Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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Because hepatocytes communicate via gap
junctions, it has been proposed that Ca2+
waves propagate through this pathway and in the process
activate Ca2+-dependent cellular
responses. We tested this hypothesis by measuring vasopressin-induced glycogenolysis in short-term cultures of rat hepatocytes. A 15-min vasopressin
(108 M) stimulation induced
a reduction of glycogen content that reached a maximum 1-3 h
later. Gap junction blockers, octanol or 18
-glycyrrhetinic acid,
reduced the effect by 70%. The glycogenolytic response
induced by Ca2+ ionophore
8-bromo-A-21387, which acts on each hepatocyte, was not
affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a
synthetic peptide homologous to a domain of the extracellular loop 1 of
the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced.
Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP
as a paracrine mediator. We propose that gap junctions allow for a
coordinated vasopressin-induced glycogenolytic response despite the
heterogeneity among hepatocytes.
cell-to-cell communication; cultured hepatocytes; glycogen content; octanol; 18-glycyrrhetinic acid
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INTRODUCTION |
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IN THE HEPATIC ACINUS, unidirectional perfusion creates different microenvironments around hepatocytes near the inlet (terminal portal venules) or outlet (terminal hepatic venules), such as periportal (PP) to pericentral (PC) gradients of oxygen and substrate concentrations (11, 16). As a result of these and other factors, parenchymal cells within the acinus show morphological, biochemical, and functional heterogeneity, which has led to the model of dynamic metabolic zonation. Thus liver acini rather than liver lobules are the units in which functional processes occur and are regulated.
Parenchymal cells of the hepatic acinus communicate extensively with each other by gap junctions (22), which are constituted by intercellular channels permeable to ions and small molecules (3). Gap junctions between hepatocytes have been proposed to play an important role in physiological responses of the liver, including hormone-induced glycogenolysis (2, 6, 20, 25, 29). Among the relevant aspects of this hypothesis is that all hepatocytes can store and break down glycogen (15, 30), but the receptors for at least two glucogenic hormones, glucagon (2) and vasopressin (20), are preferentially found in PC hepatocytes. In the latter, Ca2+ mobilization induced by vasopressin is greater than in PP hepatocytes (33). Because gap junctions are permeable to cyclic nucleotides, inositol 1,4,5-trisphosphate (IP3), and Ca2+ (23, 27), which are involved in the activation of glycogenolysis (13), it has been proposed that cAMP and IP3-dependent Ca2+ signals spread among cells by a chemical gradient-driven diffusion and a regenerative process, respectively (2). The distance that cAMP diffuses would be limited in part by the size of its concentration gradient. On the other hand, it has recently been reported that vasopressin generates Ca2+ waves that propagate in a regenerative way (constant amplitude) along the intact hepatic acinus via gap junctions (20, 25). Moreover, in the liver of certain rodents, including rats, nerve terminals are preferentially found near PP cells (24). Thus it might have been conceivable to propose that the glucogenic response induced by stimulation of the sympathetic nerves is performed preferentially by PP hepatocytes. Consistently, connexin-32 (Cx32) knockout mice that present gap junction-deficient livers show a 78% lower glucose release on nerve stimulation compared with wild-type liver (21), suggesting an important role of gap junctional communication in this metabolic response. On the other hand, liver perfusion with glucagon or norepinephrine elicits a similar glucose release in Cx32-deficient and control mice (21). As discussed elsewhere (26), the distribution of glucagon and norepinephrine receptors in mouse liver remains unknown. Moreover, Cx32-deficient mice may compensate for the lack of gap junctions by a homogeneous receptor expression throughout the acinus. Therefore, further studies are required to establish the role of gap junctional communication in metabolic responses in the adult.
The systems most frequently used to study liver glycogenolysis have been hepatocyte suspensions and the perfused organ. In both systems, it is difficult to evaluate gap junctional communication and liver metabolic responses simultaneously. Moreover, measurements of glucose release in the perfused liver correspond to the glucose yield through gluconeogenesis and glycogenolysis (13). However, glucose release could be affected by byproducts released from nonparenchymal cells, such as Kupffer cells (4). Thus a more direct way to evaluate the glycogenolytic response of hepatocytes could be the direct measurement of glycogen levels. Adult hepatocytes cultured for several hours or days lose the expression of many functional genes, including gap junctional proteins and vasopressin receptors (9, 32). Nevertheless, short-term cultures of adult hepatocytes respond to various glycogenolytic hormones (13) and are coupled by gap junctions (32). In addition, gap junctional communication can be easily altered and evaluated.
