The location of hepatocytes in the rat liver acinus determines their sensitivity to...

GASTROENTEROLOGY 1996;111:1343–1352

The Location of Hepatocytes in the Rat Liver AcinusDetermines Their Sensitivity to Calcium-Mobilizing Hormones

THIERRY TORDJMANN, BRIGITTE BERTHON, LAURENT COMBETTES, and MICHEL CLARETUnite de Recherche INSERM Unite 442, Universite Paris-Sud, Orsay, France

Background & Aims: In multicellular systems of rat he- nals.11,12 For each agonist, the Ca2/ wave always origi-patocytes and in the intact liver, inositol 1,4,5-trisphos- nates in the same hepatocyte and then propagates sequen-phate (IP3)-dependent agonists induce sequentially or- tially to the other connected cells. This sequential wavedered calcium ion signals. The mechanisms by which that propagates from cell to cell has been shown in thesequential waves are oriented from one hepatocyte to presence of vasopressin and noradrenaline11 and is alsoanother are unknown. The aim of this study was to induced by other IP3-dependent agonists such as angio-investigate the relationship between hepatocyte loca- tensin II and adenosine triphosphate (ATP).12,13 Thistion in the acinus and cellular sensitivity to noradrena-

kind of response is believed to be an intrinsic propertyline, vasopressin, adenosine triphosphate, and angio-of multiplets. The same sequential order is observed intensin II. Methods: Periportal (PP) and pericentral (PC)each train of [Ca2/]i oscillations in the continued pres-rat hepatocyte suspensions, isolated by the digitonin-ence of hormones. The sequential order of [Ca2/]i re-collagenase technique, were loaded with quin2–acet-sponses is not modified by either repeated addition ofoxymethyl ester, and hormonal responses were studied

in a spectrofluorimeter. The function of the IP3 receptor the same hormone or by variation of the hormone concen-was studied by measuring the IP3-mediated 45Ca2/ re- tration. Mechanical disruption of a cell in a central posi-lease from permeabilized PP and PC hepatocytes. Re- tion of a multiplet does not block the response of thesults: Increases in noradrenaline and vasopressin-in- next cell but lengthens its latency.11 Coordinated Ca2/

duced intracellular Ca2/ concentration were greater in waves with similar kinetic properties have also been re-PC than in PP hepatocytes. In contrast, PP cells were cently described in situ in the intact rat liver with se-more responsive than PC cells to adenosine triphos- quential responses of the hepatocytes inside the liver cellphate, and angiotensin II induced similar intracellular

plate.14,15

Ca2/ concentration increases in both hepatocyte popu-The sequential nature of Ca2/ waves in multicellularlations. In permeabilized PP and PC hepatocytes, inter-

systems of rat hepatocytes and intact liver could resultnal Ca2/ stores showed the same loading kinetics, thefrom a gradually decreasing cellular sensitivity from theresponses to IP3 were similar, and the sizes of the IP3-first to the last responding cell.12,13 This putative gradi-sensitive compartment were not different. Conclu-

sions: Hepatocyte location in the acinus determines ent could be caused by a heterogeneous distribution ofcellular sensitivity to Ca2/-mobilizing agonists. Intercel- one or several elements participating in the transductionlular Ca2/ waves in the liver could be driven by sensitiv- pathway, from the activation of hormonal receptors andity gradients along the hepatocyte plate. Ca2/ influx at the cell surface to the interaction of IP3

with its receptor on the intracellular Ca2/ store. Consis-tent with this view, hepatocytes contribute differently toI a large number of biological processes depending on their

n doublets of rat hepatocytes, which are functionallycoupled by gap junctions,1–4 inositol 1,4,5-triphos-

location in the portocentrilobular axis of the liver aci-phate (IP3 )-linked agonists, namely, vasopressin and thenus.16 Moreover, there is morphological evidence for gra-a1-adrenergic agonist phenylephrine, induce coordinateddients of vasopressin15,17 and glucagon receptors18 alongcalcium ion responses that spread from one cell to thethe hepatocyte plate.other in a constant wave-like fashion.4,5 Localized or

Thus, the position of the different hepatocytes in thepropagated variations of intracellular Ca2/ concentration([Ca2/]i ) have been studied in pancreatic acinar cells6–9

