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Laboratory of Physiology, KULeuven, B-3000 Leuven, Belgium
1Correspondence: Bernard Himpens, Laboratory of Physiology, Herestraat 49, KULeuven, B-3000 Leuven, Belgium. E-mail: Bernard.Himpens{at}med.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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Key Words: RCS rats • gap junctions • intercellular communication • protein phosphorylation • calcium wave • confocal imaging • nucleus • cytoplasm • nucleo-cytoplasmic gradient • cytoskeleton
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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). In addition to playing a major role in
the bi-directional transport of retinal between photoreceptors and
choroid, they exert phagocytotic activity on the photoreceptor outer
segments. This cell layer also transports solutes and regulates the
water movement between the choroid and the subretinal space (SRS),
thereby regulating its volume. This function is potentially disturbed
by the changes in fluid distribution in microgravity. Changes in
cellular volume might also be modulated by gravity and could play a
role in vision. For example, light flashes in cosmonauts have been
observed and attributed, with little proof, to heavy ionized cosmic
particles. RPE cells also maintain the microenvironment of
photoreceptors during light and dark adaptation (2)
.They
stabilize, for example, [Ca2+]o (free
extracellular Ca2+ concentration) of the SRS and they also
play a role in regulating [Ca2+]i (free
intracellular Ca2+ concentration) in the photoreceptors.
During light and dark adaptation, the RPE cells and SRS undergo
[K+]o-dependent volume changes. Such volume
changes deform the plasma membrane (3)
.
Little was known regarding the signal transduction pathways in RPE
cells, in spite of their important physiological functions
(3)
. We therefore wanted to obtain more information
regarding some physiological and pathological aspects of the intra- and
intercellular signal transduction pathways in RPE cells. The purpose
was to investigate the mechanism of Ca2+ signaling in rat
LE-RPE cells during mechanical stimulation, an experimental model of
localized membrane deformations in single cells. Mechanical stimulation
can be used to study the intracellular propagation of the
Ca2+ wave and to investigate cell-to-cell communication
after stimulation of an individual cell, as extensively reviewed by
Sanderson et al. (4)
. Furthermore, we wanted to study the
intercellular communication between RPE cells in pathophysiological
conditions by investigating the properties of the intercellular
[Ca2+]i-wave propagation after mechanical
stimulation (4)
and by direct measurement of the gap
junction-mediated intercellular communication using the fluorescence
recovery after photobleaching (FRAP) technique. The investigations were
performed on cells grown in primary cultures.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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, 6
). Treatment of animals conformed to the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals. After enucleation, the RPE cell layer was isolated and
cultured as described (3, 5, 6). Experiments were performed on
subconfluent cells in 3- to 7-day-old primary cultures.
Cells were loaded with the Ca2+-indicator Fluo-3. The
procedure for loading and analysis of the Ca2+ data and for
the fluorescence recovery after photobleaching experiments has been
described before (3, 5, 6). The mechanical stimulation of single LE-RPE
cells consisted of a brief deformation of the cell with a glass
micropipette (tip diameter <1 µm) mounted on a vertical
micro-injection system (3)
. The micropipette was
intermittently lowered, thereby briefly touching the plasma membrane of
the cell of interest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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, 5
).
This [Ca2+]i rise originated at the point of
pressure contact and progressed throughout the mechanically stimulated
cell (Fig. 1
).If the surrounding cells had made contact with the central cell, the
[Ca2+]i rise also occurred in the two to
three cell layers surrounding this mechanically stimulated cell. The
amplitude and the rate of rise of the [Ca2+]i
transient in the neighboring cells declined as a function of the number
of the cell layer away from the central cell (Fig. 1)
(3)
.
The time delay between the application of the stimulus and the onset of
the [Ca2+]i rise increased as a function of
distance away from the central cell.
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Intracellular Ca2+ changes after mechanical stimulation
Origin of Ca2+ rise in RPE cells
To determine the role of extracellular Ca2+ during
mechanical stimulation, we performed experiments in
Ca2+-free solution containing 2 mM EGTA (3
,
6
). After a 2-min preincubation in a Ca2+-free
solution, mechanical stimulation failed to result in a rise of
[Ca2+]i in the MS or NB cells. These data
suggest that mechanical stimulation raises
[Ca2+]i in LE-RPE cells by increasing the
Ca2+ influx.
