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HORMONES AND SIGNALING

Effects of the prostaglandins PGF₂ and PGE₂ on calcium signaling in rat hepatocyte doublets

¹Institut National de la Santé et de la Recherche Médicale Unité 442, Université Paris-Sud, Orsay, France; and ²Laboratoire de Pharmacologie et d'Hormonologie, Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin

Submitted 24 February 2005 ; accepted in final form 29 July 2005

	ABSTRACT

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Coordination of intercellular Ca²⁺ signals is important for certain hepatic functions including biliary flow and glucose output. Prostaglandins, such as PGF₂ and PGE₂, may modify these hepatocyte functions by inducing Ca²⁺ increase, but very little is known about the organization of the Ca²⁺ signals induced by these agonists. We studied Ca²⁺ signals induced by PGF₂ and PGE₂ in fura-2 AM-loaded hepatocyte doublets. Even though both prostaglandins induced Ca²⁺ oscillations, neither PGF₂ nor PGE₂ induced coordinated Ca²⁺ oscillations in hepatocyte doublets. Gap junction permeability (GJP), assessed by fluorescence recovery after photobleaching, showed that this absence of coordination was not related to a defect in GJP. Inositol (1,4,5)trisphosphate [Ins(1,4,5)P₃] assays and the increase in Ins(1,4,5)P₃ receptor sensitivity to Ins(1,4,5)P₃ observed in response to thimerosal suggested that the absence of coordination was a consequence of the very small quantity of Ins(1,4,5)P₃ formed by these prostaglandins. Furthermore, when PGE₂ and PGF₂ were added just before norepinephrine, they favored the coordination of Ca²⁺ signals induced by norepinephrine. However, GJP between hepatocyte doublets was strongly inhibited by prolonged (2 h) treatment with PGF₂, thereby preventing the coordination of Ca²⁺ oscillations induced by norepinephrine in these cells. Thus, depending on the time window, prostaglandins, specially PGF₂, may enhance or diminish the propagation of Ca²⁺ signals. They may therefore contribute to the fine tuning of Ca²⁺ wave-dependent functions, such as nerve stimulation, hormonal regulation of liver metabolism, or bile secretion, in both normal and pathogenic conditions.

calcium oscillations; gap junction permeability

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	MATERIALS AND METHODS

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Materials. Williams' medium and F-12 medium were obtained from Life Technology (Invitrogen), and collagenase type A was obtained from Boehringer (Roche Diagnostics). Prostaglandins were obtained from TEBU (BioMol). PGF₂ and PGE₂ receptor primers for RT-PCR and other chemicals were purchased from Sigma.

Hepatocyte preparation. Single hepatocytes and multicellular systems were prepared from fed female Wistar rats by limited collagenase digestion of the rat liver, as previously described (8). Experiments were conducted according to European Community directives for animal experimentation (Decree 2001-131, Official Journal 06/02/01). After isolation, rat hepatocytes were maintained (2 x 10⁶ cells/ml) at 4°C in Williams' medium E supplemented with 10% fetal calf serum, penicillin (200,000 U/ml), and streptomycin (100 mg/ml). Cell viability, assessed by trypan blue exclusion, remained >96% for 4–5 h.

WIF-B9 culture. The WIF-B9 cell line, obtained from a rat hepatoma cell line-human fibroblast fusion that forms functional bile canaliculus (23), was provided by D. Cassio (Institut National de la Santé et de la Recherche Médicale Unitè 442) and was maintained in Coon's modified F-12 medium supplemented with 5% FBS, as previously described (23).

Determination of Ca²⁺ changes in hepatocytes. Hepatocytes were plated on glass coverslips coated with type I collagen and loaded by incubation for 40 min with 3 µM fura-2 AM in modified Williams' medium (37°C, 5% CO₂). Coverslips were washed and transferred to a perfusion chamber placed on the stage of a Zeiss (Axiovert 35) inverted microscope. Ca²⁺ imaging was performed as described previously (8). Fluorescence images were collected with a charge-coupled device camera, digitized, and integrated in real time by an image processor (Metafluor). Results (R/R₀) were expressed as ratios between 340 and 380 fluorescence signals measured during a response divided by the ratio measured in resting conditions, i.e., before the addition of an agent.

