The location of hepatocytes in the rat liver acinus determines their sensitivity to calcium-mobilizing hormones

GASTROENTEROLOGY 1996;111:1343–1352

The Location of Hepatocytes in the Rat Liver Acinus Determines Their Sensitivity to Calcium-Mobilizing Hormones THIERRY TORDJMANN, BRIGITTE BERTHON, LAURENT COMBETTES, and MICHEL CLARET Unite´ de Recherche INSERM Unite´ 442, Universite´ Paris-Sud, Orsay, France

Background & Aims: In multicellular systems of rat hepatocytes and in the intact liver, inositol 1,4,5-trisphosphate (IP3 )-dependent agonists induce sequentially ordered calcium ion signals. The mechanisms by which sequential waves are oriented from one hepatocyte to another are unknown. The aim of this study was to investigate the relationship between hepatocyte location in the acinus and cellular sensitivity to noradrenaline, vasopressin, adenosine triphosphate, and angiotensin II. Methods: Periportal (PP) and pericentral (PC) rat hepatocyte suspensions, isolated by the digitonincollagenase technique, were loaded with quin2–acetoxymethyl ester, and hormonal responses were studied in a spectrofluorimeter. The function of the IP3 receptor was studied by measuring the IP3-mediated 45Ca2/ release from permeabilized PP and PC hepatocytes. Results: Increases in noradrenaline and vasopressin-induced intracellular Ca2/ concentration were greater in PC than in PP hepatocytes. In contrast, PP cells were more responsive than PC cells to adenosine triphosphate, and angiotensin II induced similar intracellular Ca2/ concentration increases in both hepatocyte populations. In permeabilized PP and PC hepatocytes, internal Ca2/ stores showed the same loading kinetics, the responses to IP3 were similar, and the sizes of the IP3sensitive compartment were not different. Conclusions: Hepatocyte location in the acinus determines cellular sensitivity to Ca2/-mobilizing agonists. Intercellular Ca2/ waves in the liver could be driven by sensitivity gradients along the hepatocyte plate.

I

n doublets of rat hepatocytes, which are functionally coupled by gap junctions,1 – 4 inositol 1,4,5-triphosphate (IP3 )-linked agonists, namely, vasopressin and the a1-adrenergic agonist phenylephrine, induce coordinated calcium ion responses that spread from one cell to the other in a constant wave-like fashion.4,5 Localized or propagated variations of intracellular Ca2/ concentration ([Ca2/]i ) have been studied in pancreatic acinar cells6 – 9 and other epithelial cells (see Sanderson et al.10 for review). More recently, we have shown that multiplets (3– 5 cells), which are more complex multicellular systems of rat hepatocytes and intact parts of the hepatocyte plate, exhibit highly organized hormone-mediated Ca2/ sig/ 5e13$$0040

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nals.11,12 For each agonist, the Ca2/ wave always originates in the same hepatocyte and then propagates sequentially to the other connected cells. This sequential wave that propagates from cell to cell has been shown in the presence of vasopressin and noradrenaline11 and is also induced by other IP3-dependent agonists such as angiotensin II and adenosine triphosphate (ATP).12,13 This kind of response is believed to be an intrinsic property of multiplets. The same sequential order is observed in each train of [Ca2/]i oscillations in the continued presence of hormones. The sequential order of [Ca2/]i responses is not modified by either repeated addition of the same hormone or by variation of the hormone concentration. Mechanical disruption of a cell in a central position of a multiplet does not block the response of the next cell but lengthens its latency.11 Coordinated Ca2/ waves with similar kinetic properties have also been recently described in situ in the intact rat liver with sequential responses of the hepatocytes inside the liver cell plate.14,15 The sequential nature of Ca2/ waves in multicellular systems of rat hepatocytes and intact liver could result from a gradually decreasing cellular sensitivity from the first to the last responding cell.12,13 This putative gradient could be caused by a heterogeneous distribution of one or several elements participating in the transduction pathway, from the activation of hormonal receptors and Ca2/ influx at the cell surface to the interaction of IP3 with its receptor on the intracellular Ca2/ store. Consistent with this view, hepatocytes contribute differently to a large number of biological processes depending on their location in the portocentrilobular axis of the liver acinus.16 Moreover, there is morphological evidence for gradients of vasopressin15,17 and glucagon receptors18 along the hepatocyte plate. Thus, the position of the different hepatocytes in the Abbreviations used in this paper: [Ca2/]i , intracellular Ca2/ concentration; EGTA, ethylene glycol-bis (b-aminoethyl ether)-N,N,N*,N*-tetraacetic acid; GS, glutamine synthetase; IP3 , inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; PC, pericentral; PP, periportal; quin2-AM, quin2–acetoxymethyl ester. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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acinus and in a multiplet directly obtained from hepatocyte plates may determine cellular sensitivity to Ca2/mobilizing agonists. To test this hypothesis, we selectively isolated rat hepatocytes from periportal (PP) and pericentral (PC) areas by digitonin-collagenase perfusion and analyzed the responses of each cell population to several Ca2/-mobilizing hormones, namely, noradrenaline, vasopressin, ATP, and angiotensin II. These four agents stimulate influx of Ca2/ from the extracellular medium19 – 21 and mobilize Ca2/ from the intracellular IP3-sensitive compartments.22 – 25 We show that PP and PC hepatocytes do not have the same sensitivity to a given agonist. Thus, the sequential pattern of Ca2/ waves in multicellular systems of rat hepatocytes and in the intact liver could correspond to gradual heterogeneity of cellular sensitivity to Ca2/-mobilizing hormones within the hepatic acinus.

