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Intracellular calcium in the isolated rat liver: correlation to glucose release, K+ balance and bile flow
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Cell Calcium (2001) 30(6), 403–412 © 2001 Harcourt Publishers Ltd
Research
doi: 10.1054/ceca.2001.0248, available online at http://www.idealibrary.com on
Intracellular calcium in the isolated rat liver: correlation to glucose release, K; balance and bile flow R. Wurzinger,1 R. Englisch,2 S. Roka,1 R. Langer,3 M. Roden,2 J. Graf1 1Department
of Pathophysiology, University of Vienna, Austria 2Department of Internal Medicine III, Division of Endocrinology and Metabolism, University Hospital Vienna, Austria 3Department of Surgery, Semmelweis University, Budapest, Hungary
Summary This study correlates whole organ measurements of intracellular calcium concentration ([Ca2;]i) with hormone-induced (epinephrine, vasopressin) changes of liver functions (glucose release, K; balance and bile flow). [Ca2;]i was measured in the isolated perfused rat liver using the sensor Fura-2 and applying liver surface fluorescence spectroscopy. The technique was improved by (i) minimizing biliary elimination of the sensor by employing a rat strain deficient in canalicular organic anion transport (TR9 mutation) and (ii) by correcting for changes of interfering intrinsic organ fluorescence that was shown to depend on the oxidation-reduction state (NAD(P)H content) of the organ. Epinephrine (50 nM) elicits an instantaneous peak rise of [Ca2;]i to approx. 400 nM, followed by a sustained elevation that depends on the presence of extracellular Ca2;. The rise of [Ca2;]i coincides with initiation of glucose release, transient K; uptake, and transient stimulation of bile flow. Vasopressin (2 nM) exerts qualitatively similar effects. The transient rise of bile flow is attributed to Ca2;-mediated contraction of the pericanalicular actin-myosin web of hepatocytes. © 2001 Harcourt Publishers Ltd
INTRODUCTION Modulation of intracellular free calcium concentration ([Ca2;]i) plays a pivotal role as a signaling pathway for a large number of hormonal effects [e.g. 1–7]. Most previous studies have been performed in isolated hepatocytes or in liver cell lines and have been correlated with hormonal regulation of complex liver functions such as regulation of various metabolic pathways, membrane transport or bile formation, mechanisms that can be easily assessed in the whole organ. It was, therefore, the aim of this study to establish a reliable method to measure [Ca2;]i in the intact isolated perfused liver and to directly correlate hormone induced Ca2; signalling with modulation of liver functions.
Received 21 February 2001 Revised 15 July 2001 Accepted 6 September 2001 Correspondence to: Jürg Graf MD, Department of Pathophysiology, University Hospital Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria. Tel.:;43 1 40400 5126; fax:;43 1 40400 5130; e-mail: [email protected]
Measurements of [Ca2;]i or intracellular pH ( pHi) in whole organs by use of fluorescent sensors have been performed already, e.g. [Ca2;]i in heart [8] and liver [9–12] or pHi in liver [13]. In principle, these methods followed the techniques applied in single cells: the fluorescent sensor is loaded into the organ in form of its membrane-permeant and non-fluorescent ester which is then cleaved by intracellular esterases. The resulting acidic form of the sensor should remain ‘trapped’ within the cell and report changes of [Ca2;]i or pHi. For measurements of [Ca2;]i in the intact liver we have chosen to use the sensor Fura-2 that combines the advantages of (i) having a dissociation constant within the physiological range of [Ca2;]i and (ii) allowing for estimating changes of [Ca2;]i from changes of the excitation fluorescence ratio 340/380 nm (emission at 510 nm), a parameter that is independent of the intracellular concentration of the sensor [4]. As previously noted with the use of the sensor Indo-1 [12], two major disadvantages became apparent during our studies. Firstly, Fura-2 is secreted into bile by being a substrate of the canalicular membrane multispecific anion transport system cMOAT (mrp2) [14,15], which results in reduction of intracellular concentration 403
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of the sensor and may increase ‘contaminating’ fluorescence signals arising from the presence of the sensor within bile canaliculi and ductules. Secondly, it appeared unfeasible (loading time, costs) to achieve intracellular concentrations of the sensor that are far above intrinsic organ fluorescence. We show, that correction for background fluorescence by subtraction of fluorescence values obtained before Fura-2 loading may be insufficent because background fluorescence (excitation: 340, 360 and 380; emission 510) changed during experimental manoeuvers, mainly due to the overlapping of Fura-2 fluorescence with the fluorescence spectrum of NAD(P)H (excitation maximum: 340–360; emission 460) [compare: 16–23]. In order to overcome these two disadvantages, we used a mutant Wistar rat strain (TR9) that is devoid of the hepatic canalicular organic anion transport system (cMOAT or mrp2) [14,15], thus furthering retention of the sensor within the cells. Secondly, we performed parallel experiments in the absence of Fura-2 to obtain appropriate background corrections and we compared these results with recordings of changes of the oxidationreduction state measured at 360–460 nm. These experimental aspects and corresponding figures are included in the Method section. As examples of applications of the technique we demonstrate effects of epinephrine and vasopressin showing the correlation in time between changes of [Ca2;]i and modulation of glucose release, K; balance, and bile flow. Besides reproducing hormonal effects on [Ca2;]i, glucose release and K; balance as anticipated from previous measurements in single cells we show that the steep initial rise of [Ca2;]i produced by epinephrine and vasopressin is associated with a rapid but transient stimulation of bile flow.
MATERIALS AND METHODS Animals and liver perfusion Male Wistar rats (BW 150 –220 g) and the mutant TR9 strain (kindly provided to us by P.L.M. Jansen, Groningen) were used as liver donors. After canulation of the common bile duct and of portal and hepatic veins the liver was removed and placed on a perspex support that contained an UV-translucent glass window and the liver was perfused in a single pass system at 37⬚C with KrebsHenseleit buffer (120 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM NaHCO3, 5 mM glucose) at a constant flow of 3.0 ml/gliver/min. The perfusion solution was gas equilibrated with 95% O2 and 5% CO2 (95% N2/5% CO2 in some experiments). After 30 min, perfusion was switched to buffer containing 1 M Fura-2-acetoxymethylester (Fura-2 AM; Lambda Fluoreszenztechnik, Graz, Austria) (prepared from stock solution Cell Calcium (2001) 30(6), 403–412
containing 1 mg Fura-2-AM in 1 ml DMSO) while surface fluorescence was monitored. Fluorescence measurements Similar to studies in single cells [4], [Ca2;]i was determined from the fluorescence ratio of Fura-2 measured at the excitation-emission pairs 340–510 and 380–510. We used the RatioMaster™ system as fluorescence spectrophotometer (PTI – Photon Technology International; obtained from Photo Med GmbH, Wedel, Germany). This system consists of motor driven excitation and emission monochromators that are computer controlled to allow sequential and repetitive measurements at preset pairs of excitation and emission wavelengths. Slit widths of monochromators were set to 4 nm (band-pass) and 10 nm for excitation and emission, respectively. The following excitation-emission pairs were used: 340–510 (for the Fura-2-Ca2;-complex), 360–510 (for [Ca2;]-independent Fura-2 concentration at the isosbestic point), 380–510 (for Ca2;-free Fura-2) and 360–460 (for NAD(P)Hsensitive background fluorescence; see below). Excitation light was guided to the liver surface through one branch of a bifurcated quartz fibre optic bundle and emitted light was collected through the other branch and emission was measured with the 710-Photomultiplier Detection System (PTI). We used the software ‘Felix’ (Version 1.1; PTI) for data acquisition and for controlling the monochromators. The liver was illuminated in an area of about 1 cm2 through the UV-translucent glass window at the bottom of the organ. This geometry was chosen because we had observed that measurements from the top free surface are seriously affected by changes of liver volume (e.g. induced by osmotic cell swelling) which result in changes of the distance between the organ surface and the port of the fibre optic bundle. We used red room illumination (9600 nm) and dimmed computer monitors throughout fluorescence measurements. Corrections for changes of intrinsic organ fluorescence We observed that intrinsic organ fluorescence decreased within the initial period of liver perfusion, particularly at the excitation-emission pairs of 360–460, 340–510 and 360–510. These changes are attributed to the recovery of the organ from its anoxic state after surgery which is accompanied by the decrease of the ratio of NAD(P)H to NAD(P) and of the oxidation-reduction pairs malateoxalacetate or lactate-pyruvate towards levels found in vivo [e.g. 24,22]. We therefore monitored NAD(P)H dependent fluorescence at excitation-emission of 360–460 throughout experimental manoeuvers as an indicator of rapid oxidation-reduction changes [e.g. 22,25]. © 2001 Harcourt Publishers Ltd
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In experiments employing Fura-2, these changes of background fluorescence may interfere with fluorescence signals that are generally solely attributed to changes of cellular Fura-2 concentration (360–510) and changes of [Ca2;]i (340/380–510 fluorescence ratio). We proved this assumption during Fura-2-AM-free perfusions by application of either ischaemia (produced by short term stopping the flow of perfusion) or by anoxia (produced by gas equilibrating the perfusate with 95% N2/5% CO2) and we observed a substantial increase of fluorescence not only at 360–460 (redox sensitive), but also at 340–510 and 360–510, wavelength pairs that are sensitive to Fura-2 concentration or to changes of [Ca2;]i after Fura-2-AM loading. We have chosen two options to correct for these changes of background fluorescence when measuring [Ca2;]i with Fura-2. In one set of experiments we studied hormonal effects in parallel experiments performed in either the absence or the presence of Fura-2 and we obtained Fura-2-specific data by subtraction. In a second set of experiments we used multiple excitation-emission pairs (360–460, 340–510, 360–510 and 380–510). We first applied a short period of hypoxia to raise intracellular NAD(P)H in the absence of Fura-2 and, after recovery from hypoxia, we loaded the organ with Fura-2. In the first step, this manoeuver revealed estimates for the contribution of an increase of NAD(P)H (measured at 360–460) to the fluorescence at the three Fura-2 sensitive wavelengths and, in the second step, for the contribution of Fura-2 fluorescence (measured at 360–510) to the NAD(P)H sensitive wavelength pair. These parameters for mutual over-spill (‘contamination’ of Fura-2 fluorescence by changes of NAD(P)H and vice versa) could be used to calculate the changes of NAD(P)H and [Ca2;]i from measurements at the four wavelength pairs after application of epinephrine or vasopressin. The first correction was independent on what may be the cause of changes of intrinsic fluorescence: we performed control experiments to show hormonal effects on background fluorescence at 340–510 and 380–510 in the absence of Fura-2. Traces from three experiments were normalized by division by the fluorescence values prior to hormone application. Examples for changes of background fluorescence after application of epinephrine or vasopressin are given in Figure 1. Experiments in the presence of Fura-2 were performed with an identical protocol and the differences in fluorescence at 340–510 and 380–510, respectively, between control livers and Fura-2 loaded livers was taken to calculate the 340/380–510 fluorescence ratio to report changes of [Ca2;]i . The second approach assumed that changes of the oxidation-reduction state (changes of the NAD(P)Hspecific fluorescence) are the major factors affecting background fluorescence at the Fura-2-sensitive wavelengths (Fig. 2). We first determined the effect of changes of the © 2001 Harcourt Publishers Ltd
Fig. 1 Effects of epinephrine and vasopressin on surface fluorescence of the isolated rat liver. Livers were perfused with a Fura-2 free medium and surface fluorescence was recorded at pairs of wavelengths used to measure [Ca2;]i with Fura-2 (nm: 340 : 510; 380 : 510; excitation : emission). Note the instantaneous increase of fluorescence upon addition of epinephrine (50 nM; upper panel) or vasopressin (2 nM; lower panel).
