BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 949-953
Vo1.173, No. 3,1990 December 31,1990
HEPOXILIN A 3 BLOCKS THE RELEASE OF NOREPINEPHRINE FROM RAT HIPPOCAMPAL SLICES
Cecil R. Pace-Asciak +,1, Lydia Wong 1 and Elias J. Corey2
IResearch Institute, Hospital for Sick Children, Toronto MSG 1XS, Canada 1Department of Pharmacology, University of Toronto, M5S IA S, Canada 2Department of Chemistry, Harvard University, Cambridge, Mass. 02138 Received November 7, 1990
Hepoxilin A3 was previously shown to display neuromodulatory actions on rat hippocampal CA1 neurons, with hyperpolarization of the membrane potential, an increase in the amplitude and duration of the post-spike train afterhyperpolarization and an increase in the inhibitory post synaptic potential. The present report describes new biochemical evidence of a presynaptic action of hepoxilin A3 in rat hippocampal slices prelabeled with [3H]-norepinephrine. Hepoxilin A3 on its own had a marginal effect on the release of label, but blocked release which was induced by 4aminopyridine (4-AP). Prostaglandin E2 also behaved in a similar way. These results demonstrate that hepoxilins modulate neurotransmission in the mamm~an CNS through both pre- and postsynaptic actions. ©1990 Academic Press, Inc.
New roles are emerging for products of the 12-1ipoxygenase pathway of arachidonic acid metabolism. Bioactive products so far detected in this pathway are 12-HETE (1-5), and the hepoxilins, 8 and 10-hydroxy-11,12-epoxide metabolites derived from 12-HPETE (6-8). We have shown that 12-HPETE is rapidly converted into the hepoxilins through ferriheme catalysis (9) so that biological actions of 12-HPETE are likely derived through its prior conversion into the hepoxilins. Hepoxilins have thus been shown to cause the release of insulin from pancreatic islets (8,10,11) and to cause hyperpolarization of the membrane potential in the mammalian CNS (12) and that of the Aplysia (13). The hepoxilins cause an increase in the transport of calcium across membranes (14) and they have been shown to raise intracellular calcium concentrations in the human neutrophil in a receptor mediated fashion (15). They have recently been shown to stimulate the release of diacylglycerol and arachidonic acid in human neutrophils in a time- and pertussistoxin dependent fashion (16). In the Aplysia, the hepoxilins have been implicated in increasing the probability of S-type K + channel opening (17). It appears that these products may play an
+ To whom correspondence should be addressed. Abbreviations used: HxA3, hepoxilin A3, 8-hydroxy- 11,12-epoxyeicosa-5Z, 10E, 14Z- trienoic acid; HxB3, hepoxilin B3, 10-hydroxy-11,12-epoxyeicosa-5Z, 8Z, 14Z-trienoic acid; PGE2, prostaglandin E2; NE, norepinephrine; 4-AP, 4-aminopyridine; ACSF, artificial cerebrospinal fluid; PK, protein kinase.
949
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
important role as modulators of a diversity of cellular events. This study describes new biochemical evidence that the hepoxilins affect the release of neurotransmitters from the hippocampus. MATERIALS AND METHODS
Materials: [7,8-3H]-NE (spec. act. 38Ci/mmo1) was purchased from Amersham (Toronto), and 4-AP from Sigma. I-IxA3 was used in the methyl ester form prepared as previously described (18). PGE2 was generously provided by Dr. John Pike, The Upjohn Co., Kalamazoo, MI. PGE2 and HxA3-methyl ester were dissolved in 1 gl DMSO and diluted appropriately with artificial cerebrospinal fluid (ACSF) (12) to a final concentration of 1 gM. Brain tissue: Male Wistar rats (200-250 g, pathogen free, Charles River, St. Constant, Quebec) were briefly anaesthetised with halothane, decapitated and the brain was rapidly dissected and placed in ice-cold oxygenated (95% 02 - 5% CO2) ACSF solution, pH 7.4. Coronal sections of the hippocampus were prepared using a vibratome (400 microns thick) and the slices were allowed to equilibrate at 30oc for 60 minutes in oxygenated ACSF. The slices were incubated at 37°C in 2ml of ACSF containing 2.106 cprn/8 slices/brain of tdtiated NE. After 15 minutes the tissue was gently washed with 2 x 5 ml ACSF and each slice was