The role of Ca2+ in stimulated bioluminescence of the dinoflagellate Lingulodinium polyedrum
Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92037-0202, USA
* Author for correspondence (e-mail: pvondass{at}ucsd.edu)
Accepted 9 July 2002
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Summary |
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Key words: luminescence, calcium, dinoflagellate, flow, mechanotransduction, shear, Lingulodinium polyedrum
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Introduction |
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Bioluminescent dinoflagellates have long provided dramatic examples of
suspended cells that are remarkably sensitive to fluid motion. In response to
stirring, bubbling or direct mechanical stimulation, luminescent species
produce very bright flashes of light, emitting between 107 photons
cell-1 and 1010 photons cell-1 (depending on
species) within a 100 ms duration (Eckert,
1965; Latz et al.,
1987
; Latz and Lee,
1995
). The luminescent response is believed to serve an
anti-predation function (Esaias and Curl,
1972
; Fleisher and Case,
1995
; Mensinger and Case,
1992
). Luminescence is also stimulated by both laminar and
turbulent flow (Latz et al.,
1994
; Latz and Rohr,
1999
), and the shear sensitivity of the luminescent response is in
the same range as the shear sensitivity of intracellular signaling events in
attached mammalian cells (exhibiting a shear threshold of 100 s-1,
which is equal to a shear stress of 0.1 N m-2;
Papadaki and Eskin, 1997
).
Because the luminescent flash of a dinoflagellate is so readily observed under
a variety of flow conditions, it is of interest to know what cellular
processes it is reporting.
The dinoflagellate flash is governed by a flash-triggering action potential
(FTP) (Eckert, 1965;
Widder and Case, 1981
). The
FTP is unique both in that it occurs across the vacuole membrane rather than
the plasma membrane (Eckert and Sibaoka,
1968
) and in that it is believed to be mediated by protons
(Nawata and Sibaoka, 1979
).
The flux of protons from the acidic vacuole into the cytoplasm activates a
unique pH-dependent luciferase (Fogel and
Hastings, 1972
; Wilson and
Hastings, 1998
) that is packaged with the luciferin in cytoplasmic
invaginations of the vacuole membrane, termed scintillons
(Fogel et al., 1972
;
Nicolas et al., 1987
). In some
species, such as Lingulodinium polyedrum, a further pH-dependent
component is the luciferin-binding protein (LBP), which prevents oxidation of
luciferin under neutral to basic conditions but dissociates from the luciferin
under acidic conditions (Fogel and
Hastings, 1971
; Wilson and
Hastings, 1998
).
There is little understanding of the mechanotransduction pathway that
initiates the FTP; it is unknown how direct mechanical stimulation or fluid
forces, which may act at the cell surface, lead to a depolarization of the
vacuole membrane. Based on four pieces of evidence from experiments conducted
mostly with L. polyedrum, Hamman and Seliger
(1972;
1982
) proposed that the signal
transduction involved an influx of Ca2+ across the plasma membrane.
First, increasing [Ca] (total concentration of calcium, bound and free) in the
external medium (to
30 mmol l-1) strongly stimulates
luminescence, although other cations, such as KCl, NH4Cl and
LaCl3, are also stimulatory. Second, upon testing cells grown in
differing [Ca] (in the range of 1-10 mmol l-1), the maximum
luminescence intensity in response to mechanical stimulation is strongly
dependent on extracellular Ca, as is growth. Third, EGTA, a specific chelator
of Ca2+, inhibits mechanical stimulation of luminescence when added
shortly before testing. Finally, high concentrations of LaCl3 (1
mmol l-1), CoCl2 (6.5 mmol l-1) and
MnCl2 (10 mmol l-1), which have been reported to block
metazoan Ca2+ channels at lower concentrations
(Hille, 1992
), inhibit
mechanical stimulation of luminescence.
These earlier results (Hamman and Seliger,
1972;
1982
) do not yet conclusively
indicate the involvement of plasma membrane Ca2+ channels and
increases in intracellular Ca2+ in the mechanical stimulation of
flash production. First, high concentrations of extracellular CaCl2
were needed to stimulate luminescence, and other cations were potent stimuli
as well. KCl and NH4Cl were reported to stimulate luminescence at
concentrations below the minimum required for stimulation by CaCl2.
Thus, the observed stimulation of luminescence might merely result from the
disturbance of ion balance across two electrically active membranes; the
plasma membrane and the vacuole membrane. Second, in separate experiments,
LaCl3 and EGTA were each reported both to stimulate and to inhibit
luminescence at similar concentrations. Finally, only a longterm requirement
for extracellular Ca over the growth phase for mechanical stimulation of
luminescence was shown.
The present study investigates the role of Ca2+ in
flow-sensitive luminescence of L. polyedrum, the most well-studied of
the luminescent dinoflagellates. Two hypotheses were tested: (1) flow
activates plasma membrane Ca2+-permeable channels and (2) the
increase in Ca2+ acts on intracellular Ca2+ sensors to
depolarize the vacuole membrane, thereby triggering the cell to flash. The
following predictions would support these hypotheses. First, specifically
facilitating the entry of Ca2+ into the cell would trigger the
production of flashes similar to those produced in response to flow or
mechanical stimuli. Second, if Ca2+ acts on a
Ca2+-specific biochemical sensor such as calmodulin
then chelation of intracellular Ca2+ would inhibit mechanical
triggering of luminescence. By contrast, if the entering Ca2+ acts
as a voltage carrier, then chelation of intracellular Ca2+ would
have either no effect or would enhance mechanical stimulation of luminescence
by removing the inactivation of Ca2+-inactivated Ca2+
channels (Hille, 1992). Third,
luminescence produced by moderate levels of steady laminar flow would be
strongly and reversibly dependent on extracellular Ca2+. The
possible involvement of Ca2+ release from intracellular stores was
also tested using pharmacological treatments targeting these stores.
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Methods |
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Luminescence measurements
Luminescence measurements were made within spherical
integrating-light-collecting chambers with an inner diameter of 11.25 cm.
There was a 3.75 cm diameter detector port, a 3.1 cm diameter port for the
sample (large enough to accommodate the Couette flow chambers; see below) and
a 0.63 cm diameter port for the flow-chamber drive shaft. The total area
exposed to ports was kept at <5% of the inner surface area of the
integrating sphere for effective light integration. A baffle between the
sample and the detector port ensured that all light was collected by multiple
reflection off the inner surface for effective light integration from the
sample. The entire inner surface was coated with highly reflective
polyurethane enamel white paint (Sherwin-Williams, Cleveland, Ohio, USA).