oxidation-reduction state by applying hypoxia (by perfusion with N2-equilibrated medium) in the absence of Fura-2. This resulted in a large rise of fluorescence at 360–460 (NAD(P)H sensitive) whereas fluorescence at 340–510, 360–510 and 380–510 rose by approximately 0.35 (␣), 0.35 () and 0.05 (␥), respectively, of the amplitude seen at 360–460. The values ␣,  and ␥ thus represent factors that relate the increase of NAD(P)H (measured at 360–460) to an increase of fluorescence at the three Fura-2-sensitive wavelengths. After recovery from hypoxia, we loaded the organ with Fura-2. This resulted in a progressive rise of fluorescence at 360–510 (and other Fura-2 sensitive wavelengths), whereas fluorescence at 360–460 rose by approximately 0.7 (␦) of that seen at 360–510, the wavelength pair sensitive to Fura-2 concentration. Thus, the fluorescence ratio 360–460/ 360–510 (␦) determines the relative contribution of Fura-2 fluorescence (measured at 360–510, the isosbestic point) Cell Calcium (2001) 30(6), 403–412
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to the fluorescence recorded at 360–460 (NAD(P)H specific). The value ␦ remained fairly constant during the loading period indicating that Fura-2 loading had little effect on the oxidation-reduction state of the organ. The following calculations involved two steps: (i) having obtained the values  and ␦ is sufficient to calculate specific changes of the oxidation-reduction state of the organ and of cellular Fura-2 concentrations from changes of the fluorescence at 360–460 and 360–510 respectively and (ii) knowing the effect of over-spill from changes of the oxidation-reduction state on actual readings at 340–510 and 380–510 (␣ and ␥ respectively), allows to correct these readings to obtain those fluorescence components that are solely attributable to changes of the concentrations of the Ca2;-Fura-2 complex and of Ca2;-free Fura-2, respectively, giving a corrected ‘true’ 340/380–510 fluorescence ratio as a measure of [Ca2;]i that is not affected by changes of the oxidation-reduction state of the organ. In the experiments reported here we have used both these approaches and obtained comparable results. Nonetheless, we preferred to use the first approach to correct for changes of background fluorescence, as this does not rely on the assumption that changes of background fluorescence during hormone application are solely attributable and comparable to changes of the oxidation-reduction state as induced by hypoxia. Calibration of [Ca2;]i from Fura-2 signals Calibration of the 340/380 fluorescence ratio in order to obtain absolute values of [Ca2;]i requires measurements of the maximal and minimal fluorescence ratio R340/380 at high and zero [Ca2;]i and of the maximal and minimal fluorescence at 380 nm excitation [4]. Modulation of [Ca2;]i is generally achieved by variation of external [Ca2;] in the presence of the calcium ionophore ionomycin (Sigma, St. Louis, MO). After loading with Fura-2-AM in control medium, the livers were perfused for 20 min with calciumfree Krebs-Henseleit buffer (10 mM EGTA, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 20 mM NaHCO3) containing 5 M ionomycin, followed by perfusion with control, calcium-containing Krebs-Henseleit buffer with addition of 5 M ionomycin. R340/380 was 1.74<0.04 (SE, n:4) during control perfusion. R340/380 decreased during calcium free perfusion to 1.21<0.02 and rose to 3.59<0.58 after Ca2; readmission with a ratio of maximal/minimal fluorescence at 380–510 of 1.54<0.17, values that revealed [Ca2;]i=126.3<30.6 nM [20]. This approach was unsatisfactory, however, because R340/380 had not attained stable values after 20 min of either Ca2; free or Ca2; containing perfusion, but was still on a falling or rising slope respectively. We made the assumption that individual fluorescence changes during Ca2; free or Ca2; Cell Calcium (2001) 30(6), 403–412
Fig. 2 Effects of hypoxia, Fura-2 loading and application of epinephrine on liver surface fluorescence recorded at multiple wavelengths pairs. Upper panel: surface fluorescence was measured at the following excitation-emission pairs (nm): 360–460 (NAD(P)H-sensitive); 360–510 (isosbestic point of Fura-2, monitoring total Fura-2 concentration); 340–510 (monitoring the concentration of the Fura-2-Ca2; complex) and 380–510 (monitoring the concentration of Ca2;-free Fura-2). Note the increase of fluorescence during oxygen-free perfusion (N2 equilibrated medium) particularly at 360–460 (NAD(P)H-sensitive) and the associated changes at Fura-2-sensitive emission (510 nm). The relative changes 340–510/360–460, 360–510/360–460 and 380–510/360–460 provide values for ␣,  and ␥, respectively (see text in method section). Perfusion with Fura-2-AM (1 M) results in fluorescence increase at all four wavelength pairs. The ratio of fluorescence increase at excitation at 360 nm: 360–460/360–510, gives an estimate of ␦, the ‘spill-over’ of Fura-2 loading on NA(P)Hsensitive fluorescence (see text in the method section). The rise of [Ca2;]i upon administration of epinephrine (50 nM) is reflected by fluorescence increase at 340–510 and decrease at 380–510. Simultaneous changes at 360–460 and 360–510 reflect changes of background fluorescence that are ascribed to an increase of NAD(P)H. These latter effects ‘contaminate’ measurements of [Ca2;]i by the 340–510/380–510 fluorescence ratio technique generally applied with the use of Fura-2. Lower panel: Changes of the oxidation-reduction state (NAD(P)H concentration) and of Fura2 concentration are calculated from traces shown in the upper panel. The insert (R340/380) shows the change of the fluorescence ratio 340–510/380–510 after correction for the simultaneous changes of the oxidation-reduction state (compare text in Method section).
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during dye loading. Single bile drops were diluted with 1 ml PBS (2.7 mM KCl, 137 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4; pH:7.4) and Fura-2 fluorescence was measured at 340 nm excitation and 510 nm emission with a spectrofluorometer (Jasco, FP-777). Fura-2 free acid was used for dye calibration (Lambda Fluoreszenztechnik, Graz, Austria). To study inhibition of biliary dye secretion we employed the anion transport inhibitor probenecid (Sigma, St. Louis, MO) at concentrations from 10 M to 5 mM in control Wistar rats. Furthermore, we analyzed bile for Fura-2 metabolites by thin-layer chromatography (TLC): bile of Wistar rats was collected after Fura-2 loading, diluted (1 : 4) with phosphate buffer (0.1 M; pH:6.8) and compared with 100 M Fura-2 free acid in phosphate buffer on TLC sheets (TLC aluminium sheets silica gel 60 F254, layer thickness 0.2 mm, Merck, Darmstadt, Germany) developed with methanol/water 0.9/0.1. TLC sheets were analyzed by fluorescence densitometry. Bile samples were also analyzed for calcium sensitivity of Fura-2 secreted into bile: 10 l of bile was diluted with 4 ml PBS, with 100 M CaCl2 added to PBS, or with PBS containing 2 mM EGTA. Fluorescence was scanned in an excitation range of 250–450 nm, emission at 510 nm. Fig. 3 Effects of epinephrine (50 nM) in the isolated perfused liver of TR9 rats. Panels (top to bottom) show: (a) Fluorescence ratio 340/380 nm as a measure of [Ca2;]i (corrected for changes of background surface fluorescence; values on the ordinate of 1, 1.5 and 2 roughly correspond to [Ca2;]i of (nM) 160, 370 and 740, respectively); (b) Glucose production; (c) K; balance (negative values represent K; uptake); and (d) Bile flow. Note the sharp rise of [Ca2;]i upon addition of epinephrine followed by a sustained increase. The rise of [Ca2;]i is associated with an increase of glucose production, K; uptake and transient increase of bile flow.
containing perfusion follow a quasi exponential slope and extrapolating the mean slopes to t = revealed values of 0.67, 5.70, and 2.60 for minimal and maximal values of R340/380 and for the relative change at 380–510 respectively. From these values control [Ca2;]i was estimated at 159.8<8.4 nM. In order not to suggest that reliable absolute values of [Ca2;]i can be obtained in individual experiments, we have chosen to present changes of [Ca2;]i as relative changes with respect to a normalized control value of R340/380 of 1. From extrapolated endpoints of titration of intracelluar [Ca2;]i values of R340/380 of 1.0, 1.5 and 2.0 given on the ordinates in Figures 3 and 4 roughly correspond to [Ca2;]i of 160, 370 and 740 nM, respectively.
