(Received for publication, January 19, 1995; and in revised form, February 6, 1995)
From the
Activation of the inositol lipid signaling system results in
cytosolic Ca oscillations and intra- and
intercellular Ca
waves in many isolated cell
preparations. However, this form of temporal and spatial organization
of signaling has not been demonstrated in intact tissues. Digital
imaging fluorescence microscopy was used to monitor Ca
at the cellular and subcellular level in intact perfused rat
liver loaded with fluorescent Ca
indicators.
Perfusion with low doses of vasopressin induced oscillations of
hepatocyte Ca
that were coordinated across entire
lobules of the liver by propagation of Ca
waves along
the hepatic plates. At the subcellular level these periodic
Ca
waves initiated from the sinusoidal domain of
cells within the periportal region and propagated radially across
cell-cell contacts into the pericentral region, or until terminated by
annihilation collision with other Ca
wave fronts.
With increasing agonist dose, the frequency but not the amplitude of
the Ca
waves increased. Intracellular Ca
wave rates were constant, but transcellular signal propagation
was determined by agonist dose, giving rise to a dose-dependent
increase in the rate at which Ca
waves spread through
the liver. At high vasopressin doses, a single Ca
wave was observed and the direction of Ca
wave
propagation was reversed, initiating in the pericentral region and
spreading to the periportal region. It is concluded that intercellular
Ca
waves may provide a mechanism to coordinate
responses across the functional units of the liver.
In common with many other cell types(1) , isolated
hepatocytes respond with oscillations or periodic spiking of cytosolic
free [Ca]
([Ca
]
) (
)when challenged with agonists coupled to the second
messenger InsP
(2, 3) . At the subcellular
level these [Ca
]
oscillations occur as waves of
[Ca
]
increase that
initiate from a discrete subcellular
locus(4, 5, 6) . Although
[Ca
]
oscillations and
waves are observed in many isolated cell preparations (1, 7) , the possibility remains that these phenomena
may not occur in intact tissues where gap junctions and other forms of
cell-cell interaction can modify signal transduction in the individual
cells. In isolated cell preparations that maintain or establish gap
junctions, [Ca
]
changes have been shown to pass between cells and this can
result in intercellular propagation of a
[Ca
]
wave(8, 9, 10, 11, 12, 13) .
However, [Ca
]
wave
propagation is not an automatic consequence of the existence of a
syncytium, since monolayers of epithelial or glial cells, which
propagate intercellular [Ca
]
waves when stimulated mechanically, demonstrate asynchronous
[Ca
]
oscillations that
do not propagate into neighboring cells(10, 11) .
Another property of [Ca
]
oscillations observed in isolated cells that may be
regulated differently in intact tissues is the control of oscillation
frequency by agonist dose. Frequency modulation of
[Ca
]
oscillations has
been proposed to play an important role in determining the extent and
targeting of cellular [Ca
]
responses(1, 14) .
Fluorescence imaging of fura2- or fluo3-loaded perfused liver
lobes at a focal plane 25-50 µm into the tissue revealed the
typical hepatic structure of sheets of hepatocytes separated by open
sinusoids (Fig. 1). Larger dark areas are due to hepatic venules
in the pericentral zones of the lobule. Identification of the lobular
organization was achieved by infusion of fluorescein-BSA, which first
appeared in periportal zones and then spread rapidly through the
sinusoids to pericentral zones. Infusion of vasopressin resulted in
increases of [Ca]
, which
occurred first in a limited number of hepatocytes in each lobule and
then spread progressively through adjacent cells. At low doses of
vasopressin, the [Ca
]
increases
occurred as periodic [Ca
]
spikes. Fig. 2A shows
[Ca
]
values calculated for one
hepatocyte in a fura2-loaded perfused liver. After an initial latent
period, 100 pM vasopressin initiated a series of
[Ca
]
oscillations, the
frequency of which was increased when the infusion of vasopressin was
stepped to 500 pM. At higher vasopressin concentrations, the
[Ca
]
increase was sustained or
decayed slowly to basal with no subsequent
[Ca
]
oscillations in the
continuing presence of the hormone (Fig. 2B). The rate
of rise and maximum amplitude of [Ca
]
increase within each cell was the same for all agonist doses, and
although the calibrated [Ca
]
values were somewhat lower than we calculated for isolated
hepatocytes, the rise time was very similar (2-6
s)(3, 4) . Also in agreement with our isolated
hepatocyte studies, the latent period to the first
[Ca
]
rise decreased with
increasing vasopressin dose. Thus, hepatocytes in the intact liver
respond to vasopressin with frequency-modulated
[Ca
]
oscillations that are
similar in character to those observed previously in isolated
hepatocytes(2, 3, 4) .