Light emission was measured using photon-counting photomultiplier detectors (PMTs) (RCA no. 8575v2). The photomultiplier discriminator produced square wave (TTL) pulses in proportion to the number of photons detected. Pulses were counted in 50 ms or 25 ms time bins using Ortec (Oak Ridge, TN, USA) ACE-MCS multichannel scalars (MCS) mounted in an IBM-compatible PC. In experiments with Couette flow, a pulse from the servomotor controller triggered the start of an MCS record (4000 bins). To determine the linear dynamic range of the detectors, an A310 Accupulser pulse-train generator (World Precision Instruments, New Haven, CT, USA) was used to control the voltage and time of pulses of light generated by a light-emitting diode (LED) inserted in front of the PMTs. The relationship between voltage and detector signal was linear when the LED light was attenuated sufficiently. Preliminary tests with night-phase cells of L. polyedrum stimulated under different conditions were made to determine the combination of neutral density filter (1.0-2.0) and aperture (3.1-1.25 cm) required to decrease the amount of light illuminating the photocathode to prevent saturation of the detector. The detectors were frequently cross-calibrated for quantum emission against a Quantalum 2000 calibrated photometer (Zefaco, Gaithesbury, MD, USA) using a C14 phosphor.
On each day of testing, measurements of background (mostly dark noise from the photocathode) were recorded before and after testing of samples and were subtracted from the records of luminescence. Measured parameters from each record were obtained using custom data analysis software and included the integrated luminescence in the total record and the integrated luminescence and maximum luminescent intensity recorded in user-defined sections of the record. For measurements of spontaneous glow, MCS data records were converted into text files and imported into DeltaGraph (SPSS, Inc., Richmond, CA, USA), where the median luminescence per time bin was calculated. Unlike the mean luminescence, median luminescence will not be influenced by rare flash events and represents a measure of the low-intensity spontaneous glow emission produced by night-phase L. polyedrum.
Solutions
Hepes-buffered filtered seawater (HbFSW) contained glass-fiber (GF/F)
filtered seawater buffered to pH 7.8 with 5 mmol l-1 Hepes and
autoclaved. Artificial seawater (ASW) contained: 449 mmol l-1 NaCl,
25.5 mmol l-1 MgSO4, 22.9 mmol l-1
MgCl2, 9.2 mmol l-1 CaCl2, 9.0 mmol
l-1 KCl, 2 mmol l-1 NaHCO3, 5 mmol
l-1 Hepes, pH 8.0. In Ca-free ASW, CaCl2 was replaced
with excess MgCl2. In some experiments, Ca-free ASW was prepared
with 1 mmol l-1 EGTA to chelate any residual Ca2+
present. In those experiments, 1 mmol l-1 EGTA was also included in
the Careplete ASW used in control samples. 2xCa ASW was prepared by
doubling the normal amount of CaCl2 in exchange for
MgCl2 and increasing the concentration of Hepes to 25 mmol
l-1. KCl saline contained 600 mmol l-1 KCl and 5 mmol
l-1 Hepes, pH 7.8 NaCl saline contained 600 mmol l-1
NaCl and 5 mmol l-1 Hepes, pH 7.8. Stocks of ionomycin free acid
and ryanodine were prepared in dimethyl sulfoxide (DMSO) at concentrations of
5 mmol l-1 or 1 mmol l-1 and stored at -20°C. 5 mmol
l-1 stock solutions of
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester (BAPTA-AM) (Calbiochem, San Diego, CA, USA) were
prepared in DMSO containing 10% Pluronic F-127 (Sigma/Aldrich, St Louis, MO,
USA) with gentle heating under hot tap water and stored frozen at -20°C.
20 mmol l-1, 5 mmol l-1, 0.5 mmol l-1 and 0.1
mmol l-1 stocks of ammoniated ruthenium oxychloride (Ruthenium Red)
were prepared in H2O and used within 24 h. To prepare working stock
solutions of BAPTA-AM, 5 mmol l-1 BAPTA-AM was diluted in the ratio
of 1:5 in hot H2O and immediately diluted in the ratios of 1:100
(10 µmol l-1 BAPTA-AM) or 1:200 (5 µmol l-1
BAPTA-AM) in HbFSW. Preliminary experiments indicated that BAPTA-AM solutions
would precipitate and lose activity within 15-30 min unless prepared in 0.2
µm-filtered HbFSW. In all subsequent experiments, cell suspensions were
prepared with 0.2 µm-filtered HbFSW, and BAPTA-AM was diluted with 0.2
µm-filtered HbFSW. All reagents were obtained from Sigma/Aldrich (St Louis,
MO, USA) unless otherwise stated.
Chemical treatments
Chemical treatments to be tested for the ability to stimulate luminescence
were prepared as 2x or 3x working stocks. Ionomycin was diluted to
2x working concentration in HbFSW, keeping DMSO at 1%. The control
solutions for ionomycin in dose-response tests were HbFSW and 1% DMSO in
HbFSW. For tests on the role of extracellular Ca2+ in stimulation
by ionomycin, 1 mmol l-1 ionomycin stock was diluted to 10 µmol
l-1 in either Ca-free ASW or 2xCa ASW. The control solutions
used in these tests were 1% DMSO in Ca-free ASW and 2xCa ASW. For
stimulation by K+, KCl saline or NaCl was diluted in HbFSW. The
concentration of HbFSW in the added solution was always 66.7% or 83.3%,
keeping [Cl-] constant.
At the end of the day-phase, L. polyedrum was diluted to 200 cells ml-1 or 500 cells ml-1 in either HbFSW, ASW or Ca-free ASW, as indicated. 7 ml glass scintillation vials were filled with 0.5 ml of diluted cell suspension and placed in the dark at the beginning of the night phase. Testing occurred between 3 h and 7 h into the night phase. Samples were placed in the integrating sphere chamber, and 0.5 ml or 1.0 ml of stimulating solution was injected into the sample through a light-proof septum. The injection was either done by hand or, in some of the experiments involving stimulation by K+, using a syringe pump (Harvard Apparatus, Holliston, MA, USA) with a volume flow rate of 10 ml min-1. Mechanical stimulation was minimized by using bent injection needles to inject the solution against the side of the vial. Tests using dyes demonstrated that the force of the injection was sufficient to mix the injected solution with the cell suspension. Measured parameters included the integrated luminescence in the first 12 s and over a 200 s period during and after treatment addition.