Measurement of Fura-2 concentration in bile of control Wistar and TR9 rats We measured the Fura-2 concentration in bile of Wistar and of TR9 rats. Single bile drops (approx. 8 mg) leaving the bile duct cannula were collected over 15 min before loading the liver with Fura-2-AM (1 M), and for 30 min © 2001 Harcourt Publishers Ltd
Measurement of potassium and glucose balance and of bile flow Potassium and glucose balance were assessed by analyzing the venous perfusate of the liver in comparison with inflow concentrations. Perfusate was collected at 30 s or 1 min intervals. K; was measured with a NOVA 9 electrolyte analyzer (Nova Biomedical, Newton, MA) and K; balance was calculated from liver weight, perfusate flow rate and from the portal to caval venous concentration differences. Uptake or release of K; is given in nmoles/gliver /s. Glucose concentrations were measured enzymatically by the hexokinase methods (Glucose liquiUV; Human GmbH, Taunusstein, Germany). Bile flow was measured from the weight of bile drops and from the frequency of their falling from the bile duct canula and interrupting an infrared light beam in a drop counter. Average values from different experiments were obtained after interpolation to comparable time points. Bile secretion is given in mg/gliver/min. Data are given as mean values
RESULTS Liver surface fluorescence and biliary secretion of Fura-2 in Wistar and TR9 rats Perfusion of isolated livers from Wistar rats with Fura-2-AM containing solution (1 M) increased surface fluorescence Cell Calcium (2001) 30(6), 403–412
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reduction of biliary concentration of Fura-2 to 33.1< 0.2 M. Probenecid exhibits a strong choleretic effect [26], which was considered unfavourable because effects of hormones on bile formation were included in our studies. Therefore, all experiments were carried out with livers from TR9 rats in order to improve liver cell loading with Fura-2 and to reduce contamination of surface fluoresence by the presence of Fura-2 in bile.
Effects of epinephrine on [Ca2;]i, glucose balance, K; balance, and bile flow
Fig. 4 Effects of vasopressin (2 nM) in the isolated perfused liver of TR9 rats. Panels (top to bottom) show: (a) Fluorescence ratio 340/380 nm as a measure of [Ca2;]i (corrected for changes of background surface fluorescence; values on the ordinate of 1, 1.5 and 2 roughly correspond to [Ca2;]i of (nM) 160, 370 and 740, respectively); (b) Glucose production; (c) K; balance (negative values represent K; uptake); and (d) Bile flow. Note the sharp rise of [Ca2;]i upon addition of vasopressin followed by a gradual decline. The rise of [Ca2;]i is associated with an increase of glucose production, K; uptake and transient increase of bile flow.
within 30 min to values approx. 30% above the autofluorescence level, measured prior to dye loading (360–510 nm). Bile flow was 0.85<0.08 mg gliver91 min91 (n:3) and remained unaffected by Fura-2 loading. In this period of time, Fura-2 concentration in bile reached values of 380<30 M. No major fluorescent metabolites were detected in bile and biliary Fura-2 fluorescence showed a regular response to changes of Ca2; concentration. In anion transport deficient TR9 rats, bile flow was 0.47<0.04 mg gliver91 min91 (n:3). In contrast to control Wistar rats, Fura-2 concentration in bile was only 26<3 M (n:3) and surface fluorescence continued to rise during ongoing application of Fura-2-AM (compare Fig. 2). With reference to an initial background fluorescence at 340–510 nm normalized to a value of 1 the increase of fluorescence within 30 min was from 1 to 1.82<0.11 at 340–510 nm and, at 380–510 nm, from 0.52<0.03 to 0.90<0.07 by 75.1<8.9% (n:9). In control Wistar rats, application of high concentration of the anion transport inhibitor probenecid (5 mM) resulted in a comparable Cell Calcium (2001) 30(6), 403–412
As already exemplified in Figure 1, epinephrine (50 nM) caused a significant increase of background fluorescence which is attributed to a rise of intracellular concentrations of NADH and NADPH). With reference to a normalized initial background fluorescence at 340–510 nm of 1 the increase of fluorescence was from 1 to 1.044<0.002 at 340–510 nm and, at 380–510 nm, from 0.485<0.026 to 0.498<0.030 by 2.53<0.14% (n:9). These changes of background fluorescence had to be corrected when measuring [Ca2;]i with Fura-2 (see Method section). Upon application of epinephrine, Fura-2 fluorescence revealed a sharp transient rise of [Ca2;]i followed by a sustained elevated level and [Ca2;]i returned to control values after removal of epinephrine (Fig. 3). Similar changes of [Ca2;]i were evoked by phenylephrine (1 M) ( peak rise of the 340/380 fluorescence ratio by a factor of 1.6) but not by isoprenaline (1 M) (data not shown). Glucose production of the isolated liver was elevated throughout the presence of epinephrine (Fig. 3). In parallel with the rise of [Ca2;]i, the continuous small release of K; was transiently reversed to K; uptake. Also in parallel with the rise of [Ca2;]i, bile flow was transiently increased which was followed by a short depression before returning to control levels. Including data from Fura-2-free experiments, epinephrine (50 nM) caused a transient rise of bile flow from 0.42<0.02 mg gliver91 min91 to 0.57<0.03 mg gliver91 min91 by 38.3<2.3% (n:10). Application of phenylephrine (1 M) similarly initiated K; uptake and transiently stimulated bile flow (data not shown). During Ca2;-free perfusion, the initial peak rise of [Ca2;]i was preserved, but [Ca2;]i returned to baseline levels within 3 min despite the continuous presence of epinephrine. In accordance with this short lived effect on [Ca2;]i the epinephrine induced glucose release and K; uptake were also abbreviated. Similarly, the rise of calculated NAD(P)H dependent fluorescence was abbreviated in comparison to the increase of background fluorescence seen in Figure 1. Bile flow ceased in the absence of extracellular Ca2; and epinephrine showed no stimulatory effect (data not shown). © 2001 Harcourt Publishers Ltd
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Effects of vasopressin on [Ca2;]i, glucose balance, K; balance, and bile flow Vasopressin caused a substantial transient rise of background fluorescence. With reference to a normalized initial background fluorescence at 340–510 nm of 1 the increase of fluorescence was from 1 to 1.090<0.012 at 340–510 nm and, at 380–510 nm, from 0.527<0.028 to 0.556<0.031 by 5.42<0.59% (n:8). Following correction for these changes, Figure 4 correlates effects of vasopressin (2 nM) on [Ca2;]i, glucose production, K; balance and bile flow. Vasopressin causes a sharp rise of [Ca2;]i followed by a slow decline. Glucose production was suddenly increased and remained elevated throughout the presence of vasopressin. Coincident with the rise of [Ca2;]i, transient K; uptake was initiated which was followed by K; release. Vasopressin caused a sudden but transient increase of bile flow from 0.46<0.02 mg gliver91 min91 to 1.02<0.09 mg gliver91 min91 by 120.4<16.1% (n:10) which was followed by a slight depression.
