Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9
* Author for correspondence (e-mail: jeff.goldberg{at}ualberta.ca)
Accepted 12 January 2004
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Summary |
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Key words: serotonin, 5-hydroxytryptamine, pond snail, Helisoma trivolvis, intracellular calcium, ciliary beating, cilia, serotonin receptor
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Introduction |
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An aspect of ciliary regulation that requires more attention is the
identification of the endogenous neurotransmitters, neuromodulators and
hormones that stimulate the aforementioned intracellular events. Serotonin
(5-hydroxytryptamine; 5-HT), one such neurotransmitter that has been shown to
be cilio-excitatory, was first identified to increase CBF in bivalve gill
cilia (Gosselin et al., 1962).
Other neurotransmitters and modulators that regulate ciliary activity include
dopamine (Wada et al., 1997
),
acetylcholine, substance P (Aiello et al.,
1991
) and adenosine triphosphate (ATP;
Morales et al., 2000
). In
order to develop an integrated understanding of ciliary activity, studies on
the endogenous extracellular regulators and their associated signal
transduction pathways from the cell membrane to the axoneme must be
complimented by analysis of ciliary cell function at the whole-animal
level.
The direct-developing embryos of the gastropod mollusc Helisoma
trivolvis present an opportunity to study neurociliary interactions
during development. Three subpopulations of ciliary cells are evident on the
surface of the embryo throughout early development: a single pedal band,
paired dorsolateral bands and numerous scattered single ciliary cells (SSCCs;
Kuang and Goldberg, 2001). The
pedal ciliary cells and the most medial of the four cells composing each
dorsolateral band are innervated by a bilateral pair of serotonergic
sensory-motor neurons, termed embryonic neurons C1 (ENC1s;
Diefenbach et al., 1991
;
Koss et al., 2003
). ENC1s
develop prior to the central nervous system and are the first neurons detected
within the embryo. The ENC1-ciliary neural circuits generate cilia-driven
rotational movements within the egg capsule in response to egg capsule oxygen
content, with hypoxia stimulating an increase in embryo rotation
(Kuang et al., 2002
). In
Helisoma ciliary cells, stimulation of a diacylglycerol-sensitive,
phorbol ester-insensitive PKC isoform and calcium influx are known to produce
an increase in CBF (Christopher et al.,
1996
,
1999
). Additionally, the
presence of nitric oxide synthase (NOS), the enzyme responsible for the
generation of NO, was detected in ENC1s and ciliary cells
(Cole et al., 2002
). Recent
studies have revealed that pharmacological manipulations of NO alter embryo
rotation by directly affecting ENC1s and the postsynaptic ciliary bands
(Cole et al., 2002
;
Doran et al., 2003
). It
appears that NO has a novel constitutive excitatory action in cilia that is
permissive to the cilio-excitatory activity of 5-HT. Thus, the use of the
Helisoma model system facilitates an examination of the neural
control of ciliary activity as well as the signal transduction pathways and
potential `cross-talk' between pathways.
In the present study, we begin to characterize some of the individual
subpopulations of Helisoma ciliary cells. We developed a technique to
culture identified ciliary cells to enable study of the pedal ciliary cells,
dorsolateral ciliary cells and SSCCs individually. Given that previous
attempts to visualize intracellular Ca2+ in these embryonic cells
using membrane-permeable indicators were unsuccessful, ciliary cells were
microinjected with impermeable Fura dextran for calcium imaging experiments.
Here, we report that 5-HT stimulates a calmodulin-mediated rapid increase in
the CBF of pedal and dorsolateral cilia and a slower increase in
[Ca2+]i. By contrast, the SSCCs do not show a reliable
change in CBF or in [Ca2+]i in response to 5-HT. These
results are supported by immunohistochemical data that suggest that the pedal
and dorsolateral ciliary cells, but not the SSCCs, express the
5-HT1Hel and 5-HT7Hel serotonin receptor proteins
(Mapara et al., 2001).
Furthermore, the SSCCs are anatomically distinct from pedal and dorsolateral
ciliary cells. These results suggest that the pedal and the dorsolateral
ciliary populations share many physiological and anatomical characteristics,
whereas the SSCCs represent a distinct ciliary subtype.
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Materials and methods |
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Chemicals and solutions
Embryonic cells were cultured in Helisoma defined medium [HDM: 50%
Liebovitz-15 (Gibco, Burlington, ON, Canada), 40 mmol l1
NaCl, 1.7 mmol l1 KCl, 4.1 mmol l1
CaCl2, 1.5 mmol l1 MgCl2, 5.0 mmol
l1 Hepes, 50 µg ml1 gentamicin, 0.015%
L-glutamine (Sigma, St Louis, MO, USA); pH 7.307.35). All
imaging and CBF experiments were performed in Helisoma saline (HS:
51.3 mmol l1 NaCl, 1.7 mmol l1 KCl, 4.1
mmol l1 CaCl2, 1.5 mmol l1
MgCl2, 5.0 mmol l1 Hepes; pH 7.337.35).
Calmidazolium chloride (Cal; Sigma) and ionomycin (free acid; Calbiochem, La
Jolla, CA, USA) were dissolved in dimethyl sulfoxide (DMSO; Sigma) and then
diluted to working concentration in HS so that the DMSO level did not exceed
0.1%. 5-HT (creatine sulfate complex; Sigma) was dissolved in HS. Fura dextran
potassium salt (fura; 10 000 Mr; Molecular Probes, Eugene,
OR, USA) was dissolved in filtered distilled water to a concentration of 2
mmol l1. All drugs were prepared on the day of use.