We investigated the mechanism of the Ca2+ influx during
mechanical stimulation by studying the effect of verapamil,
[K+]o, nickel, and gadolinium. Pre-exposure
to verapamil (10 µM), a blocker of the L-type voltage-sensitive
Ca2+ channels, did not affect the
[Ca2+]i rise induced by the mechanical
stimulus. It is therefore unlikely that the trans-plasmalemmal
Ca2+ influx induced by mechanical stimulation occurs
through L-type voltage-sensitive Ca2+ channels
(3)
. To investigate the possible involvement of changes of
the membrane potential, the cells were depolarized with a solution
containing 140 mM K+. The [Ca2+]i
transients elicited after pre-exposing the cell to the
K+-rich solution were not different from the control,
eliminating a change in membrane potential on mechanical stimulation as
the cause of the effects (3)
. Ni2+ (1 mM), a
nonspecific cation channel blocker, reduced the rise in
[Ca2+]i in the MS cell by 36%. These data
suggest that Ca2+ influx during mechanical stimulation
occurs through a Ni2+-sensitive Ca2+ pathway.
When the stretch-sensitive Ca2+-influx channels were
blocked by 10 mM gadolinium the amplitude of the Ca2+
transient on mechanical stimulation was decreased in the MS cells
(-46%) as well as in the NB cells (-40%). This suggests the
involvement of stretch-sensitive cation channels. No change was
observed in the number of responsive NB cells (6)
.
To investigate the possible contribution of Ca2+ release
from intracellular Ca2+ stores, we studied the effect of
thapsigargin and ryanodine. In the presence of 1 µM thapsigargin or
10 µM ryanodine in a Ca2+-containing solution, the
Ca2+ transients upon mechanical stimulation were markedly
reduced. These experiments provide evidence that the Ca2+
entry during mechanical stimulation in LE-RPE cells is followed by
Ca2+ release from intracellular Ca2+ stores
(6)
.
Kinetics of the Ca2+ wave propagation and subcellular
Ca2+ changes
The mechanical stimulation of an individual LE-RPE cell in a
cultured monolayer induced an intracellular
[Ca2+]i rise that was not significantly
different from that measured in RCS-RPE cells stimulated under similar
experimental conditions. However, we could not exclude the existence of
differences at the subcellular level such as differences of nuclear and
cytoplasmic Ca2+ concentration
([Ca2+]n and
[Ca2+]c), which could not be detected by the
measurement of the [Ca2+]i in the entire cell
(7
, 8
).
To measure the fluorescence rise in limited areas, we determined the
fluorescence increase in all directions of the MS cell, starting from
the point of mechanical stimulation. This was done by determining
centrifugally the averaged fluorescence in a series of linearly placed
squares (each 7 µm2) over a length of 30 µm. The
averaged amplitude of the normalized fluorescence rise in all those
polygons at identical distances away from the origin of stimulation was
determined in the mechanically stimulated (MS) cell (P. Stalmans and B.
Himpens, unpublished observations). Figure 2
represents the averaged result of eight LE and RCS cells analyzed using
this procedure. The individual data show only a limited decline of the
intensity of the [Ca2+]i signal away from the
point of stimulation in LE- and RCS-RPE cells.
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To investigate whether the intracellular Ca2+-wave propagation was dependent on the Ca2+ present in the intracellular Ca2+ stores, similar experiments were performed after exposing the cells to thapsigargin. Under these conditions, vasopressin no longer elicited a [Ca2+]i rise in Ca2+-free medium, suggesting that all InsP3-sensitive and ryanodine-sensitive Ca2+ stores were depleted (P. Stalmans and B. Himpens, unpublished observations). When experiments were performed in Ca2+-containing solutions, the depletion of the intracellular stores by thapsigargin induced a significant decrease of the amplitude of the [Ca2+]i wave, demonstrating the important contribution of the Ca2+ stores to this Ca2+ wave. Adding 10 mM gadolinium inhibited this remaining [Ca2+]i signal, presumably by blocking stretch-sensitive Ca2+ channels (6).
We also determined the rate of propagation of the intracellular
Ca2+ wave under these conditions (9
,
10
). As can be deduced from Figure 2B
, the
propagation of the Ca2+ wave declined rapidly as a function
of distance. Propagation was significantly higher in LE-RPE cells (a
median velocity for the [Ca2+]i wave was 30
µm/s) than in RCS-RPE cells (10 µm/s), independent of a
pretreatment with 1 µM thapsigargin. Because this
Ca2+-Mg2+-ATPase blocker depletes the
Ca2+ pools in RPE cells, our findings indicate that a
regenerative system of Ca2+ release from the intracellular
Ca2+ stores is necessary for the progression of the
Ca2+ wave in RPE cells (11)
. The slower
propagation of the Ca2+ wave in RCS-RPE cells compared with
that in LE-RPE cells could be due to a difference in sensitivity, in
density, or in rate of refilling of the Ca2+ stores. This
may be caused by a different IP3R isoform, a different
regulatory mechanism, or a lower InsP3 concentration
(12)
.