Materials and Methods Isolation of Hepatocytes Digitonin-collagenase perfusion. PP and PC rat hepatocytes were isolated by the digitonin-collagenase perfusion technique as described by Quistorff26 and Lindros and Penttila¨.27 Briefly, the liver was perfused for 5 minutes with Krebs’–Henselheit bicarbonate buffer, which was equilibrated with oxygen and carbon dioxide (19:1). A 4 mg/L digitonin solution was then infused at the rate of 10 mL/min for 15– 20 seconds in the antegrade direction via the portal vein or 45–55 seconds in the retrograde direction through the caudal vena cava, i.e., until a regularly scattered perivenous or PP decoloration pattern on the liver surface was obtained. Digitonin was washed out by perfusion of a Ca2/-free solution in the opposite direction for 10 minutes at a rate of 20 mL/min. The collagenase was then perfused via the portal vein in all the experiments. The subsequent steps of the isolation were identical to those for conventional dispersion of rat liver cells.28 Cell viability, assessed by trypan blue exclusion, was consistently ú90%. The enrichment of cell suspensions in PP and PC hepatocytes was monitored by measurements of glutamine synthetase (GS) (PC marker) and alanine aminotransferase (ALT) (PP marker).

Enzyme Assays GS activity was determined as previously described,29 and ALT was assayed with a commercial kit (Sigma Chemical Co., St. Louis, MO) as described by the manufacturer. Protein was assayed according to Lowry et al.30

Spectrofluorimetry After isolation, the cells were incubated in Dulbecco’s modified Eagle medium supplemented with amino acids, vitamins, and gelatin at room temperature and then gassed with O2/CO2 (19:1) at pH 7.4 until later use, as previously de-

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scribed.31 Before each experiment, cells were loaded with the Ca2/-sensitive indicator quin2–acetoxymethyl ester (AM). Briefly, an aliquot corresponding to 106 cells was incubated with 50 mmol/L quin2-AM for 180 seconds, centrifuged, washed, and resuspended in 2 mL of Dulbecco’s modified Eagle medium. The cell suspension was then transferred to the oxygenated spectrofluorimeter cuvette (under O2/CO2 , 19:1) and agitated with a magnetic barrel at 377C (JY3D fluorometer; Jobin & Yvon, Longjumeau, France). Calcium movements were measured from the observed changes in the fluorescence of quin2 (excitation wavelength, 340 nm; emission wavelength, 492 nm). At the conclusion of each experiment, the fluorescence signal was calibrated by making the cell plasma membrane permeable with 5 mmol/L digitonin (quin2 saturation by external Ca2/ gave maximal fluorescence) and then by adding 20 mmol/L ethylene glycol-bis(b-aminoethyl ether)N,N,N*,N*,-tetraacetic acid (EGTA) to the suspension of lysed cells to obtain the minimal fluorescence. Experiments in Ca2/-free medium were performed to evaluate extracellular Ca2/ entry during hormone stimulation. The Ca2/ influx was suppressed by adding EGTA (4 mmol/L final concentration) to the bathing solution. In these conditions, we imposed an extracellular Ca2/ concentration of approximately 80 nmol/L. The hormone was then quickly added to the medium. After the [Ca2/]i had increased to a maximal level, 4 mmol/L ionomycin was quickly added to estimate the total intracellular Ca2/ pool. The fluorescence signal was then calibrated as previously described.31 45