The experiments described here were designed to elucidate the role of gap junctional communication during glycogenolysis induced by vasopressin. The content of glycogen was measured after vasopressin stimulation in short-term cultures of adult rat hepatocytes treated under various conditions that block or prevent gap junctional communication. The results indicate that gap junctional communication allows a coordinated glycogenolytic response among rat hepatocytes with heterogeneous sensitivity to vasopressin.
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MATERIALS AND METHODS |
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Reagents.
Collagenase type IV,
[Arg8]vasopressin,
1-octanol, rabbit liver glycogen, 18-glycyrrhetinic acid,
suramin, and 8-bromo-A-21387 (8Br-A-21387) were from Sigma Chemical
(St. Louis, MO). Waymouth's medium and fetal bovine serum
(FBS) were from GIBCO BRL (Grand Island, NY). Phenol was from Fluka
Chemika-BioChemika (Buchs, Switzerland). Synthetic peptides with
sequence homology to regions of the extracellular loop 1 of Cx32
(residues 52-63) [Cx32-(52-63)] and the COOH
terminus of Cx37 (residues 261-276)
[Cx37-(261-276)] were synthesized by BiosChile
(Santiago, Chile). Green 5-chloromethylfluorescein diacetate
(CellTracker) was from Molecular Probes (Eugene, OR).
Isolated hepatocytes.
Adult female Sprague-Dawley rats (150-200 g) were obtained from
the Animal Institute of the Pontificia Universidad Católica de
Chile (Santiago, Chile). Rats were anesthetized with pentobarbital sodium (65 mg · ml1 · kg
body wt
1), and hepatocytes were dissociated by perfusing
the liver with collagenase type IV, as described previously (1).
Hepatocytes were plated on plastic tissue culture plates (NUNC, Delta,
The Netherlands) at a cell density of 3 × 106 cells/60-mm plate in
Waymouth's medium supplemented with 10% FBS, 50 U/ml penicillin, and
50 µg/ml streptomycin. In some experiments, cells were maintained in
suspension (4 × 106
cells/ml) in Waymouth's medium supplemented with 10% FBS and buffered
with 30 mM HEPES under continuous rotating conditions to prevent cell
reaggregation. After 2 h incubation the medium was replaced by fresh
medium (with 10% FBS) to eliminate dead cells.
Glycogen measurements. Levels of glycogen were measured using a previously reported method (8). Briefly, hepatocytes of a single culture dish (3 × 106 cells/plate) or maintained in suspension (4 × 106 cells/ml) were harvested by scraping with a rubber policeman or by centrifugation (1,000 rpm in a bench centrifuge), respectively, placed in capped glass test tubes, immersed in 0.4 ml of 30% KOH saturated with Na2SO4, and placed in a boiling water bath for 20 min. An aliquot of 20 µl/ml was taken to determine the protein concentration using the method previously described by Lowry et al. (18). The tubes were then removed from boiling water and cooled on ice, and 2 vol of 95% ethanol were added. After incubation on ice for 30 min, samples were centrifuged at 3,000 rpm for 30 min (model 8700; Kubota, Tokyo, Japan) and the supernatant was carefully aspirated. The glycogen-containing pellet was resuspended in a minimal volume of distilled water and then 1 vol of 5% phenol and 5 vol of H2SO4 were added. The absorption at 490 nm of the colored complex arising from the phenol-sulfuric acid reaction with glycogen was measured spectrophotometrically. A standard curve was obtained using glycogen solutions of known concentration. Levels of glycogen measured with this method were 51.8 ± 32.0 µg glycogen/mg protein. Therefore, results were first calculated as micrograms of glycogen per milligrams of protein and then normalized as the percentage with respect to control cells to obtain the percentage of reduction in glycogen content and also to reduce the variability between experiments.
Dye coupling. The permeability to molecules of low molecular weight was evaluated qualitatively by microinjecting one cell of a pair or a cluster of cells with lucifer yellow CH (5% lucifer yellow in 150 mM LiCl) at room temperature on a glass coverslip. The dye was injected by repeated 0.1-s, 0.1-nA current pulses (or by overcompensation of the negative capacitance amplifier) until the impaled cell was brightly fluorescent. After injection, the cells were observed for 2-3 min on a Nikon Diaphot microscope with xenon arc lamp illumination and a Nikon B filter block (excitation wavelength, 450-490 nm; emission wavelength, >520 nm) to determine whether dye transfer occurred. The incidence of dye coupling was scored as the percentage of injections that resulted in dye transfer to at least one adjacent cell.
