- Email: [email protected]
Intracerebroventricular pertussis toxin enhances sensitivity to N-methyl-D-aspartate-induced seizures in mice
Short communication
Michael
J. Durcan
and Philip F. Morgan
Received 13 December 1990. xcepted 19 March IYYI
The effect of prclreatmcn! with pcrtussis toxin on N-mcthyi-D-aspartatc (NMDAJ-induced seizures was investigated in mice. In animals ircatcd with pcrtussis toxin iij.5 pg/animal i.c.v) five days prior to testing the convulsant ED,,, of NMDA uas calculated to he 18 mg/kg whereas it was calculated to be 107 mg/kg in sham-treated animals. These rc>u!ts suggest the pcrtussis
toxin enhances sensitivity
to NMDA.
Pcrtussis
possibly via its actions on inhibiwry
toxin: NMDA
(N-methyl-D-aspartatc):
1. Introduction
G-proteins.
Seizures:
(Mouse)
with pertussis toxin (0.5 pg/animal days later their sensitivity
Many neurotransmitter effector
mechanisms
via
proteins (G-proteins)
receptors
are linked to their
guanine
nucleotide-binding
(Gilman,
monophosphate
IAJ-trisphosphate volve actions G-proteins although not
inactivate
Gi and G,,
proteins.
mechanisms
(Ui
controls.
by ADP from
were
a phenomenon Post-mortem
in animals
five
the
their
effcctor
current
ad libitum.
food and
The mice weighed
between
25 and 1X g at the time of pertussis toxin or vehicle A!!
mice
were
housed
individtially
after
pertussis toxin or vehicle treatment.
indicated
seizures were
by noise or cage move-
from administraticln rapid mice
of N-methyl-
circular
by full tonic-clonic experiment
water available
Mice were trlatcd
cause of death
Occasionally,
(i.e.
Swiss male mice were housed in groups
10 on a 1,‘: 13 h light: dark cycle with
in sham-treated
of animals
these seizures were reminiscent
acid (NMDA)
bouts often followed In
ribosylation,
Naive NlH of
to six days post
not seen
disturbed
observed
of seizures resulting D-aspartic
can
per anima! or greater, a
noted
inspection
(i.e. hind limb extension). ment. When
toxin
treatment.
that a seizure may have been a frequent observed
(G,),
with pertussis toxin (i.c.v.1 in our
at doses of 0.5 pg
of deaths
treatment,
1987).
et al., 1988).
In mice pretreated laboratory,
Pertussis
receptors
and methods
or may in-
et al.,
(Ci,) or stimulatory
also exist.
uncouple
2. Materials
(e.g. G,,) whose exact role is
understood
and therefore
number
(Worley
of
or inositol
(IP3) and diacylglycerol,
can be inhibitory
fully
(CAMP)
on ion channels
other G-proteins
seizures
was investigated.
1987). This linkage can
be via second messenger systems such as production cyclic adenosine
i.c.v.1 and then five
to NMDA-induced
running
seizures).
were
pretreated
Biological
with 0.5 c(g pertussis toxin (List
Labs. Campbell.
CA) injected in a volume of
5 ~1 directly
into the lateral
anesthetized
using chloral
made
in the scalp. and the lateral
(coordinates -3
ventricle. hydrate.
from bregma:
mm vcrtics!!.
Animals
:I small ventricle
1 mm lateral,
were
incision accessed
I mm rosrral.
Pertussis toxin was injected
at a rate
of 5 pl/min
for I min. the injection needle remained in place for a further 2 min before being slowly re-
tracted
and
Control
animals were injected with vehicle buffer (0.01
M
sodium
chloride;
the wound phosphate,
on!:/.
closed pH
using a wound
7.0 with
0.05
M
clip.
sodium
DOSE OF NMDA
(mglkg)
Fiy. 1. Pcrcrntage of pertussis toxin ( )- and sham (c)-treatrd following administration of NMDA. * * P < 0.01 animals S&ZirI~ Fi&cr’s e~ct test of proportions seizing where seizure dose-rezponw cuws overlap (i.e. 30 and 100 mg/kg doses of NMDA).
Groups of pertussis toxin-treated or control animals (N = S-11) were treated with doses of NMDA (Research Biochemicals Inc.. Natick, MA), l-200 mg/kg i.p.. at a volume of 10 ml/kg dissolved in distilled water and the number seizing within 60 min of administration was noted by an observer unaware of the drug dose or the pertussis toxin treatment.
