- Email: [email protected]
Arachidonic acid induces an increase in the cytosolic calcium concentration in single pancreatic islet beta cells
Vol.
184,
No.
April
30,
1992
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
647-653
ARACHIDONIC ACID INDUCES AN INCREASE IN THE CYTOSOLIC CALCIUM CONCENTRATION IN SINGLE PANCREATIC ISLET BETA CELLS Sasanka
Ramanadham,
Richard
Gross*,
and John Turk
Departments of Medicine and Pathology, Division of Laboratory Medicine, and *Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, St. Louis, Missouri 631 IO Received
March
12,
1992
The insulin secretagogue D-glucose induces both accumulation of nonesterified arachidonic acid (35 pM) in pancreatic islets and a rise in beta cell cytosolic [Ca++]i. Arachidonate amplifies both voltage-dependent Ca++ entry in secretory cells and depolarization-induced insulin secretion. Here, arachidonate induced a biphasic rise in [Ca++]i of Fura-2AM loaded beta cells which increased with arachidonate concentration (5-30 PM), was reversed upon washout, and was unaffected by the arachidonate oxygenase inhibitor BW755C. The sustained phase of the rise was abolished by removal of extracellular Ca++ and amplified by depolarization with KCI. The accumulation of nonesterified arachidonate in islets stimulated by D-glucose may therefore promote the D-glucose-induced 0 1992AcadrmrcPL&S,Inc. rise in beta cell [Ca++]i.
Isolated pancreatic islets secrete insulin upon metabolism of fuel secretagogues such as D-glucose (1). The metabolism of glucose generates ATP which may serve a second messenger function in islet beta cells. A beta cell K+-channel is inactivated by ATP (2,3), and its closure induces membrane depolarization (3). The rise in membrane potential activates voltage-gated Ca ++-channels, and Ca++ enters the beta cell from the extracellular space, resulting in a rise in [Ca++]i which is required for the induction of insulin secretion (4, 5). Fuel secretagogues also induce hydrolysis of beta cell membrane phospholipids, resulting in accumulation of the phospholipid-derived mediators inositol 1 ,4,5-trisphosphate, nonesterified arachidonic acid, and arachidonate metabolites (6-8). Fuel secretagogue-induced activation of beta cell phosphoinositidephospholipase C requires Ca++ influx and is prevented by Ca++-channel blockers or by removal of extracellular Ca++ (6). In contrast, a component of the fuel-secretagogue-induced accumulation of nonesterified
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
arachidonate and its metabolites in islets occurs without extracellular Ca++ and in the presence of Ca++-channel blockers (8). At concentrations which accumulate in 17 mM D-glucose-stimulated islets (8), arachidonate amplifies voltage-dependent Ca ++ entry into prolactin-secreting cells (14) and amplifies depolarization-induced insulin secretion from islets These observations suggest that fuel secretagogue-induced (8). accumulation of nonesterified arachidonate in beta cells may amplify Ca++ entry and the secretory response to small changes in secretagogue concentration. We have therefore measured the direct effects of arachidonate on cytosolic [Ca++] using individual pancreatic islet beta cells.