carefully placed in a superfusion chamber. Each chamber was superfused at 0.25rnl/rnin with ACSF at 37oc through a multichannel roller pump (Masterflex). The effluent was collected in 5 minute samples directly into scintillation vials containing 5mi Ecolite (ICN) scintillation cocktail, and the radioactivity in the vials was counted in a Beckman scintillation counter. After each experiment the radioactivity remaining in each tissue was extracted with 0.1N HC1 and counted. R e l e a s e o f [7,8-3I-I]-NE: An 80 min control perfusion was initiated to wash off any nonspeeifically bound label. By the end of this control period the amount of label in each sample of perfusate had dropped off to a steady baseline level of about 100-200 cpm/sample. At this point the following sequence of challenges with 4-AP (100 laM) and eicosanoid (llaNI) were carried out each lasting 10 min, followed by a 30 min control washout period, i.e. medium plus 4-AP ; medium plus 4-AP plus HxA 3 or PGE2 ; medium plus HxA3 or PGE2 in the absence of 4-AP; and finally medium plus 4-AP alone. Hence each tissue was exposed to four challenges only. Control experiments in which four successive 4-AP challenges were performed were also present in each assay to determine the change in response of the tissue to 4-AP. Quantitative results were obtained by subtracting the response produced by the eicosanoid from a line extrapolated from the first and last response due to 4-AP as shown in Figure 1. Results are expressed in cpms alone (Figure 1) or as a percentage of the response to 4-AP alone (Figure 2).
RESULTS AND DISCUSSION
Rat brain hippocampal slices were incubated with [7,8-3H]-NE for 15 rain at 37oc. The tissue was subsequently superfused for an 80 minute period which was necessary to obtain constant baseline levels of label in the perfusate. Before challenge, slices was estimated to contain approximately 30,000-40,000 cpm. Small amounts of label were released by challenge with 4-AP. Previous studies have utilized this technique of investigating the effects of drugs on the release of neurotransmitter with considerable success (19,20). We confirmed that the method also responded with considerable reproducibility in our hands. We noticed, as well, that with subsequent challenges with 4-AP, there was a reduction in the amount of label that was released, hence the effect of the eicosanoids (HxA3 and PGE2) was bracketed between control challenges with 4-AP to determine the rate of release of NE, and the effect of the eicosanoids in stimulating or blocking the release of NE. The amount of label recovered in the tissues at the end of the experiment was 23,100 + 1900 (n=24). Figure 1 shows data for the release of NE from A) control (4-AP) stimulation, B) the effect of PGE2 on this stimulation, and C) the effect of HxA3 on the 4-AP induced stimulation. It is clear that at the concentrations used, both PGE2 and HxA3 antagonize the 4-AP induced release of NE. Figure 2 950
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RELEASE OF 3H-NE (CPM)
1200~
RELEASE O F 3H-NE (CPM) 2000
Q
4-AP
4-AP
4-AP
m
Q
4-AP 4-AP 4-AP + P G E 2 PGE 2
1600 4-AP 1200
i
800 800 400 0
~" . . . . . . 50 100
.~
400 0
---. , - , 150 200
0
50
100
150
200
250
T I M E (min)
TIME (rnin) 3 RELEASE OF H-NE (CPM)
000
1600
© 4-AP
[ ~
1200
4-AP + 4-AP _Hx23H2A3
800 4OO
0 0
,,,,, ......... , ......... , ................... , .... 50 100 150 200 TIME (min)
Figure 1. Inhibition of the release of [7,8-3H]-NE from rat hippocampal slices by PGE2 and hepoxilin A 3. Hippocampal slices were prelabeled with NE for 15 rain at 37°C, and superfused at 0.25ml/min with artificial CSF medium. After washing out loosely bound NE for approximately 80 rain, the slices were stimulated by 4-AP (100gM) to release NE. Each tissue was challenged four times as indicated for 10 min followed with a washout period of 30 n'fin with medium alone. Panel A shows the effect of 4-AP repeated four times, panel B shows the effect of PGE2 (1JAM) on the 4-AP effect as well as on its own (second and third challenge) and panel C shows the effect of HxA3 on the 4-AP induced release of NE as well as on its own.