The dose effect curves for both control and pertussis toxin-treated mice are shown as fig. 1. The ED,,, for convulsant effect of NMDA was calculated, by linear regression analysis of log probit plots (r’s > 0.98), to be 18 mg/kg in pertussis toxin-treated animals and 107 mg/kg in sham-treated controls. Fisher’s exact probability test revealed that where the NMDA seizure dose-response curves overlapped those groups pretreated with pertussis toxin had significantly greater proportions of animals seizing than did the shamtreated animals i? = 0.002 for 30 mg/kg; and P = 0.013 for 100 mg/kg NMDA).
These results suggest that increased susceptibility to excitatory neurotransmission may explain the mortality seen at five to six days post pertussis toxin treatment. Pertussis toxin inactivates Gi and G,, G-proteins which constitute an integral and essential part of a number of
inhibitory neurotransmitter activated transduction mechanisms (Gilman, 1987; Worley et al., 1987), but has little or no action on G,-proteins coupled to excitatory neurotransmitter systems. This suggests that increased sensitivity to NMDA following i.c.v. pertussis toxin may be due to a functional inactivation of inhibitory components of neurotransmission, rather than a direct effect on G-protein-linked excitatory neurotransmission. Several neurotransmitter receptors have been reported to be linked to pertussis-sensitive Gi- or G,-proteins (see Worley et al., 1987 for review), the inactivation of which may contribute to the increased sensitivity to NMDA. Mechanisms other than the inactivation of G-proteins may, however, be responsible for the observed increase in NMDA sensitivity. Pertussis toxin is made up of six protein subunits (consisting of five different proteins as one repeats), the largest of these is the enzymatically active A-protomer, whilst the remaining pentamer forms the binding B-oligomer (Tamura et al., 1982). The A-protomer is responsible for catalyzing the ADP riboslyation of susceptible G-proteins. The Boligomer binds to the cell membrane and is required to deliver the cataljrtic subunit to the target cell. Whilst the B-oligomer has no effect on G-proteins it is not completely without biological effect (Burns, 1988) and therefore activity of this binding oligomer may possibly contribute to the enhanced sensitivity to NMDA-induced seizures seen following pertussis toxin treatment. NMDA primarily acts at the NMDA sub-set of giutamate receptors which are coupled to cation channels which principally gate the transmembrane flux of calcium (Huetter and Bean, 1988). The present findings would therefore suggest that pertussis toxin mortality may be reduced by NMDA receptor antagonists, NMDA receptor-associated cation channel blockers, or compounds inhibiting the modulatory sites of the NMDA receptor/cation channel complex such as strychnine-insensitive glycine receptors (Johnson and Ascher, 1987) or polyamine binding sites (Ransom and Stec, 1988).
References Burns, D.L., lY88, Subunit structure and enzymatic activity of pertussis toxin, Microbial. Sci. 5, 285. Gilman, A.G., 1987. G-proteins: transducers of receptor-generated signals, Ann. Rev. Biochem. 56, 615. Huetter. J.E. and B.P. Bean, 1988, Block of N-methyl-D-aspartateactivated current by the anticonvulsant MK-801: selective binding to open channels, Proc. Natl. Acad. Sci. U.S.A. 85. 1307. Johnson. J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse hrain neurons, Nature 325. 529. Ransom, R. and N. Stec, 1988, Cooperative modulation of [ ‘H]MK801 binding to the N-methyl-D-aspartate receptor-ion channel
211 complex by L-glutamate, glycine and polyamines, J. Neurochem. 51, 830. Tamura. M., K. Nogimori, S. Murai, M. Yajima, K. Ito, T. Katada. M. Ui and S. Ishii. 1982, Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model, Biochemistry 21, 5516. Ui, M.. F. Okajima, T. Katada and T. Murayama, 1988, Roles of
GTP regulatory proteins, the substrates of islet-activating protein, in receptor-mediated adenylate cyclase inhibition, phosphohpase C activation, and cell proliferation, Adv. Second Messenger Phosphoprotein Res. 21, 39. Vv’orley, P.F., J.M. Baraban and S.H. Snyder. 1987. Beyond receptors: Multiple second-messenger systems in brain, Ann. Neural. 21, 217.