Assessment of gap junction permeability. Gap junction permeability (GJP) was assayed in hepatocyte doublets and WIF-B9 cells using the fluorescence recovery after photobleaching (FRAP) method. FRAP experiments were performed on hepatocyte doublets and WIF-B9 cells loaded with calcein using an inverted confocal microscope (Nikon EZC1). Briefly, the fluorescence emitted by calcein in one of the two connected hepatocytes or in a WIF-B9 cell was bleached by focusing the laser (488 nm, 100% intensity) on this defined region. The recovery of fluorescence in the bleached cells was monitored by measuring the diffusion of calcein through gap junctions over time (see Figs. 3 and 7).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of a brief incubation with F2 and E2 on gap junction permeability (GJP) in hepatocyte doublets. Hepatocytes loaded with calcein were treated with F2 (1 µM) or E2 (1 µM) for 10 min before fluorescence recovery after photobleaching (FRAP). Cells were then imaged by confocal microscopy, and one cell was bleached as described in MATERIALS AND METHODS. Top: typical images of a hepatocyte doublet loaded with calcein before (1), immediately (2), and about 4 min (3) after photobleaching. In this case, the lower cell was photobleached. Bottom: typical fluorescence recovery (FR) curves for control hepatocyte doublets () and connected hepatocytes in the presence of F2 () or E2 (). Inset: bar graph showing means ± SE of the maximum percentages of FR in control (C) hepatocyte doublets and those in the presence of F2 or E2. The number of doublets (n) included in each experiment is given within the bars.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of a brief incubation with F2 and E2 on GJP in hepatocyte doublets. Hepatocytes loaded with calcein were treated with F2 (10 µM) or E2 (10 µM) for 30 min before FRAP. Cells were then imaged by confocal microscopy, and one cell was bleached as described in MATERIALS AND METHODS. Typical FR curves for control hepatocyte doublets () and connected hepatocytes in the presence of F2 () or E2 () are shown. Inset: bar graph showing means ± SE of the maximum percentages of FR in control hepatocyte doublets and those in the presence of F2 or E2. The number of doublets included in each experiment is given within the bars.

Ins(1,4,5)P₃ assay. Cell suspensions (5 x 10⁵ cells) were washed twice with PBS, and Ins(1,4,5)P₃ concentrations were determined in neutralized supernatants with a radiochemical-binding assay kit {[³H]Ins(1,4,5)P₃ Biotrak Assay System, Amersham Biosciences} following the manufacturer's instructions.

RT-PCR. Total RNA was extracted from freshly isolated hepatocytes and WIF-B9 cells cultured in 35-cm² dishes using Trireagent (Sigma) according to the manufacturer's recommendations. Reverse-transcribed mRNA (cDNA) was amplified by PCR in the presence of the following specific primers: EP1, forward 5'-TGT ATA CTG CAG GAC GTG CGC CC-3' and reverse 5'-GGG CAG CTG TGG TTG AAG TGA TG-3' with a product size of 537 bp; EP3, forward 5'-GCC GGG AGA GCA AAC GCA AAA A-3' and reverse 5'-ACA CCA GGG CTT TGA TGG TCG CCA GG-3' with a product size of 537 bp; and FP, forward 5'-GGC GTT TAT CTC CAC AAC-3' and reverse 5'-CTA GAT GCT TGC TTG CTG ATT-3' with a product size of 1,086 bp.