Mixed population ciliary cell culture
Embryonic ciliary cells were cultured as previously described (Christopher
et al., 1996,
1999
). Briefly, egg masses were
disinfected with 35% ethanol and the embryos were removed. Isolated embryos
were treated with 0.2% trypsin (Sigma) for 30 min and then mass dissociated by
repeatedly passing them through a 63-µm nylon mesh (Small Parts Inc.,
Miami, FL, USA). The resulting cell suspension was plated on
poly-L-lysine-coated (hydrobromide; Mr
400015 000; 1 µg ml1; Sigma) culture dishes
(Falcon 3001) for ciliary beat experiments and on
poly-L-lysine-coated glass bottom dishes for imaging experiments
and immunohistochemistry. The cultures were maintained in the dark at room
temperature (2022°C) for 1824 h to enable cells to adhere to
the substrate.
Cell culture of identified ciliary cell populations
Egg masses were incubated in antibiotic-containing HS (gentamicin sulfate;
Sigma) for 15 min prior to removal of embryos. Embryos were immersed in HS and
examined with Normarski differential interference contrast (DIC) optics on an
inverted compound microscope (Nikon, Diaphot) for identification of the pedal
and dorsolateral ciliary cells and SSCCs. An identified cluster of ciliary
cells (see Fig. 1) was gently
sucked into the 30-µm tip of a glass micropipette (World Precision
Instruments, Sarasota, FL, USA) using a micrometer syringe (Gilmont
Instruments, Barrington, IL, USA), and the remainder of the embryo was
surgically detached with a 30-gauge needle (Becton-Dickinson, Mississauga, ON,
Canada). The tissue explants containing identified ciliary cells were then
expelled onto poly-L-lysine-coated culture dishes (see above)
containing HDM. The culture dishes were maintained in the dark at room
temperature (2022°C) for 1824 h to enable the explants to
adhere to the substrate.
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Electron microscopy
For scanning electron microscopy (SEM), isolated embryos were transferred
to glass vials and fixed for 3060 min in 2% OsO4 in 0.01 mol
l1 phosphate-buffered saline (PBS), pH 7.5. Specimens were
then rinsed for 2x10 min in PBS and dehydrated in an ascending ethanol
series of 30%, 50%, 70% and 100%. After three changes of 100% ethanol, the
ethanol was replaced by adding increments of 30% isoamyl acetate to reach
saturation. Subsequently, specimens were dried via CO2
critical point drying, sputter-coated with goldpaladium and viewed with
a Cambridge Stereocan S250 scanning electron microscope.
For transmission electron microscopy (TEM), embryos were fixed for 1 h in
2.5% glutaraldehyde in 0.01 mol l1 PBS (pH 7.5), followed by
a 1 h post-fixation in 2% OsO4 in 1.25% sodium bicarbonate (pH 7.2;
Wood and Luft, 1965). They
were then dehydrated through an ethanol series and directly embedded in Spurrs
(Ted Pella Inc., Redding, CA, USA). After a polymerization period of 48 h,
silvergold sections were cut, collected on copper grids and stained
with uranyl acetate and Reynold's lead citrate
(Reynolds, 1963
) for 20 min
and 5 min, respectively. TEM sections were then observed with an FEI
transmission electron microscope (Morgagni model).
Ciliary beat frequency analysis
For the calmodulin inhibitor experiment, ciliary beating was monitored with
a CCD video camera (JVC, TK-860U) mounted on an inverted compound microscope
set up for phase-contrast optics through either a 20x or 40x
objective (Nikon, Diaphot). Using a time-lapse video recorder (VCR; Panasonic
AG-6720), ciliary activity was recorded over a 510 s interval
immediately prior to and 10 min after application of the vehicle control or
drug solution. Off-line analysis involved slowing the playback speed to 1/24
normal and manually counting ciliary beats over a 1-min interval. For this
experiment, CBF was presented as a percentage of the pre-treatment
measurement.
For the quantification of ciliary beating in identified ciliary populations, ciliary cells were viewed on an inverted microscope (Zeiss Axiovert 135; Zeiss, ON, Canada) with DIC optics and a 100x objective. Ciliary beating was recorded using either a high-speed digital videocamera (Vitana) linked to a Macintosh Powerbook containing Pixelink software (generously provided by Improvision, Inc., Quoram Technologies, Inc., Guelph, ON, Canada) or a Retiga Ex digital CCD camera (Q-Imaging; Burnaby, BC, Canada) linked to a Pentium 4 PC containing Northern Eclipse software (Empix Imaging Inc., Mississauga, ON, Canada). With both cameras, videos were collected at >50 frames s1 for 1 s segments, once every minute over the course of the experiment. Off-line analysis was performed on a Macintosh G4 computer using the public-domain NIH Image program (written by Wayne Rasband at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov) and Particle Analysis (a user-contributed macro, written by C. J. H. Wong at the University of Alberta). The displacement of the cilia from an initial position was calculated in both X and Y coordinates. The CBF was then determined by performing an autocorrelation on the resulting displacement waveform. The CBF for each time point was the result of an average of three measurements made in approximately the same location on the cell over the course of the experiment. For each cell type, a few experiments were performed at a higher temporal resolution to determine if there were undetected changes in CBF in response to drug treatments. Given that the CBF experiments with a collection rate of once every 15 s revealed no additional events, the data are presented with the collection rate at once every minute.
