Abstract:
Six serotonin antagonists, cinanserin, methiothepin, spiperone,
metergoline, ketanserin, and methysergide, were all found to
delay the first two divisions of sea urchin embryos. Three
serotonergic agonists, DOI, RU24969, and phenyl biguanide were
also found to delay cleavage, although more weakly than the
antagonists. Cleavage delays caused by 10 uM metergoline and 10
uM ketanserin were reversed by serotonin. Serotonin alone was
found to speed up cell division over controls. Cytological
studies showed the serotonin antagonists metergoline, ketanserin
and methysergide delayed karyokinesis as well as cytokinesis.
Introduction:
Serotonin, or 5-hydroxytryptamine (5-HT), a potent mammalian
neurotransmitter, was first reported in sea urchin embryos in the
late '60's (Buznikov et al, 1964, 1970). The method originally
used was a fluorescence assay (Buznikov et al., 1970; Toneby,
1977), and these findings were later confirmed with the more
specific techniques of HPLC and thin-layer chromatography (Renaud
& Parisi, 1981, Toneby, 1977). Two lines of evidence suggest
that 5-HT is a regulator of sea urchin embryo development.
First, fluctuations in 5-HT levels, coincide with early cell
divisions (Buznikov et al. 1964). Secondly, serotonin
antagonists inhibit development, and in particular cause a delay
in early cleavages (Buznikov et al, 1970; Renaud & Parisi,
1981).
Using the sea urchin species Paracentrotus lividus and
Sphaerechinus granularis, Renaud & Parisi reported reversal of
the cleavage inhibition, caused by the 5HT antagonists
metergoline and gramine, by the simultaneous addition of
serotonin to the incubating eggs. However, Denis LaRochelle in
David Epel's lab found that this was not possible with
Strongylocentrotus purpuratus eggs.
Species differences can mean variable plasma membrane
permeability. Since serotonin is a charged molecule at
physiological pH, egg permeability could indeed be the reason for
the seemingly negative results. I therefore decided to use the
technique of electrically permeabilizing the eggs, adding a
substrate, and then resealing the plasma membrane pores with low
calcium sea water (method of Robert Swezey and David Epel, 1989).
I achieved moderate success reversing the effects of 5-HT
antagonists metergoline and ketanserin with serotonin. When high
amounts of serotonin were used, cells treated with serotonin
cleaved much earlier than controls.
Renaud and Parisi (1981) had reported that gramine's effect
on cleavage only included a delay in cytokinesis, not in
karyokinesis, or chromosal replication and separation. So, in
order to investigate the nature of serotonin involvement in
development, I used 4,6-diamidino-2-phenyl-indole (DAPI) to
fluorescently stain the chromosomes and observe karyokinesis in
antagonist-treated eggs as compared to controls. I found that
metergoline, ketanserin, and methysergide all delayed
karyokinesis concurrent with the delay in cytokinesis.
Recently, a great deal of neurological research has resulted
in the characterization of three types of serotonin receptors:
5-HT1, 5-HT2, and 5-HT3 receptors. In addition, within the 5-HTI
class, there are four distinct subclasses: 5-HTIA, 5-HTIB, 5-
ITIC, and 5-HTID receptors. Each receptor type and subtype has
different affinities for different serotonergic drugs. They are
characterized by radioligand studies and by pharmacological
differences. Most 5-HT receptors bind the same drugs; the
difference is in the affinities for these drugs. I have included
as table 1, a characterization of 5-HTI and 5-HT2 receptors (from
S. Peroutka, 1988). Different receptor types have also been
correlated with different activities. For example, 5-HTIC and 5¬
T2 receptors have been proposed to affect phophotidylinositol
turnover, whereas 5-HTlA receptors have been correlated with
adenylate cyclase modulation. Although the putative
intracellular receptors in sea urchin eggs are unlikely to be the
same as any classified neural extracellular receptors, comparison
of different drug affinities for these receptors is nevertheless
a useful way of characterizing them. Therefore, I have done a