Ca2/ Release by Permeabilized PP and PC Hepatocytes 45

Ca2/ release from saponin-treated cells. IP3-in-

duced Ca2/ release was measured as previously described.32 Briefly, hepatocytes (2.5 1 106 cells/mL) were incubated at room temperature in an internal medium and permeabilized. The medium (100 mmol/L KCl, 20 mmol/L NaCl, 5 mmol/ L MgCl2 , 0.96 mmol/L NaH2PO4 , and 25 mmol/L HEPES buffer at pH 7.15 [with KOH]) was supplemented with 50 mg/mL saponin, 1.5 mmol/L Na2ATP, 5 mmol/L creatine phosphate, 5 U/mL creatine phosphokinase, and 20 mmol/L quin2. Within a few minutes, saponin permeabilized the plasma membrane but not the membrane enclosing the internal Ca2/ stores, which retained their Ca2/ accumulation activity. After this treatment, more than 98% of the cells were freely permeable to trypan blue. Uptake was initiated by addition of 2 mCi/mL 45Ca2/ tracer. External free Ca2/ was monitored continuously by observing quin2 fluorescence in the spectrofluorimeter cuvette. After steady-state loading of the cellular stores, the 45Ca2/ efflux was measured by diluting 50-mL aliquots of the cell suspension in 1 mL of the internal medium without saponin, quin2, or the regenerating system but supplemented with 40Ca2/ and EGTA to buffer the free Ca2/ concentration at pCa 7. IP3 was added 5 seconds after dilution of 45 Ca2/-loaded cells. Ten seconds after IP3 addition, cells were filtered through a Whatman GF/C glass-fiber filter (Whatman Chemical Separation Inc., Clifton, NJ), washed, and counted for radioactivity in a scintillation counter.

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Materials Collagenase was from Boehringer Mannheim GmbH (Mannheim, Germany). Digitonin was purchased from Merck (Darmstadt, Germany). Williams’ medium E was from GIBCO (Paisley, Scotland). All other chemicals were from Sigma Chemical Co.

Statistical Methods Analysis of variance (ANOVA) and Student’s t test comparisons with paired controls were used. Results are expressed as means { SE. P values of ° 0.05 were considered statistically significant.

Results Characterization of Cell Populations Isolated by Digitonin-Collagenase Perfusion Comparability. To validate the comparison of Ca2/ responses in PP and PC cells after digitonin-collagenase perfusion, several variables were measured in both cell populations. First, cell viability assessed by trypan blue exclusion (94% { 3.7%, n Å 21 for PP cells; 93% { 3.5%, n Å 21 for PC cells) and cell yields (130 { 66 1 106 PP cells per liver, n Å 21; 160 { 67 1 106 PC cells per liver, n Å 21) were similar for PP and PC cells. Second, protein content was indistinguishable in PP (2.28 { 0.52 mg/106 cells, n Å 15 in nine experiments) and PC (2.53 { 0.71 mg/106 cells, n Å 15 in nine experiments) hepatocytes. These values are consistent with previously published data.33 Third, basal [Ca2/]i measured in quin2-loaded cell suspensions was similar in PP and PC cells (178 { 6 nmol/L and 185 { 6 nmol/L, respectively; n Å 168) and comparable to the resting value of [Ca2/]i previously determined in isolated hepatocytes.31 These observations indicate that the digitonin-collagenase technique does not itself introduce any detectable modification of PP or PC hepatocytes that may amplify or diminish Ca2/ responses. Zonal purity. Standard enzymatic markers were assayed to assess the zonal purity of each cell population. GS is strictly confined to the last two or three rings of hepatocytes surrounding the central venule,34 and ALT is distributed along a gradient from the PP to the PC area.16 The PC/PP ratio of GS was 40 (1.80 { 0.7 PC cells/tissue; n Å 16). The PC/PP ratio of ALT was 0.80 (0.77 { 0.19 PC cells/tissue; n Å 16). Thus, each cell population was significantly enriched with PP or PC hepatocytes, as previously reported.33 Agonist-Evoked Calcium Responses in PP and PC Hepatocytes Noradrenaline and vasopressin. Calcium responses of PP and PC quin2-loaded hepatocyte populations were studied using spectrofluorimetry31 because / 5e13$$0040

