©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Coordination of Ca Signaling by Intercellular Propagation of Ca Waves in the Intact Liver (*)

(Received for publication, January 19, 1995; and in revised form, February 6, 1995)

Lawrence D. Robb-Gaspers Andrew P. Thomas (§)

From the Department of Anatomy, Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

In common with many other cell types(1) , isolated hepatocytes respond with oscillations or periodic spiking of cytosolic free [Ca] ([Ca]) (^1)when challenged with agonists coupled to the second messenger InsP(3)(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) .


EXPERIMENTAL PROCEDURES

Isolated Perfused Rat Liver

The livers of anesthetized male Sprague-Dawley rats (180-250 g) were perfused in situ via the hepatic portal vein with Krebs-Ringer bicarbonate buffer(3) . For most experiments the median lobe of the liver was studied and other lobes were tied off with silk thread and excised (perfusion adjusted to maintain 3.5 ml/min/g, wet weight). The remaining liver was then removed from the rat and perfused with buffer composed of (in mM): 121 NaCl, 25 HEPES, 5 NaHCO(3), 4.7 KCl, 1.2 KH(2)PO(4), 1.2 MgSO(4), 1.3 CaCl(2), 5.5 glucose, 0.5 glutamine, 3 lactate, 0.3 pyruvate, 0.2 bromosulfophthalein (BSP), 0.1% BSA, pH 7.4, equilibrated with 100% O(2) at 30 °C. BSP, a competitive anion transport inhibitor, was included to inhibit loss of Ca indicator dyes from the hepatocytes(15) . BSP did not affect liver viability or alter [Ca](i) oscillations when added to isolated hepatocytes. (^2)After 20-30 min of initial perfusion, Ca-sensitive indicators were loaded by recirculating 60 ml of perfusion buffer containing the acetoxymethyl ester (2-5 µM) plus 0.003% Pluronic F-127 and 2% BSA for 30 min.

Imaging Measurements of [Ca]

The liver was immobilized with gauze sheets anchored to a microscope imaging chamber constructed from a 100-mm Petri dish with a 24 times 50-mm glass coverslip inserted into the base. The nonrecirculating perfusion was maintained by gravity feed. Hormones and other agents were infused via a mixing chamber 15 cm upstream of the liver using a syringe pump at 1-2% of perfusate flow. Confocal fluorescence images were obtained from fluo3-loaded livers using a Bio-Rad MRC-600 laser scanning confocal microscope (488 nm excitation). Optical slice thickness determined under the experimental conditions (Bio-Rad technical bulletin 101) ranged from 5 µm using a 60times objective to 125 µm at 10times. To visualize the spatial organization of Ca signals measured with the single wavelength confocal microscope system, these image data are presented as the actual fluorescence depicted on a gray scale with the [Ca](i) change at each time point displayed as a yellowoverlay. The [Ca](i) changes were calculated by subtraction of sequential fluorescence images, and the intensity of the overlay is proportional to the fluorescence change at each point. The difference images were processed with a 3 times 3 median filter and a minimum threshold to remove high frequency noise. The fura2 fluorescence was monitored using a cooled charge-coupled device (CCD) camera and alternating excitation at 340 nm and 380 nm, as described previously (3, 16) . Values for [Ca](i) were calculated from the 340/380 nm ratio after correction for autofluorescence and out of focus information(15, 17) . Images of autofluorescence were obtained at the end of each experiment by perfusion of the liver for 10 min with 100 µM MnCl(2) in Ca-free medium. Deblurring of the nonconfocal fura2 fluorescence images was achieved using the deconvolution algorithm described by Monck et al.(18) . Similar [Ca](i) values were obtained when the fluorescence of individual cells was corrected by subtraction of fluorescence signals from neighboring sinusoidal regions. Calibration parameters for fura2 were determined in vitro, a K(d) value of 184 nM was used.


RESULTS AND DISCUSSION

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](i), 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](i) increases occurred as periodic [Ca](i) spikes. Fig. 2A shows [Ca](i) values calculated for one hepatocyte in a fura2-loaded perfused liver. After an initial latent period, 100 pM vasopressin initiated a series of [Ca](i) oscillations, the frequency of which was increased when the infusion of vasopressin was stepped to 500 pM. At higher vasopressin concentrations, the [Ca](i) increase was sustained or decayed slowly to basal with no subsequent [Ca](i) oscillations in the continuing presence of the hormone (Fig. 2B). The rate of rise and maximum amplitude of [Ca](i) increase within each cell was the same for all agonist doses, and although the calibrated [Ca](i) 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](i) rise decreased with increasing vasopressin dose. Thus, hepatocytes in the intact liver respond to vasopressin with frequency-modulated [Ca](i) 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 times 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 times 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](i) 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](i) changes between cells, such that the rising phase of each [Ca](i) oscillation spread sequentially through neighboring hepatocytes. Fig. 2C shows [Ca](i) oscillations from Fig. 2A on an expanded time scale overlaid with the [Ca](i) response of a second cell 40 µm further along the same hepatic plate. The [Ca](i) oscillations in the two hepatocytes have the same periodicity, but are phase-shifted due to the propagation of the [Ca](i) signals from one cell to the next. This intercellular propagation of the [Ca](i) oscillations resulted in a series of [Ca](i) waves (one for each oscillation) that spread through the lobule. The low magnification confocal images of Fig. 1A show the path of [Ca](i) wave propagation for several lobules. In each lobule the [Ca](i) increase was detected first in a small number of cells in the periportal zone and then a wave of [Ca](i) increase spread radially through the hepatocytes toward the central veins. At vasopressin doses that caused [Ca](i) oscillations, each oscillation started in the same periportal cells and propagated through the lobule in a similar manner. Thus, the [Ca](i) oscillation frequency of these initiating cells sets the frequency and pattern for the entire lobule.