PCR was carried out in a final volume of 25 µl using 1 µl cDNA, 1x PCR buffer (Invitrogen), 1 µmol/l forward primer, 1 µmol/l reverse primer, 0.2 mmol/l deoxynucleotide triphosphates, 2.25 mmol/l MgCl₂, and 2 units Taq DNA polymerase. PCR conditions were as follows: 94°C for 10 min and then 40 cycles at 94°C for 30s (EP1 and EP3) or 1 min (FP), 69°C for 1 min (EP1 and EP3) or 56°C for 80 s (FP), 72°C for 1 min and an additional 10 min (EP1 and EP3) or 8 min (FP) at 72°C. The final reaction products were subjected to electrophoresis in 2% agarose gels.

	RESULTS AND DISCUSSION

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Characterization of Ca²⁺ signaling induced by PGF₂ and PGE₂ in individual and connected hepatocytes. We compared the Ca²⁺ responses induced by PGF₂ and PGE₂ with those induced by norepinephrine in individual hepatocytes and hepatocyte doublets loaded with fura-2 AM (Fig. 1A). The addition of norepinephrine (1 µM), PGF₂ (5 µM), and PGE₂ (5 µM) induced Ca²⁺ oscillations in isolated hepatocytes. However, most of responses induced by these prostaglandins were simple peaks, suggesting that the efficiency of PGF₂ and PGE₂ was much lower than that of norepinephrine (data not shown). This was confirmed for various concentrations (Fig. 1B). In our experiments, more than 75% of the observed hepatocytes (110 of 136 cells in 5 different experiments) responded to 0.1 µM norepinephrine, whereas, even at 10 µM, PGF₂ and PGE₂ induced Ca²⁺ responses in only 67% and 30% of the cells, respectively (116 cells in 5 different experiments). However, the largest difference between these prostaglandins and norepinephrine was observed with hepatocyte doublets. Norepinephrine induced synchronized Ca²⁺ oscillations in most of the hepatocyte doublets (30 coordinated Ca²⁺ oscillations/44 responding doublets), whereas none of the 17 hepatocyte doublets responding to PGF₂ presented coordinated Ca²⁺ oscillations, because only one cell of the doublet responded (Fig. 2). Similarly, there was no transmission of Ca²⁺ signals and therefore no coordination of Ca²⁺ signals in the presence of PGE₂ (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Ca2+ signals induced by norepinephrine (Nor), PGF2 (F2), and PGE2 (E2) in individual hepatocytes. A: fura-2-loaded hepatocytes were challenged with Nor (1 µM), F2 (5 µM), or E2 (5 µM) for the times indicated by the horizontal bars. The traces shown are representative of the Ca2+ oscillations observed in the presence of these agonists in responding cells in 5 independent experiments. B: percentage of cells responding by an increase in cytosolic Ca2+ concentration to perfusion of 0.1, 1, and 10 µM Nor; 1, 5, and 10 µM F2; or 1, 5, and 10 µM E2. Results are from 5 independent experiments and are expressed as means ± SE.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Ca2+ oscillations induced by F2 and E2 in hepatocyte doublets are not coordinated. Doublets of hepatocytes were loaded with fura-2 and successively stimulated with Nor (0.1 µM) and F2 (5 µM). The left and right portions of the curves show successive measurements of intracellular Ca2+ in the same doublets. Unlike Nor (left), the addition of F2 induced uncoordinated Ca2+ oscillations in connected hepatocytes (right). Traces are shifted arbitrarily along the y-axis for clarity. For technical convenience, traces were interrupted during the washes (5 min). This experiment is representative of those obtained in independent experiments using 8 doublets. Similar results were observed with E2 (not shown).

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Brief treatment of hepatocyte doublets with PGF₂ and PGE₂ did not affect GJP. GJP was determined by measuring the rate and the extent of fluorescence recovery after bleaching as illustrated in Fig. 3. In control doublets, a new fluorescence steady state was reached after 200 s, with a maximal fluorescence recovery of 74 ± 5% of the initial fluorescence (n = 16). The incubation of hepatocyte doublets for 10 min in the presence of PGF₂ (1 µM) or PGE₂ (1 µM) did not affect GJP: maximum fluorescence recovery values of 72 ± 4% (n = 15) and 70 ± 5% (n = 8), respectively, were obtained (Fig. 3). We therefore investigated whether the lack of coordination of Ca²⁺ oscillations induced by these prostaglandins was due to insufficient Ins(1,4,5)P₃ production.