DISCUSSION Measurement of hepatocellular [Ca2;]i in the isolated perfused liver allows to correlate this parameter with liver cell functions that are not easily assessable in isolated hepatocytes. Principles of using fluorescent intracellular sensors for pHi and [Ca2;]i and of measuring surface fluorescence of intact organs have been presented before [9–13]. We have chosen to use Fura-2 to measure [Ca2;]i: the dissociation constant of this sensor is in the physiological range of [Ca2;]i and, using dual wavelength excitation, this sensor offers the advantage of providing signals for [Ca2;]i that are independent of the intracellular concentration of the sensor itself [4]. Furthermore, the non-fluorescent acetoxymethyl ester of Fura-2 is readily taken up from the vasculature into liver cells and hydrolyzed to the fluorescent sensor. Nonetheless, several factors limit this approach: (i) intrinsic fluorescence from the liver surface must be considered as a composite signal that arises from contributions of different cell types [including hepatocytes, endothelial cells or Kupffer cells; compare Ref. 27], from their metabolic state and storage of fluorescent material, (ii) these cells will also contribute to the increase of surface fluorescence after loading the organ with Fura-2, and (iii) anionic dyes such as Fura-2 are secreted at high concentrations into bile so that bile canalicular fluorescence will substantially contribute to the overall surface fluorescence. The specific aim of this study was to identify artifacts and limitations in measuring liver cell [Ca2;]i by surface fluorescence recording and, by using epinephrine and vasopressin, two agonists which act through Ca2; signalling, to compare these measurements with changes © 2001 Harcourt Publishers Ltd
of physiological parameters such as glucose production, K; balance and bile flow. Reducing biliary secretion of Fura-2 will result in better retention of the dye within hepatocytes and lower concentration of the fluorecent sensor in bile canaliculi. In accordance with a previous study [12] we show that secretion of Fura-2 into bile is reduced by the anion transport inhibitor probenecid but high concentrations are required (5 mM in the perfusate). At this concentration, probenecid elicits a strong choleretic effect [26] that was considered disadvantageous for studies that aim at correlating changes of [Ca2;]i with alterations of bile flow (see below). We therefore used an anion transport deficient rat strain (TR9 rats). These rats lack the canalicular anion transporter cmoat or mrp2 [14,15]. Using TR9 rats as liver donors provided for both, reduction of secretion of Fura-2 into bile (7% of control Wistar rats) and efficient loading of the sensor into hepatocytes. It is noted though that bile flow in this rat strain is low as compared to control Wistar rats, most likely as a result of reduced bile salt-independent GSH-dependent bile production [28]. In the method section, we describe two approaches to correct for changes of intrinsic background fluorescence. These corrections are required because (i) it is not feasible to load the isolated liver with Fura-2 to achieve surface fluorescence levels that are far above background fluorescence because of limitations in loading time and because of financial restrictions, and, (ii) experimental manoeuvres such as application of epinephrine or vasopressin do elicit changes in background fluorescence at the Fura-2 and [Ca2;]i sensitive excitation-emission wavelength pairs 340–510 and 380–510 (Fig. 1). One option was to perform parallel sets of experiments in the absence and presence of Fura-2. The Fura-2 and [Ca2;]isensitive signals are then obtained by subtracting background signals from data obtained in the presence of Fura-2 (compare Fig. 1). The second approach assumed that changes of intrinsic fluorescence are mainly due to changes of liver cell NAD(P)H content [16]. Changes of NAD(P)H were first generated by a short phase of hypoxia [compare 18,19,22] prior to Fura-2 loading and fluorescence was measured at pairs of excitationemission wavelength, one with preferential sensitivity to NAD(P)H (emission at 460 nm) the others preferentially sensitive to Fura-2 (emission at 510 nm). This allowed to calculate the effect of changes of NAD(P)H on the fluorescence in the Fura-2-sensitive wavelength range. Corrections for this ‘spill over’ were then continuously applied after Fura-2 loading and during manoeuvers that altered [Ca2;]i (Fig. 2). Both methods of correcting background fluorescence resulted in comparable estimates of changes [Ca2;]i as reflected by the adjusted 340/380–510 fluorescence ratio. In order to calibrate the 340/380 Cell Calcium (2001) 30(6), 403–412
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signal to obtain quantitative measurements of [Ca2;]i livers were perfused with medium containing the ionophore ionomycin at 0 and normal Ca2; concentrations. [Ca2;]i followed the changes of perfusate calcium concentrations but stable minimal and maximal endpoints of the 340/380 fluorescence ratio were not obtained within a reasonable time span. By extrapolating the quasi exponential changes of [Ca2;]i to t: a mean control value of [Ca2;]i of 160<8 nM (n:3) was estimated (see Material and Methods). Control 340/380 fluorescence ratios given in Figures 3 and 4 are normalized with the values of 1, 1.5 and 2 roughly corresonding to (nM) 160, 370 and 740 respectively. In single isolated hepatocytes application of vasopressin or ␣-adrenergic activation results in an abrupt rise of [Ca2;]i followed by oscillations of [Ca2;]i at an elevated level. The initial steep increase is effected by release of Ca2; from intracellular stores whereas the prolonged elevation results from ‘capacitative’ entry from the extracellular space [e.g. 3,6,7,29–36]. Our study shows that these two phases of changes of [Ca2;]i are also detected in the isolated perfused liver (Fig. 3) and confirms that prolonged elevation of [Ca2;]i depends on the presence of external Ca2;. The particular advantage of the technique used here is that these changes of [Ca2;]i can be directly related to the biological effects of the hormones. Figures 3 and 4 show that the early rise of [Ca2;]i following application of epinephrine or vasopressin is associated with stimulation of glucose release which remains elevated until removal of the agonist. Potassium homeostasis results from the balance between K; uptake by the Na;/K;-pump and from passive leakage of K; out of cells through ion channels [37]. The data show that this equilibrium is disturbed by epinephrine and vasopressin which both cause transient K; uptake (associated with the early rise of [Ca2;]i). This pattern is interpreted to result from initial transient activation of the Na;/K;-pump followed by increased efflux of K; [e.g. 38]. Numerous previous studies had addressed these effects of adrenergic stimulation and of vasopressin on the balance of glucose [e.g. 16,39–43] and K; balance [e.g. 39–41,43–45]. Here, we studied changes of these parameters to demonstrate their correlation with changes of [Ca2;]i measured by surface fluorescence of the isolated perfused liver. Vasopressin and ␣1-adrenergic stimulation increase the level of reduced pyridine nucleotides in liver [16,25,29,46] resulting in an increase of fluorescence at 360–460 nm. This increase in fluorescence is also produced by oxygenfree perfusion and Figure 2 shows its interference with Fura-2 fluorescence. As addressed above, appropriate corrections have to be applied when measuring [Ca2;]i at low intracellular concentrations of Fura-2. By comparing
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the changes of background fluorescence (Fig. 1) with changes of [Ca2;]i (Figs 3 & 4) we also note that the time course of changes of [Ca2;]i after application of epinephrine and vasopressin is largely paralleled by the increase of NAD(P)H fluorescence. We observed that epinephrine, phenylephrine and vasopressin elicit an instantaneous but short-lasting increase of bile flow. This effect coincided with the early rise of [Ca2;]i. On the other hand, bile flow ceased in the absence of external Ca2; and subsequent application of epinephrine was without effect (data not shown). This latter effect appears attributable to loosening of tight junctions and to an associated increase in the bile to perfusate permeability [e.g. 47]. It has been previously observed that application of hormones which act through Ca2; signalling lead to an early but transient increase of bile flow [48]. This effect appears to correlate with in vitro observations obtained in hepatocyte couplets, a preparation of liver cell secretory units that consist of non-dissociated pairs of isolated hepatocytes which enclose a bile canalicular vacuole between each other [49]. In this preparation, microinjection of Ca2; into hepatocytes [50] as well as hormonally mediated increases in [Ca2;]i [51,52] lead to a contraction of the bile secretory vacuole which is mediated by the actin-myosin web underneath the canalicular membrane [53,54]. In the intact organ, this forceful contraction of bile canaliculi appears to promote bile flow by ‘squeezing out’ a small volume of canalicular bile. It is noted though that, despite near equal peak rises of [Ca2;]i, the transient choleretic effect of vasopressin was about three times as large as that of epinephrine, suggesting that additional components of the respective signal transduction pathways may affect bile flow as well. In conclusion, this study shows that changes of hepatocellular [Ca2;]i can be reliably measured in the isolated perfused rat liver provided that biliary elimination of the fluorescent intracellular sensor (Fura-2) is minimized and that changes of background fluorescence are also monitored, either in parallel experiments or by measurements at multiple wavelengths. This method allows to correlate the pattern of changes of [Ca2;]i with changes of liver function. We demonstrate the effects of epinephrine and vasopressin in simultaneous recordings of [Ca2;]i, glucose balance, K; balance, bile flow and changes of NAD(P)H. The data are consistent with an early release of Ca2; from intracellular stores followed by uptake of extracellular Ca2;. The early effect coincides with initiation of glucose release, K; uptake and with a rise of NAD(P)H. Furthermore, the rise of [Ca2;]i is associated with a transient increase of bile flow which is attributed to contraction of the actin-myosin web in the pericanalicular cytoplasm of liver cells.