To test if reaggregated hepatocytes establish gap junctional communication, freshly dispersed hepatocytes (3 × 106 cell/ml) were labeled with CellTracker, according to the manufacturer's suggested protocol, and then mixed with unlabeled cells (6 × 106 cells/ml). Two hours after plating, cell coupling was tested by monitoring the transfer of lucifer yellow from a microinjected CellTracker-labeled cell to unlabeled neighboring cells.Statistical analysis. The F value of the Bonferroni multiple comparison test was calculated to evaluate the differences between means ± SD.
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RESULTS |
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Vasopressin induces a reduction in glycogen content of rat
hepatocytes.
The effect of different vasopressin concentrations
(1012 to
10
6 M) on the glycogen
content of plated hepatocytes was measured 3 h after stimulation. All
tested concentrations induced nearly a 50% reduction of glycogen
levels (Fig.
1A).
The reduction resulting from
10
6 to
10
8 M vasopressin was only
slightly smaller than that induced by the two lowest concentrations
tested (Fig. 1A). No detectable changes were found in the basal levels of glycogen of unstimulated hepatocytes during the 3-h incubation period (not shown).
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Gap junction blockers reduce vasopressin-induced glycogenolytic
response.
Gap junctions of adult rat hepatocytes are reversibly blocked by
octanol (32) or 18-glycyrrhetinic acid (34). Therefore, we used
these agents as a first approach to test the role of gap junctional
communication in vasopressin-induced glycogenolysis. Cells were treated
with 10
8 M vasopressin for
different periods of time, as described above, but in the presence of
500 µM octanol or 30 µM 18
-glycyrrhetinic acid, added 2-3
min before stimulation with vasopressin. When the period
of stimulation was completed, the medium was replaced by
vasopressin-free, gap junction blocker-free medium and cells were
incubated until a total of 3 h was completed. The effect induced by
15-min vasopressin stimulation was drastically reduced by both octanol
and 18
-glycyrrhetinic acid (Fig. 2). Nonetheless, the effect of the
gap junction blockers on the vasopressin-induced glycogenolysis was
progressively less pronounced at longer periods of stimulation, losing
statistical significance (P < 0.05)
after 45-60 min for 18
-glycyrrhetinic acid and after
60-180 min for octanol (Fig. 2).
Octanol and 18-glycyrrhetinic acid block gap
junction conductance during vasopressin stimulation.
To determine the degree of cell-to-cell coupling during experiments
designed to study the effect of gap junction blockers on
vasopressin-induced glycogenolysis, the incidence of dye coupling was
studied in hepatocytes under the same protocol described above. In
control cultures, the incidence of dye coupling was close to 90% (Fig.
3). Within 5 min after the addition of 500 µM octanol or 30 µM 18
-glycyrrhetinic acid, coupling between
hepatocytes was nearly 0% and remained low for the following 15 min
(Fig. 3). Soon after washing, a progressive recovery of the incidence of dye coupling was detected in hepatocytes treated with
18
-glycyrrhetinic acid but not in cells treated with octanol (Fig.
3). Cells treated with 500 µM octanol or 30 µM 18
-glycyrrhetinic
acid for 3 h showed an incidence of dye coupling of 0%.
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Intercellular contact enhances vasopressin-induced glycogenolysis in
a gap junction-dependent manner.
Gap junctional communication was reduced by maintaining the dissociated
cells in suspension under continuous shaking to prevent reaggregation.
Under this condition, the reduction in glycogen content induced by 15 min treatment with 108 M
vasopressin was 55% lower than in plated hepatocytes, in which cell-to-cell contact was favored (Fig. 4).
Moreover, 500 µM octanol or 30 µM 18
-glycyrrhetinic acid reduced
the vasopressin-induced glycogenolysis by ~68% in the plated cells
(Fig. 4) but did not affect the vasopressin-induced glycogenolytic
response in hepatocytes maintained dispersed in suspension (Fig. 4),
suggesting that these gap junction blockers do not affect steps from
the activation of vasopressin receptors to the activity of glycogen
phosphorylase. In addition, cells in suspension stimulated with
10
8 M vasopressin for 3 h
showed a reduction of glycogen content of 54.0 ± 0.8% (mean ± SD; n = 3), which was similar to that
found in plated hepatocytes stimulated for 15 min (Fig. 4). Glycogen levels were not affected by treatment with octanol or
18
-glycyrrhetinic acid alone (not shown).