MATERIALS AND METHODS. Pancreatic islets were isolated by collagenase digestion (18) and dispersed into single cells with dispase (19). Filtered (60 pm nylon screen) dispersed cells were subjected to autofluorescence-activated cell sorting (FACS) with a FACS-IV at 488 nm. Emission at instrument (16) using argon laser illumination Suspensions of single 510-550 nm reflected endogenous FAD content. beta cells (>90% purity) were so prepared, as verified by immunocytochemical staining for insulin, glucagon, somatostatin, and pancreatic polypeptide (15,16). FACS-purified beta cells (1 OS/ml) were plated onto Cell Tak-coated glass coverslips and incubated overnight at 37°C in CMRL-1066 medium containing 10% heat-inactivated fetal bovine serum, 1% L-glutamine, and 1% (w/v) each of penicillin and streptomycin under 5% CO2/95% air. CMRL 1066 medium was replaced with KRB [containing in (mM); NaCl (115), KCI (5), NaHC03 (24), CaCl2 (2.5), MgCl2 (l), HEPES (50), D-glucose (3), pH 7.41 and the cover-slip attached beta cells were loaded with Fura-2AM for 30 min as described (21), washed with KRB, and perifused with medium in a thermostatically-heated (37°C) chamber for the microfluorimetry experiments. Cytosolic [Ca++]i was measured as described (16, 17, 20, 21) with dual excitation wavelength (340 and 380 nm) microfluorimetry of single Fura-2AM loaded beta cells on a PTI DeltaScan instrument with a Nikon microscope. Emission was measured at 500-530 nm. ‘The ratio (R) of emission intensity at an excitation wavelength of 340 nm was divided by that at 380 nm to yield an index of [Ca ++]i. Absolute [Ca++]i was calculated from R after determining Rmax (R with 2 FM 4-Br-A23187) and Rmin (R following 1 mM EGTA after 4-Br-A23187) and correcting for autofluorescence (R with MnCl2) as described (17, 21). Arachidonic acid (NuChek, Elysian, MN) was dissolved in 0.1 M Na2C03 at [0.2 mM] just before use and diluted in albumin-free KRB (7). BW755C (Wellcome Research Laboratories, Kent, UK) was dissolved in DMSO, 0.15 M NaCl (1 :17, w/v) just before use. Nifedipine and EGTA were from Sigma (St. Louis, MO). 4-Br-A23187 (Calbiochem, LaJolla, CA) was dissolved in DMSO. Cell Tak was obtained from Collaborative Res. Inc., Bedford, MA. 648
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS. Exogenous arachidonic acid induced a rise in the cytosolic of individual beta cells (Figure 1). The threshold concentration was about 5 FM while no measurable effect was observed with 1 PM. At higher concentrations (1 O-30 PM) arachidonate produced a biphasic [Ca++]i response. A rapidly developing, transient rise was followed by a slowly developing, sustained increase. The magnitude of the sustained phase increased with increasing [arachidonate]. The arachidonate-induced rise in beta cell [Ca++]i was fully reversible, and the [Ca++]i returned to basal [Ca++]
levels upon removal of arachidonate from the medium and perifusion with albumin-containing buffer. Arachidonate did not, therefore, simply permeabilize the cell membrane like a detergent. The arachidonate oxygenase inhibitor BW755C at a concentration of 500 FM, which
_
7.00, B.
7.00,A.
AAl5rMl
$ 7.00 EE fc
c.
AAllO,Ml
g3.50 629 -. n d
”
r--OJ )0
I I 500 1000 TnmeIre<)
OJ,
OJ,
, 0
I I r oJ,1000 1500 2000 0 Tme (set)
I 500
0
1 1500
800
i
400
, 200
, 800
:,A G.
1200,
? c E .?
, , 400 Ttme tree)
I f 400 600 Tme (SK)
Arachbdonic Acid Concenlrotion Response Curve 0 Peak 1 . Peak 2
0 0 I
5
IO [AA]lrMl
I5
20
Fiaure 1: Arachidonic acid induces a rise in beta cell cvtosolic calcium concentration in a concentration-deDendent and reversible manner. Single beta cells were attached to coverslips and loaded with Fura-2AM as in Methods At the indicated time arachidonate (l-30 PM) was introduced in albumin ‘(BSA)-free buffer. Washout solution contained 0.1% fatty acid-free BSA (w/v) and no arachidonate. 649
Vol.
184,
No.
2,
1992
BIOCHEMICAL
1500
AND
BIOPHYSICAL
AA
RESEARCH
COMMUNICATIONS
AA + BW755C
1000 AA
AA
/Iv
400 200 0 iA
AA
AA + BW755C
Fiaure 2. The arachidonic acid-induced rise in beta cell cvtoso lid ca lcium concentration is not influenced bv the arachidonate oxvgenase inhibitor BW755C. Experiments were performed as in Fig. 1 with a fixed arachidonate concentration (15 PM) in the absence (left) or presence (right) of BW755C (500 PM). Mean (n = 8) values (_+SEM) of [Ca++]i are presented. completely inhibits islet arachidonate the rise in beta cell [Ca++]i induced
metabolism (22), did not influence by arachidonate (Figure 2). Similar
results were obtained with the selective cyclooxygenase indomethacin (10 uM, not shown). This suggests that the rise was
induced
cell
The sustained phase of the 15 pM arachidonate-induced [Ca++]i was virtually abolished by removal of extracellular
EGTA
and was slightly
nifedipine reflects sensitive transient unaffected the
by arachidonate
early
(Figure
3A),
lower
itself
and
not a metabolite.