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ACKNOWLEDGEMENT This work was supported by the Austrian Science Fund P10416-MED to M.R. REFERENCES 1. Charest R, Prpic V, Exton JH, Blackmore PF. Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2;. Biochem J 1985; 227: 79–90. 2. Clapham DE. Calcium Signaling. Cell 1997; 80: 259–268. 3. Glennon MC, Bird GStJ, Kwan CY, Putney JW Jr. Actions of vasopressin and the Ca2;-ATPase inhibitor, thapsigargin, on Ca2; signaling in hepatocytes. J Biol Chem 1992; 267: 8230–8233. 4. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2; indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–3450. 5. Hansen CA, Yang LJ, Williamson JR. Mechanisms of receptormediated Ca2; signaling in rat hepatocytes. J Biol Chem 1991; 266: 18573–18579. 6. Kawanishi T, Blank LM, Harootunian AT, Smith MT, Tsien RY. Ca2; oscillations induced by hormonal stimulation of individual fura-2-loaded hepatocytes. J Biol Chem 1989; 264: 12859–12866. 7. Nathanson MH, Schlosser SF. Calcium signaling mechanisms in liver in health and disease. Prog Liver Dis 1996; 14: 1–27. 8. Stefenelli T, Wikman-Coffelt J, Wu ST, Parmley WW. Intracellular calcium during pacing-induced ventricular fibrillation. Effects of lidocaine. J Electrocardiol 1992; 25: 221–228. 9. Motoyama K, Karl IE, Flye MW, Osborne DF, Hotchkiss RS. Effect of Ca2; agonists in the perfused liver: determination via laser scanning confocal microscopy. Am J Physiol 1999; 276: R575–R585. 10. Nathanson MH, Burgstahler AD, Mennone A, Fallon MB, Gonzalez CB, Saez JC. Ca2; waves are organized among hepatocytes in the intact organ. Am J Physiol 1995; 269: G167–G171. 11. Robb-Gaspers LD, Thomas AP. Coordination of Ca2; signaling by intracellular propagation of Ca2; waves in the intact liver. J Biol Chem 1955; 270: 8102–8107. 12. Ruttner Z, Ligeti L, Reinlib L, Hines K, McLaughlin AC. Monitoring of intracellular free calcium in perfused rat liver. Cell Calcium 1993; 14: 465–472. 13. Felberbauer FX, Graf J. Fiber-optic measurement of intracellular pH in intact rat liver using pH-sensitive dyes. Pflügers Arch 1991; 419: 215–217. 14. Jansen PL, Peters WH, Lamers WH. Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport. Hepatology 1985; 5: 573–579. 15. Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, Scheper RJ, Borst P, Oude Elferink RP. Congenital jaundice in rats with a mutation in a multidrug resistanceassociated protein gene. Science 1996; 271: 1126–1128. 16. Blackmore PF, Hughes BP, Charest R, Shuman EA, 4th Exton JH. Time course of alpha1-adrenergic and vasopressin actions on phosphorylase activation, calcium efflux, pyridine nucleotide reduction, and respiration in hepatocytes. J Bio Chem 1983; 258: 10488–10494. 17. Chance B, Williamson JR, Jamieson D, Schoener B. Properties and kinetics of reduced pyridine nucleotide fluorescence of the isolated and in vivo rat heart. Biochem Z 1965; 341: 357–377.
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Research
doi: 10.1054/ceca.2001.0248, available online at http://www.idealibrary.com on
Intracellular calcium in the isolated rat liver: correlation to glucose release, K; balance and bile flow R. Wurzinger,1 R. Englisch,2 S. Roka,1 R. Langer,3 M. Roden,2 J. Graf1 1Department
of Pathophysiology, University of Vienna, Austria 2Department of Internal Medicine III, Division of Endocrinology and Metabolism, University Hospital Vienna, Austria 3Department of Surgery, Semmelweis University, Budapest, Hungary
Summary This study correlates whole organ measurements of intracellular calcium concentration ([Ca2;]i) with hormone-induced (epinephrine, vasopressin) changes of liver functions (glucose release, K; balance and bile flow). [Ca2;]i was measured in the isolated perfused rat liver using the sensor Fura-2 and applying liver surface fluorescence spectroscopy. The technique was improved by (i) minimizing biliary elimination of the sensor by employing a rat strain deficient in canalicular organic anion transport (TR9 mutation) and (ii) by correcting for changes of interfering intrinsic organ fluorescence that was shown to depend on the oxidation-reduction state (NAD(P)H content) of the organ. Epinephrine (50 nM) elicits an instantaneous peak rise of [Ca2;]i to approx. 400 nM, followed by a sustained elevation that depends on the presence of extracellular Ca2;. The rise of [Ca2;]i coincides with initiation of glucose release, transient K; uptake, and transient stimulation of bile flow. Vasopressin (2 nM) exerts qualitatively similar effects. The transient rise of bile flow is attributed to Ca2;-mediated contraction of the pericanalicular actin-myosin web of hepatocytes. © 2001 Harcourt Publishers Ltd
INTRODUCTION Modulation of intracellular free calcium concentration ([Ca2;]i) plays a pivotal role as a signaling pathway for a large number of hormonal effects [e.g. 1–7]. Most previous studies have been performed in isolated hepatocytes or in liver cell lines and have been correlated with hormonal regulation of complex liver functions such as regulation of various metabolic pathways, membrane transport or bile formation, mechanisms that can be easily assessed in the whole organ. It was, therefore, the aim of this study to establish a reliable method to measure [Ca2;]i in the intact isolated perfused liver and to directly correlate hormone induced Ca2; signalling with modulation of liver functions.
Received 21 February 2001 Revised 15 July 2001 Accepted 6 September 2001 Correspondence to: Jürg Graf MD, Department of Pathophysiology, University Hospital Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria. Tel.:;43 1 40400 5126; fax:;43 1 40400 5130; e-mail: [email protected]
Measurements of [Ca2;]i or intracellular pH ( pHi) in whole organs by use of fluorescent sensors have been performed already, e.g. [Ca2;]i in heart [8] and liver [9–12] or pHi in liver [13]. In principle, these methods followed the techniques applied in single cells: the fluorescent sensor is loaded into the organ in form of its membrane-permeant and non-fluorescent ester which is then cleaved by intracellular esterases. The resulting acidic form of the sensor should remain ‘trapped’ within the cell and report changes of [Ca2;]i or pHi. For measurements of [Ca2;]i in the intact liver we have chosen to use the sensor Fura-2 that combines the advantages of (i) having a dissociation constant within the physiological range of [Ca2;]i and (ii) allowing for estimating changes of [Ca2;]i from changes of the excitation fluorescence ratio 340/380 nm (emission at 510 nm), a parameter that is independent of the intracellular concentration of the sensor [4]. As previously noted with the use of the sensor Indo-1 [12], two major disadvantages became apparent during our studies. Firstly, Fura-2 is secreted into bile by being a substrate of the canalicular membrane multispecific anion transport system cMOAT (mrp2) [14,15], which results in reduction of intracellular concentration 403
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of the sensor and may increase ‘contaminating’ fluorescence signals arising from the presence of the sensor within bile canaliculi and ductules. Secondly, it appeared unfeasible (loading time, costs) to achieve intracellular concentrations of the sensor that are far above intrinsic organ fluorescence. We show, that correction for background fluorescence by subtraction of fluorescence values obtained before Fura-2 loading may be insufficent because background fluorescence (excitation: 340, 360 and 380; emission 510) changed during experimental manoeuvers, mainly due to the overlapping of Fura-2 fluorescence with the fluorescence spectrum of NAD(P)H (excitation maximum: 340–360; emission 460) [compare: 16–23]. In order to overcome these two disadvantages, we used a mutant Wistar rat strain (TR9) that is devoid of the hepatic canalicular organic anion transport system (cMOAT or mrp2) [14,15], thus furthering retention of the sensor within the cells. Secondly, we performed parallel experiments in the absence of Fura-2 to obtain appropriate background corrections and we compared these results with recordings of changes of the oxidationreduction state measured at 360–460 nm. These experimental aspects and corresponding figures are included in the Method section. As examples of applications of the technique we demonstrate effects of epinephrine and vasopressin showing the correlation in time between changes of [Ca2;]i and modulation of glucose release, K; balance, and bile flow. Besides reproducing hormonal effects on [Ca2;]i, glucose release and K; balance as anticipated from previous measurements in single cells we show that the steep initial rise of [Ca2;]i produced by epinephrine and vasopressin is associated with a rapid but transient stimulation of bile flow.