series of concentration experiments with the serotonergic drugs
metergoline, methysergide, spiperone, ketanserin, methiothepin,
cinanserin, DOI, RU24969, and 1-phenyl biguanide in order to
compare drug potencies and help characterize the hypothesized
receptors. Structures of all drugs used are in tables 2 and 3.
Methods and Materials:
Fresh S. purpuratus gametes were collected by injecting 1-2 mls
of .5 M KCl into gravid sea urchins. The eggs were kept stirring
Eggs were always
at 160C. Dry sperm was kept at 400.
mechanically dejellied by passage through Nitex mesh and washed
twice before use. Eggs were diluted or concentrated as necessary
to keep a constant suspension of 28 (as measured by Bauer-Schenk
centrifuge tubes). Eggs were then fertilized, and subsequently,
the appropriate drugs added. Eggs were incubated in 24-microwell
plates, .5 ml per well. Drugs that were able to freely diffuse
through the egg membrane were simply added to the samples, the
same amount of solvent being added to the control plate. Whether
drugs were freely diffusible through the membrane was judged by
trial and error, i.e. determining whether the drug had an effect
on cleavage when simply added to the incubation plate. The two
solvents used were distilled water and dimethyl sulfoxide (DMSO).
If DMSO was used, the maximum amount added to the eggs was 2ul/ml
of egg suspension. The fertilized, treated eggs were incubated
at 1600. Samples of two-cell and four-cell stages were taken of
the developing embryos at 100, 110, 120, 155, and 165 minutes and
put into a 18 formaldehyde solution. 100 embryos were counted
for each time stop of each plate and numbers of cleaved cells
taken to be percentages.
Electric Permeabilization of Eggs
Whenever serotonin was used, it was necessary to permeabilize the
unfertilized eggs to allow the 5-HT to enter the cells. For
these experiments, after dejellying and washing the eggs, they
were resuspended in zero calcium sea water and washed twice, then
washed twice in transpermeabilization media (Trans Mix). The
transpermeabilization media is intended to mimic the cytoplasm of
the egg so that when eggs are electroporated, they do not lose
too many nutrients to their surroundings, nor do they become
activated by calcium or lysed by high sodium concentrations.
This media consists of 225 mM potassium gluconate, 185 mM
mannitol, 300 mM glycine, .5 mg/ml glutathione, 5 mM magnesium
chloride, 4 mM ATP, 10 mM spermidine (to keep the membranes
intact), 20 mM sodium chloride and 2 mM sodium bicarbonate. The
eggs were then given 300 volts of electric current, with.6
microfarads capacitance, 1 pulse every 10 seconds, for a total of
5 pulses. The eggs were then transferred to Eppendorf tubes
containing either serotonin in.058 ascorbic acid, or .058
ascorbic acid only (for controls). After 2 minutes, an excess of
low calcium sea water (9:1 ratio of zero calcium sea water to
fresh sea water) was added to each vial, the low concentration of
calcium resealing the egg pores without activating the eggs. The
eggs were washed once with 9:1 mix, then twice with fresh sea
water for a final suspension of 28. The rest of the procedure is
identical to the first method.
DAPI Staining
S.purpuratus eggs were prepared according to the above two
methods, depending on whether serotonin was used or not. Instead
of fixing eggs with formaldehyde, however, the DAPI staining
method was used to kill the eggs and stain the chromosomes for
viewing under a fluorescence microscope. At 70, 80, 90, and 100
minutes, 1 ml of egg suspension from each plate was washed twice
in KGE mix (3 mM potassium gluconate, 3.2 mM glycine, .4 mM EGTA,
and .4 mM magnesium sulfate). Eggs were then resuspended in 1 ml
of .18 Triton detergent in KGE mix. 1 ul of a 1 mg/ml solution
of 4,6-diamidino-2-phenyl-indole (DAPI) was added to each ml of
prepared eggs. In addition, formaldehyde stops were taken at 90,
100, 110, and 120 minutes in order to see cytokinesis effects and
compare them to effects on mitosis by the drugs. DAPI samples
were then viewed under a fluorescence microscope. If the results
