- Email: [email protected]
Dynorphin B inhibits thymidine incorporation into DNA in fetal rat and guinea pig brain cell aggregates
109
DYNORPHIN B INHIBITS THYMIDINE INCORPORATION INTO DNA IN FETAL R A T A N D GUINEA PIG BRAIN CELL AGGREGATES A.Gorodinsky t , J.Barg#t, M.M.Belcheva$, R.Levy#, R.J.McHalet, F.E.Johnson#, Z.Vogel#, C.J.Coscia$, $Dept. Biochem. & Mol. Biol., $Dept. Surg., St. Louis Univ. Sch. Med., St. Louis, MO, #Dept. Neurobiol., Welzmann, Rehovot, Wolfson, Tel Aviv Univ., Holon, Israel.
ABSTRACT
Numerous studies suggest that opioids have a modulatory role in the regulation of neural cell proliferation (I). We have found that ~ opioid receptor agonists inhibit thymidine incorporation into DNA of neural cells in rat fetal brain cell aggregates (2). This inhibition is culture age-dependent. Moreover, morphine inhibited glial cell proliferation in a concentration dependent manner (3). In contrast, U50488, a opiold receptor agonist, exerted either an inhibitory or stimulatory action on thymidine incorporation depending on the culture duration (4). Here we addressed the question of the effect of the endogenous ~-selectlve opioid peptide, Dynorphin B, on thymldlne incorporation in rat or guinea pig aggregates expressing more than 50% of ~ (rat) or ~ (guinea pig) receptors. In both cultures Dynorphin B inhibited DNA synthesis by 30-40%. METHODS Fetal brain cell aggregates were prepared from rat or guinea pig 15- or 25-day old embryos, respectively (2,5). Cultures were maintained for a total of 7 days, treated with opioids for the final 48 h and 3H-6-thymidine (15 Ci/mmol) for the last 23-24 h. In experiments performed with Dynorphin B, protease inhibitors were included as described (6). RESULTS Treatment of guinea pig brain cell aggregate cultures with the ~ opioid receptor agonist Dynorphin B significantly inhibited thymidlne incorporation into the DNA. A ~-selective antagonist, nor-binaltorphlmine (norBNl), reversed the agonist effect (Fig. i). As shown in figure 2, Dynorphin B significantly inhibited thymidine incorporation into the DNA in 7-day fetal rat brain cell aggregates compared to control cultures. Again norBNI reversed the effect of the Dynorphin B. DISCUSSION The role of opiolds as neurotrophic factors is supported by numerous studies (I). In general, opioids inhibit thymidine incorporation into DNA in neural cells (2-4, but see ref. 4 and 7). Here we show that a ~ opioid peptide inhibits thymidine incorporation in 7-day brain cell aggregates prepared from embryonal rat (El5) or guinea pig (E25). Although embryonal brain of different ages were used to prepare aggregates, the tissues were at a relatively similar developmental stage with respect to brain ontogeny in each species. Dynorphin B is a potent inhibitor of thymidine incorporation that acts in the nM range (8). These findings complement our previous observations with ~-endorphin which, by acting through ~ and ~ receptors, also inhibited DNA synthesis at nanomolar concentrations (6). This physiological potency suggests that ~-selective opioid peptides such as Dynorphln B may have a role in regulating neural cell proliferation during early stages of brain ontogeny.
I10
Acknowledgement:
Supported by grants from the National Institute of Drug Abuse (DA05412) and the US-Israel Binational Science Foundation. 2 ! o
~,00 © 19 --
80
.~
60
!