Abbreviations used in this paper: [Ca2/]i , intracellular Ca2/ concen-and other epithelial cells (see Sanderson et al.10 for re- tration; EGTA, ethylene glycol-bis (b-aminoethyl ether)-N,N,N*,N*-tet-

raacetic acid; GS, glutamine synthetase; IP3, inositol 1,4,5-trisphos-view). More recently, we have shown that multiplets (3–phate; IP3R, inositol 1,4,5-trisphosphate receptor; PC, pericentral;5 cells), which are more complex multicellular systemsPP, periportal; quin2-AM, quin2–acetoxymethyl ester.

of rat hepatocytes and intact parts of the hepatocyte plate, q 1996 by the American Gastroenterological Association0016-5085/96/$3.00exhibit highly organized hormone-mediated Ca2/ sig-

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1344 TORDJMANN ET AL. GASTROENTEROLOGY Vol. 111, No. 5

scribed.31 Before each experiment, cells were loaded with theacinus and in a multiplet directly obtained from hepato-Ca2/-sensitive indicator quin2–acetoxymethyl ester (AM).cyte plates may determine cellular sensitivity to Ca2/-Briefly, an aliquot corresponding to 106 cells was incubatedmobilizing agonists. To test this hypothesis, we selec-with 50 mmol/L quin2-AM for 180 seconds, centrifuged,tively isolated rat hepatocytes from periportal (PP) andwashed, and resuspended in 2 mL of Dulbecco’s modified Eaglepericentral (PC) areas by digitonin-collagenase perfusionmedium. The cell suspension was then transferred to the oxy-

and analyzed the responses of each cell population togenated spectrofluorimeter cuvette (under O2/CO2, 19:1) and

several Ca2/-mobilizing hormones, namely, noradrena- agitated with a magnetic barrel at 377C (JY3D fluorometer;line, vasopressin, ATP, and angiotensin II. These four Jobin & Yvon, Longjumeau, France). Calcium movements wereagents stimulate influx of Ca2/ from the extracellular measured from the observed changes in the fluorescence ofmedium19–21 and mobilize Ca2/ from the intracellular quin2 (excitation wavelength, 340 nm; emission wavelength,IP3-sensitive compartments.22–25 We show that PP and 492 nm). At the conclusion of each experiment, the fluores-

cence signal was calibrated by making the cell plasma mem-PC hepatocytes do not have the same sensitivity to abrane permeable with 5 mmol/L digitonin (quin2 saturationgiven agonist. Thus, the sequential pattern of Ca2/ wavesby external Ca2/ gave maximal fluorescence) and then by add-in multicellular systems of rat hepatocytes and in theing 20 mmol/L ethylene glycol-bis(b-aminoethyl ether)-intact liver could correspond to gradual heterogeneity ofN,N,N*,N*,-tetraacetic acid (EGTA) to the suspension of lysedcellular sensitivity to Ca2/-mobilizing hormones withincells to obtain the minimal fluorescence.

the hepatic acinus.Experiments in Ca2/-free medium were performed to evalu-

ate extracellular Ca2/ entry during hormone stimulation. TheMaterials and MethodsCa2/ influx was suppressed by adding EGTA (4 mmol/L final

Isolation of Hepatocytes concentration) to the bathing solution. In these conditions, weimposed an extracellular Ca2/ concentration of approximatelyDigitonin-collagenase perfusion. PP and PC rat hepa-80 nmol/L. The hormone was then quickly added to the me-tocytes were isolated by the digitonin-collagenase perfusiondium. After the [Ca2/]i had increased to a maximal level, 4technique as described by Quistorff26 and Lindros and Pent-mmol/L ionomycin was quickly added to estimate the totaltila.27 Briefly, the liver was perfused for 5 minutes withintracellular Ca2/ pool. The fluorescence signal was then cali-Krebs’–Henselheit bicarbonate buffer, which was equilibratedbrated as previously described.31

with oxygen and carbon dioxide (19:1). A 4 mg/L digitoninsolution was then infused at the rate of 10 mL/min for 15– 45Ca2/ Release by Permeabilized PP and20 seconds in the antegrade direction via the portal vein or PC Hepatocytes45–55 seconds in the retrograde direction through the caudal