PGF₂ and PGE₂ induced small increases in intracellular Ins(1,4,5)P₃ levels. To understand the role of Ins(1,4,5)P₃ in the lack of coordination of Ca²⁺ oscillations induced by PGF₂ and PGE₂, we first determined the quantity of Ins(1,4,5)P₃ produced by hepatocytes treated with a high concentration (10 µM) of these prostaglandins. In accordance with previous results (2, 11, 28, 49) and in contrast to the results obtained with norepinephrine (10 µM), very little Ins(1,4,5)P₃ was produced after treatment with PGF₂ and PGE₂ (Fig. 4A), suggesting that the quantity of Ins(1,4,5)P₃ produced in response to the two prostaglandins was indeed not sufficient to induce coordinated Ca²⁺ oscillations. We investigated whether a shift in the affinity of the Ins(1,4,5)P₃ receptor [Ins(1,4,5)P₃R] for Ins(1,4,5)P₃ could compensate for the small amount of Ins(1,4,5)P₃ present.

View larger version (20K): [in this window] [in a new window]   Fig. 4. F2- and E2-induced inositol (1,45)trisphosphate [Ins(1,4,5)P3] increase was not sufficient to ensure coordinated Ca2+ oscillations in hepatocyte doublets. A: Ins(1,4,5)P3 level was measured in control cells or in hepatocytes incubated for 1 min in the presence of Nor, F2, or E2. Results are from 3 determinations in 3 independent experiments and are expressed as means ± SE. *P < 0.05 vs. control. B and C: left and right portions of the curves show successive measurements of intracellular Ca2+ in the same doublets of fura-2-loaded hepatocytes. The addition of 5 µM F2 or E2 induced uncoordinated Ca2+ oscillations (B) or no Ca2+ response (C). The right parts of the curves show that effect of these prostaglandins was strongly potentiated in the presence of thimerosal (T; 10 µM), as the Ca2+ oscillation frequency was clearly higher. Moreover, the same concentration of F2 elicited coordinated Ca2+ oscillations (B). Traces are shifted arbitrarily along the y-axis for clarity. For technical reasons, recording of the traces was interrupted during the washing process (3 min, dashed line). Results are representative of those obtained using 6 (B) and 5 doublets (C) in 4 independent experiments.

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Under normal conditions, prostaglandins are released by perisinusoidal cells stimulated by norepinephrine and may therefore act synergistically with this agonist (3, 24). We thus studied the effects of PGF₂ and PGE₂ on Ca²⁺ signaling induced by norepinephrine in hepatocyte doublets.

Effect of short-term perfusion of PGF₂ and PGE₂ on Ca²⁺ responses induced by norepinephrine in hepatocyte doublets. As expected, application of low concentrations of norepinephrine (0.05 µM) to hepatocyte doublets induced uncoordinated Ca²⁺ oscillations, with the production of Ins(1,4,5)P₃ being insufficient to allow for efficient junctional Ins(1,4,5)P₃ diffusion in these conditions (8). After being washed, the same cells were challenged with PGF₂ (1 µM) or PGE₂ (1 µM) for 2 min and then, still in the presence of prostaglandins, the perfusion of norepinephrine was turned on again. In these conditions, the frequency of norepinephrine-induced Ca²⁺ oscillations increased significantly, especially in the presence of PGF₂ (Fig. 5A, right). Moreover, in both conditions, these Ca²⁺ oscillations were very well coordinated (Fig. 5, right).