were not obvious, plates were scored by counting 100 samples in
each plate. When high amounts of serotonin antagonists were
used, the results were obvious simply by viewing.
Results:
Delay of Cleavage by Serotonergic drugs
Table 4 shows the effective concentrations of all drugs used.
Cinanserin, a 5-HT2 antagonist, was found to be the most powerful
drug in delaying cleavage. The graph of the delay in cleavage
caused by 10 uM concentration cinanserin looks like delays caused
by 100 uM concentrations of most other drugs (see figures 1-6).
C
Other potent drugs were Methiothepin, Spiperone, Ketanserin and
Metergoline, all 5HT2 antagonists. Methysergide, a 5-HT2
antagonist, was somewhat less potent, but nevertheless
significantly delayed cleavage at a concentration of 30 uM.
Three selective agonists were used as well: RU24969, a 5-
HTIB agonist; DOI, a 5-HT2 agonist; and 1-phenyl biguanide, a
5-HT3 agonist. None of these agonists had the expected effect of
speeding up cleavage. On the contrary, as shown in figures 7-9,
they all acted as serotonin antagonists by delaying cleavage. As
shown in table 1 and figures 7-9, Phenyl biguanide was a very
weak antagonist, effective only at very high concentrations,
whereas DOI was the most potent and RU24969 showed a medium
effectiveness (effectiveness as measured by the concentration of
drug required to significantly delay cleavage).
Effects of Serotonin
As shown in figures 10-11, I was able to reverse cleavage delays
by metergoline and ketanserin with serotonin. The concentrations
of serotonin used are only approximate because, using the
electroporation technique, it is impossible to know for any given
experiment exactly how much substrate is entering into the cells.
For the metergoline experiment, I found that "100 uM
serotonin reversed the effects of 10 uM metergoline, but the 1mM
serotonin sample was not sped up over controls. In the
ketanserin experiment, however, I found that serotonin not only
reversed the 10 uM ketanserin delay, but also, as shown in
figures 11 and 12, embryos with concentrations of serotonin
higher than 10 um divided significantly ahead of controls.
Delay of Mitosis
Usin the DAPI method, I examined the effects of metergoline,
methysergide, and ketanserin on progression through mitosis. All
antagonists were found to also delay karyokinesis to the same
degree that cytokinesis was delayed. If a low concentration of
drug was used, the same percentage type delay ensued in both
cytokinesis and in karyokinesis. When a high concentration of
drug was used, drug-treated eggs were 1008 delayed over controls
in both cytokinesis and karyokinesis. Please see Table 5 for
results of each experiment.
Discussion:
Although serotonin receptors have not yet been characterized in
sea urchin eggs or embryos, all my results point to the
conclusion that serotonin receptors do exist in these embryos.
Six selective serotonin antagonists all delayed cleavage of
activated eggs. In addition serotonin appears to be involved in
early cell division, both cytokinesis and karyokinesis.
Cytological studies showed that eggs treated with high amounts of
antagonist (100 uM ketanserin and 50 uM metergoline) accumulated
in prophase, whereas controls moved on to other phases of
mitosis.
The most interesting aspect of these experiments is the
reversal of metergoline and ketanserin inhibition by serotonin,
Serotonin seems to interact competitively with these two
antagonists, acting in a concentration-dependent way. The
experiments with ketanserin especially show that the higher the
serotonin concentration used, the faster cell division occurred.
While the putative sea urchin egg serotonin receptors are
affected by all the serotonergic drugs I assayed, known to be
effective on nerve cells, these receptors do not seem to be
comparable to nerve receptors. In terms of the antagonist
affinities, sea urchin receptors seem to be comparable to 5-HT2
receptors, ordinarily found in mammalian cerebral cortex cells.
However, the potent 5-HT2 agonist DOI had the effect of acting as
an antagonist and delaying cleavage. Similar results were seen