45Ca2/ release from saponin-treated cells. IP3-in-vena cava, i.e., until a regularly scattered perivenous or PPduced Ca2/ release was measured as previously described.32

decoloration pattern on the liver surface was obtained. Digito-Briefly, hepatocytes (2.5 1 106 cells/mL) were incubated atnin was washed out by perfusion of a Ca2/-free solution in theroom temperature in an internal medium and permeabilized.opposite direction for 10 minutes at a rate of 20 mL/min. TheThe medium (100 mmol/L KCl, 20 mmol/L NaCl, 5 mmol/collagenase was then perfused via the portal vein in all theL MgCl2 , 0.96 mmol/L NaH2PO4, and 25 mmol/L HEPESexperiments. The subsequent steps of the isolation were identi-buffer at pH 7.15 [with KOH]) was supplemented with 50cal to those for conventional dispersion of rat liver cells.28 Cellmg/mL saponin, 1.5 mmol/L Na2ATP, 5 mmol/L creatine phos-viability, assessed by trypan blue exclusion, was consistentlyphate, 5 U/mL creatine phosphokinase, and 20 mmol/L quin2.ú90%.Within a few minutes, saponin permeabilized the plasma

The enrichment of cell suspensions in PP and PC hepato-membrane but not the membrane enclosing the internal Ca2/

cytes was monitored by measurements of glutamine synthetasestores, which retained their Ca2/ accumulation activity. After

(GS) (PC marker) and alanine aminotransferase (ALT) (PPthis treatment, more than 98% of the cells were freely perme-

marker).able to trypan blue. Uptake was initiated by addition of 2mCi/mL 45Ca2/ tracer. External free Ca2/ was monitored con-Enzyme Assaystinuously by observing quin2 fluorescence in the spectrofluo-

GS activity was determined as previously described,29rimeter cuvette. After steady-state loading of the cellular

and ALT was assayed with a commercial kit (Sigma Chemical stores, the 45Ca2/ efflux was measured by diluting 50-mL ali-Co., St. Louis, MO) as described by the manufacturer. Protein quots of the cell suspension in 1 mL of the internal mediumwas assayed according to Lowry et al.30

without saponin, quin2, or the regenerating system but supple-mented with 40Ca2/ and EGTA to buffer the free Ca2/ concen-

Spectrofluorimetry tration at pCa 7. IP3 was added 5 seconds after dilution ofAfter isolation, the cells were incubated in Dulbecco’s 45Ca2/-loaded cells. Ten seconds after IP3 addition, cells were

modified Eagle medium supplemented with amino acids, vita- filtered through a Whatman GF/C glass-fiber filter (Whatmanmins, and gelatin at room temperature and then gassed with Chemical Separation Inc., Clifton, NJ), washed, and counted

for radioactivity in a scintillation counter.O2/CO2 (19:1) at pH 7.4 until later use, as previously de-

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HEPATOCYTE HETEROGENEITY AND Ca2/ SIGNALS 1345November 1996

Materials [Ca2/]i signals in single hepatocytes cannot be easilycompared as a result of the large interindividual variabil-Collagenase was from Boehringer Mannheim GmbH

(Mannheim, Germany). Digitonin was purchased from Merck ity.35–37 Cells were isolated and loaded with the Ca2/-(Darmstadt, Germany). Williams’ medium E was from GIBCO sensitive indicator quin2-AM. The basal [Ca2/]i , the(Paisley, Scotland). All other chemicals were from Sigma [Ca2/]i after stimulation, and the time constant of theChemical Co. [Ca2/]i increase were measured. The magnitude of the

response was normalized as the ratio between [Ca2/]iStatistical Methodsunder hormonal stimulation and basal [Ca2/]i .Analysis of variance (ANOVA) and Student’s t test

comparisons with paired controls were used. Results are ex- The [Ca2/]i increase was plotted as a function of thepressed as means { SE. P values of ° 0.05 were considered applied noradrenaline or vasopressin concentration. Thestatistically significant. magnitude of the response to 1008 and 1007 mol/L nor-

adrenaline and to 10010, 1009, and 1008 mol/L vasopres-Resultssin was significantly greater in PC than in PP hepatocytesCharacterization of Cell Populations(Figure 1A and B), and the time constant of the [Ca2/]iIsolated by Digitonin-Collagenaseincrease was significantly less in PC than in PP hepato-Perfusioncytes (not shown). The differences were not significantComparability. To validate the comparison ofat higher concentrations. The vasopressin concentration-Ca2/ responses in PP and PC cells after digitonin-colla-response curve was bell shaped. This phenomenon maygenase perfusion, several variables were measured in bothresult from a desensitization of hormone receptors at highcell populations. First, cell viability assessed by trypanconcentrations of the agonist.38

blue exclusion (94% { 3.7%, n Å 21 for PP cells; 93%Therefore, the apparent sensitivity of hepatocytes to{ 3.5%, n Å 21 for PC cells) and cell yields (130 {

noradrenaline and vasopressin was greater in the PC than66 1 106 PP cells per liver, n Å 21; 160 { 67 1 106

in the PP area.PC cells per liver, n Å 21) were similar for PP and PCATP. The ATP-induced Ca2/ responses of PP andcells. Second, protein content was indistinguishable in