View larger version (29K): [in this window] [in a new window]   Fig. 5. F2 and E2 increased the Ca2+ response and coordinated Ca2+ oscillations induced by Nor. Hepatocyte doublets were loaded with fura-2. The presence of a low concentration of Nor (0.05 µM) for the time indicated by the bar induced uncoordinated Ca2+ oscillations (A and B). F2 (1 µM; A) or E2 (1 µM; B) were applied as indicated by the open boxes. After the addition of prostaglandins to the bath, Nor elicited high-frequency coordinated Ca2+ oscillations in both cells. For technical reasons, the recording of the traces was interrupted during the washing process (3 min, dashed line) and was shifted arbitrarily along the y-axis for clarity. Results are representative of those obtained using 4 (A) and 5 doublets (B) in independent experiments.

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In other circumstances, for example, in response to endotoxin or other proinflamatory signals, PGF₂, PGE₂, and other prostaglandins are produced in the liver in large amounts over a long time period, mainly by Kupffer cells (25, 45).

We thus studied the effects of long-term incubation with PGF₂ and PGE₂ on the Ca²⁺ signaling induced by norepinephrine in hepatocyte doublets.

Effect of long-term perfusion of PGF₂ and PGE₂ on Ca²⁺ responses induced by norepinephrine in hepatocyte doublets. Hepatocytes were incubated with or without PGF₂ or PGE₂ (1 µM) for 2 h at 37°C and then loaded with fura-2 AM for 30 min (see MATERIALS AND METHODS). Control and treated cells were washed and perfused with norepinephrine (1 µM). In contrast to what was observed with control cells and PGE₂-treated cells, norepinephrine-induced Ca²⁺ oscillations were not coordinated in PGF₂-treated cells (Fig. 6A and data not shown). To understand the cause of the loss of Ca²⁺ coordination in the presence of PGF₂, we examined the permeability of gap junctions in hepatocytes incubated for 2 h with or without PGF₂ and PGE₂. Typical kinetics of FRAP experiments are shown in Fig. 6B and summarized in Fig. 6C. It should be noted that, due to the fast disappearance of connexins in primary cultures of hepatocytes (38), the %FR was lower in control cells plated for 2 h (compare Figs. 3 and 6, B and C). Nevertheless, in contrast with PGE₂, GJP between hepatocyte doublets treated for 2 h with PGF₂ was strongly inhibited (see closed circles in Fig. 6B). The maximum fluorescence recovery for PGF₂-treated hepatocytes was 22 ± 7% (n = 12), whereas that for PGE₂-treated hepatocyte doublets was not significantly different from the control, at 40 ± 9% (n = 8) and 51 ± 3% (n = 12), respectively (Fig. 6C). Interestingly, shorter incubation times in the presence of higher concentrations of PGF₂ (10 µM) did not modify GJP (see Fig. 7), suggesting that the inhibitory effect was not due to activation of a gating mechanism. Instead, these findings indicate the existence of rate-limiting steps such as a reduction in the number of channels. Moreover, alterations in connexin32 expression in the liver have been observed in various forms of liver inflammation (13).

View larger version (35K): [in this window] [in a new window]   Fig. 6. Effect of a prolonged incubation with F2 and E2 on Nor-induced Ca2+ oscillations and GJP in hepatocyte doublets. A: hepatocytes incubated for 2 h with or without prostaglandins (1 µM) were loaded with fura-2 and stimulated with Nor (1 µM). In contrast with control (left) and E2-treated hepatocytes (data not shown), perfusion of Nor induced uncoordinated Ca2+ oscillations in F2-treated cells (right). Traces are shifted arbitrarily along the y-axis for clarity. Results are representative of those obtained using 8 (control) and 7 doublets (F2-treated cells) in 4 independent experiments. B: typical FR curves for control hepatocyte doublets () and hepatocyte doublets incubated for 2 h with E2 () or F2 (). Results are summarized in C and are expressed as means ± SE. The number of doublets included in each experiment is indicated within the bars. *P < 0.05 compared with control.