The periportal initiation of the [Ca](i) waves was observed for all livers during coordinated [Ca](i) oscillations. However, with suprathreshold doses of vasopressin that gave only a single [Ca](i) increase, the path of wave propagation was reversed and the [Ca](i) waves initiated in the pericentral zones. Although the [Ca](i) 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](i) wave propagation was dependent on agonist dose suggests that the [Ca](i) 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](i) oscillations initiating from the periportal zones. Termination of intralobular [Ca](i) waves occurred either at the hepatic venules or as a result of collision annihilation. An example of [Ca](i) wave annihilation is shown in Fig. 1B, where [Ca](i) 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](i) wave to high vasopressin.

Fig. 3shows higher magnification confocal images of [Ca](i) wave propagation at a dose of vasopressin causing [Ca](i) 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](i) oscillations within individual hepatocytes of the intact liver occurred as [Ca](i) waves initiating from discrete loci and then propagating throughout the cell. In some cases the [Ca](i) 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](i) 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](i) waves generally initiated from the membrane region in contact with the previously responding cell. There was a marked lag as [Ca](i) waves propagated between cells, such that [Ca](i) 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](i) wave propagation were the same. This suggests that certain cells acted as pacemakers for each lobule, driving the successive waves of [Ca](i) increase that underlie the entrained lobular [Ca](i) oscillations. Consistent with this, specific cells have been found to initiate the synchronized [Ca](i) 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 times 122 µm with a slice thickness of 5 µm.



We have reported previously that the rate of [Ca](i) 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](i) waves in the intact liver were also unaffected by agonist dose and fell within the same range as the [Ca](i) waves in isolated hepatocytes (Fig. 4A). In contrast to the intracellular [Ca](i) wave rates, the time required for propagation of [Ca](i) 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](i) waves spread through the liver (Fig. 4C). Under conditions giving rise to maximal [Ca](i) increases, the rate of spread through the lobule sometimes exceeded the intracellular [Ca](i) wave rate. This may reflect the existence of multiple origination points along the [Ca](i) wave path at high agonist doses, as noted above. The mean intercellular [Ca](i) wave rate for vasopressin doses giving sustained [Ca](i) 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](i) oscillations versus sustained [Ca](i) increases. Therefore, the data in Fig. 4(A-C) are from single livers. In order to combine the dose-response data for intercellular [Ca](i) 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](i) oscillations (Fig. 4D). These data indicate that the rate of intercellular [Ca](i) 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](i) 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 60times 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 10times 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](i) waves through the hepatic lobule. As discussed above, the path of [Ca](i) wave propagation is not secondary to the flow of perfusate. Moreover, the fact that the direction of the [Ca](i) 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](i) waves when CaCl(2) was omitted from the perfusate (data not shown). Previous studies have demonstrated that both Ca and InsP(3) pass through gap junctions in isolated hepatocyte couplets(8) . In addition, propagation of [Ca](i) waves between lung epithelial cells appears to depend on diffusion of InsP(3) 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](i) responses.

Despite the inconclusive data described above, the most likely form of intercellular communication underlying the spread of [Ca](i) waves in the intact liver is through gap junctions. It has been suggested that, in lung epithelial cells, intercellular [Ca](i) waves may result from InsP(3) 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(3) this might be achieved through a process of Ca activation of phospholipase C. An alternative mechanism would rely on a subthreshold elevation of InsP(3) throughout the liver (induced by the global perfusion with vasopressin), which then forms an excitable medium through which [Ca](i) waves can propagate as a result of the feedback activation of InsP(3) receptors by Ca. This is directly analogous to the process that has been proposed to underlie subcellular [Ca](i) waves in Xenopus oocytes(20, 21) . Regenerative Ca release mechanisms of this type can explain the constant rate and amplitude of [Ca](i) 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](i) 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(3) rather than Ca. However, intercellular [Ca](i) 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(3) 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](i).

The nature of the pacemaker-like cells initiating the oscillatory [Ca](i) 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](i) increases, but argues against a role for vasopressin receptor distribution in the portal origin of [Ca](i) 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(3), or a greater efficacy of InsP(3) for releasing Ca.


CONCLUSIONS

The present study demonstrates for the first time that intracellular [Ca](i) 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](i) oscillations was regulated by vasopressin dose (frequency modulation), whereas the amplitude and rate of intracellular [Ca](i) waves were not. The oscillatory [Ca](i) responses were organized in the intact liver, such that each [Ca](i) 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](i) signals may contribute to the secretion and canalicular movement of bile, which are important functions of the hepatocyte plasma membrane.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants DK38422 and AA07215 and Training Grant T32-AA07463. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Anatomy, Pathology and Cell Biology, Rm. 271 JAH, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-955-5017; Fax: 215-923-2218.

(^1)
The abbreviations used are: [Ca], cytosolic free [Ca]; InsP(3), inositol 1,4,5-trisphosphate; BSP, bromosulfophthalein; BSA, bovine serum albumin.

(^2)
L. D. Robb-Gaspers and A. P. Thomas, unpublished observations.


ACKNOWLEDGEMENTS

We thank Paul A. Anderson for his role in developing the computer software for image processing.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.