Treatment with Ruthenium Red and ryanodine began either at the end of the day phase (by diluting 100x stocks into HbFSW or cell suspensions diluted in HbFSW) or, in some experiments, during the night phase. Treatment with BAPTA-AM always began 3-6 h into the night phase. After the treatment period, samples were either subjected to stirring or Couette flow for assessing mechanical sensitivity (see below) or tested for sensitivity to chemical stimuli.
Preparation of low-Ca and Ca-free cell suspensions
Even mild centrifugation triggered the formation of pellicle cysts, so
low-Ca and Ca-free cell suspensions were prepared by gentle filtration. 50 ml
volumes of cell culture were placed under a 10 µm or 20 µm Nitex filter
and gently diluted by addition of 200-250 ml of Ca-free ASW (with or without
EGTA, as indicated). 200-250 ml of fluid that passed through the filter (free
of cells) was then slowly removed, leaving the cells behind. This process was
repeated a total of 3-4 times; after the final volume of cells was returned to
50 ml, samples were taken for cell counting, and the cell suspension was
diluted in Ca-free ASW to the desired final cell concentration. When cells
were resuspended in Ca-free ASW without EGTA, the cell suspensions were
considered to be low-Ca rather than Ca-free, owing to a small amount of Ca
carried over with the cells. When 1 mmol l-1 EGTA was present, the
remaining free Ca2+ was chelated, and cell suspensions were
considered to be Ca-free.
Flow fields for mechanical stimulation of suspended cells
Both stirring and Couette flow were used to subject populations of
suspended cells to flow stimuli. It was possible to process large sample
numbers using stirring to screen different treatments for effects on
mechanical sensitivity. Also, while it was not possible to load the Couette
flow chambers (see below) without causing substantial mechanical stimulation
to the samples to be tested, the insertion of the stirrer into the
scintillation vials did not cause visible luminescence and, thus, no
pre-stimulation of luminescence occurred prior to testing. Use of the stirrer
therefore allowed mechanical stimulation to be tested almost immediately after
treatment addition when necessary. Unfortunately, stirring does not provide a
defined flow field so that the nature and level of the flow stimulus is not
known.
A curved metal rod operating at 2500 revs min-1 stirred the contents of a 7 ml glass scintillation vial containing the sample to be tested. The vial, screwed into the stirrer/motor mount, was placed inside the light-collection chamber. The timing of the start of the motor was controlled manually, approximately 13 s into each record of light emission. Measured parameters for stirring assays are shown in Table 1.
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Couette flow allowed testing of samples using quantified laminar shear. In
simple Couette flow, fluid contained in the gap between two concentric
cylinders is sheared by rotating the outer cylinder while keeping the inner
cylinder still, thus ensuring a stable flow field
(Taylor, 1936;
van Duuren, 1968
). Also,
non-swimming cells or cells that swim weakly can be gently kept in suspension
by rotating the outer cylinder prior to testing at a rate slow enough that the
shear stress in the gap is well below the threshold for stimulation of
luminescence. Several small-volume Couette devices were constructed with a
polished cast acrylic outer cylinder (inner radius: 12.7 mm) and a white
delrin inner cylinder (outer radius: 12.07 mm), yielding a gap width of 0.635
mm. The rotation of the outer cylinder was driven by a servomotor (SilverMax
QCI 17-3-E; QuickSilver Controls, Inc., Covina, CA, USA) controlled by an
IBM-compatible PC through preset commands in the program QuickControl
(QuickSilver Controls, Inc.). The time scale for diffusion of momentum across
the gap,
, can be estimated from
h2v-1, where h is the gap width
and v is the kinematic viscosity of seawater (1.047x10-6
m2 s-1 at 20°C). For the present chamber
configuration,
0.4 s, which is much greater than the 30 ms time scale
for production of a dinoflagellate flash in response to abrupt mechanical
stimulation (Eckert, 1965
;
Widder and Case, 1981
).
Therefore, the luminescent response should begin before the flow becomes fully
developed. Rapid acceleration of the outer cylinder would lead to transient
shear stresses that are much greater than the final steady shear stress near
the outer wall as momentum diffused across the gap. To avoid the development
of transient shear stresses significantly higher than the final shear stress,
the outer cylinder was accelerated to the final test velocity over 1 s (i.e.
slower than the rate of diffusion of momentum across the gap).
The time-dependent shear stress was calculated as follows. The assumption
was made that the inner and outer cylinder walls represented infinite parallel
plates, where the outer one accelerated at a constant rate and the inner one
stayed still, because it was not possible to solve the corresponding equations
in cylindrical coordinates. During the phase of the flow when the outer
cylinder is accelerating, the velocity u as a function of position
x from the inner cylinder towards the outer cylinder and time
t [u(x,t)] was modeled by solving the partial
differential equation:
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The typical flow profile involved a programmed 8 s delay between the start
of the PMT record and the servomotor. The outer cylinder was first accelerated
to a final velocity of 0.05 revs s-1 (equivalent to a shear stress
of 0.0065 N m-2) over 1 s, and the velocity was held constant for
49 s (time -50 s to 0 s). This warm-up rotation period served to mix any
non-swimming cells from off the wall of the outer cylinder and to lubricate
the chamber bearings with seawater to promote smooth acceleration. The steady
shear stress of 0.0065 N m-2 during the warm-up period was well
below the response threshold for this species
(Latz et al., 1994;
Latz and Rohr, 1999
). The
outer cylinder was then accelerated over 1 s to the test velocity (either 3.2
revs s-1=0.43 N m-2 or 4.8 revs s-1=0.65 N
m-2, as indicated), which was maintained for 19 s. Finally, the
outer cylinder was accelerated over 1 s to a final velocity of 8 revs
s-1 (shear stress=1.1 N m-2). Luminescence parameters
measured during the flow protocol are shown in
Table 1 and included: (1) rate
of spontaneous emission prior to flow, (2) mean integrated luminescence over
two 12 s periods during the sub-threshold warm-up phase, (3) integrated
luminescence over the first 12s of the test phase when shear stress was either
0.43 N m-2 or 0.65 N m-2, (4) time to reach 50%
cumulative luminescence during the test phase, representing the median
response time, and (5) the total shear-stimulated luminescence integrated over
the entire flow protocol. Because total shear-stimulated luminescence
generally paralleled the luminescence during the first 12 s of the test phase,
only the latter parameter is reported in this study.