Ratiometric Ca2+ imaging and Ca2+ calibration
Fura dextran (Molecular Probes) was dissolved in filtered distilled water
to a concentration of 2 mmol l1. Fura dextran was utilized
instead of the free acid form of the indicator because it is considered to be
less toxic to the cell and more resistant to subcellular compartmentalization
(Tombal et al., 1999). The dye
was backloaded into custom-made pulled micropipettes (1.0 mm glass with
filament; World Precision Instruments, Inc.). An Eppendorf Femtojet Rapid
injection system (Brinkmann, Mississauga, ON, Canada) mounted on an inverted
Nikon Eclipse microscope was used to microinject cultured ciliated cells under
100x DIC optics. Loaded ciliary cells were imaged with a 100x
oil-immersion objective (1.3 N.A. Fluor) on an inverted microscope (Axiovert
135; Zeiss) with excitation at 340 nm and 380 nm from an HgXe arc lamp
(Hamamatsu, Hamamatsu, Japan). Emission fluorescence at 510 nm was collected
using an intensified charge-coupled device (ICCD) video camera (Paultek
Imaging, Grass Valley, CA, USA). Neutral density filters (Omega Optical,
Brattleboro, VT, USA) were used to ensure that fluorescent images were within
the sensitivity range of the camera. Data were collected as 8-bit images using
custom software kindly provided by Dr. S. Kater (University of Utah). Captured
images were digitized through a QuickCapture frame grabber board (Data
Translation, Mississauga, ON, Canada) and saved to a computer (Macintosh
Quadra 950) for off-line analysis. Vehicle or drug-containing solutions were
perfused into the culture dish using a gravity-driven perfusion set-up at
approximately 1 ml min1 with a latency of 15 s (Warner
Instruments Corp., Holliston, MA, USA). Images were analyzed for whole-cell
fluorescence intensity on a Macintosh G4 computer using the public-domain NIH
Image program and Ca2+Ratiometrics (a user-contributed macro,
written by C. J. H. Wong at the University of Alberta).
The 340/380 ratios, which provide a relative measure of cytoplasmic free
calcium concentration, were converted to estimates of
[Ca2+]i using the equation
[Ca2+]i=KD(RRmin)/(RmaxR)(Fo/Fs),
where R is the 340/380 ratio, KD is the
dissociation constant and Fo and Fs
are the fluorescence values obtained at minimal and saturating
[Ca2+], respectively, at the 380 nm excitation
(Grynkiewicz et al., 1985).
The KD was determined to be 0.586 µmol
l1 using thin-wall glass capillary tubes (20 nm in width;
VitroCom Inc., Mt. Lks., NJ, USA) filled with one of 11 different
Ca2+ buffer solutions (Ca2+ concentration ranged from 0
µmol l1 to 39 µmol l1; calcium
calibration buffer kit with magnesium II; Molecular Probes) and 50 µmol
l1 fura dextran. Fluorescence values acquired from the
images taken with excitation at both 340 nm and 380 nm for all 11
Ca2+ buffer solutions were entered into a computer program
available on the Molecular Probes website
(www.probes.com/resources/calc/kd.html)
to determine the value of KD. This value for
KD is consistent with other in vitro estimates
for fura dextran (Konishi and Watanabe,
1995
; Tombal et al.,
1999
). The values for Rmin,
Rmax, Fo and Fs
were determined in situ using ciliary cells loaded with fura dextran.
Cells were permeabilized with 50 µmol l1 ionomycin in the
presence of either Ca2+-free HS with 1.0 mmol l1
ethylene glycol-bis(ß-aminoethyl ether) N,N,N,N-tetraacetic acid
(EGTA) for the determination of Rmin and
Fo or unaltered HS for the determination of
Rmax and Fs.
Immunohistochemistry
Identified populations of ciliary cells cultured on glass-bottom Petri
dishes were fixed in 4% paraformaldehyde in 0.01 mol l1
phosphate-buffered saline (PBS) at 4°C for 1 h. The cells were washed for
3x10 min in PBS. This and subsequent steps were performed at room
temperature with agitation unless otherwise stated. Embryos were washed in PBS
containing 3% horse serum (Sigma) and 0.3% Triton X-100 for 1 h. This was
followed by incubation in rabbit anti-5-HT1Hel or
anti-5-HT7Hel antiserum diluted 1:1000 in PBS containing 1% horse
serum and 0.4% Triton X-100 for 12 h at 4°C. The cells were then washed
for 4x7 min with 0.4% Triton X-100 in PBS and then incubated for 1 h
with goat anti-rabbit immunoglobulin G conjugated to Alexa 488 (Molecular
Probes) that was diluted in a solution of 1% horse serum and 0.3% Triton X-100
in PBS. This was followed by 4x5 min washes in 0.3% Triton X-100 in PBS
and 2x10 min washes in PBS. The cells were examined in 80% glycerol in
PBS. In control experiments, pre-immune serum from the same rabbit as the one
used to generate the primary antibody was used in place of the primary
antibody. In addition, control experiments in which the primary antibody was
excluded were also performed.
Preparation of antibodies to 5-HT1Hel and 5-HT7Hel
Antibodies to 5-HT1Hel and 5-HT7Hel were raised
against peptide derived from intracellular loop sequence that had a high
antigenicity. Peptide 1Hel (Residues 409423; YSRTREKLELKRERK) and
Peptide 7Hel (Residues 246261; YFKIWRVSSKIAKAEA) were prepared by
Washington Biotechnology (Baltimore, MD, USA). Peptides were synthesized,
coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits. Sera
were collected when the antibody gave a positive reaction against the antigen
at a titer of >100 000 in an ELISA.