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Impediment of gap junction formation reduces vasopressin-induced glycogenolysis. We first tested whether reaggregated hepatocytes establish gap junctional communication. Freshly dissociated hepatocytes suspended in Ca2+-free Leaffert's solution were filtered through a 33-µm nylon mesh that allowed only single cells or cell pairs to pass. In one experiment the number of single cells and cell pairs was evaluated before filtering and after plating. Before filtration, 74% of the hepatocytes were single cells and 26% were pairs, but 1 h after plating in Ca2+-containing medium only 14% of the cells were single, whereas all other cells had formed cell aggregates of higher order than two, suggesting that reaggregation had occurred. In addition, an aliquot of cells filtered through a 33-µm mesh was labeled with CellTracker, mixed with unlabeled hepatocytes, and plated onto plastic culture dishes. Dye coupling was studied by microinjecting lucifer yellow into a CellTracker-labeled cell, and the spread of the dye to adjacent unlabeled hepatocytes was observed (Fig. 5). The incidence of dye transfer from CellTracker-labeled to -contacting unlabeled cells was 40%, suggesting that in primary cell cultures hepatocytes from different zones of the hepatic acinus might couple to each other.
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Glycogenolysis induced by vasopressin is not mediated by ATP. Because intercellular communication in cultured hepatocytes can occur via ATP secretion (31), we investigated the possible involvement of this pathway during vasopressin-induced glycogenolysis. Addition of the purinergic receptor blocker suramin (1 µM suramin) 2-3 min before the addition of vasopressin caused only a minor reduction in the glycogenolysis induced by stimulation with the vasopressin for 15 or 180 min (Fig. 7). Moreover, suramin alone did not affect the basal levels of glycogen after its application for 15 or 180 min (not shown).
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DISCUSSION |
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In 1986 (29), it was proposed that gap junctional communication between rat hepatocytes would favor hormone-elicited metabolic responses such as glycogenolysis by allowing intercellular transfer of second messengers. Recently, gap junctional communication between hepatocytes has been implicated as the pathway that allows intercellular propagation of vasopressin-induced Ca2+ waves in intact liver (20, 25). We now report that although all rat liver parenchymal cells can exhibit a vasopressin-induced glycogenolytic response, hepatocytes with rather different vasopressin sensitivities can be distinguished. Nevertheless, these different types of hepatocytes showed a coordinated glycogenolytic response when they communicated to each other by gap junctions, supporting the hypothesis that intercellular propagation of Ca2+ waves activates Ca2+-dependent metabolic responses. Our results indicate that gap junctional communication maximizes Ca2+-dependent metabolic responses in cultured hepatocytes, and presumably in the liver, induced by activation of heterogeneously distributed hormone receptors.
The metabolic zonation of liver carbohydrate metabolism has been
documented extensively (11, 16). The time and rate of both glycogen
synthesis and degradation differ in PP and PC hepatocytes (30),
suggesting that the activation of these metabolic responses follows an
order of hierarchy among parenchymal cells. We now find that
hepatocytes maintained under conditions that prevent the exchange of
intercellular signals, under either the effect of gap junction blockers
[octanol, 18-glycyrrhetinic acid, and Cx32-(52-63)
peptide] or constant shaking to maintain them in a dispersed
state, display a glycogenolytic response to vasopressin that is only
~30% of that observed in plated cells, in which cell reaggregation
occurred and cell coupling was close to 90%. Nonetheless, after long
periods of stimulation (2-3 h) under conditions that prevented
cell communication, either under the effect of gap junction blockers or
constant shaking in suspension, a response equivalent to that observed
under conditions that favor cell-to-cell coupling was observed.
Therefore, all hepatocytes have the potential to show a glycogenolytic
response but with rather different kinetics. After a minimum of ~15
min of vasopressin stimulation, gap junctional communication allows
less responsive hepatocytes to show a coordinated response with the
more responsive cells. In isolated hepatocyte couplets, gap junctional
communication allows for a coordinated increase in intracellular
Ca2+ concentration
([Ca2+]i)
elicited by vasopressin (19). Moreover, and consistent with a cell
hierarchy for activation of glycogenolysis, the repeated application of
different vasopressin concentrations to isolated rat hepatocyte
triplets or quadruplets does not alter the position of the pacemaker
cell in which the intercellular
[Ca2+]i
wave is initiated (5).
