Document 2jOoqZ0LZg0dYZYpYmKXMJ6ob

THEJOURNALOF BIOLOGICACLHEMISTRY 0 1989 by The American Society for Biochemistry and MolecularBiology, Inc. Vol. 264, No. 22, Issue of August 5, pp. 12859-12866,1989 Printed in U.S.A. Ca2+ Oscillations Induced by Hormonal StimulatiofnIndividual Fura-2-loaded Hepatocytes* (Receivedfor publication, December 7, 1988) Toru KawanishiSQ, LogaMn. Blankll, Alec T. HarootunianS, Martyn T. Smithli, and Roger Y. TsienS11 From the Devartments of fPhy-siobg"y-Anat0m.yand TBiomedicaland Environmental Health Sciences, University of California, Berkeley, Caiifornin94720' Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 Isolated rat hepatocytes were loaded with the Ca" dependent coding of information roughly analogous to action indicator fura-2 to measure cytosolifcree Ca2+ concen- potentials in excitable cells. However, oscillations are not trations([Ca"+]i)inindividual cells by digitalratio observed under all conditions of stimulation, and even when imagingmicroscopy.Stimulationwith 0.1 nM vaso- oscillations are most prevalent, not every cell in the popula- pressin, 0.5 p~ phenylephrine, or 0.5 p~ ATP caused tion participates. Incontrasttothe pioneering results of repetitivespikes of high [Ca2+Ii in a high percenotfageWoods et al. (4,5) with aequorin in isolated hepatocytes, cells, in agreement with Woods et al. (Woods, N. M., Monck et al. (12) were hardly able to detect any clear Ca2+ fisWtaC4nrult5.uoegri7qrtle(tlh5eMuih3abs)ea1m.eonatRr9ncnst,ycoetsd6hkodn0neb,tu,sJ0h,upcJ.-Katti.6Ritko.0Rne.tnS,2hos. R.t)rei(,enR1etesbhy9.une,une8ltxattro8tnayurl)ddfmanJroscClop.,fiemEklolBeilebt.uxiubotlEdodatrolhfu.a.rel,[dectmCTCrhe,eahhplPe"sloeuiu.]nrmslnlHagptda.sr.ies2koo(c,C6fe1frAs3ae9M,.s,a2i8unPosa+46gten.f5r),dgfcaN6eakecc9ntaseeht-td-stl-elulaItwtosrhhfiirspniaecstpghoitrlll,lefeetautsshhtspriepoaioosi-nnnfk2eessetfemeihfdnsseealbcotnyaotdwoccwemlvkocyoanaimusndytolraeiedipnlcbclrgasyhueuyatpssgiensinhgmiigeansensmistleootoa.srutrahMgpcgahheepotlenlClnavnsecatayrbek2arlyaie+tauiebupnt,tushigowifarnfnilCieng.trhoea(if21nfacu+2gtothro)tnaemsos-scp2boiiise.unlstlctfteauIefdtsnerlimaofrsoiettnfnaeerslgdeaa.ld, only the rate of replenishment of those stores. Mem- level might be an interesting way to probe those mechanisms. brane depolarization failed to elevate [Ca2+]i and had On the other hand, we have found (10) in the rat embryo an effect similar to removal of extracellular Ca2+ in fibroblast cell line REF52 that repetitive Ca2+oscillations are decreasing the frequency of agonist-evoked [Caos2-+Iireadily observable with fura-2 if the cells are simultaneously cillations or inhibiting them altogethera,rguing depolarized and stimulated with mitogens, whereas with against anysignificant role for voltage-operated Ca2+either stimulus alone the response consists merely of a single channels. spike. This result promptedus to ask whether the oscillations seen by Woods et al. (4,5) may have resulted from microin- jection damage or whether fura-2 buffering of Ca2+ really prevents oscillations in hepatocytes. We now report that Ca2+ The concentration of cytosolic free Ca2+is a key parameter oscillations are induced by phenylephrine, vasopressin, and in regulating many cellular processes (1, 2). Inthe liver, ATP infura-2 loaded hepatocytes. Depolarization is not a stimulants thatactivate glycogenolysis are known to increase prerequisite for these oscillations, but rather inhibits them. the concentration of cytosolic Ca2+,[Ca2+Ii(3). Woods et al. Moreover, spike frequency but notamplitude is lowered with (4,5) reported that in single isolated hepatocytes microin- decreasing extracellular Ca2+concentrations. jected with aequorin, the increase in [Ca2+Iidue to agonist stimulation is a periodic train of spikes. Oscillations in extra- EXPERIMENTALPROCEDURES cellular Ca2+concentration representing periodic releases of Materiak-Collagenase (grade 11) and gramicidin D were obtained Caz+from hormone-stimulated perfused liver have also been from Boehringer Mannheim. Bovine serum albumin, ATP, glucose, observed (6).In addition, Capiod et al. (7) found in guinea pig phenylephrine, vasopressin, and