The effect of PKC activation on the [Ca2+]n
and [Ca2+]c in RPE cells was also determined.
Compared with LE-RPE cells, the [Ca2+]i rise
in the cytoplasm of pathological strain (RCS-RPE) cells in control
conditions was significantly lower, whereas it was not different in the
nucleus (7
, 8
, 13
,
14
). PKC activation reduced the
[Ca2+]n rise but had no further decreasing
effect on [Ca2+]c. A down-regulation of the
PKC activity did not alter [Ca2+]n rise, but
increased [Ca2+]c rise to a level found in
control LE-RPE cells (5, 6, and P. Stalmans and B. Himpens, unpublished
observations) (Fig. 3
).These findings indicate that in RCS-RPE cells, in contrast to LE-RPE
cells, the rise of [Ca2+]n and
[Ca2+]c are both PKC sensitive and could
suggest that an increased level of PKC-dependent phosphorylation in the
cytoplasm is responsible for the decreased
[Ca2+]i rise (5, 6, and P. Stalmans and B.
Himpens, unpublished observations). On a molecular level, this
difference between LE-RPE cells and RCS-RPE cells could be explained by
a different IP3R subtype or by a different level of
InsP3 metabolism between the cells (12)
.
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Intercellular communication (IC)
Mechanical stimulation of a single RPE cell in the presence of
external Ca2+ induced a spreading intra- and intercellular
rise of the [Ca2+]i. The occurrence of
mechanically induced intercellular Ca2+ waves is now well
established in several cell types (4)
. IC occurs through
gap junctions (GJ). GJ are intercellular channels composed of different
classes of transmembrane-spanning proteins, the connexins
(15)
. Via cDNA-cloning studies it is known that at least
12 different connexin isoforms can be found (15)
. These
connexins are identified by the molecular mass of their polypeptide
chain (Cx26, Cx32, Cx43...). Connexins can be present as mono- or
heteromeres and their synthesis and function is controlled by the cell
adhesion molecules such as the cadherins. The connexins form a partial
cytoplasmic continuity with selective permeability for secondary
messengers like Ca2+, InsP3, cAMP, etc.
(4
, 15
). The important role of these channels
in propagating Ca2+ signaling has been demonstrated for
various non-excitable cells by pharmacologically closing the GJ or by
transfecting cell lines with connexins, which increased the speed of
the wave propagation (4)
. We wanted to investigate the
pathway of intercellular communication during mechanical stimulation in
RPE cells during mechanical stimulation and how it could be modulated.
Blocking of the intercellular communication during mechanical
stimulation
The properties of intercellular communication developed in
monolayers were investigated by treating the cells with 6 mM halothane,
a gap junction blocker (3)
. On stimulation of the MS cell
after superfusion with halothane for 10 min, more than 80% of the NB
cells failed to show a [Ca2+]i rise. A small
percentage of the NB cells presented a
[Ca2+]i rise that was reduced by 53%.
Halothane did not affect significantly the rise of
[Ca2+]i in the MS cells. This effect was
reversible (3)
.
Activation of PKC by incubation of the cells for 30 min with PMA
resulted in a strong inhibition of
[Ca2+]i-wave propagation. This inhibition did
not depend on the oxidizing effects of PMA because the addition of
glaucine, a known antioxidant, did not prevent the inhibition
(5)
. Stimulation or inhibition of protein kinase A
activity by incubating LE-RPE cells with Sp-cAMP or Rp-cAMP,
respectively, or inhibition of tyrosine kinase activity with herbimycin
A did not alter the intercellular communication on mechanical
stimulation. We showed that Cx43 in LE-RPE presented an enhanced double
phosphorylation level after PKC stimulation (unpublished observations).
This increase could be inhibited by PKC down-regulation. This could
indicate that this enhanced phosphorylation (P2 form) level contributes
to the diminished IC.