in the presence
indicating
that
inhibitor in [Ca++]i
this
rise in beta Ca++ with
of the Ca++-channel phase
of the
rise
blocker in [Ca++]
extracellular Ca++ entry, possibly mediated in part by voltageCa+ +-channels. In contrast, the magnitude of the early, phase of the 15 pM arachidonate-induced rise in [Ca++]i was by removal rise
may
of extracellular reflect
Ca ++
release
of
or nifedipine, Ca++
from
suggesting that intracellular
sequestration sites. Arachidonate (5-30 PM) has similarly been reported to induce a biphasic rise in [Ca ++]i in a T-lymphocyte cell line with an early
transient
phase
reflecting
mobilization
of
intracellular
Ca++
and a
space, later sustained phase reflecting Ca++ entry from the extracellular induced by arachidonate itself and not its metabolite(s) (23). Depolarizing concentrations (lo-40 mM) of KCI also induced a rise in the beta cell [Ca++]i (Figure 3B). The 15 ,LLM arachidonate-induced rise in beta cell [Ca++]i was potentiated by KCI (lo-40 mM) (Figure 3B). Arachidonate may 650
Vol.
184,
No.
2,
1992
BIOCHEMICAL
A
AND
ARACHIDONIC
BIOPHYSICAL
ACID/KC1
i
EGTA
RESEARCH
COMMUNICATIONS
or NIFEDIPINE
1500 = 15 !a
AA (Peak 2)
1200 iz
--
c
900
F .z 00
600
0, 300
0 1
2
3
1
B
2
ARACHIDONIC
3
1
ACID
2
3
t KCI
1600/
0
10
20 WI1
30
40
(mW
Fiaure 3: The sustained chase of the arachidonic acid-induced rise in beta cell cvtosolic calcium concentration derives from extracellular calcium and is amolified bv deoolarization with KCI. Panel A studies were performed as in Fig. 1. Values represent mean (n = 8) differences (+SEM) between basal [Ca++]i and the sustained phase of the stimulus-induced rise. The [EGTA] was 1 mM and the [nifedipine] 400 nM. Panel E3 cells were exposed to the indicated [KCI] and [arachidonate] (0 or 15 PM) simultaneously. Values represent mean (n = 8) differences (+SEM) between [Ca ++]i at 5 mM KCI with 0 or 15 PM and [Ca++ arachidonate ]i at the indicated [KCI] with 0 or 15 FM arachidonate during the sustained phase of the [Ca++]i response to arachidonate. therefore
amplify
prolactin-secreting l7lSCUSSION. arachidonate from
voltage-dependent GH3
cells
Ca++
entry
in beta cells,
as it does
in
(14).
The observations beta cell membrane
that glucose induces release of free phospholipids by a process that
does not require Ca++ entry (8, 24), that glucose induces a rise in the beta cell [Ca++]i reflecting Ca++ entry (4, 5), and that exogenous arachidonate in concentrations which accumulate in glucose-stimulated induces Ca++ entry into beta cells (Figures l-3) suggest that 651
islets also arachidonate
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
may participate in the glucose-induced rise in beta cell [Ca++]i. Inhibition of glucose-induced phospholipid hydrolysis and arachidonate release might therefore blunt the rise in beta cell [Ca++]i and insulin secretion. A Ca++independent phospholipase A2 which is selective for arachidonic acid and whose activity is regulated by ATP has been identified in canine myocardium (g-12). A similar activity has also recently been identified in pancreatic islets (13). Recently, a haloenol lactone suicide substrate (HELSS) which is a selective, mechanism-based inhibitor of this Ca++independent phospholipase A2 enzyme has been identified. We have recently observed that the islet phospholipase A2 activity is inhibited by low uM concentrations of HELSS which do not have an affect on islet oxidation of [14C]-glucose to [l4C]O2 (13). The 17 mM D-glucose-induced rise in beta cell [Ca++]i was inhibited by 66 f 13% by 3-10 uM HELSS, although the rise in beta cell [Ca++]i induced by depolarization was unaffected, and HELSS also suppressed both arachidonate release and insulin secretion from D-glucose-stimulated islets (13). Thus, Ca++independent phospholipase AZ-mediated accumulation of nonesterified arachidonate in islets stimulated by D-glucose may promote Ca++ influx and the rise in beta cell [Ca++]i required for the induction of insulin secretion.