with the 5-HTIB agonist RU24969 and the 5-HT3 agonist phenyl
biguanide, indicating that sea urchin receptors cannot be 5-
ITIB, 5-HT2, or 5-HT3 receptors. The only other characterized
receptor types are 5-HTIA, 5-HTIC and 5-HTID. However, the fact
that the cinanserin, ketanserin and spiperone all potently
inhibited development would discourage the conclusion that sea
urchin egg receptors could be one of these three remaining
subtypes-these three antagonists have very low, if any affinity
Most nerve receptors are
for these three subtypes.
extracellular. But this study indicates sea urchin egg receptors
are probably intracellular; in order to get an effect from
serotonin, it was necessary to permeabilize the egg so that the
substrate could enter the cell. Therefore, sea urchin receptors
may be a novel receptor type or subtype, as yet uncharacterized.
An interesting finding is that the only agonist that has
been found to act on these receptors is serotonin itself. The
receptors appear to bind similar substrates, but not be activated
by them in the same way as its intended substrate, serotonin. As
shown in figure 12, serotonin not only reverses the effect of the
antagonists ketanserin and metergoline, but by itself, speeds up
development.
Further studies on serotonin's role in sea urchin egg
development could clarify many remaining questions. The use of
5-HTIA, 5-HTIC, and 5-HTID agonists would confirm whether or not
sea urchin egg receptors are comparable to known nerve cell
receptors. Radiolabeling, antibodies, or other methods could
help confirm the existence of the receptors, and possibly locate
them in the cell.
I did in fact attempt a standard radiolabeling experiment in
Steve Peroutka's lab at Stanford University, but found no
receptor activity. However, these results are not conclusive
since this test is designed for high density nervous tissue, such
as rat brain. In fact this test does not find activity in lower
density receptor tissue such as cardiac tissue, which has known,
characterized serotonin receptors (S. Peroutka, personal
communication). So, a different radioassay would have to be
designed to definitively find activity.
Finally, as noted in the introduction, serotonin receptors
in other systems are known to affect phosphatidylinositol
turnover, and adenylate cyclase activity. Therefore, studies
measuring the effect of serotonin on calcium and cAMP levels in
sea urchin embryos, or phosphotidylinositol turnover could help
10
c
clarify the mechanism by which serotonin affects development. A
most intriguing result, given the apparent intracellular nature
of these receptors, is that the mode of action may be by some yet
undescribed mechanism.
table
5-HT RECEPTOR SUBTYPES
A summary of drug potencies at each of the five known S-HT
binding site subtypes is provided in Table 2. These data are discussed in
greater detail in the following section.
Table 1 Characteristics of 5-HT, S-HT», 5-HT,c, 5-HTp, and 5-HT, binding sites
S-HT
5-HTc
S-HTp
5-HT,
5-HTA
H-S-HT
H-S-HT
H-S-HI
H-Spiperone
H-S-HT
Radiolabeled
1231.CYI
H-Mesulergine
H-Mesulergine
H-8-OH¬
DPAT
Linans
1231-LSD
1231.LSD
H-Ipsapirone
(rat and
H-Ketanserin
H-WB 4101
mouse only)
Mianserin
H-Buspirone
H-N-methylspiperone
'H-PAPP
1231. Methyl-LSD
H-DOB
Layer IV
Raphe nuclei Substantia
Choroid
Basal
High
plexus
nigra
ganglia cortex
density
Hippocampus Globus
pallidus
Table 2 Drug affinities for 5-HTA, S-HTI», 5-HTIC, S-HTD, and 5-HT, receptors
Drug
potencies
S-HT
S-HT
5-HTp
5-HT,
S-HT
(K., nM)
5.CT
10
RU 24969
Mesulergine
Spiperone
5.CI
5-CT
5-HT
Mesulergine
8-OH-DPAT
Metergoline
5S-HT
5-HT
Methysergide
Methysergide
Metergoline
RU 24969
Metergoline
Mianserin
d-LSD
d-LSD
Methysergide
Metergoline
Metergoline
Mianserin
10-1000
Methysergide
5-HT
Mianserin
Methysergide
RU 24969
8-OH-DPAT
d-LSD
Spiperone
5-CT
d-LSD
Mesulergine
d-LSD
RU 24969
RU 24969
Mesulergine
» 1000 Mianserin
Mianserin
Spiperone
5-HT
8-OH-DPAT
Spiperone
Spiperone
8-OH-DPAT
Mesulergine
8-OH-DPAT
Data given are derived from Peroutka & Snyder (1979), Peroutka (1986), Hoyer et al (1985b), Heuring
—
Metergoline.
Cen,