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[Ca2/]i signals in single hepatocytes cannot be easily compared as a result of the large interindividual variability.35 – 37 Cells were isolated and loaded with the Ca2/sensitive indicator quin2-AM. The basal [Ca2/]i , the [Ca2/]i after stimulation, and the time constant of the [Ca2/]i increase were measured. The magnitude of the response was normalized as the ratio between [Ca2/]i under hormonal stimulation and basal [Ca2/]i . The [Ca2/]i increase was plotted as a function of the applied noradrenaline or vasopressin concentration. The magnitude of the response to 1008 and 1007 mol/L noradrenaline and to 10010, 1009, and 1008 mol/L vasopressin was significantly greater in PC than in PP hepatocytes (Figure 1A and B), and the time constant of the [Ca2/]i increase was significantly less in PC than in PP hepatocytes (not shown). The differences were not significant at higher concentrations. The vasopressin concentrationresponse curve was bell shaped. This phenomenon may result from a desensitization of hormone receptors at high concentrations of the agonist.38 Therefore, the apparent sensitivity of hepatocytes to noradrenaline and vasopressin was greater in the PC than in the PP area. ATP. The ATP-induced Ca2/ responses of PP and PC hepatocytes also differed; however, in contrast to noradrenaline- and vasopressin-induced increases of [Ca2/]i , PC cells were less sensitive to ATP than PP cells. The magnitude of the increases of [Ca2/]i as a function of ATP concentration was significantly greater in PP than in PC hepatocytes (Figure 2A). The differences were statistically significant for 2.0 1 1007 and 1006 mol/L ATP but not at higher concentrations. The time constant of the ATP [Ca2/]i increase was less in PP than in PC hepatocytes (not shown). The same hepatocyte population (either PP or PC cells) responded inversely to the agonists ATP and vasopressin in the presence of extracellular Ca2/ (Figure 3). The magnitude of vasopressinmediated Ca2/ response was less but the magnitude of ATP-mediated Ca2/ response was greater in PP than in PC cells (Figure 3). Thus, the ATP-stimulated hepatocytes in the PP area were more sensitive to the agonist than those in the PC area. Angiotensin II. [Ca2/]i changes in PP and PC hepatocytes in response to angiotensin II were also studied by spectrofluorimetry. Angiotensin II induced similar [Ca2/]i increases in both cell populations (Figure 2B). The time constant of angiotensin II–induced [Ca2/]i increases was similar in PP and PC hepatocytes (not shown). Thus, the sensitivity of hepatocytes of these two cell WBS-Gastro

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populations to the agonist angiotensin II appeared to be similar. Calcium responses in Ca2/-free medium. The different sensitivities of PP and PC cells to Ca2/-mobilizing agonists may result from an intrinsic difference in the transduction pathway between the cell surface (hormone receptor, Ca2/ influx) to the intracellular Ca2/ stores (IP3 receptor [IP3R], Ca2/ content). We first investigated whether the differences in hormonal response between

PP and PC cells were observed in the absence of extracellular Ca2/. We also measured the hormone-sensitive and ionomycin-sensitive Ca2/ pools. EGTA was added to the bathing medium, and the agonist and then ionomycin were also quickly added to prevent any Ca2/ loss from the internal stores to the extracellular medium (see Materials and Methods). The agonist concentrations used were those at which significant differences in the [Ca2/]i in-

Figure 1. Effect of noradrenaline and vasopressin on [Ca2/]i in PP and PC hepatocytes in the presence of extracellular Ca2/. The magnitude of the [Ca2/]i increase was estimated as a relative [Ca2/]i increase (see Results). The values are plotted against hormone concentration. The data are means { SE from n (number in parentheses on the x-axis) separate experiments in each cell population. *P Å 0.05; **P Å 0.02; ***P Å 0.01; ****P Å 0.001. (A ) Noradrenaline-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 162 { 11 nmol/L in PP (n Å 34) and 175 { 12 nmol/L in PC (n Å 34) hepatocytes. (B ) Vasopressin-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 178 { 9.6 nmol/L in PP (n Å 70) and 188 { 9.1 nmol/L in PC (n Å 70) hepatocytes.

Figure 2. Effect of ATP and angiotensin II on [Ca2/]i in PP and PC hepatocytes in the presence of extracellular Ca2/. The magnitude of the [Ca2/]i increase was estimated as a relative [Ca2/]i increase (see Results). The values are plotted against hormone concentration. The data are means { SE from n (number in parentheses on the x-axis) separate experiments in each cell population. *P Å 0.05; **P Å 0.02; ***P Å 0.01; ****P Å 0.001. (A ) ATP-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 148 { 7.7 nmol/L in PP (n Å 31) and 185 { 13.7 nmol/L in PC (n Å 31) hepatocytes. (B ) Angiotensin II–induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 213 { 10.2 nmol/L in PP (n Å 24) and 193 { 14.1 nmol/L in PC (n Å 24) hepatocytes.