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Prostaglandin receptors are involved in the inhibitory action of PGF₂ on GJP. We tested the involvement of FP receptors using WIF-B cells. This hepatoma hybrid cell line expresses functional connexin32, which is the most abundant connexin in hepatocytes and communicates efficiently (7, 18) (Fig. 8, B and C). As shown in Fig. 8A, WIF-B cells do not express the FP, EP1, and EP3 prostaglandin receptors normally expressed in hepatocytes (16).

View larger version (47K): [in this window] [in a new window]   Fig. 8. F2 does not modify GJP in cells lacking FP receptors. A: representative RT-PCR analysis of EP1, EP3, and FP mRNA in hepatocytes and WIF-B cells. B: WIF-B cells incubated for 2 h in the absence or presence of F2 (1 µM) were loaded with calcein and imaged by confocal microscopy. FRAP experiments were then performed as described in MATERIALS AND METHODS. Typical images of WIF-B cells loaded with calcein before (1), immediately (2), and about 4 min (3) after photobleaching are shown. *Bleached cell. As shown in C, FR was the same for WIF-B cells incubated in the absence ( and open bar) or presence of F2 ( and shaded bar). In contrast, the addition of octanol (O; 300 µM, 10 min) very efficiently inhibited FR ( and filled bar). Histograms shows means ± SE from 8 independent experiments.

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In summary, we showed that prostaglandins are unable to induce coordinated Ca²⁺ oscillations in hepatocyte doublets but that the presence for a short period of PGE₂, and especially PGF₂, increases the efficiency of norepinephrine to induce Ca²⁺ increases and facilitates the synchronization of Ca²⁺ oscillations induced by this agonist. However, when present for a longer period of time, PGF₂, by inhibiting gap junction permeability, disorganizes Ca²⁺ signals induced in hepatocytes by norepinephrine. These two conditions (contact of the cells with PGE₂ and PGF₂ for a short or long period of time) are reminiscent of well-known physiological and physiopathological situations: stimulation of hepatic sympathetic nerves and inflammation of the liver, respectively. In the first case, full glucose output induced by nerve activation requires the release of both norepinephrine and prostaglandins, especially PGF₂, together with functional gap junctions (24, 32, 40, 42). Potentiation and improvement of coordination of the Ca²⁺ signals induced by norepinephrine in the presence of PGE₂ and PGF₂ may be important because Ca²⁺ signal propagation is crucial for glucose output (15, 32, 41).

Our results are also consistent with observations made in the course of the inflammatory response induced by lipolysaccharides (LPS). Indeed, LPS cause the release of prostaglandins from Kupffer cells, thereby increasing glucose output in early or mild endotoxemia (6, 27). This increase is followed by hypoglycemia and cholestasis in prolonged endotoxemia (25), and it has been suggested that, in such cases, a decrease in GJP may be involved in liver dysfunction (10, 12, 13, 18, 43). This inhibition may be mediated by the release of inflammatory cytokines such as tumor necrosis factor-, interleukin-1, and interleukin-6 by Kupffer cells (18). Our results suggest that PGF₂ may participate in this effect.

Thus PGF₂, depending on the time window of its action, may enhance or diminish the propagation of Ca²⁺ signals and may therefore play a key role in the fine tuning of Ca²⁺ wave-dependent functions, such as nerve stimulation (32), the hormonal regulation of liver metabolism (15, 41), and bile secretion (31), in both normal and pathogenic conditions.

	GRANTS

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O. Koukoui and A. Sezan were supported by an exchange program between Université Paris Sud-XI (France) and Abomey-Calavi (Benin). This work was supported by Association pour la Recherche sur le Cancer Grants Nos. 5457 and 5674.

	ACKNOWLEDGMENTS

 
We thank J. Sappa for the help in editing the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Combettes, Institut National de la Santé et de la Recherche Médicale Unité 442, Bâtiment 443, Université Paris-Sud, 15 rue Georges Clémenceau, 91405 Orsay cedex, France (e-mail: laurent.combettes{at}ibaic.u-psud.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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