Figure 1:
Lobular
organization of intercellular Ca waves fronts in
perfused rat liver. Confocal images of fluo3 fluorescence are shown on
a linear grayscale, with the fluorescence changes at
each time point superimposed in yellow (calculated as
described under ``Experimental Procedures''). Numbers in the upperleftcorner of each image
represent the time (seconds) after initiating vasopressin infusion
(corrected for perfusion dead time). A, the first in a series
of oscillatory [Ca
]
waves is shown propagating across several lobules during
perfusion with 0.2 nM vasopressin. Periportal (PP)
and pericentral (PC) regions determined from the path of
fluorescein-BSA perfusion are labeled on the initial image. Image
dimensions are 1134
756 µm with a slice thickness of 125
µm. B, collision and annihilation of intercellular
[Ca
]
wave fronts
during perfusion with 0.5 nM vasopressin, which gave a single
nonoscillatory [Ca
]
increase in this liver. The imaged area is centered between
two central veins, located near the upperleft and lowerrightcorners. Image dimensions are
709
473 µm with a slice thickness of 50
µm.
Figure 2:
[Ca]
responses of individual hepatocytes within the perfused
liver. Perfusion of fura2-loaded livers with the indicated
concentrations of vasopressin (VP) was initiated at the arrows. Each trace shows the
[Ca
]
response of a
single hepatocyte calculated from the ratio of 340 and 380 nm
fluorescence intensities extracted from the image time series, as
described under ``Experimental Procedures.'' Panel C shows the trace from panel A on an expanded time scale (solidline), together with the
[Ca
]
response of a cell displaced 40 µm along the same hepatic plate (brokenline). The data of panel B are
derived from a different liver.
The
[Ca]
oscillations of individual
cells in the intact liver did not occur independently. Remarkably, all
of the hepatocytes visible in a single lobule (400-600 cells)
were entrained to the same frequency and pattern of oscillation.
Underlying this phenomenon was a propagation of the
[Ca
]
changes between cells,
such that the rising phase of each
[Ca
]
oscillation spread
sequentially through neighboring hepatocytes. Fig. 2C shows [Ca
]
oscillations
from Fig. 2A on an expanded time scale overlaid with the [Ca
]
response of a second cell 40 µm further along the same
hepatic plate. The [Ca
]
oscillations in the two hepatocytes have the same periodicity,
but are phase-shifted due to the propagation of the
[Ca
]
signals from one cell to
the next. This intercellular propagation of the
[Ca
]
oscillations resulted in a
series of [Ca
]
waves (one for
each oscillation) that spread through the lobule. The low magnification
confocal images of Fig. 1A show the path of
[Ca
]
wave propagation for
several lobules. In each lobule the
[Ca
]
increase was detected
first in a small number of cells in the periportal zone and then a wave
of [Ca
]
increase spread
radially through the hepatocytes toward the central veins. At
vasopressin doses that caused [Ca
]
oscillations, each oscillation started in the same periportal
cells and propagated through the lobule in a similar manner. Thus, the
[Ca
]
oscillation frequency of
these initiating cells sets the frequency and pattern for the entire
lobule.