PC hepatocytes also differed; however, in contrast to nor-PP (2.28 { 0.52 mg/106 cells, n Å 15 in nine experi-adrenaline- and vasopressin-induced increases of [Ca2/]i ,ments) and PC (2.53 { 0.71 mg/106 cells, n Å 15 inPC cells were less sensitive to ATP than PP cells. Thenine experiments) hepatocytes. These values are consis-magnitude of the increases of [Ca2/]i as a function oftent with previously published data.33 Third, basalATP concentration was significantly greater in PP than[Ca2/]i measured in quin2-loaded cell suspensions wasin PC hepatocytes (Figure 2A). The differences were sta-similar in PP and PC cells (178 { 6 nmol/L and 185 {tistically significant for 2.0 1 1007 and 1006 mol/L ATP6 nmol/L, respectively; n Å 168) and comparable to thebut not at higher concentrations. The time constant ofresting value of [Ca2/]i previously determined in isolated

hepatocytes.31 These observations indicate that the dig- the ATP [Ca2/]i increase was less in PP than in PCitonin-collagenase technique does not itself introduce any hepatocytes (not shown). The same hepatocyte popula-detectable modification of PP or PC hepatocytes that tion (either PP or PC cells) responded inversely to themay amplify or diminish Ca2/ responses. agonists ATP and vasopressin in the presence of extracel-

Zonal purity. Standard enzymatic markers were lular Ca2/ (Figure 3). The magnitude of vasopressin-assayed to assess the zonal purity of each cell population. mediated Ca2/ response was less but the magnitude ofGS is strictly confined to the last two or three rings of ATP-mediated Ca2/ response was greater in PP than inhepatocytes surrounding the central venule,34 and ALT PC cells (Figure 3).is distributed along a gradient from the PP to the PC Thus, the ATP-stimulated hepatocytes in the PP areaarea.16 The PC/PP ratio of GS was 40 (1.80 { 0.7 PC were more sensitive to the agonist than those in the PCcells/tissue; n Å 16). The PC/PP ratio of ALT was 0.80 area.(0.77 { 0.19 PC cells/tissue; n Å 16). Thus, each cell Angiotensin II. [Ca2/]i changes in PP and PC he-population was significantly enriched with PP or PC patocytes in response to angiotensin II were also studiedhepatocytes, as previously reported.33

by spectrofluorimetry. Angiotensin II induced similarAgonist-Evoked Calcium Responses in PP [Ca2/]i increases in both cell populations (Figure 2B).and PC Hepatocytes The time constant of angiotensin II–induced [Ca2/]i in-

creases was similar in PP and PC hepatocytes (notNoradrenaline and vasopressin. Calcium re-shown).sponses of PP and PC quin2-loaded hepatocyte popula-

tions were studied using spectrofluorimetry31 because Thus, the sensitivity of hepatocytes of these two cell

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populations to the agonist angiotensin II appeared to be PP and PC cells were observed in the absence of extracel-lular Ca2/. We also measured the hormone-sensitive andsimilar.

Calcium responses in Ca2/-free medium. The ionomycin-sensitive Ca2/ pools. EGTA was added to thebathing medium, and the agonist and then ionomycindifferent sensitivities of PP and PC cells to Ca2/-mobiliz-

ing agonists may result from an intrinsic difference in the were also quickly added to prevent any Ca2/ loss fromthe internal stores to the extracellular medium (see Mate-transduction pathway between the cell surface (hormone

receptor, Ca2/ influx) to the intracellular Ca2/ stores rials and Methods). The agonist concentrations used werethose at which significant differences in the [Ca2/]i in-(IP3 receptor [IP3R], Ca2/ content). We first investigated

whether the differences in hormonal response between

Figure 1. Effect of noradrenaline and vasopressin on [Ca2/]i in PP Figure 2. Effect of ATP and angiotensin II on [Ca2/]i in PP and PChepatocytes in the presence of extracellular Ca2/. The magnitude ofand PC hepatocytes in the presence of extracellular Ca2/. The magni-