nifedipine were purchased from hepatocytes that norepinephrine elicited cyclic fluctuations of K+ conductance which could be due to Ca2+-activatedK+ channels. [Ca"], oscillations have also been observed ina wide variety of other cell types (see Refs. 8-10 for reviews). Their prevalence has led to speculations (4,5, 8, 9) that Sigma. Fura-2 acetoxymethyl ester (fura-B/AM)' and fura-2 were from Molecular Probes. Acridine orange, rhodamine 123, and Percoll were obtained from Aldrich, Eastman Kodak, and Pharmacia LKB Biotechnology Inc., respectively. The (+) and (-) enantiomers of 202-791 werea gift from Dr. Richard W. Tsien (Yale University). All other reagents were commercial products of the highest available repetitive [Ca2+Isi piking may bea general mode of frequency- grade of purity. Preparation of Zsoluted Hepatocytes-Isolated hepatocytes were * This work was supported by a fellowship from the Science and prepared by the method of Moldeus et al. (13) and the suspension Technology Agencyof Japan (to T. K.), by National Institutes of was then centrifuged through a mixture of Ca2+-freeand M$+-free Health Grants GM31004 and EY04372 (to R. Y. T.), and by a grant Hank's solution and Percoll (final density 1.06 g/ml) to eliminate from the National Foundation for Cancer Research (to M. T. S.). dead cells. Cell viability, judged by trypan blue exclusion, was more The costs of publication of this article were defrayed in part by the than 90%. It should be noted that imaging of fura-2 in single cells payment of page charges. This article must therefore be hereby provided two further protections against contributions from damaged marked "advertisement" in accordance with 18 U.S.C. Section 1734 cells: leakycells lost their fura-2and were excluded from the analysis; solely to indicate this fact. 5 Present address: Laboratory for Cell Biology,Dept. of Anatomy, The abbreviations used are: fura-2/AM, fura-2penta(acetoxy- School of Medicine, University of North Carolina, Chapel Hill, NC methyl) ester; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul- 27514. 1) To whom correspondence should be addressed. fonic acidM; e,SO, dimethyl sulfoxide; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraaceticacid. 12859 12860 Ca2+Oscillations in Isolated Hepatocytes Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 also, the cells were continually monitored by simultaneous infrared transmitted illumination viewedby an infrared video camera, and any showing a pathological appearance, for example surface blebs, were likewise omitted. The cells were incubated in modified KrebsHenseleit buffer containing 12.5 mM HEPES (13), 20 mM glucose, and 1 mM methionine at 37 "C under 95% 02,5% COz for 30 min, washed, and resuspended in anothermodified Krebs-Henseleit buffer containing 20 mM HEPES, 5.5 mM glucose, and no methionine (KH buffer) at lo6cells/ml, then stored in arefrigerator (7-8 "C) and used within 6 h. Fura-2 Loading of Cells-An aliquot of hepatocytes was preincu- bated in KH buffer for 30-60 min at 37 "C under 95% 02,5% Con. Fura-2 loading was started by adding 1ml of the hepatocyte suspen- sion (5 X lo' cells/ml) to the mixture of fura-P/AM (2 pl of 1 mM solution in Me2SO)and 1pl of 25% Pluronic F127 in Me2S0.Loading was carried out for 40 min a t room temperature. After loading, intracellular fura-2 concentrations were250-350 pM. These values were obtained by comparing the fluorescence of fura-2 released from the cells treated with 0.2% Triton X-100 with that of solutions of known concentrations of fura-2. At these loadings, the autofluorescence of unloaded cells was at most 10-20% of the signal from dyeloaded cells. Moreover, the 350:385nm excitation ratios obtained from unloaded cells showed no change and no oscillations even when adjacent loaded cells in the same field werestimulated to oscillate by addition of agonists. Therefore changes in autofluorescence, for example due to reduced pyridine nucleotides, did not contribute to the fura-2 [Ca2+Isiignals. Measurement of Intracellular Ca2+ in Individual Cells-Fura-2 loaded cells were adhered to glass coverslips lightly coated with polylysine. After 