Assay for total luminescent capacity
The total luminescent capacity (TLC), i.e. the chemical capacity of cells
to produce light, was assayed by acidification to directly activate luciferase
and release luciferin (Fogel and Hastings,
1972; Sullivan and Swift,
1995
). After stimulation by stirring or chemical addition, a 0.5-1
ml volume of 1 mol l-1 acetic acid, pH 4.7, was slowly added to
samples to gradually lower the pH to <5 while integrated luminescence was
measured over 200 s. TLC represents the sum of the integrated luminescence
over the record during which luminescence was stimulated (by stirring or
treatment addition) and the record during which luminescence was activated by
acidification.
Measurement of total Ca
Samples of low-Ca and Ca-free cell suspensions were centrifuged for 10-15
min at >500 g to remove cells. The total [Ca] in the
supernatant was measured using an Optima 3000 DV induced coupled plasma-atomic
emission spectrometer (ICP-AES; PerkinElmer, Shelton, CT, USA).
Statistical analysis
All values represent means ± S.D. Statistical tests included
Student's t-test or analysis of variance (ANOVA) followed by Fisher's
PLSD post-hoc test for pairwise comparisons (Statview, SAS Institute,
Inc.). Statistical significance was based on a P value of 0.05.
Results from experiments performed on separate days were pooled. For testing
of ratios, values were arcsine/squareroot-transformed. For testing of maximum
luminescent intensity normalized to total luminescent capacity, values were
ln-transformed. Calculations of e-folding times were based on least-squares
regressions of 6 s integrated mechanically sensitive luminescence
versus time.
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Results |
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To examine the role of Ca2+ in ionomycin stimulation, Ca-free cell suspensions were tested with 5 µmol l-1 ionomycin prepared in Ca-free ASW or in 2xCa ASW (to raise the final [Ca] to normal levels upon addition to the samples). 5 µmol l-1 ionomycin added with Ca rapidly stimulated sustained production of flashes. When added without Ca, ionomycin stimulation was less and occurred over longer time scales (Fig. 1C,D). The slow stimulation in the absence of extracellular Ca2+ might result from release of Ca2+ from intracellular stores.
Digitonin is a detergent that generally increases plasma membrane permeability without known selectivity among ions. At 2.5 µmol l-1 and 5 µmol l-1, digitonin stimulated luminescence with a similar dependence on extracellular Ca2+ as ionomycin (data not shown). This suggests that the triggering mechanism for luminescence is more sensitive to Ca2+ than to disruption in the balance of other ions across the plasma membrane. Stimulation by 25 µmol l-1 ionomycin or 12.5 µmol l-1 digitonin was much less dependent on extracellular Ca2+ (data not shown). High concentrations of digitonin or ionomycin might cause direct release of Ca2+ from intracellular stores, or disruption of ionic balance across the plasma membrane might stimulate flashing through some other mechanism.
After prolonged treatment with either ionomycin or digitonin, cells lost
motility and formed pellicle cysts, which is a response to stressful
conditions (Lewis and Hallett,
1997). Ionomycin treatment also induces encystment in the
dinoflagellate Alexandrium tamarense
(Tsim et al., 1997
). Pellicle
cysts are formed through ecdysis, a process in which a new plasma membrane is
formed and the old plasma membrane is discarded as thecal vesicles are
exocytosed (Netzel and Dürr,
1984
). Exocytosis is triggered by a rise in
[Ca2+]i (Dodge and
Rahamimoff, 1967
; Littleton et
al., 1994
; Thomas and
Elferink, 1998
), so this effect of ionomycin was expected.
Stimulation of luminescence by stirring and by Couette flow
Most of the luminescent response to stirring occurred within the first 200
ms (Fig. 2A). Continuous
stirring for 180 s typically stimulated 60-80% of the TLC, while nearly 90% of
the mechanically sensitive luminescence was released within the first 6 s.
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The strain of L. polyedrum used in this study responded mostly
during the developing phase of Couette flow, when the outer cylinder was being
accelerated (Fig. 2B,C). The
minimal response that occurred during the warm-up rotation period in untreated
samples was attributed to low-level vibrations that are sometimes produced by
the servomotor when operating at low speed. The threshold shear stress
required for stimulation was 0.05 N m-2, which is similar to the
previously reported threshold for a different strain of this species in a
larger Couette flow chamber (Latz et al.,
1994). Further characterization of the luminescent response of
different strains of L. polyedrum to developing Couette flow and
other developing flow fields, and a more complete description of the
developing Couette flow field used here, are described elsewhere
(von Dassow and Latz,
2002
).
Effects of the intracellular Ca2+ chelator BAPTA-AM
The intracellular Ca2+ chelator BAPTA-AM is membrane-permeant;
once in the cytosol, the AM ester is cleaved by intracellular esterases to
release the membrane impermeant Ca2+-specific chelator BAPTA
(Kao, 1994). Treatment with 5
µmol l-1 BAPTA-AM caused an exponential loss of mechanical
sensitivity with time when assayed by stirring (e-folding time=10.7 min)
(Fig. 3A). After 32 min (three
e-folding times), integrated luminescence stimulated in the first 6 s of
stirring (1st 6 s MSL) in BAPTA-AM-treated samples
(0.1±0.07x108 photons cell-1;
N=43) was significantly different (t=-16.8,
P<0.001) from that of the controls
(2.7±1.0x108 photons cell-1;
N=31). Integrated luminescence of 0.6±0.3x108
photons cell-1 (N=43) over the entire 200 s of stirring
(TMSL) in BAPTA-AM-treated samples was also significantly different
(t=-16.8, P<0.001) from the controls
(3.6±1.0x108 photons cell-1,
N=31). Mechanically sensitive luminescence of samples treated with
BAPTA-AM was also compared visually with paired control samples stimulated
together by a gentle shake. Control samples produced visual flashes, while
BAPTA-AM-treated samples produced no visible light.