tude of the [Ca2/]i increase was estimated as a relative [Ca2/]i in- the [Ca2/]i increase was estimated as a relative [Ca2/]i increase (seeResults). The values are plotted against hormone concentration. Thecrease (see Results). The values are plotted against hormone concen-

tration. The data are means { SE from n (number in parentheses on data are means { SE from n (number in parentheses on the x-axis)separate experiments in each cell population. *P Å 0.05; **P Åthe 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-in- 0.02; ***PÅ 0.01; ****PÅ 0.001. (A) ATP-induced [Ca2/]i changesin PP and PC hepatocytes. Basal [Ca2/]i was 148 { 7.7 nmol/L 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 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.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) 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.and 188 { 9.1 nmol/L in PC (n Å 70) hepatocytes.

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crease were observed in the presence of extracellular Ca2/ significance at the same concentrations compared withthe differences in the presence of extracellular Ca2/ (Fig-(Figures 1 and 2). Maximal concentrations of agonists

were used to measure the hormone-sensitive Ca2/ pool. ures 1 and 2). In contrast, the differences in response tonoradrenaline stimulation in the presence of Ca2/ wereThe magnitude of the agonist-induced [Ca2/]i increase

was calculated as the ratio of increases between the hor- not observed in Ca2/-free conditions (Figure 4A). Angio-tensin II–induced [Ca2/]i responses were similar in bothmone-induced and basal [Ca2/]i . Ionomycin-induced

[Ca2/]i increases were similar in PP and PC cells (Figures cell populations (Figure 5B). Table 1 shows the particularincreases of [Ca2/]i at maximal agonist concentration in4–6). In Ca2/-free conditions, the magnitude of the

[Ca2/]i increase in response to vasopressin was greater inPC than in PP cells (Figure 4B). A downturn phenome-non was observed in the vasopressin concentration-re-sponse curve, which was similar to that in the presenceof extracellular Ca2/. Moreover, ATP induced [Ca2/]i

responses of greater magnitude in PP than in PC hepato-cytes (Figure 5A). The same cell population, either PPor PC, responded inversely to ATP and vasopressin inthe absence of extracellular Ca2/ (Figure 6). The observeddifferences in the increase of [Ca2/]i reached statistical

Figure 4. Effect of noradrenaline and vasopressin on [Ca2/]i in PPand PC hepatocytes in Ca2/-free conditions. The [Ca2/]i increase isplotted against hormone concentration. The data points are means{ SE from n (number in parentheses on the x-axis) separate experi-ments for each cell population. *PÅ 0.05; **PÅ 0.02; ***PÅ 0.01;****P Å 0.001. For agonist-mediated [Ca2/]i increases, ionomycin(IONO) data are expressed as the ratio between ionomycin [Ca2/]iincrease after agonist treatment and basal [Ca2/]i . (A) Noradrenaline-induced [Ca2/]i changes in PP and PC hepatocytes. Basal [Ca2/]i wasFigure 3. Ca2/ responses of two PP and PC hepatocyte suspensions

to hormone in the presence of extracellular Ca2/ during the same 129 { 5.4 nmol/L in PP (n Å 32) and 132 { 6.3 nmol/L in PC (n Å32) hepatocytes. (B) Vasopressin-induced [Ca2/]i changes in PP andspectrofluorimetry experiment. Submaximal concentrations of ATP

(1006 mol/L) and vasopressin (1008 mol/L) were used. VP, vasopres- PC hepatocytes. Basal [Ca2/]i was 139 { 11.1 nmol/L in PP (n Å16) and 109 { 5.2 nmol/L in PC (n Å 16) hepatocytes.sin.

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the absence and presence of extracellular Ca2/ for each to the [Ca2/]i increase. In contrast, the similarity of PPand PC cell responses to noradrenaline in Ca2/-free con-agonist.ditions indicates that the Ca2/ influx is partly responsibleThus, the differences between PP and PC hepatocytefor the differences observed in the presence of extracellu-responses to vasopressin and ATP observed in the pres-lar Ca2/. In addition, unequal [Ca2/]i responsivenessesence of extracellular Ca2/ were also found in Ca2/-freein PP and PC cells did not appear to be caused by anyconditions, which suggests that the differences were notquantitative disparity in the size of intracellular Ca2/solely caused by unequal contributions of the Ca2/ influxstores sensitive to the ionomycin and agonists tested.