MATERIALS AND METHODS Animals and liver perfusion Male Wistar rats (BW 150 –220 g) and the mutant TR9 strain (kindly provided to us by P.L.M. Jansen, Groningen) were used as liver donors. After canulation of the common bile duct and of portal and hepatic veins the liver was removed and placed on a perspex support that contained an UV-translucent glass window and the liver was perfused in a single pass system at 37⬚C with KrebsHenseleit buffer (120 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM NaHCO3, 5 mM glucose) at a constant flow of 3.0 ml/gliver/min. The perfusion solution was gas equilibrated with 95% O2 and 5% CO2 (95% N2/5% CO2 in some experiments). After 30 min, perfusion was switched to buffer containing 1 M Fura-2-acetoxymethylester (Fura-2 AM; Lambda Fluoreszenztechnik, Graz, Austria) (prepared from stock solution Cell Calcium (2001) 30(6), 403–412
containing 1 mg Fura-2-AM in 1 ml DMSO) while surface fluorescence was monitored. Fluorescence measurements Similar to studies in single cells [4], [Ca2;]i was determined from the fluorescence ratio of Fura-2 measured at the excitation-emission pairs 340–510 and 380–510. We used the RatioMaster™ system as fluorescence spectrophotometer (PTI – Photon Technology International; obtained from Photo Med GmbH, Wedel, Germany). This system consists of motor driven excitation and emission monochromators that are computer controlled to allow sequential and repetitive measurements at preset pairs of excitation and emission wavelengths. Slit widths of monochromators were set to 4 nm (band-pass) and 10 nm for excitation and emission, respectively. The following excitation-emission pairs were used: 340–510 (for the Fura-2-Ca2;-complex), 360–510 (for [Ca2;]-independent Fura-2 concentration at the isosbestic point), 380–510 (for Ca2;-free Fura-2) and 360–460 (for NAD(P)Hsensitive background fluorescence; see below). Excitation light was guided to the liver surface through one branch of a bifurcated quartz fibre optic bundle and emitted light was collected through the other branch and emission was measured with the 710-Photomultiplier Detection System (PTI). We used the software ‘Felix’ (Version 1.1; PTI) for data acquisition and for controlling the monochromators. The liver was illuminated in an area of about 1 cm2 through the UV-translucent glass window at the bottom of the organ. This geometry was chosen because we had observed that measurements from the top free surface are seriously affected by changes of liver volume (e.g. induced by osmotic cell swelling) which result in changes of the distance between the organ surface and the port of the fibre optic bundle. We used red room illumination (9600 nm) and dimmed computer monitors throughout fluorescence measurements. Corrections for changes of intrinsic organ fluorescence We observed that intrinsic organ fluorescence decreased within the initial period of liver perfusion, particularly at the excitation-emission pairs of 360–460, 340–510 and 360–510. These changes are attributed to the recovery of the organ from its anoxic state after surgery which is accompanied by the decrease of the ratio of NAD(P)H to NAD(P) and of the oxidation-reduction pairs malateoxalacetate or lactate-pyruvate towards levels found in vivo [e.g. 24,22]. We therefore monitored NAD(P)H dependent fluorescence at excitation-emission of 360–460 throughout experimental manoeuvers as an indicator of rapid oxidation-reduction changes [e.g. 22,25]. © 2001 Harcourt Publishers Ltd
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In experiments employing Fura-2, these changes of background fluorescence may interfere with fluorescence signals that are generally solely attributed to changes of cellular Fura-2 concentration (360–510) and changes of [Ca2;]i (340/380–510 fluorescence ratio). We proved this assumption during Fura-2-AM-free perfusions by application of either ischaemia (produced by short term stopping the flow of perfusion) or by anoxia (produced by gas equilibrating the perfusate with 95% N2/5% CO2) and we observed a substantial increase of fluorescence not only at 360–460 (redox sensitive), but also at 340–510 and 360–510, wavelength pairs that are sensitive to Fura-2 concentration or to changes of [Ca2;]i after Fura-2-AM loading. We have chosen two options to correct for these changes of background fluorescence when measuring [Ca2;]i with Fura-2. In one set of experiments we studied hormonal effects in parallel experiments performed in either the absence or the presence of Fura-2 and we obtained Fura-2-specific data by subtraction. In a second set of experiments we used multiple excitation-emission pairs (360–460, 340–510, 360–510 and 380–510). We first applied a short period of hypoxia to raise intracellular NAD(P)H in the absence of Fura-2 and, after recovery from hypoxia, we loaded the organ with Fura-2. In the first step, this manoeuver revealed estimates for the contribution of an increase of NAD(P)H (measured at 360–460) to the fluorescence at the three Fura-2 sensitive wavelengths and, in the second step, for the contribution of Fura-2 fluorescence (measured at 360–510) to the NAD(P)H sensitive wavelength pair. These parameters for mutual over-spill (‘contamination’ of Fura-2 fluorescence by changes of NAD(P)H and vice versa) could be used to calculate the changes of NAD(P)H and [Ca2;]i from measurements at the four wavelength pairs after application of epinephrine or vasopressin. The first correction was independent on what may be the cause of changes of intrinsic fluorescence: we performed control experiments to show hormonal effects on background fluorescence at 340–510 and 380–510 in the absence of Fura-2. Traces from three experiments were normalized by division by the fluorescence values prior to hormone application. Examples for changes of background fluorescence after application of epinephrine or vasopressin are given in Figure 1. Experiments in the presence of Fura-2 were performed with an identical protocol and the differences in fluorescence at 340–510 and 380–510, respectively, between control livers and Fura-2 loaded livers was taken to calculate the 340/380–510 fluorescence ratio to report changes of [Ca2;]i . The second approach assumed that changes of the oxidation-reduction state (changes of the NAD(P)Hspecific fluorescence) are the major factors affecting background fluorescence at the Fura-2-sensitive wavelengths (Fig. 2). We first determined the effect of changes of the © 2001 Harcourt Publishers Ltd
Fig. 1 Effects of epinephrine and vasopressin on surface fluorescence of the isolated rat liver. Livers were perfused with a Fura-2 free medium and surface fluorescence was recorded at pairs of wavelengths used to measure [Ca2;]i with Fura-2 (nm: 340 : 510; 380 : 510; excitation : emission). Note the instantaneous increase of fluorescence upon addition of epinephrine (50 nM; upper panel) or vasopressin (2 nM; lower panel).
oxidation-reduction state by applying hypoxia (by perfusion with N2-equilibrated medium) in the absence of Fura-2. This resulted in a large rise of fluorescence at 360–460 (NAD(P)H sensitive) whereas fluorescence at 340–510, 360–510 and 380–510 rose by approximately 0.35 (␣), 0.35 () and 0.05 (␥), respectively, of the amplitude seen at 360–460. The values ␣,  and ␥ thus represent factors that relate the increase of NAD(P)H (measured at 360–460) to an increase of fluorescence at the three Fura-2-sensitive wavelengths. After recovery from hypoxia, we loaded the organ with Fura-2. This resulted in a progressive rise of fluorescence at 360–510 (and other Fura-2 sensitive wavelengths), whereas fluorescence at 360–460 rose by approximately 0.7 (␦) of that seen at 360–510, the wavelength pair sensitive to Fura-2 concentration. Thus, the fluorescence ratio 360–460/ 360–510 (␦) determines the relative contribution of Fura-2 fluorescence (measured at 360–510, the isosbestic point) Cell Calcium (2001) 30(6), 403–412
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to the fluorescence recorded at 360–460 (NAD(P)H specific). The value ␦ remained fairly constant during the loading period indicating that Fura-2 loading had little effect on the oxidation-reduction state of the organ. The following calculations involved two steps: (i) having obtained the values  and ␦ is sufficient to calculate specific changes of the oxidation-reduction state of the organ and of cellular Fura-2 concentrations from changes of the fluorescence at 360–460 and 360–510 respectively and (ii) knowing the effect of over-spill from changes of the oxidation-reduction state on actual readings at 340–510 and 380–510 (␣ and ␥ respectively), allows to correct these readings to obtain those fluorescence components that are solely attributable to changes of the concentrations of the Ca2;-Fura-2 complex and of Ca2;-free Fura-2, respectively, giving a corrected ‘true’ 340/380–510 fluorescence ratio as a measure of [Ca2;]i that is not affected by changes of the oxidation-reduction state of the organ. In the experiments reported here we have used both these approaches and obtained comparable results. Nonetheless, we preferred to use the first approach to correct for changes of background fluorescence, as this does not rely on the assumption that changes of background fluorescence during hormone application are solely attributable and comparable to changes of the oxidation-reduction state as induced by hypoxia. Calibration of [Ca2;]i from Fura-2 signals Calibration of the 340/380 fluorescence ratio in order to obtain absolute values of [Ca2;]i requires measurements of the maximal and minimal fluorescence ratio R340/380 at high and zero [Ca2;]i and of the maximal and minimal fluorescence at 380 nm excitation [4]. Modulation of [Ca2;]i is generally achieved by variation of external [Ca2;] in the presence of the calcium ionophore ionomycin (Sigma, St. Louis, MO). After loading with Fura-2-AM in control medium, the livers were perfused for 20 min with calciumfree Krebs-Henseleit buffer (10 mM EGTA, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 20 mM NaHCO3) containing 5 M ionomycin, followed by perfusion with control, calcium-containing Krebs-Henseleit buffer with addition of 5 M ionomycin. R340/380 was 1.74<0.04 (SE, n:4) during control perfusion. R340/380 decreased during calcium free perfusion to 1.21<0.02 and rose to 3.59<0.58 after Ca2; readmission with a ratio of maximal/minimal fluorescence at 380–510 of 1.54<0.17, values that revealed [Ca2;]i=126.3<30.6 nM [20]. This approach was unsatisfactory, however, because R340/380 had not attained stable values after 20 min of either Ca2; free or Ca2; containing perfusion, but was still on a falling or rising slope respectively. We made the assumption that individual fluorescence changes during Ca2; free or Ca2; Cell Calcium (2001) 30(6), 403–412
Fig. 2 Effects of hypoxia, Fura-2 loading and application of epinephrine on liver surface fluorescence recorded at multiple wavelengths pairs. Upper panel: surface fluorescence was measured at the following excitation-emission pairs (nm): 360–460 (NAD(P)H-sensitive); 360–510 (isosbestic point of Fura-2, monitoring total Fura-2 concentration); 340–510 (monitoring the concentration of the Fura-2-Ca2; complex) and 380–510 (monitoring the concentration of Ca2;-free Fura-2). Note the increase of fluorescence during oxygen-free perfusion (N2 equilibrated medium) particularly at 360–460 (NAD(P)H-sensitive) and the associated changes at Fura-2-sensitive emission (510 nm). The relative changes 340–510/360–460, 360–510/360–460 and 380–510/360–460 provide values for ␣,  and ␥, respectively (see text in method section). Perfusion with Fura-2-AM (1 M) results in fluorescence increase at all four wavelength pairs. The ratio of fluorescence increase at excitation at 360 nm: 360–460/360–510, gives an estimate of ␦, the ‘spill-over’ of Fura-2 loading on NA(P)Hsensitive fluorescence (see text in the method section). The rise of [Ca2;]i upon administration of epinephrine (50 nM) is reflected by fluorescence increase at 340–510 and decrease at 380–510. Simultaneous changes at 360–460 and 360–510 reflect changes of background fluorescence that are ascribed to an increase of NAD(P)H. These latter effects ‘contaminate’ measurements of [Ca2;]i by the 340–510/380–510 fluorescence ratio technique generally applied with the use of Fura-2. Lower panel: Changes of the oxidation-reduction state (NAD(P)H concentration) and of Fura2 concentration are calculated from traces shown in the upper panel. The insert (R340/380) shows the change of the fluorescence ratio 340–510/380–510 after correction for the simultaneous changes of the oxidation-reduction state (compare text in Method section).