Data analysis
Results are presented as means ± standard error
(S.E.M.) unless otherwise stated. The
significance of differences among groups in the calmodulin inhibitor
experiment was evaluated using analysis of variance (ANOVA) followed by a
Fisher's Protected Least Significant Difference (PLSD) test. A Student's
paired t-test was used to determine if 5-HT stimulated a significant
increase in CBF in pedal and dorsolateral ciliary cells, where the mean CBF
during the 2 min prior to 5-HT perfusion was compared with the mean CBF during
minutes 14 after the start of the 5-HT perfusion.
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Results |
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Structurally, pedal and dorsolateral ciliary cells appeared identical in
profile and contents. Cilia were arranged in an organized fashion and ranged
in number from 600 to 800 per cell (Fig.
2B,C). In sectional profile, pedal and dorsolateral cells were
long, thin, flattened cells with an elliptical nucleus, containing a large
nucleolus (Fig. 2D; see
Koss et al., 2003). Microvilli
surrounded the base of each cilium (Fig.
2B,C). The cilia possessed a typical (9x2)+2 arrangement of
microtubules (data not shown), and a basal foot arose from one side of the
basal body. A lengthy primary rootlet extended into the cell from the base of
the cilium, and an accessory rootlet arose from the opposite side of the basal
body; both rootlets were striated (Fig.
2D). Each ciliary cell contained many prominent mitochondria that
were localized beneath the ciliary basal bodies
(Fig. 2D). Granular endoplasmic
reticulum was abundant and tended to be concentrated towards the cell
periphery. Pedal and dorsolateral ciliary cells consistently displayed
extensive patches of cytoplasm that appeared to be devoid of organelles,
except for some small electron-translucent vesicles ranging in size from 0.03
µm to 0.06 µm in diameter (Fig.
2D). A previous study has revealed that these regions contain
vesicles that display 5-HT immunoreactivity
(Koss et al., 2003
).
In comparison with the cells of the pedal and dorsolateral ciliary bands,
SSCCs differed in profile and content. Apically, only 3550 cilia
emerged from the cell surface. The SSCC cilia displayed a more random
orientation and a greater length than the pedal or dorsolateral cilia
(Fig. 2E). Furthermore, SSCCs
were smaller, had a wedge or cuboidal shape and a centrally located nucleus.
The fine structure of individual cilia was similar to pedal and dorsolateral
cilia, including a (9x2)+2 arrangement of microtubules
(Fig. 2F, inset). However,
there were no accessory ciliary rootlets arising laterally from the basal body
(Fig. 2F). The cytoplasm of the
SSCCs contained less prominent mitochondria and an absence of
electron-translucent regions. Finally, the SSCCs had no basal cellular
extensions such as were found on innervated pedal and dorsolateral ciliary
cells, as previously described (Koss et
al., 2003). Thus, the different morphology and fine structure of
the SSCCs, in comparison to the pedal and dorsolateral ciliary cells, suggest
that ciliary subtypes may exhibit different physiological profiles.
Examination of unidentified ciliary cells in vitro
Earlier studies revealed that Ca2+ influx is required to
stimulate an increase in CBF in Helisoma cilia
(Christopher et al., 1996).
This prompted us to examine whether alterations in
[Ca2+]i act through the calcium-binding protein
calmodulin. A 10 min application of 100 µmol l1 5-HT, a
dose known to maximally stimulate Helisoma cilia
(Christopher et al., 1996
),
stimulated a significant increase in CBF to 125.3±4.6% of the control
(P<0.05, N=7; Fig.
3). Co-application of the calcium/calmodulin-dependent enzyme
inhibitor calmidazolium (Cal; 2 µmol l1) with 100 µmol
l1 5-HT blocked the stimulatory effect of 5-HT
(Fig. 3). Application of 2
µmol l1 Cal alone or the DMSO vehicle did not produce a
change in CBF (Fig. 3). These
data suggest that calmodulin mediates the cilio-excitatory action of 5-HT in
Helisoma ciliary cells.
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The necessity of Ca2+ influx in cilio-excitation
(Christopher et al., 1996),
taken together with the results of the calmodulin inhibitor experiment,
prompted us to examine whether 5-HT, the primary cilio-excitatory
neurotransmitter in Helisoma embryos, induces a change in
[Ca2+]i. Using fast microinjection, unidentified ciliary
cells in culture were loaded with fura-2 dextran. Whereas the calcium
ionophore ionomycin (10 µmol l1) caused an increase in
the 340/380 ratio in all cells examined, only four of eight cells responded to
100 µmol l1 5-HT with an increase in
[Ca2+]i (Fig.
4). This finding prompted the hypothesis that one or more of the
subpopulations of ciliary cells in mass-dissociated cultures does not respond
to 5-HT with a change in [Ca2+]i.
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Examination of identified ciliary cells in vitro
Both the calcium imaging results described above and previous in
vivo measurements of CBF (Kuang and
Goldberg, 2001) suggest that the different ciliary populations
display distinct physiological properties. To test this, we first examined the
effect of exogenous 5-HT on CBF in identified ciliary cells in culture. In
pedal ciliary cells, perfusion with 100 µmol l1 5-HT
produced a rapid, statistically significant increase in CBF
(Fig. 5A). The pedal ciliary
cells roughly doubled the rate of ciliary beating within 1 min of the start of
5-HT perfusion. This high CBF was maintained for 10 min, with only marginal
desensitization (Fig. 5A). A
5-min washout with HS produced a partial recovery in CBF, and a challenge with
10 µmol l1 ionomycin increased CBF to values similar to
those induced by 100 µmol l1 5-HT.