ACKNOWLEDGMENTS. We acknowledge support by NIH grants HL34389, DK-34388 and DK-01553 and by the Monsanto Co. and the superb technical assistance of Alan Bohrer, Mary Mueller, Kelly Kruszka and Lori Zupan.
REFERENCES
1.I 2.1
3.) 4.) 5.1 6.)
7.1 8.)
9.1
Meglasson, M.D., and Matschinsky, F. (1986) Diabetes/Metab. Rev. 2, 63-214. Cook, D.L., and Hales, C.N. (1984) Nature 311, 271-273. Sturgess, N.C., Cook, D.L., Ashford, M.L.J., and Hales, C.N. (1985) Nature 312, 446-448. Arkhammar, P., Nilsson, T., Rorsman, P., and Berggren, P.O. (1987) J. Biol. Chem. 262, 5448-5454. Gylfe, E. (1988) J. Biol. Chem. 262, 5044-5048. Biden, T.J., Peter-Riesch, B., Schlegel, W., and Wollheim, C.B. (1987) J. Biol. Chem. 262, 446-448. Wolf, B.A., Turk, J., Sherman, W.R., and McDaniel, M.L. (1986) J. Biol. Chem. 262, 3501-3510. Wolf, B.A., Pasquale, S.M., and Turk, J. (1991) Biochemistry 30, 6372-6379. Wolf, R.A., and Gross, R.W. (1985) J. Biol. Chem. 260, 72957303. 652
Vol.
10.) 11 .) 12.) 13.) 14.) 15.) 16.) 17.) 18.) 19.) 20.) 21 .) 22.) 23.) 24.)
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Hazen, S.L., Ford, D.A., and Gross, R.W. (1991) J. Biol. Chem. 266, 5629-5633. Hazen, S.L., and Gross, R.W. (1991) J. Biol. Chem. 266, 14526-14534. Hazen, S.L., Zupan, L.A., Weiss, R.H., Getman, D.P., and Gross, R.W. (1991) J. Biol. Chem. 266, 7227-7232. Turk, J., Gross, R.W., and Ramanadham, S. (1992) ADA abstract, Diabetes, in press. Vacher, P., McKenzie, J., and Dufy, B. (1989) Am. J. Physiol. 257 E203-E211. Pipeleers, D.G. (1984) In Methods in Diabetes Research (Larner, J., ed.) pp. 185211, John Wiley and sons, New York. Wang, J.-L., and McDaniel, M.L. (1990) Biochem. Biophys. Res. Commun. 166, 813-818. Tsien, R.Y., Rink, T., and Poenie, M. (1985) Cell Calcium 6, 145- 157. McDaniels, ML., Colca , J.R., Kotagal, N., and Lacy, P.E. (1983) Methods Enzymol. 98, 182-200. Ono, J., Takaki, R., Fukuma, M. (1977) Endocrinol. Japon. 24, 265270. Goligorsky, M., Loftus, D., and Hruska, K. (1986) Am. J. Physiol. 251, F938-F944. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Turk, J., Colca, J.R., and McDaniel, M.L. (1985) Biochim. Biophys. Acta 834, 23-36. Chow, S.K., and Jondal, M. (1990) J. Biol. Chem. 265, 902-907. Turk, J., Mueller, M., Bohrer, A., and Ramanadham, S. (1992) Biochim. Biophys. Acta, in press.