5 min, unattached cells were washed off with KH buffer. Then, 3 mlof the buffer was added and the plastic holders were put into a thermostated holder on the stage of a Zeiss IM-35 microscope. The temperature was kept at 32 "C because intracellular fluorescence of fura-2 decreased rapidly at 37 "C, probably by dye leakage or extrusion. Fluorescence ratio imageswere obtained and calibrated as described elsewhere (10, 14-16). The images were taken every 5-10 s. The Ca2+values were averages over spots of more than 6.25pm diameter centered over the cells. Intracellular Location of Fura-2"The quality of loading and degree of dye compartmentalization were assessed by comparison of the release of acridine orange, fura-2, lactate dehydrogenase (a cytoplasmic marker enzyme), and rhodamine 123 (15, 17, 18), at 5-100 p~ digitonin. The total contents of these substances were assumed to be releasable by 0.2% Triton X-100. Isolated hepatocytes were incubated with 10 p~ acridine orange, or 10 pg/ml rhodamine 123for 30 min at 37 "C, or fura-P/AM and Pluronic F127 under the loading conditions described above. The cells were washed twice with Ca2+free KH buffer containing 1.0 mM EGTA and lysed by a 3-min exposure to Ca2+-freeKH buffer containing 1.0 mM EGTA with 5, 10,20 p ~or, 100 p~ digitonin or 0.2% Triton X-100. After centrifugation, supernatants were obtained and assayed by fluorescence for content of acridine orange (500 nm excitation, 530nm emission), rhodamine 123 (535 nm excitation, 580 nm emission), or fura-2 (342 nm excitation, 510 nm emission). Lactate dehydrogenase activity was measured as described previously (15). ElectrophysiologicalMeasurements of Membrane Potential-Resting potentials of isolated hepatocytes were measured by direct microelectrode penetrations roughly followingthe method of Petzinger and Bigalke (19). Fine-tipped microelectrodes were pulled from aluminosilicate glass on a microprocessor-controlled puller with jet cooling (P-80, Sutter Instruments, San Rafael, CA) and were filled with 4 M potassium acetate, giving tip resistances of70-100 megohms. The electrodes were advanced with a remote hydraulic micromanipulator (MO-102,Narishige Scientific Instruments, Tokyo). Potentials were recorded with an 8800 electrometer (Dagan Corp., Minneapolis, MN) and chart recorder. Cells were isolated, in some cases loaded with fura-2, adhered to the floor of the chamber, and superfused with normal KH buffer (5.9mM K+) as described above. RESULTS Quality ofFura-2 Loading-In [Ca2+],measurements using fura-2, incomplete hydrolysis of theester fura-2/AM and compartmentalization of dye fluorescence can be troublesome (15, 17, 20-24). In pilot experiments we found significant variations between different lots of fura-S/AM, all from Mo- lecular Probes. Analysis of four lots of fura-Z/AM by reversephase high pressure liquid chromatography showed that some were contaminated by two extra componentseluting just before and after authentic fura-2/AM but showing a similar absorbance spectrum. Fig. la compares the chromatograms of the best and worst lots. If the contaminants are assumed to have the same extinction coefficient at 254 nm as real fura2/AM, the sum of the contaminantsranged from 1to 20% of 10 min b n Ia W .- +n N 0 0 U L 0 200 400 600 800 1000 Time (sec) HEPO5 cella 0 & G FIG. 1. Impurities in fura-S/AM and their effect on hepatocyte [Ca2+Iri esponses. a, two batches of fura-P/AM (Molecular Probes) were compared by high performance liquid chromatography under isocratic conditions on a reverse phase octadecyl silica column (Supelcosil LC18, 250 X 4.6 mm diameter, Supelco, Inc., Bellefonte, PA), eluting with methanol/water 8020 (v/v) a t 1.0 ml/min. Absorbance was monitored at 254 nm. The first event marker shows the injection of 5 pl of 1mM fura-2/AM lot 8A in Me2S0(DMSO).After elution of three small peaks probably due to Me2S0 and its aging products, fura-P/AM appears closely flanked by two impurity peaks. This lot of fura-Z/AM consistently gave lowapparent [Ca2+Irieadings and small non-oscillatory responses to hormones (see b). The next injection was of 0.5 p~ of 10 mM fura-L/AM lot 7C. The