RELEASE OF 3H.NE (% relative to 4-AP) 100 80
60 40 n=7
20 0
i
n=9 i
1111111111119
n=7
n=9
-20 -40 -60 -80 *
-100 drug (1 I.tM)
HxA 3
PGE 2
4-AP (100 ~M)
HxA 3 +
p
Vo1.173, No. 3,1990 December 31,1990
HEPOXILIN A 3 BLOCKS THE RELEASE OF NOREPINEPHRINE FROM RAT HIPPOCAMPAL SLICES
Cecil R. Pace-Asciak +,1, Lydia Wong 1 and Elias J. Corey2
IResearch Institute, Hospital for Sick Children, Toronto MSG 1XS, Canada 1Department of Pharmacology, University of Toronto, M5S IA S, Canada 2Department of Chemistry, Harvard University, Cambridge, Mass. 02138 Received November 7, 1990
Hepoxilin A3 was previously shown to display neuromodulatory actions on rat hippocampal CA1 neurons, with hyperpolarization of the membrane potential, an increase in the amplitude and duration of the post-spike train afterhyperpolarization and an increase in the inhibitory post synaptic potential. The present report describes new biochemical evidence of a presynaptic action of hepoxilin A3 in rat hippocampal slices prelabeled with [3H]-norepinephrine. Hepoxilin A3 on its own had a marginal effect on the release of label, but blocked release which was induced by 4aminopyridine (4-AP). Prostaglandin E2 also behaved in a similar way. These results demonstrate that hepoxilins modulate neurotransmission in the mamm~an CNS through both pre- and postsynaptic actions. ©1990 Academic Press, Inc.
New roles are emerging for products of the 12-1ipoxygenase pathway of arachidonic acid metabolism. Bioactive products so far detected in this pathway are 12-HETE (1-5), and the hepoxilins, 8 and 10-hydroxy-11,12-epoxide metabolites derived from 12-HPETE (6-8). We have shown that 12-HPETE is rapidly converted into the hepoxilins through ferriheme catalysis (9) so that biological actions of 12-HPETE are likely derived through its prior conversion into the hepoxilins. Hepoxilins have thus been shown to cause the release of insulin from pancreatic islets (8,10,11) and to cause hyperpolarization of the membrane potential in the mammalian CNS (12) and that of the Aplysia (13). The hepoxilins cause an increase in the transport of calcium across membranes (14) and they have been shown to raise intracellular calcium concentrations in the human neutrophil in a receptor mediated fashion (15). They have recently been shown to stimulate the release of diacylglycerol and arachidonic acid in human neutrophils in a time- and pertussistoxin dependent fashion (16). In the Aplysia, the hepoxilins have been implicated in increasing the probability of S-type K + channel opening (17). It appears that these products may play an
+ To whom correspondence should be addressed. Abbreviations used: HxA3, hepoxilin A3, 8-hydroxy- 11,12-epoxyeicosa-5Z, 10E, 14Z- trienoic acid; HxB3, hepoxilin B3, 10-hydroxy-11,12-epoxyeicosa-5Z, 8Z, 14Z-trienoic acid; PGE2, prostaglandin E2; NE, norepinephrine; 4-AP, 4-aminopyridine; ACSF, artificial cerebrospinal fluid; PK, protein kinase.
949
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
important role as modulators of a diversity of cellular events. This study describes new biochemical evidence that the hepoxilins affect the release of neurotransmitters from the hippocampus. MATERIALS AND METHODS
Materials: [7,8-3H]-NE (spec. act. 38Ci/mmo1) was purchased from Amersham (Toronto), and 4-AP from Sigma. I-IxA3 was used in the methyl ester form prepared as previously described (18). PGE2 was generously provided by Dr. John Pike, The Upjohn Co., Kalamazoo, MI. PGE2 and HxA3-methyl ester were dissolved in 1 gl DMSO and diluted appropriately with artificial cerebrospinal fluid (ACSF) (12) to a final concentration of 1 gM. Brain tissue: Male Wistar rats (200-250 g, pathogen free, Charles River, St. Constant, Quebec) were briefly anaesthetised with halothane, decapitated and the brain was rapidly dissected and placed in ice-cold oxygenated (95% 02 - 5% CO2) ACSF solution, pH 7.4. Coronal sections of the hippocampus were prepared using a vibratome (400 microns thick) and the slices were allowed to equilibrate at 30oc for 60 minutes in oxygenated ACSF. The slices were incubated at 37°C in 2ml of ACSF containing 2.106 cprn/8 slices/brain of tdtiated NE. After 15 minutes the tissue was gently washed with 2 x 5 ml ACSF and each slice was carefully placed in a superfusion