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After 32 min, the total spontaneous luminescence, including spontaneous flashes and low-intensity glow, of BAPTA-AM-treated samples (1.2±0.7x104 photons cell-1 s-1; N=19) was not significantly different (t=0.078, P=0.94) from the controls (1.1±1.2x104 photons cell-1 s-1; N=16). However, BAPTA-AM treatment increased spontaneous glow emission (Fig. 3B). After 32 min, the spontaneous glow of BAPTA-AM-treated samples was 8.0±6.7x103 photons cell-1 s-1, while that of control samples was 1.4±1.9x103 photons cell-1 s-1 (t=3.76, P<0.01). The increase in spontaneous glow due to BAPTA-AM treatment may have been offset by a decrease in the production of spontaneous flashes.
To test the effect of BAPTA-AM treatment on the response to Couette flow during the night phase, small aliquots containing 500 cells were loaded into flow chambers containing either 5 µmol l-1 BAPTA-AM or a control solution. Prior to testing, samples were allowed to recover from handling for a minimum of 40 min in the dark. As described in the methods, following warm-up, cells were accelerated over 1 s to a final shear stress of 0.65 N m2, held for 20 s and, in most cases, then accelerated to a final shear stress of 1.1 N m2 and held for 20 s. A total of four BAPTA-AM-treated samples were tested in experiments performed on separate days and, in every case, the BAPTA-AM samples were completely unresponsive to the flow protocol (Fig. 3C). Integrated luminescence in the first 12 s, when the final shear stress was 0.65 N m2, was 2.2±2.0x105 photons cell-1 in BAPTA-AM-treated samples and 8.9±0.3x107 photons cell-1 in control samples; these values were significantly different (t=-5.9, P=0.0011). Loading of samples into the flow chamber probably resulted in depletion of mechanically sensitive luminescence. However, the control-treated samples run in parallel always displayed strong recovery of shear-sensitive luminescence over the same time period.
Other experiments investigated the effect of BAPTA-AM treatment on other
luminescent or physiological parameters. Addition of BAPTA-AM did not
stimulate more luminescence than the addition of control solutions and, after
treatment for 35-45 min, TLC (assayed by addition of acetic acid to directly
activate the luciferase; Fogel and
Hastings, 1972; Sullivan and
Swift, 1995
) of BAPTA-AM-treated samples
(3.3±0.5x108 photons cell-1, N=4)
was not significantly different (t=-0.74, P=0.49) from that
of control samples (3.5±0.5x108 photons
cell-1, N=4) (Fig.
4A). Ionomycin-stimulated luminescence integrated over 200 s in
BAPTA-AM-treated samples (4.9±0.7x108 photons
cell-1, N=4) was modestly increased compared with control
samples (3.7±0.6x108 photons cell-1,
N=4) (t=2.57; P=0.042)
(Fig. 4B); similar results were
obtained for digitonin treatment (data not shown). Presumably, the rise of
[Ca2+]i overwhelmed buffering by intracellular BAPTA.
The modest increase in 200 s-integrated ionomycin-sensitive luminescence in
BAPTA-AM-treated samples may be due to protection of luminescent capacity from
mechanical stimulation during handling. The time to 50% cumulative
luminescence of BAPTA-AM-treated samples (23.7±1.1 s; N=4) was
greater than control samples (18.4±0.8 s; N=4)
(t=7.9; P=0.0002). The delay in the response of
BAPTA-AM-treated samples compared with control samples might be partly
attributed to the time required for the ionomycin-induced increase of
[Ca2+]i to overwhelm buffering by cytosolic BAPTA and
partly attributed to the lack of mechanical stimulation upon injection in
BAPTA-AM-treated samples. Treatment with 5µmol l-1 BAPTA-AM did
not induce ecdysis and there was no trend observed for an effect on cell
motility. Overall, these results suggest that BAPTA-AM treatment affects
mechanically sensitive luminescence via the pathway leading from the
mechanoreceptor to elements regulating vacuole membrane potential.
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Effects of decreasing extracellular Ca2+
Owing to the motility of L. polyedrum, its tendency to form
pellicle cysts upon rigorous handling (e.g. centrifugation or prolonged
mechanical stress) and the circadian rhythm of mechanically sensitive
luminescence, it is difficult to remove extracellular Ca2+ from the
medium surrounding L. polyedrum without adversely affecting cell
physiology or exhausting luminescent capacity. L. polyedrum does not
grow in ASW made with 1 mmol l-1 CaCl2
(Hamman and Seliger, 1982
),
although it grows well in Ca-replete ASW (P. von Dassow and M. I. Latz,
unpublished results). Therefore, experiments were performed in which low-Ca
cell suspensions were prepared at the end of the day phase and allowed to
recover in Ca-free ASW or Ca-replete ASW inside the test chambers for the
first 3-5 h of the night phase prior to testing. Parallel experiments were
conducted with samples prepared for testing by Couette flow and by stirring.
Based on ICP-AES analysis of samples prepared in Ca-free ASW, the final
extracellular [Ca] after testing was 20.0±12.5 µmol l-1
in the Couette flow experiments and 3.8±3.3 µmol l-1 in
the stirring experiments; thus, these are considered to be low-Ca rather than
Ca-free conditions. A separate set of experiments was performed using samples
prepared in Ca-free ASW that contained the Ca2+-specific chelator
EGTA at a concentration of 1 mmol l-1. In these experiments, the
final [Ca] was 19.9±2.9 µmol l-1 in Couetteflow samples
and 14.3±2.5 µmol l-1 in stirred samples. The presence of
1 mmol l-1 EGTA effectively eliminated remaining free
Ca2+, so these are considered to be Ca-free conditions. 1 mmol
l-1 EGTA was also included in the corresponding Careplete condition
for these experiments, as a control for other possible effects of EGTA, as it
was not sufficient to chelate the majority of extracellular [Ca2+]
in Ca-replete ASW ([Ca]=9.2 mmol l-1).