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To ensure that microinjection with fura dextran did not alter the physiological responses of these cells, the ciliary response to 5-HT and ionomycin was examined in pedal ciliary cells that had been loaded with fura dextran. These cells exhibited a nearly identical CBF response to 5-HT and ionomycin as did pedal ciliary cells that were not loaded with fura dextran (Fig. 5B). Perfusion with 100 µmol l1 5-HT produced a rapid, statistically significant increase in CBF, followed by a partial recovery in response to HS washout. Perfusion of 10 µmol l1 ionomycin produced an increase in CBF to approximately the same amplitude as 5-HT. The results of this experiment suggest that microinjection of fura dextran does not impair the cell's ability to respond to 5-HT or ionomycin with changes in CBF.
A similar effect was observed in dorsolateral ciliary cells, as 100 µmol
l1 5-HT produced a rapid, statistically significant increase
in the rate of ciliary beating within 1 min of 5-HT perfusion
(Fig. 5C). These observations
are consistent with in vivo results on the effect of 5-HT on
dorsolateral and pedal ciliary cells
(Kuang and Goldberg, 2001).
Washout with HS produced a partial recovery in CBF over 5 min, and 10 µmol
l1 ionomycin again produced an increase in CBF that was
similar to the 5-HT response. As with pedal cilia, ionomycin produced a change
in the ciliary beat mechanics in dorsolateral cilia, and in one of the cells
the ionophore treatment killed the cell (data not shown).
The SSCCs are small, cuboidal cells with longer cilia (Fig. 2E; Figs 9C, 10E) and different ciliary beat profiles compared with the pedal and dorsolateral ciliary cells. These cells exhibited higher basal rates of ciliary activity, an unstable ciliary beat profile and no discernible response to 100 µmol l1 5-HT (Fig. 5D). Furthermore, 10 µmol l1 ionomycin did not appear to produce a change in CBF (Fig. 5D). These data suggest that ciliary beating in SSCCs may be regulated differently from that in pedal and dorsolateral ciliary cells.
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To further examine the physiological differences in the subpopulations of Helisoma ciliary cells and to determine why only a percentage of mass-dissociated cells in culture respond to 5-HT with an increase in [Ca2+]i, we imaged intracellular Ca2+ in identified ciliary cells in vitro. For both pedal and dorsolateral ciliary cells, the average baseline [Ca2+]i was between 50 nmol l1 and 150 nmol l1. In pedal ciliary cells, two different types of Ca2+ responses were evident during perfusion with 100 µmol l1 5-HT. These included an initial peak, with an average increase of approximately 350 nmol l1, followed by a sustained rise in [Ca2+]i of 150 nmol l1 above baseline (N=5 cells; Fig. 6A) or a gradual increase in [Ca2+]i to 150 nmol l1 above baseline (N=5 cells; Fig. 6B). In both cases, perfusion with 10 µmol l1 ionomycin produced a relatively rapid increase in [Ca2+]i that was highly variable in amplitude, ranging from 300 nmol l1 to 1300 nmol l1 (Fig. 6A,B). A similar result was observed with the dorsolateral cells, as they exhibited either an initial peak followed by a plateau (N=4 cells; Fig. 7A) or a gradual increase in [Ca2+]i (N=6 cells; Fig. 7B). The magnitude of these 5-HT responses was also similar to that observed in pedal ciliary cells. Dorsolateral ciliary cells displayed a variable ionomycin-induced increase in [Ca2+]i ranging in amplitude from 250 nmol l1 to 600 nmol l1. In both pedal and dorsolateral cells, little or no recovery of [Ca2+]i was observed after washout of 5-HT over the time frame examined. These data indicate that 5-HT does elicit an increase in [Ca2+]i within pedal and dorsolateral ciliary cells, but with a slower time course than observed during the CBF measurements.
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Consistent with the CBF results, intracellular calcium measurements revealed large differences between the SSCCs and the other ciliary subtypes. In four of nine SSCCs examined, the resting [Ca2+]i was stable at approximately 100 nmol l1, 5-HT perfusion did not stimulate a notable change in [Ca2+]i, and 10 µmol l1 ionomycin produced a relatively small rise in [Ca2+]i (Fig. 8A). By contrast, five of nine SSCCs exhibited irregular spikes in [Ca2+]i, both in HS and during 5-HT perfusion (Fig. 8B). The timing, duration and amplitude of these events were highly variable depending on the cell. In most of these cells, 10 µmol l1 ionomycin only produced a transient increase in [Ca2+]i. These data reinforce the notion that the SSCCs display different physiological characteristics from the pedal and dorsolateral cells, including insensitivity to 5-HT.
|
Given that some of the ciliary subtypes responded to 5-HT with changes in
CBF and [Ca2+]i, we tested whether identified ciliary
cells express the two 5-HT receptors whose genes were recently cloned from
Helisoma trivolvis. Isolated ciliary tissues were exposed to
antibodies raised against the cloned 5-HT receptors, 5-HT1Hel and
5-HT7Hel (Mapara et al.,
2001). All pedal ciliary cells expressed 5-HT1Hel
immunoreactivity strongly over the apical surface of the cells, with weaker
immunoreactivity throughout the remainder of the cell (N=15 cells;
Fig. 9A). A majority of
dorsolateral ciliary cells also expressed 5-HT1Hel
immunoreactivity, with 76% of cells (N=25 cells) exhibiting strong
surface expression and weaker distribution throughout the cell
(Fig. 9B). In the SSCCs, 0% of
cells exhibited 5-HT1Hel immunoreactivity within the cell
(N=23 cells; Fig. 9C).
