagogue effects of the EETs may be mediated through a Ca2+-dependent mechanism.
Although previous attempts to detect cytochrome P450 in pancreatic microsomal fractions by means of conventional spectrophotometric techniques have been unsuccessful, published studies report that the pancreas can, in fact, catalyze the oxidative metabolism of a variety of xenobiotics, such as nitrosamines and aromatic hydrocarbons (41, 42). The demonstration here of EETs as endogenous constituents of rat and human pancreas provides direct evidence to support the in vivo pancreatic cytochrome P450 metabolism of arachidonic acid. To our knowledge, this is the first report documenting the presence of EETs in pancreatic tissue. Previous attempts to identify EETs from preparations of isolated pancreatic islets or from incubations of microsomal fractions prepared from islet cells with arachidonic acid have been unsuccessful (19, 20). We were also unable to observe significant arachidonic acid metabolism by pancreatic microsomes, perhaps due to the abundance of proteases present in the pancreas that partially degrade and/or inactivate the pancreatic P450 enzyme(s) during microsomal preparation. Compared to rat liver and kidney, rat pancreas contains approximately 63% and 45% less total EETs per g tissue, respectively (43, 44). Furthermore, the regio- and stereochemical compositions of rat pancreas EETs are unique to this tissue. Thus, although 14,15-EET is the predominant regioisomer recovered from rat liver and kidney, 8,9-EET is the principle metabolite isolated from rat pancreas (43, 44). Rat pancreas is the only tissue reported to primarily biosynthesize 14(R),15(S)-EET and 11(S),12(R)-EET and produce racemic 8,9-EET (43, 44). Human pancreas contained less total EETs than human liver and kidney cortex, but more than human heart (21, 45, 46). Interestingly, the quantity of EETs recovered from human pancreatic tissues was variable from specimen to specimen and correlated well with the amount of immunoreactive CYP2J2 present in microsomal fractions prepared from the specimen. These data provide indirect evidence that CYP2J2 is one of the primary epoxygenases present in human pancreas. Pancreas tissue is known to undergo rapid autolysis. Special care was taken to avoid autolysis during handling of the human tissue samples, including rapid freezing of specimens after collection and use of protease inhibitors during microsomal preparation. Furthermore, histopathological examination of the human specimens used failed to reveal evidence of sample autolysis. Hence, it is unlikely that the observed interindividual differences in pancreatic CYP2J2 expression were due to variations in the degree of autolysis in different specimens.
Other investigators have detected products of 5-, 12-, and
15-lipoxygenases during incubations of isolated pancreatic islets or
the 10,000 x g supernatant of isolated pancreatic
islet homogenate with exogenous arachidonic acid (17, 47). The most
abundant lipoxygenase product formed is 12-HETE, which is produced in
the amount of 
2 ng/1000 islets (47). The synthesis of 12-HETE by
cultured pancreatic islets from endogenous arachidonic acid has also
been quantified (18). To our knowledge, however, there are no previous
reports quantifying the amount of 12-HETE present in human or rat
pancreatic tissue. Thus, it is difficult to compare the relative
amounts of lipoxygenase and epoxygenase products present in the
pancreas.
EET hydration by cytosolic epoxide hydrolase (48) and microsomal epoxide hydrolase is highly regio- and stereoselective (Zeldin, D. C., and J. H. Capdevila, unpublished observations). EET acylation to cellular phospholipids is also stereoselective (49). The regio- and stereochemical compositions of endogenous pancreatic EETs represent the sum of the following: 1) regio- and stereoselective EET production by pancreatic epoxygenase(s), 2) regio- and stereoselective EET removal by pancreatic epoxide hydrolases, and 3) stereoselective lysolipid EET acylation. Thus, the regio- and stereochemical compositions of EETs recovered from human and rat pancreas do not necessarily reflect the regio- and stereoselectivity of EET production by human CYP2J2 and rat CYP2J3.
In summary, we have provided immunologic data demonstrating that human CYP2J2 and rat CYP2J3 are abundantly expressed in the pancreas and highly localized to specific cells within the islets of Langerhans. We also present biochemical data showing that CYP2J products, the EETs, are found in vivo in the pancreas. We conclude that in addition to the cyclooxygenase and lipoxygenase pathways, the P450 epoxygenase pathway is an important member of the human pancreas arachidonic acid metabolic cascade. Based on these data and previous work showing that the EETs stimulate glucagon and insulin release from pancreatic islets at low concentrations, we speculate that epoxygenase products may be involved in stimulus-secretion coupling in the pancreas.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Received August 15, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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