Under both low-Ca and Ca-free conditions, the response to the test shear stress of 0.43 N m-2 for the Couette flow protocol was about half that of the corresponding Ca-replete condition (without or with EGTA, respectively), although the difference was only significant between low-Ca and Ca-replete conditions (without EGTA) (Table 2). Curiously, the amount of luminescence stimulated during the warm-up phase of the flow protocol, when the shear stress (0.0065 N m-2) was well below the known threshold for this species, was modestly increased under both low-Ca and Ca-free conditions compared with the corresponding Ca-replete condition, although this increase was not significant (Table 2). The increased luminescence during the warm-up period for low-Ca or Ca-free conditions was due to an increase in spontaneous luminescence and also a decrease in cell motility, which caused cells to settle on the outer cylinder wall where they were subject to stimulation by vibrations transmitted from the motor to the outer cylinder at very low speeds.
|
Stirring experiments tested the reversibility of the dependence of stimulated luminescence on extracellular Ca2+, because Ca2+ could be replaced by gently adding 2xCa ASW to low-Ca or Ca-free cell suspensions. TLC was not significantly decreased in low-Ca or Ca-free conditions compared with the corresponding Ca-replete condition (Table 3). Mechanically sensitive luminescence, assayed by three parameters normalized to the TLC of each sample, decreased under both low-Ca and Ca-free conditions (Table 3). However, approximately 40-67% of luminescent capacity was mechanically sensitive even under Ca-free conditions. The partial dependence on extracellular Ca2+ was not immediately reversible; in fact, replacement of Ca2+ decreased all three parameters of mechanically sensitive luminescence in Ca-free samples (Table 3). This decrease was probably not due to stimulation of mechanically sensitive luminescence during replacement of Ca2+, as previous experiments showed that adding Ca to Ca-free cell suspensions did not stimulate luminescence (Fig. 1D) and the final TLC was not significantly decreased after Ca2+ replacement (Table 3). The effect of Ca2+ replacement on mechanical sensitivity was opposite to that for ionomycin in Ca-free cell suspensions, where replacement of Ca2+ caused an immediate recovery of sensitivity, as discussed above.
|
Mechanically sensitive luminescence of low-Ca and Ca-free cell suspensions was also compared visually with paired samples of the corresponding Ca-replete condition during stimulation together by a gentle shake. Unlike the results observed with BAPTA-AM treatment, both low-Ca and Ca-free cell suspensions produced visible flashes when stimulated mechanically and were usually not noticeably different from the corresponding Ca-replete condition.
Effect of inhibitors affecting intracellular Ca2+
stores
Ruthenium Red and ryanodine block release of Ca2+ from
intracellular stores in metazoan tissues
(Zucchi and Ronca-Testoni,
1997). At
5 µmol l-1, Ruthenium Red decreased
mechanical sensitivity of luminescence
(Fig. 5A,B). Ruthenium Red
significantly affected TLC (Fig.
5A; F=22.1, P<0.0001) and motility
(Fig. 5C; F=58.5;
P<0.0001). However, samples treated with Ruthenium Red were
inhibited from undergoing ecdysis, as seen from the lack of empty theca
(Fig. 5C; F=11.4,
P<0.0001). The effect on motility was significant at
5 µmol
l-1 Ruthenium Red, and the effect on empty theca was significant at
50 µmol l-1 Ruthenium Red (Fisher's PLSD post-hoc
test, P<0.05). Empty theca were attributed to ecdysis rather than
mortality, as the concentration of pigmented cells remained the same during
the experiment. Despite the decrease in TLC, 84±7% of the luminescent
capacity stimulated by ionomycin after treatment with 50 µmol
l-1 Ruthenium Red (N=3) was similar to 82±5% of the
luminescent capacity stimulated in controls (N=4) (t=0.48,
P=0.65). Likewise, solutions of high [K+] stimulated
Ruthenium-Red-treated and control-treated samples equivalently (see below).
Ruthenium Red, which stains cells red, may attenuate some of the light
emission from cells, resulting in an apparent decrease in luminescent capacity
of Ruthenium-Red-treated cells.
|
Because of the apparent reduction in TLC, mechanically sensitive
luminescent parameters were normalized to TLC for statistical analysis. There
was a significant effect on TMSL for 50 µmol l-1 Ruthenium
Red (one-way ANOVA, F=15.5, P<0.0001; Fisher's PLSD
post-hoc for 50 µmol l-1 and 200 µmol l-1
Ruthenium Red compared with control, P=0.0003 and <0.0001,
respectively). Ruthenium Red had a significant effect on 1st 6 s MSL
normalized to TLC (F=99.8, P<0.0001) and maximum
intensity of luminescence (Max I; F=94.6, P<0.0001). The
difference was significant for
5 µmol l-1 Ruthenium Red
(Fisher's PLSD post-hoc test, P<0.05). Ruthenium Red
acted slowly, and effects on luminescence were not observed until
4 h
after treatment addition. Inhibition was observed both when treatment was
added at the end of the day phase and when treatment was added 3 h into the
night phase (data not shown), indicating that Ruthenium Red was probably not
acting by affecting circadian regulated changes in mechanical sensitivity. The
effects of Ruthenium Red on the response to Couette flow
(Fig. 5D) were similar to the
effects on the response to stirring, although the effect on spontaneous
luminescence was not determined. 1.25 µmol l-1, 25 µmol
l-1 and 50 µmol l-1 ryanodine had no effect on any
parameter of mechanically sensitive luminescence when assayed by stirring
(data not shown).
Stimulation by increasing [K+]
Increasing [K+] in the medium was a rapid and potent trigger of
luminescent flashing (Fig. 6).
Because much of the response to K+ occurred during the injection of
the solution into the sample, especially at high [K+], the response
was characterized relative to the [K+] in the added solution rather
than the final [K+] in the sample. With this in mind, the
EC50 for stimulation by [K+] was 25-30 mmol
l-1 (Fig. 6B).
|
To examine the role of cytosolic Ca2+ in K+-sensitive luminescence, night-phase cell suspensions were first treated for 40-50 min with 5 µmol l-1 BAPTA-AM and then stimulated by high or low [K+] solutions. Treatment with BAPTA-AM almost completely abolished K+-sensitive luminescence (Fig. 7A). Results of a two-way ANOVA showed that the effect of BAPTA-AM treatment was significant (F=193, P<0.0001), the effect of [K+] was significant (F=31, P<0.0001) and the interaction between BAPTA-AM treatment and [K+] was also significant (F=24.8, P<0.0001). Inhibition by BAPTA-AM was significant at all levels of KCl tested (Fisher's PLSD post-hoc test, P<0.001). However, while BAPTA-AM-treated samples responded much less than control-treated samples, there was evidence that BAPTA-AM samples were still affected by increasing [K+]. When KCl saline (diluted in HbFSW) was added to BAPTA-AM-treated samples, the glow intensity was substantially increased although no flashes occurred. This effect was not seen when NaCl saline (diluted in HbFSW) was added (data not shown). Integrated luminescence of BAPTA-AM-treated samples in response to high [K+] was greater than in response to low [K+] (Fisher's PLSD post-hoc test, P<0.05).