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crease were observed in the presence of extracellular Ca2/ (Figures 1 and 2). Maximal concentrations of agonists were used to measure the hormone-sensitive Ca2/ pool. The magnitude of the agonist-induced [Ca2/]i increase was calculated as the ratio of increases between the hormone-induced and basal [Ca2/]i . Ionomycin-induced [Ca2/]i increases were similar in PP and PC cells (Figures 4–6). In Ca2/-free conditions, the magnitude of the [Ca2/]i increase in response to vasopressin was greater in PC than in PP cells (Figure 4B). A downturn phenomenon was observed in the vasopressin concentration-response curve, which was similar to that in the presence of extracellular Ca2/. Moreover, ATP induced [Ca2/]i responses of greater magnitude in PP than in PC hepatocytes (Figure 5A). The same cell population, either PP or PC, responded inversely to ATP and vasopressin in the absence of extracellular Ca2/ (Figure 6). The observed differences in the increase of [Ca2/]i reached statistical

Figure 3. Ca2/ responses of two PP and PC hepatocyte suspensions to hormone in the presence of extracellular Ca2/ during the same spectrofluorimetry experiment. Submaximal concentrations of ATP (1006 mol/L) and vasopressin (1008 mol/L) were used. VP, vasopressin.

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significance at the same concentrations compared with the differences in the presence of extracellular Ca2/ (Figures 1 and 2). In contrast, the differences in response to noradrenaline stimulation in the presence of Ca2/ were not observed in Ca2/-free conditions (Figure 4A). Angiotensin II–induced [Ca2/]i responses were similar in both cell populations (Figure 5B). Table 1 shows the particular increases of [Ca2/]i at maximal agonist concentration in

Figure 4. Effect of noradrenaline and vasopressin on [Ca2/]i in PP and PC hepatocytes in Ca2/-free conditions. The [Ca2/]i increase is plotted against hormone concentration. The data points are means { SE from n (number in parentheses on the x-axis) separate experiments for each cell population. *P Å 0.05; **P Å 0.02; ***P Å 0.01; ****P Å 0.001. For agonist-mediated [Ca2/]i increases, ionomycin (IONO) data are expressed as the ratio between ionomycin [Ca2/]i increase after agonist treatment and basal [Ca2/]i . (A ) Noradrenalineinduced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 129 { 5.4 nmol/L in PP (n Å 32) and 132 { 6.3 nmol/L in PC (n Å 32) hepatocytes. (B ) Vasopressin-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 139 { 11.1 nmol/L in PP (n Å 16) and 109 { 5.2 nmol/L in PC (n Å 16) hepatocytes.

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the absence and presence of extracellular Ca2/ for each agonist. Thus, the differences between PP and PC hepatocyte responses to vasopressin and ATP observed in the presence of extracellular Ca2/ were also found in Ca2/-free conditions, which suggests that the differences were not solely caused by unequal contributions of the Ca2/ influx

to the [Ca2/]i increase. In contrast, the similarity of PP and PC cell responses to noradrenaline in Ca2/-free conditions indicates that the Ca2/ influx is partly responsible for the differences observed in the presence of extracellular Ca2/. In addition, unequal [Ca2/]i responsivenesses in PP and PC cells did not appear to be caused by any quantitative disparity in the size of intracellular Ca2/ stores sensitive to the ionomycin and agonists tested. IP3-Induced 45Ca2/ Release in PP and PC Permeabilized Cells IP3-induced Ca2/ responses in permeabilized cells from both acinar origins were analyzed to investigate whether the differences of the Ca2/ signals in PP and PC cells resulted from differences in IP3R sensitivity. In these cells, all the steps upstream from the IP3R occupancy were bypassed. The loading kinetics of the intracellular Ca2/ stores and the size of IP3- and ionomycinsensitive Ca2/ pools in both cell populations were analyzed. PP and PC hepatocytes were permeabilized with saponin as previously described.32 The half-time of 45Ca2/ uptake was similar in PP (2.40 { 0.23 minutes) and PC (2.49 { 0.21 minutes) hepatocytes (n Å 5; data not

Figure 5. Effect of ATP and angiotensin II on [Ca2/]i in PP and PC hepatocytes in Ca2/-free conditions. The [Ca2/]i increase is plotted against hormone concentration. The data points are means { SE from n (number in parentheses on the x-axis) separate experiments for each cell population. *P Å 0.05; **P Å 0.02; ***P Å 0.01; ****P Å 0.001. For agonist-mediated [Ca2/]i increases, ionomycin (IONO) data are expressed as the ratio between ionomycin [Ca2/]i increase after agonist treatment and basal [Ca2/]i . (A ) ATP-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 164 { 8.1 nmol/L in PP (n Å 19) and 153 { 7.4 nmol/L in PC (n Å 19) hepatocytes. (B ) Angiotensin II–induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i was 178 { 14.7 nmol/L in PP (n Å 13) and 139 { 13 nmol/L in PC (n Å 13) hepatocytes.