IP3-Induced 45Ca2/ Release in PP and PCPermeabilized Cells

IP3-induced Ca2/ responses in permeabilized cellsfrom both acinar origins were analyzed to investigatewhether the differences of the Ca2/ signals in PP andPC cells resulted from differences in IP3R sensitivity.In these cells, all the steps upstream from the IP3Roccupancy were bypassed. The loading kinetics of the in-tracellular Ca2/ stores and the size of IP3- and ionomycin-sensitive Ca2/ pools in both cell populations were ana-lyzed. PP and PC hepatocytes were permeabilized withsaponin 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 PChepatocytes in Ca2/-free conditions. The [Ca2/]i increase is plottedagainst hormone concentration. The data points are means { SEfrom n (number in parentheses on the x-axis) separate experimentsfor 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/]iincrease after agonist treatment and basal [Ca2/]i . (A) ATP-induced Figure 6. Ca2/ responses in two suspensions of PP and PC hepato-

cytes to hormone, during the same spectrofluorimetry experiment, in[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) the absence of extracellular Ca2/. Submaximal concentrations of ATP

(1006 mol/L) and vasopressin (1008 mol/L) were used. The magnitudehepatocytes. (B) Angiotensin II– induced [Ca2/]i changes in PP andPC hepatocytes. Basal [Ca2/]i was 178 { 14.7 nmol/L in PP (n Å of ionomycin-induced [Ca2/]i increase was similar in PP and PC cells.

VP, vasopressin; Iono, ionomycin.13) and 139 { 13 nmol/L in PC (n Å 13) hepatocytes.

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Table 1. Effect of Ca2/ Removal From the Extracellular Medium on Agonist-Induced [Ca2/]i Increases in PP and PCHepatocytes Stimulated With Maximal Agonist Concentrations

Agonist-mediated [Ca2/]i increase (nmol/L)

CaN CaO

Agonist PP n PC n PP n PC n

Noradrenaline (10 mmol/L) 610 { 166 5 669 { 140 5 310 { 25 12 284 { 16 12Vasopressin (100 nmol/L) 770 { 97 12 920 { 74 12 220 { 22 8 179 { 14 8ATP (10 mmol/L) 582 { 44 4 590 { 82 4 396 { 50 4 371 { 37 4Angiotensin II (1 mmol/L) 1149 { 44 4 959 { 143 4 374 { 50 4 318 { 6 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 Ca2/ signals along the hepatocyte plate in the intact liverhave also been described.14,15 Moreover, V1a vasopressinisolated by conventional collagenase perfusion.39 The IP3

dose-response curves (Figure 7A) were very similar for receptor messenger RNAs, detected by in situ hybridiza-tion in the rat liver, are predominantly found in theboth PP and PC hepatocyte populations (50% effective

concentration, 105 { 22 nmol/L and 125 { 36 nmol/ perivenular zone,17 and a similar distribution has beendescribed for the glucagon receptor by autohistoradiogra-L, respectively; n Å 4). These values are in agreement

with previous observations of permeabilized rat hepato- phy.18 These different in situ morphological studies sug-gest a gradient in the number of receptors from one acinarcytes.23,32 The size of the IP3-sensitive compartment was

similar in PP and PC cells and represented 26% { 2.2% zone to the opposite one, but the hormone-mediated Ca2/

mobilization activity in PP and PC hepatocytes has neverand 31.7%{ 4.2%, respectively, of the ionomycin-sensi-tive compartment (n Å 4), i.e., 2.17 { 0.23 Ca2//mg been directly investigated.protein for PP cells and 2.03 { 0.18 Ca2//mg protein In this study, hepatocytes isolated from different zonesfor PC cells (n Å 4) (Figure 7B). of the liver acinus after digitonin-collagenase perfusion