Effect of high glucose solutions
We showed that the gap-junction conductance of LE-RPE cells was
modified by experimental conditions with increased glucose
concentration as in diabetes (5)
. Our experiments indicate
that the transmission of the [Ca2+]i wave
elicited by mechanical stimulation in LE-RPE cells was also inhibited
by a medium containing a glucose concentration of 14 mM glucose (224
mg%) or higher (Fig. 4
).These glucose concentrations did not affect the
[Ca2+]i rise in the mechanically stimulated
cells but a glucose-dependent decrease of the amplitude of the
[Ca2+]i rise in the neighboring cells and of
the number of communicating neighboring cells was observed. Because it
is known that increased glucose levels can enhance PKC activity, we
investigated whether this mechanism could explain the observed changes
of the gap-junction conductance of LE-RPE cells. Adding 1 µM PMA to a
growth medium containing either 25 or 50 mM glucose for 72 h
resulted in a normal [Ca2+]i wave propagation
from the mechanical to the neighboring cells (5)
. These
results can be explained by the down-regulation of the PKC activity by
the prolonged exposure of the cells to PMA.
|
To determine whether the observed changes were due to alterations in
GJC, FRAP experiments were performed in control conditions after a
30-min incubation with 1 µM PMA and in cells cultured in 50 mM
glucose in the presence and in the absence of 1 µM PMA. The results
of the gap junction conductance measurements (5)
were
consistent with a PKC-dependent inhibition of intercellular
Ca2+-wave propagation by high glucose concentrations.
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DISCUSSION AND CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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. In
addition, microgravity has been shown to alter the actin cytoskeleton
and to reduce the number of stress fibers, elements that appear to be
important in the mechanotransduction (20
,
21
). The cytoskeleton affects the way cells sense their
extracellular environment and respond to the mechanical stimuli
(recently reviewed in ref. 20
). It is a viscoelastic structure that
provides a continuous mechanical coupling throughout the cell, which
changes as the cytoskeleton remodels. In this respect it has, for
example, already been shown that spaceflights alters microtubules and
increases apoptosis in human lymphocytes (22)
. Mechanical
stress can influence ion channel activity, eventually via the
cytoskeleton, and may be conducted to internal organelles
(20)
. In this work we have presented in epithelial cells pathways of intra- and intercellular Ca2+ signaling after mechanical stimulation. The force distribution exerted by our method of mechanical stimulation of the cells is obviously different from the one exerted by changes in gravity. However, it is conceivable that opening of stretch-activated channels as seen in our experiments can be caused by a large variety of deformations. We demonstrated that mechanical stimulation of RPE cells triggers Ca2+ influx, mediated by stretch-sensitive cation channels. This is then followed by Ca2+ release from intracellular Ca2+ stores. A regenerative [Ca2+]i wave with a decreasing rate of propagation was found in the stimulated cell. The rate of propagation was significantly lower in dystrophic RCS-RPE cells compared with LE-RPE cells. It could be increased by down-regulation of the PKC activity with phorbol esters. Incubation with thapsigargin significantly lowered the propagation rate in both LE and RCS-RPE cells.
Mechanical stimulation increased [Ca2+]i in the mechanically stimulated (MS) cell and resulted in a centrifugal propagation of an intercellular Ca2+ wave in the adjacent layers of neighboring (NB) cells. The propagation of the [Ca2+]i wave could be blocked by gap junction blockers, suggesting functional gap junctions. Activation of PKC resulted in a strong inhibition of [Ca2+]i wave propagation. An inhibition of the wave propagation similar to that induced by halothane could be observed in cells grown in 14 mM glucose or higher. Cells grown for 72 h in glucose-rich medium in which all PKC activity was down-regulated did not develop the inhibitory effect on the [Ca2+]i-wave propagation that was normally induced by elevated glucose levels. This was confirmed by FRAP measurements.
Experiments on mechanically induced Ca2+ signaling in
rabbit tracheal epithelium demonstrated that the propagated
Ca2+ rise was primarily due to mobilization of
Ca2+ from intracellular stores, and possibly also to influx
of extracellular Ca2+ (23)
. Simulated
microgravity, however, did not affect the mechanically stimulated
Ca2+ signaling (23)
, and the authors suggested
that intercellular Ca2+ signaling was not compromised in
microgravity. Because the pathways of Ca2+ mobilization in
tracheal epithelium are different from RPE cells, it will be important
to investigate in further experiments whether the effect of gravity on
RPE cells has different characteristics from those observed in tracheal
cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 12, 1999.
Revision received February 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSION AND CONCLUSIONSREFERENCES |
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