NO
C3
Spiperone.
)e-a,d,
N



KETANSERIN
table 2
ANTAGONISTS
Methysergid(e).

G, ,
don


N
N—
W
e 5
ch,
METHIOT HEPIN

ci
CINANSERIN
E
Serotonin.


Sachane
R(-)-DOI
NH,
cn.
e
oen,
Phenyl Biguanide.
NH NH
CgligNHCNHCM,
RU 24969
cn,o

table 3
AGONISTS
TABLE 4
RESULTS OF SEROTONERGIC DRUG CONCENTRATION EXPERIMENTS
SEROTONERGIC DRUG
LOWEST EFFECTIVE CONCENTRATION/POTENCY
——————
———————————.
CINANSERIN
10 uM (PROBABLY LESS)/VERY HIGH
METHIOTHEPIN
10 uM / HIGH
METERGOLINE
10 uM / HIGH
SPIPERONE
10 uM / HIGH
KETANSERIN
10 uM / MEDIUM
ZTHYSERGIDE
30 uM / LOW
10-50 uM / MEDIUM
DOI (agonist)
100 uM/ LOW
RU24969 (agonist
1mM / VERY LOW
PHENYL BIGUANIDE
(agonist)
TABLE 5
RESULTS OF KARYOKINESIS (DAPI STAINING) EXPERIMENTS
EXPERIMENT/PLATE
MITOSIS STAGE
——————
——-——---———-
-
I. KETANSERIN (20 uM)
408 PROMETAPHASE
+1 / CONTROL/ 90 MINUTES
608 METAPHASE
708 PROMETAPHASE
+1 / 20 uM KETANSERIN/ 90 MIN
308 METAPHASE
78 PROMETAPHASE
+1 / CONTROL/ 100 MINUTES
208 METAPHASE
698 ANAPHASE
88 PROMETAPHASE
+1 / 20 uM/ 100 MINUTES
528 METAPHASE
418 ANAPHASE
———
II. METHYSERGIDE
38 PROMETAPHASE
+2 / CONTROL/ 90 MINUTES
908 ANAPHASE
78 TELOPHASE
128 PROMETAPHASE
+2 / 100 uM METHYSERGIDE/ 90 MIN
148 METAPHASE
738 ANAPHASE
18 TELOPHASE
—————
—————— — — ———
-———
III. KETANSERIN (100 uM)
+3 / CONTROL/ 80 MINUTES
ALL IN PROMETAPHASE
AND METAPHASE
+3/ 100 uM KETANSERIN/ 80 MIN
ALL IN PROPHASE
+3 / CONTROL/ 90 MINUTES
ALL IN ANAPHASE AND METAPHASE
MOST IN PROPHASE
+3 / 100 uM KETANSERIN/ 90 MIN
SOME IN PROMETAPHASE
MOST IN TELOPHASE
+3 / CONTROL/ 100 MINUTES
SOME IN ANAPHASE
MOST IN METAPHASE
+3 / 100 uM KETANSERIN/ 100 MIN
SOME IN PROMETAPHASE
----—-
——
IV. METERGOLINE
+4 / CONTROL/ 90 MINUTES
1008 IN METAPHASE
1008 IN PROPHASE
#4 / 50 uM METRGOLINE/ 90 MIN
+4 / CONTROL/ 100 MINUTES
1008 IN ANAPHASE
1008 IN PROPHASE
+4 / 50 uM METERGOLINE/ 100 MIN
FIGURE LEGENDS
1. Delay of cell division by 10 uM cinanserin, 2-cell stage.
2. Delay of cell division by 10 and 20 uM spiperone, 2 and 4¬
cell stages.
3.
Delay of cell division by 10 and 50 uM methiothepin, 2- and
4-cell stages.
4.
Delay of cell division by 10 uM ketanserin, 2 and 4-cell
stages.
5. Delay of cell division by 10 uM metergoline, 2-cell stage.
6.
Delay of cell division by 30 and 100 uM methysergide, 2 and
4-cell stages.
7.
Delay of cell division by 10, 50, and 100 uM DOI, a 5-HT2
agonist.
Delay of cell division by 1 mM phenyl biguanide compared to
delay by 10 uM metergoline, 2 and 4-cell stages.
Delay of cell division by 20, 30, 50, and 100 uM RU24969, a
5-HTIB agonist, 2 and 4-cell stages.
10. Reversal of the effects of 10 uM metergoline by serotonin,
2-cell stage.
11. Reversal of the effects of 10 uM ketanserin by serotonin,
2-cell stage.
12. Serotonin speeds up cell division, lmM concentration, 2 and
4-cell stages.
CINANSERIN STRONGLY DELAYS CLEAVAGE
110
100
——e ———0
90+
80+
70
5
20
•—• CONTROL
A--A 10 UM CINANSERIN
09
90 100 110 120 | 130 | 140 | 150 | 160
MINUTES POST-FERTILIZATION
figure
EFFECT OF SPIPERONE ON CLEAVAGE
O—O CONTRO
A--A 10 HM SPIPERONE
D 20 AM SPIPERONE
——
—
75