Thus, the different sensitivities to Ca2/-mobilizing were tested for heterogeneous cellular sensitivity. Thehormones of hepatocytes from PP and PC areas do not apparent cellular sensitivity to the agonists was not iden-appear to be caused by differences concerning IP3-IP3R tical in PP and PC hepatocyte suspensions. Also, one cellinteractions or IP3-sensitive compartment size. Presum- population could be more sensitive to one agonist butably, the upstream steps (i.e., mainly hormone-receptor less sensitive to another. PC cells were more sensitiveinteractions, G protein activation, and phospholipase C- than PP cells to noradrenaline and vasopressin, but PPb activity) are involved in this heterogeneity of cellular cells were more responsive than PC cells to ATP. PP andresponses. Moreover, our data suggest that there are simi- PC cells were equally sensitive to angiotensin II.lar kinetics for the Ca2/ loading of intracellular stores The differences between PP and PC hepatocyte popu-and similar sizes of ionomycin-sensitive Ca2/ pools in lations in the magnitude and the rate of [Ca2/]i increasePP and PC hepatocytes. were significant mostly at low and intermediate agonist

concentrations. At the highest doses used, PP and PCDiscussion cells were equally responsive. This suggests that the dif-

ferences for intermediate concentrations are not causedHepatocytes contribute unequally to various liverby a subset of cells from the PP (for noradrenaline andfunctions depending on their location in the lobulus.16

vasopressin) or PC (for ATP) area that could not respondHepatocyte heterogeneity within the liver cell plate isto the agonist whatever the concentration. Also, equalparticularly well established for the metabolism of carbo-responsiveness to the highest agonist concentrations indi-hydrates,40 amino acids,41 and ammonia.41,42 Ca2/ metab-cates that PP and PC hepatocytes have the same abilityolism and hormone-mediated Ca2/ movements pre-to respond to maximal hormonal stimulation. This isviously have not been investigated in association withconsistent with a previous [Ca2/]i imaging study on iso-hepatocyte heterogeneity. We suggested previously11,12

lated rat hepatocytes in which all the cells in a videomi-that sequential hormone-mediated Ca2/ signals in multi-croscope field were responsive to the hormone, providedcellular systems were caused by an in situ gradual hetero-a high concentration was used.11geneity of one or more elements in the hormone signal

transduction pathway. Vasopressin-evoked sequential Hepatocyte sensitivity to two different Ca2/-mobiliz-

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in rat hepatocyte multiplets, which suggests intercellularheterogeneity in the number and/or the affinity of hor-monal receptors.11,12,44

We examined the apparent sensitivity of the IP3R inpermeabilized cells in which all the transduction stepsupstream from the binding of IP3 to its receptor werebypassed. IP3 dose-response curves were very similar inboth PP and PC hepatocyte populations, suggesting anequal sensitivity of the IP3R for IP3 in PP and PC cells(Figure 7). We also investigated intracellular Ca2/ com-partments in PP and PC cells. The size of hormone-sensitive (in intact cells), IP3-sensitive (in permeabilizedcells), and ionomycin-sensitive (in both intact and per-meabilized cells) Ca2/ stores were similar in the twohepatocyte populations. The loading kinetics of the Ca2/

pools (estimated by the half-time of 45Ca2/ uptake) wasalso similar in PP and PC cells, suggesting that Ca2/,Mg2/–adenosine triphosphatase of the endoplasmic re-ticulum was equally active in both hepatocyte popula-tions. Therefore, the observed acinar heterogeneity incellular responses to Ca2/-mobilizing agonists is presum-ably due to a heterogeneous distribution of the IP3 pro-duction (affecting, for example, hormonal receptor den-sity and/or affinity). Any step in the production of IP3

could be involved in this sensitivity gradient, althoughFigure 7. IP3-induced 45Ca2/ release in PP and PC permeabilized he- no data suggest a heterogeneous distribution of G pro-patocytes. PP (●) and PC (s) cells were isolated, permeabilized, and

teins, phosphatidylinositol 4,5-bisphosphate, or phos-loaded with 45Ca2/ in the spectrofluorimeter cuvette at 207C as de-pholipase C-b in the liver lobulus. These results, in addi-scribed in Materials and Methods. After equilibration, aliquots were

transferred to a cytosol-like efflux medium buffered at pCa7. Ca2/ tion to [Ca2/]i imaging in multicellular systems of ratrelease was measured after addition of various concentrations of IP3 hepatocytes4,5,11,12 and in the intact liver,14,15 suggest thatto the efflux medium 5 seconds after cell dilution (see Materials

this hepatocyte heterogeneity is fine (subtle cell-to-celland Methods). Data are means { SE of four determinations in fourexperiments in each cell population. (A) IP3 dose–response curves differences) and gradual (sequential responses). Hepato-for PP and PC hepatocyte populations were very similar (PP, EC50 Å cytes possessing a more efficient IP3 production mecha-105 { 22 nmol/L; PC, 125 { 36 nmol/L). IP3-sensitive Ca2/ pools