653
184,
No.
April
30,
1992
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
647-653
ARACHIDONIC ACID INDUCES AN INCREASE IN THE CYTOSOLIC CALCIUM CONCENTRATION IN SINGLE PANCREATIC ISLET BETA CELLS Sasanka
Ramanadham,
Richard
Gross*,
and John Turk
Departments of Medicine and Pathology, Division of Laboratory Medicine, and *Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, St. Louis, Missouri 631 IO Received
March
12,
1992
The insulin secretagogue D-glucose induces both accumulation of nonesterified arachidonic acid (35 pM) in pancreatic islets and a rise in beta cell cytosolic [Ca++]i. Arachidonate amplifies both voltage-dependent Ca++ entry in secretory cells and depolarization-induced insulin secretion. Here, arachidonate induced a biphasic rise in [Ca++]i of Fura-2AM loaded beta cells which increased with arachidonate concentration (5-30 PM), was reversed upon washout, and was unaffected by the arachidonate oxygenase inhibitor BW755C. The sustained phase of the rise was abolished by removal of extracellular Ca++ and amplified by depolarization with KCI. The accumulation of nonesterified arachidonate in islets stimulated by D-glucose may therefore promote the D-glucose-induced 0 1992AcadrmrcPL&S,Inc. rise in beta cell [Ca++]i.
Isolated pancreatic islets secrete insulin upon metabolism of fuel secretagogues such as D-glucose (1). The metabolism of glucose generates ATP which may serve a second messenger function in islet beta cells. A beta cell K+-channel is inactivated by ATP (2,3), and its closure induces membrane depolarization (3). The rise in membrane potential activates voltage-gated Ca ++-channels, and Ca++ enters the beta cell from the extracellular space, resulting in a rise in [Ca++]i which is required for the induction of insulin secretion (4, 5). Fuel secretagogues also induce hydrolysis of beta cell membrane phospholipids, resulting in accumulation of the phospholipid-derived mediators inositol 1 ,4,5-trisphosphate, nonesterified arachidonic acid, and arachidonate metabolites (6-8). Fuel secretagogue-induced activation of beta cell phosphoinositidephospholipase C requires Ca++ influx and is prevented by Ca++-channel blockers or by removal of extracellular Ca++ (6). In contrast, a component of the fuel-secretagogue-induced accumulation of nonesterified
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
arachidonate and its metabolites in islets occurs without extracellular Ca++ and in the presence of Ca++-channel blockers (8). At concentrations which accumulate in 17 mM D-glucose-stimulated islets (8), arachidonate amplifies voltage-dependent Ca ++ entry into prolactin-secreting cells (14) and amplifies depolarization-induced insulin secretion from islets These observations suggest that fuel secretagogue-induced (8). accumulation of nonesterified arachidonate in beta cells may amplify Ca++ entry and the secretory response to small changes in secretagogue concentration. We have therefore measured the direct effects of arachidonate on cytosolic [Ca++] using individual pancreatic islet beta cells.