However, neighboring nonciliary cells in the surrounding epithelia were
consistently immunoreactive (Fig.
9C). Rabbit pre-immune serum, used in parallel controls to examine
specificity of the antibody staining, did not exhibit any immunoreactivity
(Fig. 9D).
5-HT7Hel immunoreactivity was more inconsistent than that of 5-HT1Hel for pedal and dorsolateral ciliary cells. 5-HT7Hel was expressed in only 40% of pedal cells examined (N=25 cells; Fig. 10A). In most of these cells, there was strong expression on the apical surface of the cell, with a small percentage displaying a punctate distribution on the basal surface (data not shown). The majority of pedal cells examined (60%) did not demonstrate any 5-HT7Hel expression (Fig. 10B). Similarly, only 46% of dorsolateral ciliary cells expressed 5-HT7Hel strongly on the apical surface and weakly throughout the remainder of the cell (N=24 cells; Fig. 10C), while the majority (54%) did not exhibit any 5-HT7Hel expression (Fig. 10D). Finally, there was no expression of 5-HT7Hel within any of the SSCCs, but there was expression in the surrounding epithelial cells (N=22 cells; Fig. 10E). Taken together, these data implicate the 5-HT1Hel receptors, and possibly 5-HT7Hel receptors, in the signal transduction pathway for 5-HT-induced cilio-excitation in both pedal and dorsolateral cells.
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Discussion |
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Mixed populations of Helisoma ciliary cells in culture
A wide variety of signal transduction elements have been shown to
participate in regulating ciliary beating in the array of organisms examined.
However, changes in [Ca2+]i appear to be a possible
universal mechanism in altering CBF. Ca2+ regulates ciliary
activity in invertebrate systems, such as ctenophore larvae
(Tamm and Terasaki, 1994),
Paramecium (Eckert,
1972
) and Mytilus edilus
(Murakami and Machemer, 1982
),
and in almost all vertebrate systems examined, including the salamander
Necturus maculosus (Murakami and
Eckert, 1971
), the frog esophagus
(Levin et al., 1997
), the
rabbit trachea (Lansley and Sanderson,
1999
) and human respiratory epithelium
(Di Benedetto et al., 1991
). It
appears that Ca2+ is either required to bind to a
Ca2+-binding protein at the level of the cilium or is necessary in
the activation of other signal transduction cascades that result in
phosphorylation of a target protein within the cilium. The
Ca2+-binding protein calmodulin has been shown in both
invertebrates (Nakaoka et al.,
1984
) and vertebrates (Di
Benedetto et al., 1991
) to participate in mediating
cilio-excitation. Likewise, in Helisoma ciliary cells, calmodulin
appears to be a necessary component in the cilio-excitatory response to 5-HT,
as the calcium/calmodulin-dependent enzyme inhibitor calmidazolium inhibited
this response in unidentified ciliary cells. As application of the inhibitor
alone had no effect on basal CBF, it appears that calmodulin is recruited in
the 5-HT response but does not contribute to basal ciliary beating. This is
consistent with experiments on tracheal ciliary cells from rabbit suggesting
that basal ciliary activity was independent of [Ca2+]i
(Ma et al., 2002
). This
profile of calmodulin activity may also provide clues about the NOS isoform
thought to be expressed in embryonic cilia
(Cole et al., 2002
). Since NO
has been demonstrated to have a constitutive cilio-excitatory action in
Helisoma ciliary cells (Doran et
al., 2003
), the ineffectiveness of calmidazolium on basal ciliary
beating suggests that embryonic ciliary cells contain a
Ca2+/calmodulin-independent isoform of NOS. Whether this isoform is
a unique member of the inducible (i) NOS family, or is a novel isoform,
remains to be determined.
Although it has been demonstrated that Ca2+ is required to
stimulate an increase in CBF in Helisoma ciliary cells (Christopher
et al., 1996,
1999
), direct imaging of
intracellular Ca2+ had not been previously performed because
embryonic Helisoma cells do not readily accept or retain the
acetoxymethyl ester indicators. In the present study, we loaded embryonic
tissue using pressure microinjection of fura-2 dextran. An interesting finding
from these initial Ca2+ imaging experiments on unidentified ciliary
cells was that half of those cells examined did not exhibit an increase in
Ca2+ in response to 5-HT. Previous experiments on unidentified
ciliary cells in culture revealed that a smaller percentage of cells did not
exhibit an increase in CBF in response to 5-HT
(Christopher et al., 1996
).
This finding, as well as results from CBF experiments in vivo
(Kuang and Goldberg, 2001
),
suggests that the observed variations in the CBF and Ca2+ responses
from cells in culture may be the result of population differences within the
mass-dissociated cells in culture. This conclusion was further supported by
the electron microscopy analysis of ciliary populations in the present study.
Thus, in some ciliary subtypes, 5-HT may stimulate ciliary beating without
producing a rise in intracellular Ca2+, whereas other subtypes may
show no response at all.