Michael
J. Durcan
and Philip F. Morgan
Received 13 December 1990. xcepted 19 March IYYI
The effect of prclreatmcn! with pcrtussis toxin on N-mcthyi-D-aspartatc (NMDAJ-induced seizures was investigated in mice. In animals ircatcd with pcrtussis toxin iij.5 pg/animal i.c.v) five days prior to testing the convulsant ED,,, of NMDA uas calculated to he 18 mg/kg whereas it was calculated to be 107 mg/kg in sham-treated animals. These rc>u!ts suggest the pcrtussis
toxin enhances sensitivity
to NMDA.
Pcrtussis
possibly via its actions on inhibiwry
toxin: NMDA
(N-methyl-D-aspartatc):
1. Introduction
G-proteins.
Seizures:
(Mouse)
with pertussis toxin (0.5 pg/animal days later their sensitivity
Many neurotransmitter effector
mechanisms
via
proteins (G-proteins)
receptors
are linked to their
guanine
nucleotide-binding
(Gilman,
monophosphate
IAJ-trisphosphate volve actions G-proteins although not
inactivate
Gi and G,,
proteins.
mechanisms
(Ui
controls.
by ADP from
were
a phenomenon Post-mortem
in animals
five
the
their
effcctor
current
ad libitum.
food and
The mice weighed
between
25 and 1X g at the time of pertussis toxin or vehicle A!!
mice
were
housed
individtially
after
pertussis toxin or vehicle treatment.
indicated
seizures were
by noise or cage move-
from administraticln rapid mice
of N-methyl-
circular
by full tonic-clonic experiment
water available
Mice were trlatcd
cause of death
Occasionally,
(i.e.
Swiss male mice were housed in groups
10 on a 1,‘: 13 h light: dark cycle with
in sham-treated
of animals
these seizures were reminiscent
acid (NMDA)
bouts often followed In
ribosylation,
Naive NlH of
to six days post
not seen
disturbed
observed
of seizures resulting D-aspartic
can
per anima! or greater, a
noted
inspection
(i.e. hind limb extension). ment. When
toxin
treatment.
that a seizure may have been a frequent observed
(G,),
with pertussis toxin (i.c.v.1 in our
at doses of 0.5 pg
of deaths
treatment,
1987).
et al., 1988).
In mice pretreated laboratory,
Pertussis
receptors
and methods
or may in-
et al.,
(Ci,) or stimulatory
also exist.
uncouple
2. Materials
(e.g. G,,) whose exact role is
understood
and therefore
number
(Worley
of
or inositol
(IP3) and diacylglycerol,
can be inhibitory
fully
(CAMP)
on ion channels
other G-proteins
seizures
was investigated.
1987). This linkage can
be via second messenger systems such as production cyclic adenosine
i.c.v.1 and then five
to NMDA-induced
running
seizures).
were
pretreated
Biological
with 0.5 c(g pertussis toxin (List
Labs. Campbell.
CA) injected in a volume of
5 ~1 directly
into the lateral
anesthetized
using chloral
made
in the scalp. and the lateral
(coordinates -3
ventricle. hydrate.
from bregma:
mm vcrtics!!.
Animals
:I small ventricle
1 mm lateral,
were
incision accessed
I mm rosrral.
Pertussis toxin was injected
at a rate
of 5 pl/min
for I min. the injection needle remained in place for a further 2 min before being slowly re-
tracted
and
Control
animals were injected with vehicle buffer (0.01
M
sodium
chloride;
the wound phosphate,
on!:/.
closed pH
using a wound
7.0 with
0.05
M
clip.
sodium
DOSE OF NMDA
(mglkg)
Fiy. 1. Pcrcrntage of pertussis toxin ( )- and sham (c)-treatrd following administration of NMDA. * * P < 0.01 animals S&ZirI~ Fi&cr’s e~ct test of proportions seizing where seizure dose-rezponw cuws overlap (i.e. 30 and 100 mg/kg doses of NMDA).
Groups of pertussis toxin-treated or control animals (N = S-11) were treated with doses of NMDA (Research Biochemicals Inc.. Natick, MA), l-200 mg/kg i.p.. at a volume of 10 ml/kg dissolved in distilled water and the number seizing within 60 min of administration was noted by an observer unaware of the drug dose or the pertussis toxin treatment.