different pattern of MezSO-derivedpeaks is probably due to thelesser amount but greater age of Me2S0 injected. Note the relative absence of impurities immediately surrounding the main fura-B/AM peak. This batch was used for almost all the biological results reported in Fig. 2 and subsequent figures. The final injection was of 5 p1 Me2S0 to verify which peaks are attributable to it. (b),response to phenylephrine and ionomycin of hepatocytes loaded with impure fura-2/AM. Hepatocytes were loaded by the usual procedure with lot 8A of fura2/AM, whosechromatogram is shown in the first part of a. The cells weresuccessively challenged with 10 p~ phenylephrine then 3 p M ionomycin (iono) at thetimes indicated. Note the relatively low basal [Ca2+Iis, mall amplitude of response to phenylephrine, lack of oscillations, and weaksluggish response to a highdose of the Ca2+ ionophore. Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 OscillaCtiao2n+s HinepIastoolcayteteds 12861 the desired material. Batches with higher contents of the contaminants gave low 350:385 nm ratios in resting hepato- cytes, which increased little upon hormone or ionophore stim- ulation and showed no oscillations (Fig. lb), whereas purer lots gave higher, more agonist-responsive 350385 nm ratios and were used for all the work reported below. Although we have not yet determined the structure of the contaminants or their mechanism of interference, it is worth noting that REF52 fibroblasts seemed much less sensitive than hepato- cytes to the presence of the contaminants. Therefore a batch of dye that is satisfactory for one cell type may not be pure enough for another. Completeness of fura-Z/AM hydrolysis was checked in two ways. Addition of several micromolar ionomycin to loaded hepatocytes in KHmedium gave350385 nm ratios more than 8 times that of fura-2 in EGTA even if the autofluorescence of the cells was not subtracted. If the autofluorescence was measured (by addition of Mn2+to quench the fura-2) and deducted for each cell, the ratios exceeded 12 times that of fura-2 in EGTA. The quality of the fura-2 formed in hepato- cytes was more quantitatively assessed by lysing them with 0.2% Triton X-100 in EGTA medium, removing cell debris by centrifugation, antditratintghseupernatant with K,CaEGTA solution to known buffered levels of [Ca"]. The family of fluorescence spectra obtained (Fig. 2) were quite similar to those of reference solutions of fura-2 (25), not only at very low and very high [Caz+],but also at intermediate values. The correspondence not only of spectral parameters but also of apparent Ca2+ dissociation constants between hepatocyte lysate and genuine fura-2 suggests that hydrolysis of fura-P/AM was functionally nearly complete; a small com- ponent of the fluorescence, perhaps 5-lo%, did not respond until [Ca"] exceeded several micromolar and may represent a bitof incompletely hydrolyzed ester. Compartmentalization of fura-2 was assessed (15, 17) by comparison with those of acridine orange, rhodamine 123, and lactate dehydrogenase (Fig. 3). Digitonin at 20 PM released 87% of the intracellular fura-2, more than 95% of the total lactate dehydrogenase, but only 4% of the total rhodamine 123, which is accumulated selectively by intact mitochondria (18).These results suggest that this digitonin concentration permeabilized the plasma membrane but left mitochondria largely intact, and that fura-2 accumulation in mitochondria was very small. On the other handd, igitonin at 20 and 100 PM released 24 and 54% of the total acridine orange, which accumulates in acidic organelles. However, there isno difference between the amounts of fura-2 released by digitonin at 20 and 100pM. Therefore, the 10-15% of fura2 that seemed to be in organelles did not appear to be mainly in compartmentsthat accumulated acridine orange. Resting [Caz+Ji and Transients Induced by Phenylephrine, Vasopressin, and ATP-Cytosolic [Ca2+]levels in individual resting hepatocytes formed a unimodal distribution (Fig. 4) with a mean of 247 nM and standarddeviation of 53 nM (n = 255). This mean value is in reasonable agreement with or lD0l .T LDH fura-2 { - /acridine