Innervated Helisoma ciliary cells
The pedal and dorsolateral ciliary bands on the Helisoma embryo
are innervated by the serotonergic ENC1s
(Kuang and Goldberg, 2001;
Koss et al., 2003
). These
postsynaptic ciliary cells exhibited the typical morphology exhibited by cells
with motile cilia (Sleigh,
1962
). This includes a (9x2)+2 microtubule arrangement and
an extensive primary and accessory rootlet structure in close proximity to
numerous mitochondria. These cells also exhibited regions of cytoplasm that
were electron translucent. In an earlier study, these regions were shown to
contain 5-HT-immunoreactive vesicles that were proposed to be products of
endocytotic 5-HT uptake by postsynaptic ciliary cells
(Koss et al., 2003
). Taken
together, these morphological data support the conclusion that the pedal and
dorsolateral ciliary cells have the necessary machinery to exhibit
physiological responses to a chemical regulator.
With the development of the novel cell isolation techniques described in
the present study, we gained the ability to examine the effects of 5-HT on
identified ciliary cells in culture. Both pedal and dorsolateral cilia respond
to 5-HT perfusion with a significant increase in CBF, which reaches a maximum
within 45 s. Thus, there appears to be a functional 5-HT receptor on the
surface of both these cell types. Prompted by previous evidence that
Ca2+ is required for the 5-HT-stimulated increases in CBF and the
fact that ionomycin was able to increase the rate of ciliary beating, we
imaged intracellular Ca2+ in response to exogenous 5-HT. Both pedal
and dorsolateral cells exhibited two different types of Ca2+
responses to 5-HT. Whereas the CBF consistently increased rapidly following
5-HT application, imaging experiments revealed that detectable changes in
Ca2+ lagged behind the changes in CBF. The peak and plateau
response is suggestive of the profile seen in other ciliary cells where there
is an initial release from intracellular Ca2+ stores followed by a
sustained Ca2+ influx (Salathe
et al., 1997; Korngreen and
Priel, 1994
). By contrast, the slower rising change in
[Ca2+]i is less typical. All of these results are
consistent with an early rise in calcium occurring in a restricted cellular
compartment, possibly immediately below the cilia
(Lansley and Sanderson, 1999
),
which is not detectable in our whole-cell analysis. Through diffusion and
Ca2+-buffering mechanisms, the localized early signal may be
transformed into the delayed peak and plateau signal or slow-rising signal
that we observed in whole cell measurements. An alternative, albeit less
likely, explanation is that the initial ciliary response to 5-HT is not
dependent on a rise in [Ca2+]i. We hope to distinguish
between these possibilities in a detailed spatial analysis of calcium signals
and a study on the interaction of Ca2+ and PKC
(Christopher et al., 1999
),
both of which are currently underway.
An interesting finding from this study is the slow decay of both the CBF
and [Ca2+]i following washout of the 5-HT. In contrast
to these experimentally induced responses, the ciliary responses to
endogenously released 5-HT in intact embryos are transient, producing periodic
increases in the embryo rotation (Kuang
and Goldberg, 2001; Cole et
al., 2002
; Diefenbach et al.,
1991
). Thus, maintained exposure to elevated concentrations of
5-HT causes long-term changes in ciliary activity, the function of which is
not fully understood. Similar maintained increases in CBF were observed after
prolonged laser stimulation of ENC1 in intact embryos
(Kuang and Goldberg, 2001
).
Furthermore, repeated exposure of embryos to environmental hypoxia caused a
facilitation of the embryonic rotation response, a form of plasticity that is
likely to be related to the slow response decay observed herein
(Kuang et al., 2002
). Perhaps
the slowly rising Ca2+ signal observed in this study functions
primarily to produce a maintained state of elevated CBF, rather than mediating
the initial increases in CBF observed during transient exposure to 5-HT.
The pedal and dorsolateral ciliary cells showed similar immunoreactivity to
the antibodies raised against the recently cloned Helisoma 5-HT
receptors. The 5-HT1Hel protein was strongly expressed on the
apical surface, with weaker expression throughout the other regions of the
cells. The surface expression may indicate the presence of 5-HT in the
intracapsular fluid that may contribute to the tonic rotation displayed by the
embryos. Pulsatile release of 5-HT from ENC1 is believed to be responsible for
the generation of periodic surges in rotation that are superimposed upon a
slow basal rate of spinning (Kuang and
Goldberg, 2001; Diefenbach et
al., 1991
). Thus, either a different 5-HT receptor or a lower
concentration of the same receptor is expressed on the basal surface of the
pedal and dorsolateral ciliary cells to mediate ENC1ciliary cell
communication. Since punctate 5-HT1Hel-like immunoreactivity was
sometimes observed at the basal ciliary surfaces, it seems likely that the
5-HT1Hel receptor is the primary cilio-excitatory receptor in
Helisoma embryos.
In contrast to the consistent ciliary expression of the 5-HT1Hel
receptor, the 5-HT7Hel protein was expressed in only a minority of
pedal and dorsolateral cells. There was either strong, specific expression
that resembled the 5-HT1Hel pattern or no expression at all. This
inconsistent expression, together with significant differences in the
molecular structure between the 5-HT1Hel-encoding and
5-HT7Hel-encoding genes (Mapara
et al., 2001) suggest that the 5-HT7Hel receptors play
a different role from the 5-HT1Hel receptors. Furthermore, the
appearance of the 5-HT7Hel immunoreactivity in only some cells may
suggest that this protein only begins to be expressed around stage E25, with a
more complete expression pattern occurring at later stages. In order to fully
investigate this hypothesis, more studies are required to examine embryos at
different stages of embryonic and postembryonic development.