og
—
110 125 140 155 170 185
MINUTES POST-FERTILIZATION
figure 2
EFFECT OF METHIOTHEPIN ON CLEAVAGE
O—O CONTROI
100
A--A 10 AM METHIOTHEPIN
□—D 50 AM METHIOTHEPIN
o
A

110 125
140
155
MINUTES POST-FERTILIZATO
EFFECT OF 10 uM KETANSERIN ON CLEAVAGE
• CONTRO
100
•--° 10 AM KETANSERIN
6 75
25+
00
9-2-----4
100
140
120
MINUTES POST-FERTILIZATION
figure 4
EFFECT OF 10 MM METERGOLINE ON CLEAVAGE
100
O
804
O—O CONTROL
- 10 AM METERGOLINE
o
100 120 140 160 180
200
MINUTES POST-FERTILIZATION
figure 5
EFFECT OF METHYSERGIDE ON CLEAVAGE
O—O CONTROL
100
A--A 30 MM METHYSERGIDE
D—D 100 AM METINSERCRE
6 75

50
O
2
o

110 125 140
155
DOI, A 5HT2 AGONIST, ANTAGONIZES
O CONTROL
A—A TOAM D0
D— 50 AM D0
8—2
•—• 100 AM DO

4—2
175
MINUTES POST-FERTILIZATION
figure 7
PHENVL BIGUANIDE, A 5HT3 AGONISI
COMPARED TO METERGOLINE, A GENERAL 5HT ANTAGONISI
110
9—0 CONTROL
A—A 10 AM METERGOLINE
•-- ImM PHENYL BIGUANIDE
2


180
MINUTES POST-FERTILIZATION
figure 8
RU24969, A 5-HTIB AGONIST, ANTAGONIZES
•—• CONTROL
100
—• 20 M RU24969
O— 30 AM RU24969
A—A 50 UM RU24969
80
D—D 100 AM RU24969
40

2
110
125 140 155
MINUTES POST-FERTILIZATION
ure
110

REVERSAL OF METERGOLINE EFFECTS BY SEROTONIN
100
80
8
60+
O-O CONTROL
40
•— 10 AM METERGOLINE
d 20
A-A 10 EM METERGOLINE
+ 100 MM SEROTONIN
—
100 120 140 160 180
200
MINUTES POST-FERTILIZATION
figure IO
REVERSAL OF KETANSERIN BY SEROTONIN
110
•— CONTROL
•-- 10 AM KETANSERIN
Ooneansgen
+IOAM SEROTONIN
A—A 1 mM SEROTONIN

MINUTES POST-FERTILIZATION
figure1
EFFECT OF SEROTONIN ON DIVISION
0—e CONTRO
A—A 1 mM SEROTONIN

06 2—
MINUTES POST-FERTILIZATION
110
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