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Figure 6. Ca2/ responses in two suspensions of PP and PC hepatocytes to hormone, during the same spectrofluorimetry experiment, in the absence of extracellular Ca2/. Submaximal concentrations of ATP (1006 mol/L) and vasopressin (1008 mol/L) were used. The magnitude of ionomycin-induced [Ca2/]i increase was similar in PP and PC cells. VP, vasopressin; Iono, ionomycin.

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Table 1. Effect of Ca2/ Removal From the Extracellular Medium on Agonist-Induced [Ca2/]i Increases in PP and PC Hepatocytes Stimulated With Maximal Agonist Concentrations Agonist-mediated [Ca2/]i increase (nmol/L) CaN Agonist Noradrenaline (10 mmol/L) Vasopressin (100 nmol/L) ATP (10 mmol/L) Angiotensin II (1 mmol/L)

PP 610 770 582 1149

{ { { {

CaO

n 166 97 44 44

5 12 4 4

PC 669 920 590 959

{ { { {

n 140 74 82 143

5 12 4 4

PP 310 220 396 374

{ { { {

n 25 22 50 50

12 8 4 4

PC 284 179 371 318

{ { { {

n 16 14 37 6

12 8 4 4

NOTE. Values are expressed as nanomoles per liter [Ca2/]i . Data are means { SE for n experiments in each population. CaN, in the presence of extracellular Ca2/ (1.8 mmol/L); CaO, Ca2/-free conditions (see Materials and Methods).

shown) and consistent with the values for rat hepatocytes isolated by conventional collagenase perfusion.39 The IP3 dose-response curves (Figure 7A) were very similar for both PP and PC hepatocyte populations (50% effective concentration, 105 { 22 nmol/L and 125 { 36 nmol/ L, respectively; n Å 4). These values are in agreement with previous observations of permeabilized rat hepatocytes.23,32 The size of the IP3-sensitive compartment was similar in PP and PC cells and represented 26% { 2.2% and 31.7% { 4.2%, respectively, of the ionomycin-sensitive compartment (n Å 4), i.e., 2.17 { 0.23 Ca2//mg protein for PP cells and 2.03 { 0.18 Ca2//mg protein for PC cells (n Å 4) (Figure 7B). Thus, the different sensitivities to Ca2/-mobilizing hormones of hepatocytes from PP and PC areas do not appear to be caused by differences concerning IP3-IP3R interactions or IP3-sensitive compartment size. Presumably, the upstream steps (i.e., mainly hormone-receptor interactions, G protein activation, and phospholipase Cb activity) are involved in this heterogeneity of cellular responses. Moreover, our data suggest that there are similar kinetics for the Ca2/ loading of intracellular stores and similar sizes of ionomycin-sensitive Ca2/ pools in PP and PC hepatocytes.

Discussion Hepatocytes contribute unequally to various liver functions depending on their location in the lobulus.16 Hepatocyte heterogeneity within the liver cell plate is particularly well established for the metabolism of carbohydrates,40 amino acids,41 and ammonia.41,42 Ca2/ metabolism and hormone-mediated Ca2/ movements previously have not been investigated in association with hepatocyte heterogeneity. We suggested previously11,12 that sequential hormone-mediated Ca2/ signals in multicellular systems were caused by an in situ gradual heterogeneity of one or more elements in the hormone signal transduction pathway. Vasopressin-evoked sequential / 5e13$$0040

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Ca2/ signals along the hepatocyte plate in the intact liver have also been described.14,15 Moreover, V1a vasopressin receptor messenger RNAs, detected by in situ hybridization in the rat liver, are predominantly found in the perivenular zone,17 and a similar distribution has been described for the glucagon receptor by autohistoradiography.18 These different in situ morphological studies suggest a gradient in the number of receptors from one acinar zone to the opposite one, but the hormone-mediated Ca2/ mobilization activity in PP and PC hepatocytes has never been directly investigated. In this study, hepatocytes isolated from different zones of the liver acinus after digitonin-collagenase perfusion were tested for heterogeneous cellular sensitivity. The apparent cellular sensitivity to the agonists was not identical in PP and PC hepatocyte suspensions. Also, one cell population could be more sensitive to one agonist but less sensitive to another. PC cells were more sensitive than PP cells to noradrenaline and vasopressin, but PP cells were more responsive than PC cells to ATP. PP and PC cells were equally sensitive to angiotensin II. The differences between PP and PC hepatocyte populations in the magnitude and the rate of [Ca2/]i increase were significant mostly at low and intermediate agonist concentrations. At the highest doses used, PP and PC cells were equally responsive. This suggests that the differences for intermediate concentrations are not caused by a subset of cells from the PP (for noradrenaline and vasopressin) or PC (for ATP) area that could not respond to the agonist whatever the concentration. Also, equal responsiveness to the highest agonist concentrations indicates that PP and PC hepatocytes have the same ability to respond to maximal hormonal stimulation. This is consistent with a previous [Ca2/]i imaging study on isolated rat hepatocytes in which all the cells in a videomicroscope field were responsive to the hormone, provided a high concentration was used.11 Hepatocyte sensitivity to two different Ca2/-mobilizWBS-Gastro