Non-innervated Helisoma ciliary cells
In stage E25 Helisoma embryos, at least three groups of ciliary
cells are not directly innervated by ENC1 or other identified neurons: the
lateral cells of the dorsolateral bands, the SSCCs and the ciliary cells
lining the gut. Since the gut cells are not accessible to the microdissection
techniques introduced in the present study, their physiological
characteristics remain unclear.
The dorsolateral ciliary bands each contain four cells, with only the most
medial one in each band receiving innervation
(Koss et al., 2003). Despite
this arrangement, all dorsolateral band cells display similar 5-HT receptor
immunoreactivity and responsiveness to 5-HT. Thus, paracrine actions of 5-HT
and electrical or chemical signals passing through gap junctions
(Koss et al., 2003
) likely
contribute to the ENC1-induced stimulation of these non-innervated cells
(Kuang and Goldberg,
2001
).
Kuang and Goldberg (2001)
identified the SSCCs (previously referred to as isolated tufts of cilia) as a
third subtype of ciliary cells expressed on the surface of the embryo during
development. These non-innervated cells have a different morphology from
either the pedal or dorsolateral ciliary cells and do not respond to 5-HT
in vivo. While the SSCCs do exhibit the (9x2)+2 microtubular
arrangement indicative of motile cilia
(Sleigh, 1962
), these cells
differ from the pedal and dorsolateral cilia in their less organized ciliary
arrangement, a simpler rootlet structure, less prominent mitochondria and
absence of electron-translucent regions. The SSCCs are not innervated by ENC1
and do not show any anatomical evidence of neuriteciliary cell
apposition sites. These morphological differences support the findings that
the SSCCs are physiologically distinct from the pedal and dorsolateral ciliary
cells. The present study confirmed that 5-HT does not stimulate a change in
CBF in these cells in vitro. Thus, it is likely that these SSCCs and
the uncharacterized gut ciliary cells contributed to the populations of
non-responsive cells observed in mass-dissociated cultures
(Christopher et al., 1996
).
The SSCCs have been observed to exhibit two states of activity: a quiescent
phase, with slow ciliary beating and abbreviated ciliary beat strokes, and an
active beating phase, with very rapid ciliary beating and full ciliary beat
strokes (data not shown). Neither the pedal nor the dorsolateral ciliary cells
exhibited either of these states under basal conditions. The mechanics of the
ciliary beating in the SSCCs was also different from that exhibited by the
other two populations; the SSCCs beat in a flagellar fashion whereas the other
two subtypes exhibited more typical ciliary wave-like beating. This
observation is consistent with the finding that the SSCCs exhibited longer
cilia with a different rootlet structure. The Ca2+ profiles in
these cells also support the idea of two different states of activity, with
some cells demonstrating a relatively flat baseline with no response to 5-HT
and other cells showing an unstable baseline and intermittent calcium spikes,
both in the presence and absence of 5-HT. It may be that the fluctuations in
[Ca2+]i are responsible for maintaining the high rate of
ciliary beating demonstrated by these cells. Oscillations in
[Ca2+]i have been shown to regulate the CBF in rabbit
airway epithelium (Evans and Sanderson,
1999) and ovine tracheal epithelial cells
(Salathe and Bookman, 1995
).
Simultaneous CBF and Ca2+ imaging experiments need to be performed
to determine if the type of Ca2+ activity correlates to the ciliary
beat state of the cell. In some SSCCs examined, ionomycin perfusion produced a
small amplitude change in intracellular Ca2+ that was often
transient. This response to calcium ionophore further suggests that the SSCCs
regulate intracellular Ca2+ through different mechanisms than the
pedal and dorsolateral ciliary cells.
The absence of CBF and Ca2+ responses to 5-HT and
5-HT1Hel and 5-HT7Hel immunoreactivity suggests that the
SSCCs may be responsive to a non-neuronal cue. One possibility is that these
cells are activated by mechanical stimuli. This is suggested by the finding
that the start of saline perfusion triggered some SSCCs to switch from the
quiescent to active phase of ciliary beating, a result not observed in the
other ciliary cell types. A possible role for mechanosensitive ciliary cells
in the Helisoma embryo would be to keep the surface of the animal
free of debris during development. This may be especially important on the
posterior aspect of the embryo to ensure that particulate matter does not
interfere with the deposition of shell matrix. Mechanical stimulation of the
cell membrane has been identified to regulate ciliary activity in
Paramecium and the lateral gill cilia of Mytilus edilus
(Eckert, 1972;
Murakami and Takahashi, 1975
;
Murakami and Machemer, 1982
).
In these systems, mechanical stimuli generate a change in membrane potential
that leads to alterations in ion conductances, specifically enabling
Ca2+ entry, which in turn modifies ciliary activity. Alternatively,
the SSCCs may be immature at the embryonic stages examined in the present
study, differentiating at later stages under the influence of posterior
embryonic neurons, such as those described by Croll and Voronezhskaya
(1996
) in other gastropod
species. Although similar dopamine- and FMRFamide-containing neurons have yet
to be found in stage E25 Helisoma embryos
(Goldberg, 1995
), further
studies are necessary to determine whether the SSCCs eventually become
innervated in Helisoma embryos.
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