MATERIALS AND METHODS. Pancreatic islets were isolated by collagenase digestion (18) and dispersed into single cells with dispase (19). Filtered (60 pm nylon screen) dispersed cells were subjected to autofluorescence-activated cell sorting (FACS) with a FACS-IV at 488 nm. Emission at instrument (16) using argon laser illumination Suspensions of single 510-550 nm reflected endogenous FAD content. beta cells (>90% purity) were so prepared, as verified by immunocytochemical staining for insulin, glucagon, somatostatin, and pancreatic polypeptide (15,16). FACS-purified beta cells (1 OS/ml) were plated onto Cell Tak-coated glass coverslips and incubated overnight at 37°C in CMRL-1066 medium containing 10% heat-inactivated fetal bovine serum, 1% L-glutamine, and 1% (w/v) each of penicillin and streptomycin under 5% CO2/95% air. CMRL 1066 medium was replaced with KRB [containing in (mM); NaCl (115), KCI (5), NaHC03 (24), CaCl2 (2.5), MgCl2 (l), HEPES (50), D-glucose (3), pH 7.41 and the cover-slip attached beta cells were loaded with Fura-2AM for 30 min as described (21), washed with KRB, and perifused with medium in a thermostatically-heated (37°C) chamber for the microfluorimetry experiments. Cytosolic [Ca++]i was measured as described (16, 17, 20, 21) with dual excitation wavelength (340 and 380 nm) microfluorimetry of single Fura-2AM loaded beta cells on a PTI DeltaScan instrument with a Nikon microscope. Emission was measured at 500-530 nm. ‘The ratio (R) of emission intensity at an excitation wavelength of 340 nm was divided by that at 380 nm to yield an index of [Ca ++]i. Absolute [Ca++]i was calculated from R after determining Rmax (R with 2 FM 4-Br-A23187) and Rmin (R following 1 mM EGTA after 4-Br-A23187) and correcting for autofluorescence (R with MnCl2) as described (17, 21). Arachidonic acid (NuChek, Elysian, MN) was dissolved in 0.1 M Na2C03 at [0.2 mM] just before use and diluted in albumin-free KRB (7). BW755C (Wellcome Research Laboratories, Kent, UK) was dissolved in DMSO, 0.15 M NaCl (1 :17, w/v) just before use. Nifedipine and EGTA were from Sigma (St. Louis, MO). 4-Br-A23187 (Calbiochem, LaJolla, CA) was dissolved in DMSO. Cell Tak was obtained from Collaborative Res. Inc., Bedford, MA. 648
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS. Exogenous arachidonic acid induced a rise in the cytosolic of individual beta cells (Figure 1). The threshold concentration was about 5 FM while no measurable effect was observed with 1 PM. At higher concentrations (1 O-30 PM) arachidonate produced a biphasic [Ca++]i response. A rapidly developing, transient rise was followed by a slowly developing, sustained increase. The magnitude of the sustained phase increased with increasing [arachidonate]. The arachidonate-induced rise in beta cell [Ca++]i was fully reversible, and the [Ca++]i returned to basal [Ca++]
levels upon removal of arachidonate from the medium and perifusion with albumin-containing buffer. Arachidonate did not, therefore, simply permeabilize the cell membrane like a detergent. The arachidonate oxygenase inhibitor BW755C at a concentration of 500 FM, which
_
7.00, B.
7.00,A.
AAl5rMl
$ 7.00 EE fc
c.
AAllO,Ml
g3.50 629 -. n d
”
r--OJ )0
I I 500 1000 TnmeIre<)
OJ,
OJ,
, 0
I I r oJ,1000 1500 2000 0 Tme (set)
I 500
0
1 1500
800
i
400
, 200
, 800
:,A G.
1200,
? c E .?
, , 400 Ttme tree)
I f 400 600 Tme (SK)
Arachbdonic Acid Concenlrotion Response Curve 0 Peak 1 . Peak 2
0 0 I
5
IO [AA]lrMl
I5
20
Fiaure 1: Arachidonic acid induces a rise in beta cell cvtosolic calcium concentration in a concentration-deDendent and reversible manner. Single beta cells were attached to coverslips and loaded with Fura-2AM as in Methods At the indicated time arachidonate (l-30 PM) was introduced in albumin ‘(BSA)-free buffer. Washout solution contained 0.1% fatty acid-free BSA (w/v) and no arachidonate. 649
Vol.
184,
No.
2,
1992
BIOCHEMICAL
1500
AND
BIOPHYSICAL
AA
RESEARCH
COMMUNICATIONS
AA + BW755C
1000 AA
AA
/Iv
400 200 0 iA
AA
AA + BW755C
Fiaure 2. The arachidonic acid-induced rise in beta cell cvtoso lid ca lcium concentration is not influenced bv the arachidonate oxvgenase inhibitor BW755C. Experiments were performed as in Fig. 1 with a fixed arachidonate concentration (15 PM) in the absence (left) or presence (right) of BW755C (500 PM). Mean (n = 8) values (_+SEM) of [Ca++]i are presented. completely inhibits islet arachidonate the rise in beta cell [Ca++]i induced
metabolism (22), did not influence by arachidonate (Figure 2). Similar
results were obtained with the selective cyclooxygenase indomethacin (10 uM, not shown). This suggests that the rise was
induced
cell
The sustained phase of the 15 pM arachidonate-induced [Ca++]i was virtually abolished by removal of extracellular
EGTA
and was slightly
nifedipine reflects sensitive transient unaffected the
by arachidonate
early
(Figure
3A),
lower
itself
and
not a metabolite.