Figure i. Dynorphin B inhibits thymidine incorporation into DNA in fetal rat brain cell aggregates. Seven-day cultures were treated with 1 #M Dynorphin B and/or 1 ~M norBNI in the presence of protease inhibitors for the last 48 h and 3H-thymidine (0.5 ~Ci/dish) was included for the final 24 h. Data is expressed as the mearh%SEM from 3-5 experiments. *significantly different from all others, P
C12 ~ 40
~
~ I 212 e~
20
~
0
: CONT
I)YN
norBN[
DYN norl3Ni
i
o
100 -]
•
Figure 2. Inhibition of sH-thymidine incorporation by Dynorphin B in 7-day fetal guinea pig brain cell aggregates. Cultures were treated with 1 ~M Dynorphin B and/or 1 #M norBNI in the presence of protease inhibitors for the final 48 h and 3Hthymidine (0.5 ~Ci/dish) was included for the final 23 h. Data are the mean/+SEM of 3 experiments. *significantly different from all others, P<0.01.
Y _
o Ls 80 x o
~ o
6o
~
4o
z
20
~
0 CONT
I
DYN
norBNl
DYN norBNI
REFERENCES i.
R.P. Hammer, Jr., K.F. Hauser (1992) In: Develooment of the Central Nervous System, ed., M.W. Miller, Wiley-Liss, New York, NY, pp. 319-339.
2.
J. Barg, M.M. Belcheva, C.J. Coscia (1992) J. Neurochem.
3.
J. Barg, M.M. Belcheva, R. Levy, R.J. McHale, J.A. McLachlan, F.E. Johnson, Z. Vogel, C.J. Coscia (1994) Glia, I__O0,10-15.
4.
J. Barg, M.M. Belcheva, J. Rowinski, C.J. Coscia (1993) J. Neurochem. 6__00,15051511.
5.
J. Barg, R. Levy, R. Simantov (1989) Int. J. Dev. Neurosci.
6.
J. Barg, M.M. Belcheva, R.J. McHale, R. Levy, Z. Vogel, C.J. Coscia (1993) J. Neurochem. 6__O0,765-767.
7.
J. Barg, S.-Y. Nah, R. Levy, D. Saya, Z. Vogel (1993) B r a i n Res.
8.
A. Gorodinsky, J, Barg, M.M. Belcheva, R. Levy, R.J. McHale, Vogel, C.J. Coscia, manuscript in preparation.
5__99,1145-1152.
7, 173-179.
629, 109.
F.E. Johnson, Z.
DYNORPHIN B INHIBITS THYMIDINE INCORPORATION INTO DNA IN FETAL R A T A N D GUINEA PIG BRAIN CELL AGGREGATES A.Gorodinsky t , J.Barg#t, M.M.Belcheva$, R.Levy#, R.J.McHalet, F.E.Johnson#, Z.Vogel#, C.J.Coscia$, $Dept. Biochem. & Mol. Biol., $Dept. Surg., St. Louis Univ. Sch. Med., St. Louis, MO, #Dept. Neurobiol., Welzmann, Rehovot, Wolfson, Tel Aviv Univ., Holon, Israel.
ABSTRACT
Numerous studies suggest that opioids have a modulatory role in the regulation of neural cell proliferation (I). We have found that ~ opioid receptor agonists inhibit thymidine incorporation into DNA of neural cells in rat fetal brain cell aggregates (2). This inhibition is culture age-dependent. Moreover, morphine inhibited glial cell proliferation in a concentration dependent manner (3). In contrast, U50488, a opiold receptor agonist, exerted either an inhibitory or stimulatory action on thymidine incorporation depending on the culture duration (4). Here we addressed the question of the effect of the endogenous ~-selectlve opioid peptide, Dynorphin B, on thymldlne incorporation in rat or guinea pig aggregates expressing more than 50% of ~ (rat) or ~ (guinea pig) receptors. In both cultures Dynorphin B inhibited DNA synthesis by 30-40%. METHODS Fetal brain cell aggregates were prepared from rat or guinea pig 15- or 25-day old embryos, respectively (2,5). Cultures were maintained for a total of 7 days, treated with opioids for the final 48 h and 3H-6-thymidine (15 Ci/mmol) for the last 23-24 h. In experiments performed with Dynorphin B, protease inhibitors were included as described (6). RESULTS Treatment of guinea