nism (for example, more or higher affinity hormonal re-(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 ceptors) should display the highest rate of IP3 generation,4) cells. (B) The IP3-sensitive Ca2/ pools calculated as a percentage highest [Ca2/]i oscillation frequency, shortest hormonalof ionomycin-sensitive Ca2/ pools were also similar in PP (31.7 {

delay, and consequently, greatest sensitivity to the ago-42; n Å 4) and PC (26 { 2.2; n Å 4) hepatocytes. The Ca2/-loadingnist.35,44–46 Consequently, different apparent cellular sen-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 sitivities of PP and PC cells should correlate with dispari-minutes; n Å 5) hepatocytes. ties in hormonal latency along the hepatocyte plate.

Angiotensin II induced similar Ca2/ signals in the twocell populations, suggesting a homogeneous hepatocyteing agonists (vasopressin and ATP) is opposite in PP andsensitivity to this agonist in the acinus. However, angio-PC areas (Figures 3 and 6). If vasopressin and ATP sharetensin II–mediated Ca2/ signals in multicellular systemsthe same intracellular mechanism for Ca2/ mobiliza-are sequential.12,13 Possibly, there is a very shallow sensi-tion,22–25,35,43 the differences between these populationstivity gradient through the acinus, only detectable byprobably involve extracellular steps of the transduction[Ca2/]i imaging, which thus reveals very small cell-to-pathway (i.e., hormonal receptor activation). These re-cell differences.sults confirmed that hepatocyte responsiveness was not

affected by the isolation technique because PP and PC In the intact liver, vasopressin-mediated intercellularCa2/ waves are initiated at one extremity of the hepato-cells retain their characteristic sensitivity to different ag-

onists. Also consistent with these results, different ago- cyte plate and then spread across the acinus toward theopposite extremity; the orientation (PP to PC or PC tonists can produce opposite sequences of Ca2/ responses

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PP) of the wave remains controversial.14,15 Our results heterogeneity, making these systems different from otherepithelia in which only diffusion of the intracellular mes-with isolated PP and PC hepatocytes suggest that the in

situ vasopressin-mediated intercellular Ca2/ wave should senger IP3 through gap junctions is considered essen-tial.10 Hepatic gap junctions are freely permeable to ionsbe initiated in the PC area by intermediate hormone

concentrations, which was found by one in situ study.15 and small molecules.1–3,5 Therefore, the diffusion of anintercellular messenger through these junctions probablyHowever, another in situ study reported a more complex

pattern of vasopressin-elicited Ca2/ waves in which the contributes to the coordination of [Ca2/]i oscillations inrat hepatocyte multiplets.11,5 Thus, orientation of theorientation of the wave was dependent on the applied

hormone concentration.14 PP to PC waves were observed wave by a sensitivity gradient and coordination of [Ca2/]i

oscillations by intercellular diffusion of IP3 may combineat low agonist concentration, and PC to PP waves wereobserved at high hormone concentration. We suggest to organize complex Ca2/ signals in the liver lobulus. In

the hepatocyte plate, the gradient of cellular sensitivitythat hepatocyte sensitivity to Ca2/-mobilizing agonistsis a major factor that determines the orientation of the to the hormones along the portocentrilobular axis may

be a driving force for the different Ca2/-mediated pro-intercellular wave in the acinus, but the results reportedpreviously14 underline a probably more complex in situ cesses that occur in an oriented manner, mainly bile

canaliculi network motility50 and bile secretion. Hepato-regulation of intercellular Ca2/ signaling. Moreover, ourresults suggest that during maximal vasopressin stimula- cyte heterogeneity in hormonal delay in the intact liver

may allow the successive activation of the cells along thetion, all the hepatocytes in the plate would be expectedto respond at the same time with multiple initiations of plate, even if the circulating hormone has already occu-

pied the surface receptors of the whole hepatocyte cord.[Ca2/]i increase and possibly multidirectional waves.Also, intermediate concentrations of vasopressin and Furthermore, inversely proportional metabolic capacity

and cellular sensitivity to the agonists in the same hepa-ATP could generate Ca2/ waves in opposite directionsacross the acinus. tocyte area may allow the acinus to function in an optimal

and coordinated manner.Gluconeogenesis enzymes are preferentially localizedin PP zone,16 but there is no consensus about the glyco-

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