The periportal initiation of the
[Ca]
waves was observed for all
livers during coordinated [Ca
]
oscillations. However, with suprathreshold doses of vasopressin
that gave only a single [Ca
]
increase, the path of wave propagation was reversed and the
[Ca
]
waves initiated in the
pericentral zones. Although the [Ca
]
increase still spread through the lobule as a wave under these
conditions, discontinuities along the wave path suggested initiation at
multiple loci through the lobule or propagation from cells outside the
focal plane. The fact that the path of
[Ca
]
wave propagation was
dependent on agonist dose suggests that the
[Ca
]
waves are not secondary to
the perfusion flow through the liver. This is supported by the
maintenance of the wave path for many oscillation cycles during
continuous vasopressin perfusion. In addition, when a liver was first
perfused with low vasopressin via the portal vein and then the flow was
reversed by perfusion from the hepatic vein, infusion of vasopressin
from both directions caused [Ca
]
oscillations initiating from the periportal zones. Termination of
intralobular [Ca
]
waves
occurred either at the hepatic venules or as a result of collision
annihilation. An example of [Ca
]
wave annihilation is shown in Fig. 1B, where
[Ca
]
wave fronts propagating
from the two sides of the imaged region (first shown at 17.3 s) are
seen to collide in the images of 23.1-26.0 s. This example is
from a liver giving a single pericentral to periportal
[Ca
]
wave to high vasopressin.
Fig. 3shows higher magnification confocal images of
[Ca]
wave propagation at a dose
of vasopressin causing [Ca
]
oscillations. The arrows superimposed on the initial
image show the propagation paths for three parallel hepatic plates near
to the portal tract. In agreement with our studies in isolated
hepatocytes(4, 5) , the
[Ca
]
oscillations within
individual hepatocytes of the intact liver occurred as
[Ca
]
waves initiating from
discrete loci and then propagating throughout the cell. In some cases
the [Ca
]
increase in the first
cell to respond in a hepatic plate initiated from the sinusoidal
membrane (see cell at leftcenter responding
31.6-37.3 s in Fig. 3). In others the first
[Ca
]
increase spread from the
center of the cell (see cell at upperleft responding
at 43.1 s in Fig. 3), suggesting propagation from a cell out of
the focal plane. For subsequent cells in each hepatic plate, the
[Ca
]
waves generally initiated
from the membrane region in contact with the previously responding
cell. There was a marked lag as [Ca
]
waves propagated between cells, such that
[Ca
]
wave fronts arriving at
cell-cell boundaries often did not appear in the next cell for several
seconds (e.g. from 37.3 to 45.9 s for the first and second
cells in the middle of the three hepatic plates indicated in Fig. 3). This lag presumably reflects the time taken for the
propagating message to pass between cells. During each oscillation in a
series, the same cell in each hepatic plate responded initially, and
the inter- and intracellular paths of
[Ca
]
wave propagation were the
same. This suggests that certain cells acted as pacemakers for each
lobule, driving the successive waves of
[Ca
]
increase that underlie the
entrained lobular [Ca
]
oscillations. Consistent with this, specific cells have been
found to initiate the synchronized
[Ca
]
oscillations in isolated
groups of 2-4 hepatocytes(13) .
Figure 3:
Intracellular and intercellular pathways
of [Ca]
wave
propagation through hepatocytes of the perfused liver. Figure shows a
confocal image time series from a fluo3-loaded liver with fluorescence
intensity depicted on a grayscale and
[Ca
]
changes at each
time point indicated by the yellowoverlay. Images
show the first in a series of vasopressin-induced
[Ca
]
oscillations,
with the time point (seconds) in the upperrightcorner. The arrows on the first image indicate
the paths of [Ca
]
wave
propagation. Image dimensions are 148
122 µm with a slice
thickness of 5 µm.