in the presence
indicating
that
inhibitor in [Ca++]i
this
rise in beta Ca++ with
of the Ca++-channel phase
of the
rise
blocker in [Ca++]
extracellular Ca++ entry, possibly mediated in part by voltageCa+ +-channels. In contrast, the magnitude of the early, phase of the 15 pM arachidonate-induced rise in [Ca++]i was by removal rise
may
of extracellular reflect
Ca ++
release
of
or nifedipine, Ca++
from
suggesting that intracellular
sequestration sites. Arachidonate (5-30 PM) has similarly been reported to induce a biphasic rise in [Ca ++]i in a T-lymphocyte cell line with an early
transient
phase
reflecting
mobilization
of
intracellular
Ca++
and a
space, later sustained phase reflecting Ca++ entry from the extracellular induced by arachidonate itself and not its metabolite(s) (23). Depolarizing concentrations (lo-40 mM) of KCI also induced a rise in the beta cell [Ca++]i (Figure 3B). The 15 ,LLM arachidonate-induced rise in beta cell [Ca++]i was potentiated by KCI (lo-40 mM) (Figure 3B). Arachidonate may 650
Vol.
184,
No.
2,
1992
BIOCHEMICAL
A
AND
ARACHIDONIC
BIOPHYSICAL
ACID/KC1
i
EGTA
RESEARCH
COMMUNICATIONS
or NIFEDIPINE
1500 = 15 !a
AA (Peak 2)
1200 iz
--
c
900
F .z 00
600
0, 300
0 1
2
3
1
B
2
ARACHIDONIC
3
1
ACID
2
3
t KCI
1600/
0
10
20 WI1
30
40
(mW
Fiaure 3: The sustained chase of the arachidonic acid-induced rise in beta cell cvtosolic calcium concentration derives from extracellular calcium and is amolified bv deoolarization with KCI. Panel A studies were performed as in Fig. 1. Values represent mean (n = 8) differences (+SEM) between basal [Ca++]i and the sustained phase of the stimulus-induced rise. The [EGTA] was 1 mM and the [nifedipine] 400 nM. Panel E3 cells were exposed to the indicated [KCI] and [arachidonate] (0 or 15 PM) simultaneously. Values represent mean (n = 8) differences (+SEM) between [Ca ++]i at 5 mM KCI with 0 or 15 PM and [Ca++ arachidonate ]i at the indicated [KCI] with 0 or 15 FM arachidonate during the sustained phase of the [Ca++]i response to arachidonate. therefore
amplify
prolactin-secreting l7lSCUSSION. arachidonate from
voltage-dependent GH3
cells
Ca++
entry
in beta cells,
as it does
in
(14).
The observations beta cell membrane
that glucose induces release of free phospholipids by a process that
does not require Ca++ entry (8, 24), that glucose induces a rise in the beta cell [Ca++]i reflecting Ca++ entry (4, 5), and that exogenous arachidonate in concentrations which accumulate in glucose-stimulated induces Ca++ entry into beta cells (Figures l-3) suggest that 651
islets also arachidonate
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
may participate in the glucose-induced rise in beta cell [Ca++]i. Inhibition of glucose-induced phospholipid hydrolysis and arachidonate release might therefore blunt the rise in beta cell [Ca++]i and insulin secretion. A Ca++independent phospholipase A2 which is selective for arachidonic acid and whose activity is regulated by ATP has been identified in canine myocardium (g-12). A similar activity has also recently been identified in pancreatic islets (13). Recently, a haloenol lactone suicide substrate (HELSS) which is a selective, mechanism-based inhibitor of this Ca++independent phospholipase A2 enzyme has been identified. We have recently observed that the islet phospholipase A2 activity is inhibited by low uM concentrations of HELSS which do not have an affect on islet oxidation of [14C]-glucose to [l4C]O2 (13). The 17 mM D-glucose-induced rise in beta cell [Ca++]i was inhibited by 66 f 13% by 3-10 uM HELSS, although the rise in beta cell [Ca++]i induced by depolarization was unaffected, and HELSS also suppressed both arachidonate release and insulin secretion from D-glucose-stimulated islets (13). Thus, Ca++independent phospholipase AZ-mediated accumulation of nonesterified arachidonate in islets stimulated by D-glucose may promote Ca++ influx and the rise in beta cell [Ca++]i required for the induction of insulin secretion.