orange 1 /u- Rhodamine 123 AA 5 10 20 Digitonin concentration (@A) 100 FIG.3. Subcellular location of fura-2 assessed by digitonin lysis. Parallel batches of hepatocytes werelysed with increasing concentrations of digitonin in EGTA medium, centrifuged, and the supernatants assayed for lactate dehydrogenase (LDH;open circles), fura-2 (solid circles), acridine orange (squares), and rhodamine 123 (triangles), all expressed as percentages of that releasable by 0.2% Triton X-100. Note the logarithmic scale on digitonin concentrations. Error bars denote the standarderrors of replicate measurements; where they are notvisible, they are within the size of the symbol. 388.00 Wave) e n g t h [nml 400.00 *OOT FIG.2. CaZ+titration of fura-2 released from hepatocytes. Cells were loaded by the standard protocol, lysed with 0.2% Triton X-100 in Ca2+-freeKH medium containing 1mM EGTA at pH7.05. Cell debris was removed by centrifugation, and the excitation spec- trum of the supernatantwas recorded at 11free [Ca2+]levels attained by successive additions of 1.00 M K2CaEGTA(tracesA-J), concluded z by addition of 2 mM CaClz for trace K. The free [Ca2'] values, calculated as in Ref. 25, were as follows: trace A , 0 nM; trace B, 19 nM; trace C,38 nM; trace D, 76 nM; trace E, 151 nM; trace F, 303 nM; trace G, 630nM; trace H , 1.26 pM; trace I , 1.87 PM;trace J , 22 p ~ ; 50 1 trace K , 1 mM. Emission was collected a t 510 nm; bandwidths of excitation and emission were 9 nm. The autofluorescence spectrum 0.0 L....,.... 0.5 1 .o 1.5 of the lysate (measured by quenching the fura-2 in a separatealiquot of cell supernatant using excess MnC12)has been digitally subtracted [C02+Ii OtM) from all the traces shown, whichwere then smoothed with the 9- FIG.4. Population distribution of [Caz+]ivalues in resting point Savitsky-Golay algorithm supplied with the Spex Datamate hepatocytes. The mean andstandard deviation of the 255 cells fluorometer. analyzed were 0.247 and 0.053 pM, respectively. 12862 Ca2+OscillationsHinepIastoolcaytetdes slightly higher than previous measurements of hepatocytes (4,26-29). Hormones elicited transient elevations, which showed considerable heterogeneity between neighboring cells. Some examples are shown in Fig. 5,A and B , in which a group of hepatocytes were exposed to 0.1 nM vasopressin. In cells A, B, and E, clear repetitive Ca2+spikes were observed after a lag, though in cell A the timing was irregular. In 47 cells from five runs using cells from five rats, 0.1 nM vasopressin pro- duced repetitive Ca2+transients in 57% of the cells, a single Ca2+spike (similar to cells C and D in Fig. 1) in 37%, and no response in 6%(e.g. cell F). In thoescillating cells, the average frequency was about one spike every 3 min. Fig. 6 is a histogram of the distribution of peak responses to 0.1 nM vasopressin, with the population mean near 700 nM [Ca2+Ii. Fig. 7 shows typical responses to 0.5 p~ phenylephrine. In 84% of 198 cells from 27 runs derived from eight animals, repetitive Ca2+transients began within 5 min after the addition of phenylephrine, as in cell A. However, in 15% a single Ca2+spike or sustained Ca2+rise, as in cell B, was observed. The oscillation frequency averaged just over one spike/min I' ' I b ' ' ' ' 2 0 Time (minl FIG. 7. Various patterns of [Caa+]gresponses in individual phenylephrine-stimulated hepatocytes. Typical transients resulting from 0.5 pM phenylephrine are shown. In cell A (solid line), repetitive spikes began promptly, whereas in cell B (dashed line), a step-like rise to a sustained plateau was observed, although small oscillations began about 8 min after agonist addition. 'OI Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 Time ( m i n ) 0.0 0.5 1.o Peak [Ca2+Ii (pM) 1.5 FIG.8. Population distribution of peak [Caz+]iattained in response to 0.5 PM phenylephrine. The mean and standarddeviation of the 208 cells were 0.75 and 0.17 WMr,espectively. T~me ( m n ) FIG.5. Various patterns of [Ca2+Iiresponse in individual vasopressin-stimulated hepatocytes. The [Ca2+];transients for six different hepatocytes (A-F) simultaneously stimulated with 0.1 nM vasopressin are shown in two panels, (a and b), separated only for clarity. 