We have reported
previously that the rate of [Ca]
wave propagation within isolated hepatocytes (20-25
µm/s) is the same for a variety of agonists and is independent of
agonist dose(4, 5, 16) . The intracellular
[Ca
]
waves in the intact liver
were also unaffected by agonist dose and fell within the same range as
the [Ca
]
waves in isolated
hepatocytes (Fig. 4A). In contrast to the intracellular
[Ca
]
wave rates, the time
required for propagation of [Ca
]
increases between cells (the transcellular lag period) was
sensitive to agonist concentration, and decreased with increasing
vasopressin dose (Fig. 4B). This gave rise to a
dose-dependent increase in the rate at which
[Ca
]
waves spread through the
liver (Fig. 4C). Under conditions giving rise to
maximal [Ca
]
increases, the
rate of spread through the lobule sometimes exceeded the intracellular
[Ca
]
wave rate. This may
reflect the existence of multiple origination points along the
[Ca
]
wave path at high agonist
doses, as noted above. The mean intercellular
[Ca
]
wave rate for vasopressin
doses giving sustained [Ca
]
increases was 39.9 ± 4.5 µm/s (n = 7
livers). There was considerable variation between livers in the
vasopressin dose range that gave [Ca
]
oscillations versus sustained
[Ca
]
increases. Therefore, the
data in Fig. 4(A-C) are from single livers. In
order to combine the dose-response data for intercellular
[Ca
]
waves, the rates at which
the waves spread through the lobule were plotted against the frequency
for all vasopressin treatments that gave rise to
[Ca
]
oscillations (Fig. 4D). These data indicate that the rate of
intercellular [Ca
]
wave
propagation was determined by vasopressin dose, and this can be
ascribed to the effect of vasopressin concentration on the
transcellular lag period for [Ca
]
wave propagation between adjacent cells.
Figure 4:
Effect of vasopressin dose on rates of
intracellular and intercellular
[Ca]
wave propagation
in the perfused liver. Wave rates were calculated by measuring the time
offset in the rising phase of
[Ca
]
at half-peak
height over known distances either within individual cells (A)
or over multicellular regions (C and D), as described
previously for [Ca
]
waves in isolated hepatocytes(4, 16) . A, intracellular [Ca
]
wave rates were measured from images obtained using a
60
objective for a fura2-loaded liver perfused with each of the
indicated concentrations of vasopressin (n =
15-23 cells). B, transcellular lag times between
adjoining pairs of cells were measured as the time offset of
[Ca
]
rise at sites in
the two cells close to the region of cell-cell contact. Values are from
the same data sets depicted in panelA (n = 9-13 for each condition; all vasopressin doses
significantly different from the other two with p < 0.05). C, intercellular [Ca
]
wave propagation rates were measured over distances of at
least 100 µm from confocal images of a fluo3-loaded liver obtained
with a 10
objective (n = 10-16
measurements). D, mean values for intercellular
[Ca
]
wave rates are
plotted against mean [Ca
]
oscillation frequency at each vasopressin dose. Data are
combined from seven separate perfused
livers.
We have investigated
a number of possible mechanisms for the propagation of
[Ca]
waves through the hepatic
lobule. As discussed above, the path of
[Ca
]
wave propagation is not
secondary to the flow of perfusate. Moreover, the fact that the
direction of the [Ca
]
waves
remains the same when the flow is reversed suggests that release of an
extracellular paracrine signal is unlikely to be involved. A wave of
activated Ca
influx at the plasma membrane can also
be excluded, because vasopressin still induced both intracellular and
intercellular [Ca
]
waves when
CaCl
was omitted from the perfusate (data not shown).