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during dye loading. Single bile drops were diluted with 1 ml PBS (2.7 mM KCl, 137 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4; pH:7.4) and Fura-2 fluorescence was measured at 340 nm excitation and 510 nm emission with a spectrofluorometer (Jasco, FP-777). Fura-2 free acid was used for dye calibration (Lambda Fluoreszenztechnik, Graz, Austria). To study inhibition of biliary dye secretion we employed the anion transport inhibitor probenecid (Sigma, St. Louis, MO) at concentrations from 10 M to 5 mM in control Wistar rats. Furthermore, we analyzed bile for Fura-2 metabolites by thin-layer chromatography (TLC): bile of Wistar rats was collected after Fura-2 loading, diluted (1 : 4) with phosphate buffer (0.1 M; pH:6.8) and compared with 100 M Fura-2 free acid in phosphate buffer on TLC sheets (TLC aluminium sheets silica gel 60 F254, layer thickness 0.2 mm, Merck, Darmstadt, Germany) developed with methanol/water 0.9/0.1. TLC sheets were analyzed by fluorescence densitometry. Bile samples were also analyzed for calcium sensitivity of Fura-2 secreted into bile: 10 l of bile was diluted with 4 ml PBS, with 100 M CaCl2 added to PBS, or with PBS containing 2 mM EGTA. Fluorescence was scanned in an excitation range of 250–450 nm, emission at 510 nm. Fig. 3 Effects of epinephrine (50 nM) in the isolated perfused liver of TR9 rats. Panels (top to bottom) show: (a) Fluorescence ratio 340/380 nm as a measure of [Ca2;]i (corrected for changes of background surface fluorescence; values on the ordinate of 1, 1.5 and 2 roughly correspond to [Ca2;]i of (nM) 160, 370 and 740, respectively); (b) Glucose production; (c) K; balance (negative values represent K; uptake); and (d) Bile flow. Note the sharp rise of [Ca2;]i upon addition of epinephrine followed by a sustained increase. The rise of [Ca2;]i is associated with an increase of glucose production, K; uptake and transient increase of bile flow.
containing perfusion follow a quasi exponential slope and extrapolating the mean slopes to t = revealed values of 0.67, 5.70, and 2.60 for minimal and maximal values of R340/380 and for the relative change at 380–510 respectively. From these values control [Ca2;]i was estimated at 159.8<8.4 nM. In order not to suggest that reliable absolute values of [Ca2;]i can be obtained in individual experiments, we have chosen to present changes of [Ca2;]i as relative changes with respect to a normalized control value of R340/380 of 1. From extrapolated endpoints of titration of intracelluar [Ca2;]i values of R340/380 of 1.0, 1.5 and 2.0 given on the ordinates in Figures 3 and 4 roughly correspond to [Ca2;]i of 160, 370 and 740 nM, respectively.
Measurement of Fura-2 concentration in bile of control Wistar and TR9 rats We measured the Fura-2 concentration in bile of Wistar and of TR9 rats. Single bile drops (approx. 8 mg) leaving the bile duct cannula were collected over 15 min before loading the liver with Fura-2-AM (1 M), and for 30 min © 2001 Harcourt Publishers Ltd
Measurement of potassium and glucose balance and of bile flow Potassium and glucose balance were assessed by analyzing the venous perfusate of the liver in comparison with inflow concentrations. Perfusate was collected at 30 s or 1 min intervals. K; was measured with a NOVA 9 electrolyte analyzer (Nova Biomedical, Newton, MA) and K; balance was calculated from liver weight, perfusate flow rate and from the portal to caval venous concentration differences. Uptake or release of K; is given in nmoles/gliver /s. Glucose concentrations were measured enzymatically by the hexokinase methods (Glucose liquiUV; Human GmbH, Taunusstein, Germany). Bile flow was measured from the weight of bile drops and from the frequency of their falling from the bile duct canula and interrupting an infrared light beam in a drop counter. Average values from different experiments were obtained after interpolation to comparable time points. Bile secretion is given in mg/gliver/min. Data are given as mean values
RESULTS Liver surface fluorescence and biliary secretion of Fura-2 in Wistar and TR9 rats Perfusion of isolated livers from Wistar rats with Fura-2-AM containing solution (1 M) increased surface fluorescence Cell Calcium (2001) 30(6), 403–412
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reduction of biliary concentration of Fura-2 to 33.1< 0.2 M. Probenecid exhibits a strong choleretic effect [26], which was considered unfavourable because effects of hormones on bile formation were included in our studies. Therefore, all experiments were carried out with livers from TR9 rats in order to improve liver cell loading with Fura-2 and to reduce contamination of surface fluoresence by the presence of Fura-2 in bile.
Effects of epinephrine on [Ca2;]i, glucose balance, K; balance, and bile flow
Fig. 4 Effects of vasopressin (2 nM) in the isolated perfused liver of TR9 rats. Panels (top to bottom) show: (a) Fluorescence ratio 340/380 nm as a measure of [Ca2;]i (corrected for changes of background surface fluorescence; values on the ordinate of 1, 1.5 and 2 roughly correspond to [Ca2;]i of (nM) 160, 370 and 740, respectively); (b) Glucose production; (c) K; balance (negative values represent K; uptake); and (d) Bile flow. Note the sharp rise of [Ca2;]i upon addition of vasopressin followed by a gradual decline. The rise of [Ca2;]i is associated with an increase of glucose production, K; uptake and transient increase of bile flow.
within 30 min to values approx. 30% above the autofluorescence level, measured prior to dye loading (360–510 nm). Bile flow was 0.85<0.08 mg gliver91 min91 (n:3) and remained unaffected by Fura-2 loading. In this period of time, Fura-2 concentration in bile reached values of 380<30 M. No major fluorescent metabolites were detected in bile and biliary Fura-2 fluorescence showed a regular response to changes of Ca2; concentration. In anion transport deficient TR9 rats, bile flow was 0.47<0.04 mg gliver91 min91 (n:3). In contrast to control Wistar rats, Fura-2 concentration in bile was only 26<3 M (n:3) and surface fluorescence continued to rise during ongoing application of Fura-2-AM (compare Fig. 2). With reference to an initial background fluorescence at 340–510 nm normalized to a value of 1 the increase of fluorescence within 30 min was from 1 to 1.82<0.11 at 340–510 nm and, at 380–510 nm, from 0.52<0.03 to 0.90<0.07 by 75.1<8.9% (n:9). In control Wistar rats, application of high concentration of the anion transport inhibitor probenecid (5 mM) resulted in a comparable Cell Calcium (2001) 30(6), 403–412
As already exemplified in Figure 1, epinephrine (50 nM) caused a significant increase of background fluorescence which is attributed to a rise of intracellular concentrations of NADH and NADPH). With reference to a normalized initial background fluorescence at 340–510 nm of 1 the increase of fluorescence was from 1 to 1.044<0.002 at 340–510 nm and, at 380–510 nm, from 0.485<0.026 to 0.498<0.030 by 2.53<0.14% (n:9). These changes of background fluorescence had to be corrected when measuring [Ca2;]i with Fura-2 (see Method section). Upon application of epinephrine, Fura-2 fluorescence revealed a sharp transient rise of [Ca2;]i followed by a sustained elevated level and [Ca2;]i returned to control values after removal of epinephrine (Fig. 3). Similar changes of [Ca2;]i were evoked by phenylephrine (1 M) ( peak rise of the 340/380 fluorescence ratio by a factor of 1.6) but not by isoprenaline (1 M) (data not shown). Glucose production of the isolated liver was elevated throughout the presence of epinephrine (Fig. 3). In parallel with the rise of [Ca2;]i, the continuous small release of K; was transiently reversed to K; uptake. Also in parallel with the rise of [Ca2;]i, bile flow was transiently increased which was followed by a short depression before returning to control levels. Including data from Fura-2-free experiments, epinephrine (50 nM) caused a transient rise of bile flow from 0.42<0.02 mg gliver91 min91 to 0.57<0.03 mg gliver91 min91 by 38.3<2.3% (n:10). Application of phenylephrine (1 M) similarly initiated K; uptake and transiently stimulated bile flow (data not shown). During Ca2;-free perfusion, the initial peak rise of [Ca2;]i was preserved, but [Ca2;]i returned to baseline levels within 3 min despite the continuous presence of epinephrine. In accordance with this short lived effect on [Ca2;]i the epinephrine induced glucose release and K; uptake were also abbreviated. Similarly, the rise of calculated NAD(P)H dependent fluorescence was abbreviated in comparison to the increase of background fluorescence seen in Figure 1. Bile flow ceased in the absence of extracellular Ca2; and epinephrine showed no stimulatory effect (data not shown). © 2001 Harcourt Publishers Ltd
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Effects of vasopressin on [Ca2;]i, glucose balance, K; balance, and bile flow Vasopressin caused a substantial transient rise of background fluorescence. With reference to a normalized initial background fluorescence at 340–510 nm of 1 the increase of fluorescence was from 1 to 1.090<0.012 at 340–510 nm and, at 380–510 nm, from 0.527<0.028 to 0.556<0.031 by 5.42<0.59% (n:8). Following correction for these changes, Figure 4 correlates effects of vasopressin (2 nM) on [Ca2;]i, glucose production, K; balance and bile flow. Vasopressin causes a sharp rise of [Ca2;]i followed by a slow decline. Glucose production was suddenly increased and remained elevated throughout the presence of vasopressin. Coincident with the rise of [Ca2;]i, transient K; uptake was initiated which was followed by K; release. Vasopressin caused a sudden but transient increase of bile flow from 0.46<0.02 mg gliver91 min91 to 1.02<0.09 mg gliver91 min91 by 120.4<16.1% (n:10) which was followed by a slight depression.