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Figure 7. IP3-induced 45Ca2/ release in PP and PC permeabilized hepatocytes. PP (●) and PC (s) cells were isolated, permeabilized, and loaded with 45Ca2/ in the spectrofluorimeter cuvette at 207C as described in Materials and Methods. After equilibration, aliquots were transferred to a cytosol-like efflux medium buffered at pCa7. Ca2/ release was measured after addition of various concentrations of IP3 to the efflux medium 5 seconds after cell dilution (see Materials and Methods). Data are means { SE of four determinations in four experiments in each cell population. (A ) IP3 dose–response curves for PP and PC hepatocyte populations were very similar (PP, EC50 Å 105 { 22 nmol/L; PC, 125 { 36 nmol/L). IP3-sensitive Ca2/ pools (nmol Ca2//mg protein) were similar in PP (2.17 { 0.23 nmol Ca2// mg protein; n Å 4) and PC (2.03 { 0.18 nmol Ca2//mg protein; n Å 4) cells. (B ) The IP3-sensitive Ca2/ pools calculated as a percentage of ionomycin-sensitive Ca2/ pools were also similar in PP (31.7 { 42; n Å 4) and PC (26 { 2.2; n Å 4) hepatocytes. The Ca2/-loading kinetics, measured by the half-time of 45Ca2/ uptake at 207C, was similar in PP (2.49 { 0.21 minutes; n Å 5) and PC (2.40 { 0.23 minutes; n Å 5) hepatocytes.

ing agonists (vasopressin and ATP) is opposite in PP and PC areas (Figures 3 and 6). If vasopressin and ATP share the same intracellular mechanism for Ca2/ mobilization,22 – 25,35,43 the differences between these populations probably involve extracellular steps of the transduction pathway (i.e., hormonal receptor activation). These results confirmed that hepatocyte responsiveness was not affected by the isolation technique because PP and PC cells retain their characteristic sensitivity to different agonists. Also consistent with these results, different agonists can produce opposite sequences of Ca2/ responses / 5e13$$0040

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in rat hepatocyte multiplets, which suggests intercellular heterogeneity in the number and/or the affinity of hormonal receptors.11,12,44 We examined the apparent sensitivity of the IP3R in permeabilized cells in which all the transduction steps upstream from the binding of IP3 to its receptor were bypassed. IP3 dose-response curves were very similar in both PP and PC hepatocyte populations, suggesting an equal sensitivity of the IP3R for IP3 in PP and PC cells (Figure 7). We also investigated intracellular Ca2/ compartments in PP and PC cells. The size of hormonesensitive (in intact cells), IP3-sensitive (in permeabilized cells), and ionomycin-sensitive (in both intact and permeabilized cells) Ca2/ stores were similar in the two hepatocyte populations. The loading kinetics of the Ca2/ pools (estimated by the half-time of 45Ca2/ uptake) was also similar in PP and PC cells, suggesting that Ca2/, Mg2/ –adenosine triphosphatase of the endoplasmic reticulum was equally active in both hepatocyte populations. Therefore, the observed acinar heterogeneity in cellular responses to Ca2/-mobilizing agonists is presumably due to a heterogeneous distribution of the IP3 production (affecting, for example, hormonal receptor density and/or affinity). Any step in the production of IP3 could be involved in this sensitivity gradient, although no data suggest a heterogeneous distribution of G proteins, phosphatidylinositol 4,5-bisphosphate, or phospholipase C-b in the liver lobulus. These results, in addition to [Ca2/]i imaging in multicellular systems of rat hepatocytes4,5,11,12 and in the intact liver,14,15 suggest that this hepatocyte heterogeneity is fine (subtle cell-to-cell differences) and gradual (sequential responses). Hepatocytes possessing a more efficient IP3 production mechanism (for example, more or higher affinity hormonal receptors) should display the highest rate of IP3 generation, highest [Ca2/]i oscillation frequency, shortest hormonal delay, and consequently, greatest sensitivity to the agonist.35,44 – 46 Consequently, different apparent cellular sensitivities of PP and PC cells should correlate with disparities in hormonal latency along the hepatocyte plate. Angiotensin II induced similar Ca2/ signals in the two cell populations, suggesting a homogeneous hepatocyte sensitivity to this agonist in the acinus. However, angiotensin II–mediated Ca2/ signals in multicellular systems are sequential.12,13 Possibly, there is a very shallow sensitivity gradient through the acinus, only detectable by [Ca2/]i imaging, which thus reveals very small cell-tocell differences. In the intact liver, vasopressin-mediated intercellular Ca2/ waves are initiated at one extremity of the hepatocyte plate and then spread across the acinus toward the opposite extremity; the orientation (PP to PC or PC to WBS-Gastro