|
The dependence of K+-sensitive luminescence on extracellular Ca2+ was tested by measuring the response of Ca-free cell suspensions (prepared at the end of the day phase) to K+ stimulation. Low [K+] and high [K+] solutions were prepared in Ca-free ASW or in 2xCa ASW, both containing 1 mmol l-1 EGTA, and added to Ca-free suspensions during the night phase (Fig. 7B). Results of a two-way ANOVA showed that the effect of extracellular [Ca2+] on 200 s integrated luminescence was significant (F=22.3, P=0.0002), the effect of [K+] on 200 s integrated luminescence was significant (F=130, P<0.0001), and the interaction between extracellular [Ca2+] and [K+] was also significant (F=28.5, P<0.0001). Integrated luminescence over 200 s stimulated by high [K+] in the absence of extracellular [Ca2+] was slightly less than half that in the presence of extracellular [Ca2+] (Fisher's PLSD post-hoc test, split by [K+]; P<0.0001) but was still much greater than stimulation by low [K+] (P=0.0006). These results suggest that K+-induced luminescence might rely, in part, on intracellular Ca2+ stores.
To further examine the possible role of intracellular Ca2+ stores in K+-sensitive luminescence, samples treated for 4-6 h with 50 µmol l-1 Ruthenium Red were stimulated with solutions containing a range of [K+] (Fig. 8). Results of a two-way ANOVA showed that the effect of Ruthenium Red treatment was significant (F=20.2, P<0.0001), the effect of [K+] was significant (F=102, P<0.0001), and the interaction between Ruthenium Red treatment and [K+] was significant (F=6.30, P=0.001). The response of samples treated with Ruthenium Red to moderately increased [K+] was less than that of controls (when normalized to TLC; Fisher's PLSD post-hoc test, split by [K+], P<0.01), but the response of Ruthenium-Red-treated samples to the highest [K+] was slightly increased relative to control samples (Fisher's PLSD post-hoc test, split by [K+], P=0.033).
|
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Discussion |
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In the present study, low to moderate concentrations of calcium ionophores rapidly stimulated luminescent flashing with a strong dependence on extracellular Ca2+. At low concentrations, digitonin also stimulated flash production that was dependent on extracellular Ca2+. At higher concentrations, stimulation of flashing by both ionophores and digitonin was not strongly dependent on extracellular Ca2+. This may be explained in part from the ability of these reagents to release Ca2+ from intracellular stores. Alternatively, the vacuole membrane might be sensitive to general disruption of ionic balance across the plasma membrane. However, the results clearly support the hypothesis that an increase in cytosolic Ca2+ resulting from Ca2+ entry is a potent stimulus for flash production.
Treatment with BAPTA-AM, which accumulates in the cell as the Ca2+-specific chelator BAPTA after cleavage by cytoplasmic esterase activity, strongly inhibited mechanical stimulation of luminescence. BAPTA-AM did not affect the luminescent chemistry itself, as samples treated with BAPTA-AM and control-treated samples produced equivalent amounts of light when acidified to directly activate the luciferase. Also, BAPTA-AM treatment did not appear to decrease the ability of the vacuole membrane to produce the FTP, because BAPTA-AM-treated samples could produce vigorous flashing upon stimulation with ionomycin or digitonin. Thus, the target of BAPTA-AM treatment appears to lie between the mechanosensor and generation of the FTP.
An increase in cytosolic Ca2+ can occur via several mechanisms.
In mechanosensory cells such as hair cells, Ca2+ entry through
stretch-activated cation channels in the plasma membrane is considered to be
the source of increased [Ca2+]i upon mechanical
stimulation (Lumpkin and Hudspeth,
1995). In contrast, mechanically induced
[Ca2+]i increases in plants appear to result from
release of intracellular Ca2+ stores
(Haley et al., 1995
).
Removing extracellular Ca2+ from suspended L. polyedrum proved to be surprisingly difficult. Cells lost motility and underwent ecdysis after brief and gentle centrifugation. The process of rinsing cells to replace the extracellular medium with Ca2+-free ASW was also stressful to L. polyedrum. Recovery of motility after this treatment was dependent on extracellular Ca2+, and mechanical sensitivity of rinsed cells was much lower than that of non-rinsed cells, regardless of whether extracellular Ca2+ was present. Luminescent capacity in the night phase following the rinsing procedure was slightly decreased in samples resuspended in Ca-free rather than Ca-replete ASW. However, the mechanical sensitivity of rinsed cells appeared to be much lower under both Ca-free and Ca-replete conditions. These observations highlight the difficulty in conducting certain apparently simple experiments using an extremely mechanically sensitive cell type.
There was a partial and irreversible dependence of mechanically sensitive luminescence on extracellular Ca2+. Removal of extracellular Ca2+ decreased the response elicited by stirring (an unquantifiable but high stimulus) and Couette flow (moderate, defined fluid forces) compared with that of control samples prepared in the same manner but using Ca2+-replete ASW. However, even when extracellular Ca2+ was decreased to very low levels and chelation by EGTA lowered free Ca2+ to essentially zero, a substantial level of mechanically sensitive luminescence remained. The irreversibility of the observed dependence of mechanically sensitive luminescence on extracellular Ca2+ contrasts with the rapidly reversible dependence of ionomycin-sensitive luminescence on extracellular Ca2+ and is inconsistent with the hypothesis that influx of extracellular Ca2+ is required for mechanical stimulation of flash production. Therefore, mechanical sensitivity appears to be, at most, only partially dependent on the influx of extracellular Ca2+, and intracellular Ca2+ stores may be important.
The possible involvement of intracellular Ca2+ stores was also
supported by the strong inhibition by Ruthenium Red, which inhibits release of
Ca2+ from intracellular stores in metazoan tissues such as skeletal
muscle (Zucchi and Ronca-Testoni,
1997). The slow time course of inhibition is consistent with it
acting on a site inside the cell. Ruthernium Red also caused a loss of
motility and a modest decrease in TLC; however, the majority of the effect on
mechanical sensitivity appears to be due to a decoupling of the mechanosensor
from the effector system, because Ruthenium-Red-treated samples still
responded to ionomycin. Ryanodine had no apparent effect on the response to
stirring at concentrations as high as 50 µmol l-1. However,
ryanodine does not affect intracellular Ca2+ dynamics in the
ciliate Paramecium tetraurelia
(Plattner and Klauke, 2001
)
and even muscle cells of certain animals are insensitive to ryanodine,
although they still appear to utilize intracellular Ca2+ stores in
excitation-contraction coupling (Lin and
Spencer, 2001
).