DISCUSSION Measurement of hepatocellular [Ca2;]i in the isolated perfused liver allows to correlate this parameter with liver cell functions that are not easily assessable in isolated hepatocytes. Principles of using fluorescent intracellular sensors for pHi and [Ca2;]i and of measuring surface fluorescence of intact organs have been presented before [9–13]. We have chosen to use Fura-2 to measure [Ca2;]i: the dissociation constant of this sensor is in the physiological range of [Ca2;]i and, using dual wavelength excitation, this sensor offers the advantage of providing signals for [Ca2;]i that are independent of the intracellular concentration of the sensor itself [4]. Furthermore, the non-fluorescent acetoxymethyl ester of Fura-2 is readily taken up from the vasculature into liver cells and hydrolyzed to the fluorescent sensor. Nonetheless, several factors limit this approach: (i) intrinsic fluorescence from the liver surface must be considered as a composite signal that arises from contributions of different cell types [including hepatocytes, endothelial cells or Kupffer cells; compare Ref. 27], from their metabolic state and storage of fluorescent material, (ii) these cells will also contribute to the increase of surface fluorescence after loading the organ with Fura-2, and (iii) anionic dyes such as Fura-2 are secreted at high concentrations into bile so that bile canalicular fluorescence will substantially contribute to the overall surface fluorescence. The specific aim of this study was to identify artifacts and limitations in measuring liver cell [Ca2;]i by surface fluorescence recording and, by using epinephrine and vasopressin, two agonists which act through Ca2; signalling, to compare these measurements with changes © 2001 Harcourt Publishers Ltd
of physiological parameters such as glucose production, K; balance and bile flow. Reducing biliary secretion of Fura-2 will result in better retention of the dye within hepatocytes and lower concentration of the fluorecent sensor in bile canaliculi. In accordance with a previous study [12] we show that secretion of Fura-2 into bile is reduced by the anion transport inhibitor probenecid but high concentrations are required (5 mM in the perfusate). At this concentration, probenecid elicits a strong choleretic effect [26] that was considered disadvantageous for studies that aim at correlating changes of [Ca2;]i with alterations of bile flow (see below). We therefore used an anion transport deficient rat strain (TR9 rats). These rats lack the canalicular anion transporter cmoat or mrp2 [14,15]. Using TR9 rats as liver donors provided for both, reduction of secretion of Fura-2 into bile (7% of control Wistar rats) and efficient loading of the sensor into hepatocytes. It is noted though that bile flow in this rat strain is low as compared to control Wistar rats, most likely as a result of reduced bile salt-independent GSH-dependent bile production [28]. In the method section, we describe two approaches to correct for changes of intrinsic background fluorescence. These corrections are required because (i) it is not feasible to load the isolated liver with Fura-2 to achieve surface fluorescence levels that are far above background fluorescence because of limitations in loading time and because of financial restrictions, and, (ii) experimental manoeuvres such as application of epinephrine or vasopressin do elicit changes in background fluorescence at the Fura-2 and [Ca2;]i sensitive excitation-emission wavelength pairs 340–510 and 380–510 (Fig. 1). One option was to perform parallel sets of experiments in the absence and presence of Fura-2. The Fura-2 and [Ca2;]isensitive signals are then obtained by subtracting background signals from data obtained in the presence of Fura-2 (compare Fig. 1). The second approach assumed that changes of intrinsic fluorescence are mainly due to changes of liver cell NAD(P)H content [16]. Changes of NAD(P)H were first generated by a short phase of hypoxia [compare 18,19,22] prior to Fura-2 loading and fluorescence was measured at pairs of excitationemission wavelength, one with preferential sensitivity to NAD(P)H (emission at 460 nm) the others preferentially sensitive to Fura-2 (emission at 510 nm). This allowed to calculate the effect of changes of NAD(P)H on the fluorescence in the Fura-2-sensitive wavelength range. Corrections for this ‘spill over’ were then continuously applied after Fura-2 loading and during manoeuvers that altered [Ca2;]i (Fig. 2). Both methods of correcting background fluorescence resulted in comparable estimates of changes [Ca2;]i as reflected by the adjusted 340/380–510 fluorescence ratio. In order to calibrate the 340/380 Cell Calcium (2001) 30(6), 403–412
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signal to obtain quantitative measurements of [Ca2;]i livers were perfused with medium containing the ionophore ionomycin at 0 and normal Ca2; concentrations. [Ca2;]i followed the changes of perfusate calcium concentrations but stable minimal and maximal endpoints of the 340/380 fluorescence ratio were not obtained within a reasonable time span. By extrapolating the quasi exponential changes of [Ca2;]i to t: a mean control value of [Ca2;]i of 160<8 nM (n:3) was estimated (see Material and Methods). Control 340/380 fluorescence ratios given in Figures 3 and 4 are normalized with the values of 1, 1.5 and 2 roughly corresonding to (nM) 160, 370 and 740 respectively. In single isolated hepatocytes application of vasopressin or ␣-adrenergic activation results in an abrupt rise of [Ca2;]i followed by oscillations of [Ca2;]i at an elevated level. The initial steep increase is effected by release of Ca2; from intracellular stores whereas the prolonged elevation results from ‘capacitative’ entry from the extracellular space [e.g. 3,6,7,29–36]. Our study shows that these two phases of changes of [Ca2;]i are also detected in the isolated perfused liver (Fig. 3) and confirms that prolonged elevation of [Ca2;]i depends on the presence of external Ca2;. The particular advantage of the technique used here is that these changes of [Ca2;]i can be directly related to the biological effects of the hormones. Figures 3 and 4 show that the early rise of [Ca2;]i following application of epinephrine or vasopressin is associated with stimulation of glucose release which remains elevated until removal of the agonist. Potassium homeostasis results from the balance between K; uptake by the Na;/K;-pump and from passive leakage of K; out of cells through ion channels [37]. The data show that this equilibrium is disturbed by epinephrine and vasopressin which both cause transient K; uptake (associated with the early rise of [Ca2;]i). This pattern is interpreted to result from initial transient activation of the Na;/K;-pump followed by increased efflux of K; [e.g. 38]. Numerous previous studies had addressed these effects of adrenergic stimulation and of vasopressin on the balance of glucose [e.g. 16,39–43] and K; balance [e.g. 39–41,43–45]. Here, we studied changes of these parameters to demonstrate their correlation with changes of [Ca2;]i measured by surface fluorescence of the isolated perfused liver. Vasopressin and ␣1-adrenergic stimulation increase the level of reduced pyridine nucleotides in liver [16,25,29,46] resulting in an increase of fluorescence at 360–460 nm. This increase in fluorescence is also produced by oxygenfree perfusion and Figure 2 shows its interference with Fura-2 fluorescence. As addressed above, appropriate corrections have to be applied when measuring [Ca2;]i at low intracellular concentrations of Fura-2. By comparing
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the changes of background fluorescence (Fig. 1) with changes of [Ca2;]i (Figs 3 & 4) we also note that the time course of changes of [Ca2;]i after application of epinephrine and vasopressin is largely paralleled by the increase of NAD(P)H fluorescence. We observed that epinephrine, phenylephrine and vasopressin elicit an instantaneous but short-lasting increase of bile flow. This effect coincided with the early rise of [Ca2;]i. On the other hand, bile flow ceased in the absence of external Ca2; and subsequent application of epinephrine was without effect (data not shown). This latter effect appears attributable to loosening of tight junctions and to an associated increase in the bile to perfusate permeability [e.g. 47]. It has been previously observed that application of hormones which act through Ca2; signalling lead to an early but transient increase of bile flow [48]. This effect appears to correlate with in vitro observations obtained in hepatocyte couplets, a preparation of liver cell secretory units that consist of non-dissociated pairs of isolated hepatocytes which enclose a bile canalicular vacuole between each other [49]. In this preparation, microinjection of Ca2; into hepatocytes [50] as well as hormonally mediated increases in [Ca2;]i [51,52] lead to a contraction of the bile secretory vacuole which is mediated by the actin-myosin web underneath the canalicular membrane [53,54]. In the intact organ, this forceful contraction of bile canaliculi appears to promote bile flow by ‘squeezing out’ a small volume of canalicular bile. It is noted though that, despite near equal peak rises of [Ca2;]i, the transient choleretic effect of vasopressin was about three times as large as that of epinephrine, suggesting that additional components of the respective signal transduction pathways may affect bile flow as well. In conclusion, this study shows that changes of hepatocellular [Ca2;]i can be reliably measured in the isolated perfused rat liver provided that biliary elimination of the fluorescent intracellular sensor (Fura-2) is minimized and that changes of background fluorescence are also monitored, either in parallel experiments or by measurements at multiple wavelengths. This method allows to correlate the pattern of changes of [Ca2;]i with changes of liver function. We demonstrate the effects of epinephrine and vasopressin in simultaneous recordings of [Ca2;]i, glucose balance, K; balance, bile flow and changes of NAD(P)H. The data are consistent with an early release of Ca2; from intracellular stores followed by uptake of extracellular Ca2;. The early effect coincides with initiation of glucose release, K; uptake and with a rise of NAD(P)H. Furthermore, the rise of [Ca2;]i is associated with a transient increase of bile flow which is attributed to contraction of the actin-myosin web in the pericanalicular cytoplasm of liver cells.
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