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PP) of the wave remains controversial.14,15 Our results with isolated PP and PC hepatocytes suggest that the in situ vasopressin-mediated intercellular Ca2/ wave should be initiated in the PC area by intermediate hormone concentrations, which was found by one in situ study.15 However, another in situ study reported a more complex pattern of vasopressin-elicited Ca2/ waves in which the orientation of the wave was dependent on the applied hormone concentration.14 PP to PC waves were observed at low agonist concentration, and PC to PP waves were observed at high hormone concentration. We suggest that hepatocyte sensitivity to Ca2/-mobilizing agonists is a major factor that determines the orientation of the intercellular wave in the acinus, but the results reported previously14 underline a probably more complex in situ regulation of intercellular Ca2/ signaling. Moreover, our results suggest that during maximal vasopressin stimulation, all the hepatocytes in the plate would be expected to respond at the same time with multiple initiations of [Ca2/]i increase and possibly multidirectional waves. Also, intermediate concentrations of vasopressin and ATP could generate Ca2/ waves in opposite directions across the acinus. Gluconeogenesis enzymes are preferentially localized in PP zone,16 but there is no consensus about the glycogenolytic capacity of the different areas in the acinus.47,48 In vivo experiments show that ATP and eicosanoids mobilize glucose mainly from the PP zone.49 In this study greater sensitivity to noradrenaline and vasopressin was shown in cells where neoglucogenesis enzymes are weakly expressed (i.e., PC), and less sensitivity was shown in cells with strong neoglucogenesis enzymatic activities (i.e., PP). In the hepatocyte plate, this reciprocal pattern of Ca2/ signaling efficiency and metabolic capacity could provide a means by which the cells in one area are recruited for a metabolic function preferentially performed by a different zone. The ATP-induced [Ca2/]i increase was greater in PP than in PC cells, suggesting that in this case, metabolic capacity and Ca2/ signaling efficiency were similarly distributed in the acinus. We report that hepatocytes from PP and PC areas have different sensitivities to the Ca2/-mobilizing agonists noradrenaline, vasopressin, and ATP. This cellular heterogeneity could be caused by an unequal distribution in the lobulus of one or several elements participating in the IP3 production. Therefore, the sequential pattern of hormone-mediated Ca2/ signals in multicellular systems of rat hepatocytes4 – 6,11,12 and in the intact liver14,15 presumably results from a heterogeneous cellular sensitivity to the Ca2/-mobilizing hormones in the hepatic acinus. In the multicellular systems of rat hepatocytes, the intercellular Ca2/ wave may be oriented by this gradual / 5e13$$0040

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heterogeneity, making these systems different from other epithelia in which only diffusion of the intracellular messenger IP3 through gap junctions is considered essential.10 Hepatic gap junctions are freely permeable to ions and small molecules.1 – 3,5 Therefore, the diffusion of an intercellular messenger through these junctions probably contributes to the coordination of [Ca2/]i oscillations in rat hepatocyte multiplets.11,5 Thus, orientation of the wave by a sensitivity gradient and coordination of [Ca2/]i oscillations by intercellular diffusion of IP3 may combine to organize complex Ca2/ signals in the liver lobulus. In the hepatocyte plate, the gradient of cellular sensitivity to the hormones along the portocentrilobular axis may be a driving force for the different Ca2/-mediated processes that occur in an oriented manner, mainly bile canaliculi network motility50 and bile secretion. Hepatocyte heterogeneity in hormonal delay in the intact liver may allow the successive activation of the cells along the plate, even if the circulating hormone has already occupied the surface receptors of the whole hepatocyte cord. Furthermore, inversely proportional metabolic capacity and cellular sensitivity to the agonists in the same hepatocyte area may allow the acinus to function in an optimal and coordinated manner.

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Received April 22, 1996. Accepted July 22, 1996. Address requests for reprints to: Michel Claret, Ph.D., Unite´ de Recherche INSERM Unite´ 442, Ba ˆtiment 443, Universite´ Paris-Sud, 91405 Orsay Cedex, France. Fax: (33) 1-69-41-05-74. The authors thank Raymonde Leuillet and Denise Reuter for excellent technical assistance and A. Edelman for his help in editing the manuscript.

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