As reported by Hamman and Seliger
(1982), increasing
[K+] is a more potent stimulus than increasing [Ca2+]
when added to the extracellular medium. In this study, increasing
[K+] stimulated flash production very rapidly with an
EC50 of about 25 mmol l-1, which is in the range of
reported total intracellular [K] in night-phase L. polyedrum
(Sweeney, 1974
). Stimulation
by [K+] requires an increase in [Ca2+]i, as
stimulation by K+ was very strongly inhibited by pre-treatment with
BAPTA-AM. At present, it is not clear whether stimulation by K+
results from depolarization of the plasma membrane (which may result in
Ca2+ entry through voltage-gated channels) or from inhibition of
the K+-dependent sodiumcalcium exchanger, an important
Ca-extrusion mechanism found in many cell types
(Blaustein and Lederer, 1999
).
The observation that K+ stimulated an increase in low-intensity
spontaneous glow without stimulating flashes in BAPTA-AM-treated samples has
two possible explanations. First, the K+-induced rise in
[Ca2+]i may still be sufficient to trigger a flux of
H+ across the vacuole membrane that is strong enough to modestly
increase the activity of the luciferase but is not sufficient to cross the
threshold for triggering of the flash-triggering vacuolar action potential. It
might also be that the K+-induced rise in
[Ca2+]i replaced H+ from intracellular BAPTA
accumulated in the cell, and the resulting drop in cytoplasmic pH moderately
increased the activity of the luciferase. K+ stimulation could
occur in the absence of extracellular free Ca2+. Stimulation by
moderately increased [K+] was inhibited by Ruthenium Red, but
stimulation by high [K+] was not. This suggests that, at low
concentrations, K+ stimulation involves the release of
intracellular Ca2+ stores but that, at higher concentrations,
K+-induced Ca2+ entry is sufficient when intracellular
Ca2+ release is inhibited.
Current attempts to demonstrate a mechanically induced rise in
[Ca2+] have had very limited success. Three difficulties were
encountered. First, while L. polyedrum cells take up AM
(acetoxymethyl ester) forms of Ca2+-sensitive dyes, the dye tends
to be compartmentalized and/or expelled from the cells upon attempts to rinse
the cells to remove extracellular dye. Second, a good system for observing a
dilute cell suspension under still and flow conditions has not yet been
developed. Finally, Ca2+-sensitive dyes are also pH sensitive
(Eberhard and Erne, 1991;
Lattanzio and Bartschat,
1991
); the FTP results in a transient acidification of the
cytoplasm that can dramatically decrease the Ca2+ fluorescent
signal. Attempts to simply stir a stained cell suspension using magnetic stir
bars in a standard spectrofluorometer often yielded a transient apparent
increase in fluorescence at the start of stirring. The same response was also
seen in subsamples of stained suspensions that were tested with the addition
of ionophore or digitonin. Similar changes were rarely seen in control,
unstained samples prepared and tested in parallel. However, because of the
difficulty in repeating these experiments and in ruling out artifacts (such as
from resuspension of non-swimming cells or light-scattering effects due to the
formation of a vortex as stirring begins), these results must be considered
preliminary.
The results of this study are consistent with the hypothesis that the
mechanosensor initiates the flash-triggering action potential through a rise
in cytosolic Ca2+. The partial dependence on extracellular
Ca2+ and the inhibitory effect of Ruthenium Red both suggest that
mechanically sensitive luminescence also involves activation of intracellular
Ca2+ stores. The lack of strong dependence on extracellular
Ca2+ might also be explained if the site of Ca2+ entry
was in the pusule, a tubular or sac-like invagination of the plasma membrane
beneath the thecal vesicles found in dinoflagellates
(Dodge and Greuet, 1987;
Schmitter, 1971
). Washing with
Ca-free ASW might not have completely removed Ca2+ from this
organelle. There is currently no evidence that the pusule is involved in
mechanotransduction, but the possibility should be considered because the
pusule appears to be a unique cellular location where the plasma and vacuole
membranes are closely associated (Dodge and
Greuet, 1987
).
Alternatively, mechanotransduction in dinoflagellates may involve
second-messenger-mediated release of intracellular Ca2+ stores or
it may utilize some form of direct coupling between plasma membrane
stimulation and intracellular Ca2+ release. In skeletal muscle, the
dihydropyridine receptor (a Ca2+ channel) in the plasma membrane is
physically coupled to Ca2+ release through the ryanodine receptor
in the sarcoplasmic reticulum membrane
(Franzini-Armstrong, 1999;
Lamb, 2000
;
Melzer et al., 1995
). A
similar mechanism has been proposed to explain mechanically induced
excitation-contraction coupling in the ciliate Vorticella
(Katoh and Kikuyama, 1997
;
Katoh and Naitoh, 1994
).
Likewise, chemically induced trichocyst discharge in the ciliate P.
tetraurelia is mediated by a rise in [Ca2+]i,
caused first by a rapid Ca2+ release from alveolar (cortical)
vesicles (sites of Ca2+ storage;
Erxleben et al., 1997
) and then
by an influx of extracellular Ca2+
(Plattner and Klauke, 2001
).
In this well-studied system, the initial release of Ca2+ from
alveolar vesicles is thought to be mediated by some direct coupling to a
plasma membrane receptor, although direct ultrastructural homologs to the
dihydropyridine receptor/ryanodine receptor complexes of vertebrate skeletal
muscle have not been found in P. tetraurelia
(Plattner and Klauke, 2001
).
The alveolar vesicles enclosing the thecal plates in dinoflagellates are
likely to be homologous to the alveolar vesicles of ciliates, as these two
groups are both members of the Alveolates
(Kumar and Rzhetsky, 1996
);
thus, the thecal vesicles might serve as sites of Ca2+ storage in
dinoflagellates. If the mechanosensory elements involved in dinoflagellate
mechanotransduction reside on the cell surface, the signal must be rapidly
transduced past tightly joined alveolar vesicles to reach the vacuole
membrane. Perhaps these vesicles might actually serve to mediate this
communication. A mechanism involving direct coupling between the plasma
membrane and Ca2+ release in dinoflagellates might explain the
sensitivity to stimulation by K+ and the partial dependence of
mechanical or K+ stimulation on extracellular Ca2+.
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Acknowledgments |
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