chamber. Each chamber was superfused at 0.25rnl/rnin with ACSF at 37oc through a multichannel roller pump (Masterflex). The effluent was collected in 5 minute samples directly into scintillation vials containing 5mi Ecolite (ICN) scintillation cocktail, and the radioactivity in the vials was counted in a Beckman scintillation counter. After each experiment the radioactivity remaining in each tissue was extracted with 0.1N HC1 and counted. R e l e a s e o f [7,8-3I-I]-NE: An 80 min control perfusion was initiated to wash off any nonspeeifically bound label. By the end of this control period the amount of label in each sample of perfusate had dropped off to a steady baseline level of about 100-200 cpm/sample. At this point the following sequence of challenges with 4-AP (100 laM) and eicosanoid (llaNI) were carried out each lasting 10 min, followed by a 30 min control washout period, i.e. medium plus 4-AP ; medium plus 4-AP plus HxA 3 or PGE2 ; medium plus HxA3 or PGE2 in the absence of 4-AP; and finally medium plus 4-AP alone. Hence each tissue was exposed to four challenges only. Control experiments in which four successive 4-AP challenges were performed were also present in each assay to determine the change in response of the tissue to 4-AP. Quantitative results were obtained by subtracting the response produced by the eicosanoid from a line extrapolated from the first and last response due to 4-AP as shown in Figure 1. Results are expressed in cpms alone (Figure 1) or as a percentage of the response to 4-AP alone (Figure 2).
RESULTS AND DISCUSSION
Rat brain hippocampal slices were incubated with [7,8-3H]-NE for 15 rain at 37oc. The tissue was subsequently superfused for an 80 minute period which was necessary to obtain constant baseline levels of label in the perfusate. Before challenge, slices was estimated to contain approximately 30,000-40,000 cpm. Small amounts of label were released by challenge with 4-AP. Previous studies have utilized this technique of investigating the effects of drugs on the release of neurotransmitter with considerable success (19,20). We confirmed that the method also responded with considerable reproducibility in our hands. We noticed, as well, that with subsequent challenges with 4-AP, there was a reduction in the amount of label that was released, hence the effect of the eicosanoids (HxA3 and PGE2) was bracketed between control challenges with 4-AP to determine the rate of release of NE, and the effect of the eicosanoids in stimulating or blocking the release of NE. The amount of label recovered in the tissues at the end of the experiment was 23,100 + 1900 (n=24). Figure 1 shows data for the release of NE from A) control (4-AP) stimulation, B) the effect of PGE2 on this stimulation, and C) the effect of HxA3 on the 4-AP induced stimulation. It is clear that at the concentrations used, both PGE2 and HxA3 antagonize the 4-AP induced release of NE. Figure 2 950
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RELEASE OF 3H-NE (CPM)
1200~
RELEASE O F 3H-NE (CPM) 2000
Q
4-AP
4-AP
4-AP
m
Q
4-AP 4-AP 4-AP + P G E 2 PGE 2
1600 4-AP 1200
i
800 800 400 0
~" . . . . . . 50 100
.~
400 0
---. , - , 150 200
0
50
100
150
200
250
T I M E (min)
TIME (rnin) 3 RELEASE OF H-NE (CPM)
000
1600
© 4-AP
[ ~
1200
4-AP + 4-AP _Hx23H2A3
800 4OO
0 0
,,,,, ......... , ......... , ................... , .... 50 100 150 200 TIME (min)
Figure 1. Inhibition of the release of [7,8-3H]-NE from rat hippocampal slices by PGE2 and hepoxilin A 3. Hippocampal slices were prelabeled with NE for 15 rain at 37°C, and superfused at 0.25ml/min with artificial CSF medium. After washing out loosely bound NE for approximately 80 rain, the slices were stimulated by 4-AP (100gM) to release NE. Each tissue was challenged four times as indicated for 10 min followed with a washout period of 30 n'fin with medium alone. Panel A shows the effect of 4-AP repeated four times, panel B shows the effect of PGE2 (1JAM) on the 4-AP effect as well as on its own (second and third challenge) and panel C shows the effect of HxA3 on the 4-AP induced release of NE as well as on its own.
RELEASE OF 3H.NE (% relative to 4-AP) 100 80
60 40 n=7
20 0
i
n=9 i
1111111111119
n=7
n=9
-20 -40 -60 -80 *
-100 drug (1 I.tM)
HxA 3
PGE 2
4-AP (100 ~M)
HxA 3 +
p