2o T TABLEI Average parameters of agonist-induced Caz+oscillations The highest oscillation frequency and amplitude elicited by 0.1nM vasopressin and 0.5 WM phenylephrine were determined for each individual cell and the means and standard deviations calculated. A cell wascounted as responsive if it produced at least one transient of at least 80 nM amplitude. It was deemed oscillatory if it produced at least two separate spikes of that amplitude within a single period of agonist exposure. The amplitude was defined as thechange in [Ca2+Ii from the trough to thepeak of the oscillation. The frequency was the number of full oscillations per minute. Agonist Cells Ressivpeon- Oscillating Oscillation treated cells cells amplitude Oscillation frequency Vasopressin 47 Phenylephrine 198 % nM rnin" 94 57 307 k 112 0.36 k 0.16 99 81 288 k 126 1.11k 0.63 0 0.0 0.5 1.o Peak [CaP+Ii (pM) 1.5 FIG.6. Population distribution of peak [CaZ+lai ttained in response to 0.1nM vasopressin. The mean and standarddeviation of the 47 cells were 0.71 and 0.15 wM, respectively. at 0.5 p M phenylephrine and increased with agonist dose. In some cells an initial rise in free Ca2+lasting 1-2 min was fdlowed by transientsas reported byWoods et al. (4,5 ) whereas ic other cells only a sustainedrise was observed (data not shown). Histograms of peak [Ca2+Iiattained in response to vasopressin and phenylephrine (Fig. 8) show population means of around 700 nM, similar to thatfor vasopressin (Fig. 6). The incidence and properties of the oscillations are summarized in Table I. We also found repetitive Ca2+transients in response to ATP (Fig. 9), though the oscillations were irregular. Effect of K+ Depolarization and Gramicidin on Ca2' Tran- sients-Direct microelectrode measurements of the resting Ca2+Oscillations in Isolated Hepatocytes 12863 Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 U 13 TimeIminI FIG.9. Typical patterns of [Ca2+]iresponses to 0.6 p~ ATP. Repetitive spikes were frequently observed, but they were relatively irregular in waveform and timing. T ~ m o (mtn~ FIG.10. Effect of K+ depolarization on [Ca2+Iroscillations induced by phenylephrine. Addition of0.5 p~ phenylephrine to hepatocytes in KH buffer containing 131 mM Na+ and 5.9 mM K+ caused [Ca2+],oscillations as usual. Then 50% of the 3.0-ml KH buffer in the chamber was removedand replacedwith KC1-substituted KH buffer also containing 0.5 p~ phenylephrine, to give a net K+ concentration of 71 mM. Then, one third of the medium (1ml) was exchanged with normal KH buffer containing agonist to give a net K+ concentration of49mM. 1 mlof normal KH buffer was then added to give a final K+ concentration of38.5mM, all while maintaining the phenylephrine concentration. Two typical response patterns areshown. potential of hepatocytes isolated under our conditions were made to verify their viability and conformance to previous detailed electrophysiological investigations (19). When finetipped microelectrodes filled with 4 M potassium acetate were used, as advocated by Petzinger and Bigalke (19),resting potentials of -69 k 9 mV (mean f S.D., n = 6) were obtained within a few minutes after impalement, in good agreement with the previous results (-74 f 8 mV) at thesame extracellular K+ concentration, 5.9 mM (19). Two of the six cells in the present experiments had been loaded with fura-2; their potentials, -65 and -85 mV, respectively, gave no sign that fura-2 had affected the resting potential significantly. Isotonic substitutions of KC1 for NaCl were used to see the effect of membrane depolarizations on Ca2+oscillations. Fig. 10 shows an experiment in which Ca2+ oscillations were started with phenylephrine, and then50% of the NaCl in KH was replaced byKC1 ([K+] = 71.3mM) in the continued presence of the agonist. This much K+ should have depolarized the cells to -19mV (19). [Ca2+]ifell immediately, and the Ca2+oscillations wereslowed or stopped (Fig. 10). The effect of K+ depolarization was reversible, since partial removal of the KC1 increased the frequency or caused them to resume. In otherexperiments (not shown), the 137mM