pig brain cell aggregate cultures with the ~ opioid receptor agonist Dynorphin B significantly inhibited thymidlne incorporation into the DNA. A ~-selective antagonist, nor-binaltorphlmine (norBNl), reversed the agonist effect (Fig. i). As shown in figure 2, Dynorphin B significantly inhibited thymidine incorporation into the DNA in 7-day fetal rat brain cell aggregates compared to control cultures. Again norBNI reversed the effect of the Dynorphin B. DISCUSSION The role of opiolds as neurotrophic factors is supported by numerous studies (I). In general, opioids inhibit thymidine incorporation into DNA in neural cells (2-4, but see ref. 4 and 7). Here we show that a ~ opioid peptide inhibits thymidine incorporation in 7-day brain cell aggregates prepared from embryonal rat (El5) or guinea pig (E25). Although embryonal brain of different ages were used to prepare aggregates, the tissues were at a relatively similar developmental stage with respect to brain ontogeny in each species. Dynorphin B is a potent inhibitor of thymidine incorporation that acts in the nM range (8). These findings complement our previous observations with ~-endorphin which, by acting through ~ and ~ receptors, also inhibited DNA synthesis at nanomolar concentrations (6). This physiological potency suggests that ~-selective opioid peptides such as Dynorphln B may have a role in regulating neural cell proliferation during early stages of brain ontogeny.
I10
Acknowledgement:
Supported by grants from the National Institute of Drug Abuse (DA05412) and the US-Israel Binational Science Foundation. 2 ! o
~,00 © 19 --
80
.~
60
!
Figure i. Dynorphin B inhibits thymidine incorporation into DNA in fetal rat brain cell aggregates. Seven-day cultures were treated with 1 #M Dynorphin B and/or 1 ~M norBNI in the presence of protease inhibitors for the last 48 h and 3H-thymidine (0.5 ~Ci/dish) was included for the final 24 h. Data is expressed as the mearh%SEM from 3-5 experiments. *significantly different from all others, P
C12 ~ 40
~
~ I 212 e~
20
~
0
: CONT
I)YN
norBN[
DYN norl3Ni
i
o
100 -]
•
Figure 2. Inhibition of sH-thymidine incorporation by Dynorphin B in 7-day fetal guinea pig brain cell aggregates. Cultures were treated with 1 ~M Dynorphin B and/or 1 #M norBNI in the presence of protease inhibitors for the final 48 h and 3Hthymidine (0.5 ~Ci/dish) was included for the final 23 h. Data are the mean/+SEM of 3 experiments. *significantly different from all others, P<0.01.
Y _
o Ls 80 x o
~ o
6o
~
4o
z
20
~
0 CONT
I
DYN
norBNl
DYN norBNI
REFERENCES i.
R.P. Hammer, Jr., K.F. Hauser (1992) In: Develooment of the Central Nervous System, ed., M.W. Miller, Wiley-Liss, New York, NY, pp. 319-339.
2.
J. Barg, M.M. Belcheva, C.J. Coscia (1992) J. Neurochem.
3.
J. Barg, M.M. Belcheva, R. Levy, R.J. McHale, J.A. McLachlan, F.E. Johnson, Z. Vogel, C.J. Coscia (1994) Glia, I__O0,10-15.
4.
J. Barg, M.M. Belcheva, J. Rowinski, C.J. Coscia (1993) J. Neurochem. 6__00,15051511.
5.
J. Barg, R. Levy, R. Simantov (1989) Int. J. Dev. Neurosci.
6.
J. Barg, M.M. Belcheva, R.J. McHale, R. Levy, Z. Vogel, C.J. Coscia (1993) J. Neurochem. 6__O0,765-767.
7.
J. Barg, S.-Y. Nah, R. Levy, D. Saya, Z. Vogel (1993) B r a i n Res.
8.
A. Gorodinsky, J, Barg, M.M. Belcheva, R. Levy, R.J. McHale, Vogel, C.J. Coscia, manuscript in preparation.
5__99,1145-1152.
7, 173-179.
629, 109.
F.E. Johnson, Z.