ACKNOWLEDGMENTS. We acknowledge support by NIH grants HL34389, DK-34388 and DK-01553 and by the Monsanto Co. and the superb technical assistance of Alan Bohrer, Mary Mueller, Kelly Kruszka and Lori Zupan.
REFERENCES
1.I 2.1
3.) 4.) 5.1 6.)
7.1 8.)
9.1
Meglasson, M.D., and Matschinsky, F. (1986) Diabetes/Metab. Rev. 2, 63-214. Cook, D.L., and Hales, C.N. (1984) Nature 311, 271-273. Sturgess, N.C., Cook, D.L., Ashford, M.L.J., and Hales, C.N. (1985) Nature 312, 446-448. Arkhammar, P., Nilsson, T., Rorsman, P., and Berggren, P.O. (1987) J. Biol. Chem. 262, 5448-5454. Gylfe, E. (1988) J. Biol. Chem. 262, 5044-5048. Biden, T.J., Peter-Riesch, B., Schlegel, W., and Wollheim, C.B. (1987) J. Biol. Chem. 262, 446-448. Wolf, B.A., Turk, J., Sherman, W.R., and McDaniel, M.L. (1986) J. Biol. Chem. 262, 3501-3510. Wolf, B.A., Pasquale, S.M., and Turk, J. (1991) Biochemistry 30, 6372-6379. Wolf, R.A., and Gross, R.W. (1985) J. Biol. Chem. 260, 72957303. 652
Vol.
10.) 11 .) 12.) 13.) 14.) 15.) 16.) 17.) 18.) 19.) 20.) 21 .) 22.) 23.) 24.)
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Hazen, S.L., Ford, D.A., and Gross, R.W. (1991) J. Biol. Chem. 266, 5629-5633. Hazen, S.L., and Gross, R.W. (1991) J. Biol. Chem. 266, 14526-14534. Hazen, S.L., Zupan, L.A., Weiss, R.H., Getman, D.P., and Gross, R.W. (1991) J. Biol. Chem. 266, 7227-7232. Turk, J., Gross, R.W., and Ramanadham, S. (1992) ADA abstract, Diabetes, in press. Vacher, P., McKenzie, J., and Dufy, B. (1989) Am. J. Physiol. 257 E203-E211. Pipeleers, D.G. (1984) In Methods in Diabetes Research (Larner, J., ed.) pp. 185211, John Wiley and sons, New York. Wang, J.-L., and McDaniel, M.L. (1990) Biochem. Biophys. Res. Commun. 166, 813-818. Tsien, R.Y., Rink, T., and Poenie, M. (1985) Cell Calcium 6, 145- 157. McDaniels, ML., Colca , J.R., Kotagal, N., and Lacy, P.E. (1983) Methods Enzymol. 98, 182-200. Ono, J., Takaki, R., Fukuma, M. (1977) Endocrinol. Japon. 24, 265270. Goligorsky, M., Loftus, D., and Hruska, K. (1986) Am. J. Physiol. 251, F938-F944. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Turk, J., Colca, J.R., and McDaniel, M.L. (1985) Biochim. Biophys. Acta 834, 23-36. Chow, S.K., and Jondal, M. (1990) J. Biol. Chem. 265, 902-907. Turk, J., Mueller, M., Bohrer, A., and Ramanadham, S. (1992) Biochim. Biophys. Acta, in press.
653