Previous studies have demonstrated that both Ca
and
InsP
pass through gap junctions in isolated hepatocyte
couplets(8) . In addition, propagation of
[Ca
]
waves between lung
epithelial cells appears to depend on diffusion of InsP
through gap junctions(11) . Gap junction coupling can be
blocked in these preparations with chemical agents such as octanol,
carbeneoxolene, and halothane. However, infusion of these compounds
into the liver either had no effect (perhaps due to buffering or other
forms of uptake by cells proximal to the inflow of perfusate) or caused
substantial damage to the areas of the liver visible to the imaging
system and abolished all [Ca
]
responses.
Despite the inconclusive data described above, the
most likely form of intercellular communication underlying the spread
of [Ca]
waves in the intact
liver is through gap junctions. It has been suggested that, in lung
epithelial cells, intercellular [Ca
]
waves may result from InsP
diffusion from a single
activated cell(19) . In view of the much greater distances and
three-dimensional structure involved in the intact liver, such a
mechanism would almost certainly require some form of regeneration of
the diffusing signal. For InsP
this might be achieved
through a process of Ca
activation of phospholipase
C. An alternative mechanism would rely on a subthreshold elevation of
InsP
throughout the liver (induced by the global perfusion
with vasopressin), which then forms an excitable medium through which
[Ca
]
waves can propagate as a
result of the feedback activation of InsP
receptors by
Ca
. This is directly analogous to the process that
has been proposed to underlie subcellular
[Ca
]
waves in Xenopus oocytes(20, 21) . Regenerative Ca
release mechanisms of this type can explain the constant rate and
amplitude of [Ca
]
waves within
hepatocytes, independent of agonist dose, and the modulation of
oscillation frequency can be explained by a dose-dependent increase in
the rate of activation of the trigger Ca
pool(7) . The fact that the time required to propagate
[Ca
]
waves between cells in the
perfused liver (transcellular lag period) was inversely dependent on
agonist dose suggests that the concentration of message passing between
hepatocytes is controlled by the agonist, which would implicate
InsP
rather than Ca
. However,
intercellular [Ca
]
waves could
also be driven by Ca
passing through the gap
junctions if the amount of trigger Ca
required is
determined by the level of InsP
in the next cell, or if
Ca
activation of phospholipase C is involved. Another
alternative is that agonist dose controls gap junction permeability,
independently of [Ca
]
.
The
nature of the pacemaker-like cells initiating the oscillatory
[Ca]
waves from the periportal
zones of the liver remains to be elucidated. One possibility is a
difference in the signal transduction system that results in a greater
sensitivity to low vasopressin levels. However, in situ hybridization measurements of vasopressin V1a receptor mRNA
expression have shown a gradient that is highest in pericentral
hepatocytes and decreases toward the periportal cells(22) .
This distribution could explain the pericentral initiation of
non-oscillatory [Ca
]
increases,
but argues against a role for vasopressin receptor distribution in the
portal origin of [Ca
]
oscillations. Nevertheless, the initiating hepatocytes of the
periportal zone could be established by enhanced sensitivity due to
other elements of the signaling system, including the
G-protein/phospholipase C system, a higher basal level of
InsP
, or a greater efficacy of InsP
for
releasing Ca
.
The present study demonstrates for the first time that
intracellular [Ca]
oscillations
and waves can occur in an integral tissue, composed of multiple cell
types and a circulatory system in the configuration existing in
vivo. The frequency of the hepatocyte
[Ca
]
oscillations was regulated
by vasopressin dose (frequency modulation), whereas the amplitude and
rate of intracellular [Ca
]
waves were not. The oscillatory
[Ca
]
responses were organized
in the intact liver, such that each
[Ca
]
oscillation initiated in
the periportal zone and spread throughout the lobule as an
intercellular wave. This coordination of Ca
signaling
may have important consequences for hepatic function. Previous studies
have shown oscillations of Ca
release into the
perfusate from intact perfused rat liver(23) , raising the
possibility that the metabolic function of the liver may oscillate. It
is also possible that coordination of lobular
[Ca
]
signals may contribute to
the secretion and canalicular movement of bile, which are important
functions of the hepatocyte plasma membrane.