The dose effect curves for both control and pertussis toxin-treated mice are shown as fig. 1. The ED,,, for convulsant effect of NMDA was calculated, by linear regression analysis of log probit plots (r’s > 0.98), to be 18 mg/kg in pertussis toxin-treated animals and 107 mg/kg in sham-treated controls. Fisher’s exact probability test revealed that where the NMDA seizure dose-response curves overlapped those groups pretreated with pertussis toxin had significantly greater proportions of animals seizing than did the shamtreated animals i? = 0.002 for 30 mg/kg; and P = 0.013 for 100 mg/kg NMDA).
These results suggest that increased susceptibility to excitatory neurotransmission may explain the mortality seen at five to six days post pertussis toxin treatment. Pertussis toxin inactivates Gi and G,, G-proteins which constitute an integral and essential part of a number of
inhibitory neurotransmitter activated transduction mechanisms (Gilman, 1987; Worley et al., 1987), but has little or no action on G,-proteins coupled to excitatory neurotransmitter systems. This suggests that increased sensitivity to NMDA following i.c.v. pertussis toxin may be due to a functional inactivation of inhibitory components of neurotransmission, rather than a direct effect on G-protein-linked excitatory neurotransmission. Several neurotransmitter receptors have been reported to be linked to pertussis-sensitive Gi- or G,-proteins (see Worley et al., 1987 for review), the inactivation of which may contribute to the increased sensitivity to NMDA. Mechanisms other than the inactivation of G-proteins may, however, be responsible for the observed increase in NMDA sensitivity. Pertussis toxin is made up of six protein subunits (consisting of five different proteins as one repeats), the largest of these is the enzymatically active A-protomer, whilst the remaining pentamer forms the binding B-oligomer (Tamura et al., 1982). The A-protomer is responsible for catalyzing the ADP riboslyation of susceptible G-proteins. The Boligomer binds to the cell membrane and is required to deliver the cataljrtic subunit to the target cell. Whilst the B-oligomer has no effect on G-proteins it is not completely without biological effect (Burns, 1988) and therefore activity of this binding oligomer may possibly contribute to the enhanced sensitivity to NMDA-induced seizures seen following pertussis toxin treatment. NMDA primarily acts at the NMDA sub-set of giutamate receptors which are coupled to cation channels which principally gate the transmembrane flux of calcium (Huetter and Bean, 1988). The present findings would therefore suggest that pertussis toxin mortality may be reduced by NMDA receptor antagonists, NMDA receptor-associated cation channel blockers, or compounds inhibiting the modulatory sites of the NMDA receptor/cation channel complex such as strychnine-insensitive glycine receptors (Johnson and Ascher, 1987) or polyamine binding sites (Ransom and Stec, 1988).
References Burns, D.L., lY88, Subunit structure and enzymatic activity of pertussis toxin, Microbial. Sci. 5, 285. Gilman, A.G., 1987. G-proteins: transducers of receptor-generated signals, Ann. Rev. Biochem. 56, 615. Huetter. J.E. and B.P. Bean, 1988, Block of N-methyl-D-aspartateactivated current by the anticonvulsant MK-801: selective binding to open channels, Proc. Natl. Acad. Sci. U.S.A. 85. 1307. Johnson. J.W. and P. Ascher, 1987, Glycine potentiates the NMDA response in cultured mouse hrain neurons, Nature 325. 529. Ransom, R. and N. Stec, 1988, Cooperative modulation of [ ‘H]MK801 binding to the N-methyl-D-aspartate receptor-ion channel
211 complex by L-glutamate, glycine and polyamines, J. Neurochem. 51, 830. Tamura. M., K. Nogimori, S. Murai, M. Yajima, K. Ito, T. Katada. M. Ui and S. Ishii. 1982, Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model, Biochemistry 21, 5516. Ui, M.. F. Okajima, T. Katada and T. Murayama, 1988, Roles of
GTP regulatory proteins, the substrates of islet-activating protein, in receptor-mediated adenylate cyclase inhibition, phosphohpase C activation, and cell proliferation, Adv. Second Messenger Phosphoprotein Res. 21, 39. Vv’orley, P.F., J.M. Baraban and S.H. Snyder. 1987. Beyond receptors: Multiple second-messenger systems in brain, Ann. Neural. 21, 217.