During normal feeding rhythms, glycogen can be stored in all hepatocytes (30). Moreover, PP and PC hepatocytes show similar glycogen phosphorylase content and sensitivity to Ca2+-dependent activation (15). Complementary to these findings, we observed that in plated cells stimulated with vasopressin for increasing periods of time in the presence of gap junction blockers or in dispersed cells stimulated for 3 h, compared with those stimulated for 15 min, the reduction in glycogen content was time dependent, indicating that the glycogenolytic response among hepatocytes occurs with different time courses. The latter is consistent with the finding that the increases in [Ca2+]i induced by vasopressin are greater in PC than in PP hepatocytes (33). These differences are likely to be explained by the greater vasopressin V1 receptor density found in PC than in PP cells (20). We consistently found that bypassing differences in receptor density by increasing the [Ca2+]i in each hepatocyte using a Ca2+ ionophore (8Br-A-21387) resulted in similar reductions in glycogen content in vasopressin-stimulated reaggregated and dispersed hepatocytes, both of which were close to the reduction elicited by vasopressin in reaggregated cells. Moreover, the response elicited by the Ca2+ ionophore either in plated or dispersed hepatocytes was not affected by gap junction blockers, suggesting that intercellular communication is not required if the threshold in [Ca2+]i required to elicit maximal glycogenolytic response is attained in each cell.
Mechanically induced cell deformation initiates signaling that is mediated in part by release of ATP, which increases [Ca2+]i (31) and induces glycogenolysis through activation of purinergic receptors (14). The lack of inhibition of vasopressin-induced glycogenolysis observed in hepatocytes treated with suramin suggests that those steps of our experimental procedure that might cause cell deformation (e.g., medium wash out, centrifugation, or constant shaking) did not cause significant ATP release, so that these procedures were appropriate to evaluate the role of gap junctions in vasopressin-induced glycogenolysis.
Maximal increase in [Ca2+]i in plated hepatocytes and activation of glycogen phosphorylase in dissociated hepatocytes in suspension each occur within a few minutes after vasopressin stimulation (5, 15, 19). In short-term cultures of rat hepatocytes, we found a ~35% reduction of glycogen content 1 h after a single episode of vasopressin stimulation. Similarly, in previous work using the perfused rat liver it was found that vasopressin induces a ~30-35% reduction in glycogen content within 1 h after addition of a single dose of vasopressin to the perfusate (12), suggesting that vasopressin induces a similar glycogenolytic response in both systems. Moreover, in hepatocytes in culture we found a 25-35% reduction in glycogen 3 h after 5- or 10- min stimulation with vasopressin, but a minimum of 15 min of treatment was required to induce a maximal effect (50%), indicating that even though activation of glycogen phosphorylase occurs within 1 min after vasopressin stimulation (15), a stimulus with duration well above 1 min is required to induce maximal glycogen degradation. In addition, even though a significant reduction in glycogen levels occurred 30 min after vasopressin stimulation, maximal reduction was found 1-3 h later, suggesting that glycogen phosphorylase remained active for 1-3 h after stimulation with vasopressin for 15 min.
The lack of response beyond the observed maximal (~50%) reduction of glycogen content could at least partially be explained by downregulation of the vasopressin receptors (9). We consistently found that a second addition of vasopressin did not further reduce hepatocyte glycogen content. Alternatively, the glucose released could have inhibited glycogen phosphorylase activity (13) and induced glycogen synthesis (13), thus decreasing the net vasopressin-induced reduction in glycogen levels. Nonetheless, it has been reported previously that vasopressin inhibits glycogen accumulation (12), so the reduction in glycogen content measured within 3 h after stimulation with vasopressin should primarily have reflected glycogenolysis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Nathanson for the thoughtful comments.
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FOOTNOTES |
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This work was partially supported by Fondo Nacional de Ciencia y Technología Grants 3930011 (to C. G. Sáez), 1960559 (to J. C. Sáez), and 1971124 (L. Accatino).
A portion of this work has been previously published in abstract form (see Ref. 28).
Address for reprint requests: J. C. Sáez, Dept. de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.
Received 9 December 1997; accepted in final form 18 February 1998.
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