extracellular Na+was first completely replaced by K', which should have depolarized the cells to -8 mV (19). This substitution had no effect on [Ca2+Ii,but subsequent addition of 0.5 p~ phenylephrine induced Ca2+oscillations in areduced percentage of cells and at a lower than normal frequency (0.1-0.4/ min). The effects of partial substitution of KC1 for NaCl are summarized in Table 11. A few experiments with other substitutes for NaCl were equivocal:partial replacement of NaCl by choline somewhat slowed oscillations, whereas partial replacement by sucrose had littleeffect. We also examined the effect of gramicidin, which equilibrates intra- andextracellular Na+ and K+ andshould also induce membrane depolarization. 270 nM gramicidin seemed to decrease the frequency of the Ca2+oscillations in some cells (5 cells out of 12 stimulated with phenylephrine, and 2 cells out of 8 treated with vasopressin). Gramicidin concentrations of 1 pM or greater stopped the oscillations in almost all of the 61 cells examined, as in Fig. 11. Some pharmacological antagonists and agonists of L-type voltage-operated Ca2+channels were tested, but their effects were ambiguous. Nifedipine at 10 p~ decreased oscillation amplitude but not frequency of phenylephrine-induced oscillations, whereas a reputedly more potent channel blocker, (-)202-791 (30, 31) at 1-5 p~ decreased the frequency with little effect on amplitude. The (+) enantiomer of 202-791, which is reported to promote channel opening (30,31), had a similar effect to the (-) enantiomer, so that the mechanism by which these drugs affect hepatocyte oscillations seems complex or nonspecific. Effect of Extracellular Ca2+Concentrations on Ca2+Transients-Fig. 12 shows the effect on Ca2+oscillations of changing from KH buffer to Ca2+-freeKH buffer containing 1mM EGTA but still containingphenylephrine. In 7 of the 12 cells from two separate runs from two rats, Caz+oscillations produced by 0.5 p~ agonist were stopped. In the otherfive cells, Ca2+oscillations persisted even in Ca2+-freemedium though at a much reduced frequency (e.g. Fig. 12). Restoration of normal Ca2+started the [Ca2+]ioscillations again at normal frequency in most cells, althougha few failed to resume. Interestingly, in all the cells that resumed spiking, the first such transient occurred 25-40 s after thereaddition of extracellular Ca2+,even though the period between subsequent spikes was much greater. It would seem that the protocol of Ca2+ deprivation then restorationresets the phase of the oscillation mechanism to a stagoef incipient spike generation. When Ca2+deprivation with 1 mM EGTA in Ca2+-freeKH was begun 1 min before addition of 1 p~ phenylephrine, a small number of Ca2+transients were produced for a short period (Fig. 13A), and the first peak heights were almost the same as those in KH buffer (Fig. 13B). In seven out of nine cells ( e g . cell Ain Fig. 13A), only one spike or cluster of spikes could be observed after Ca2+removal, whereas in the remaining two (e.g. cell B in Fig. 13A), further spikes could be observed as late as 10 min after Ca2+withdrawal. Fig. 14 shows the dependency of frequency of Ca2+oscillations on intermediate extracellularCa2+concentrations. Ca2+ oscillations were stopped after the exchange of extracellular solution to Ca2+-freeKH buffer. Upon readdition of0.5-1.0 mM Ca2+ the oscillations began again andthe frequency increased depending on the concentrations of extracellular Ca2+.Surprisingly, once the spikes restarted, their amplitude did not increase with increasing extracellular Ca2+. DISCUSSION In our experiments clear Ca2+oscillations were detected by fura-2 ratio imaging in more than 50% of single rat liver cells in response to phenylephrine, vasopressin, and ATP. In our hands, phenylephrine was the most effective inducer of Ca2+ oscillations. The high percentage of oscillating cells and their temporal waveform is in general agreement with the results ofWoods et al. (4, 5), who used microinjected aequorin as Ca2+detector, but contrasts with the paucity of sustained spiking reported by Monck et al. (12) using fura-2 imaging. Monck et al. (12) hypothesized that fura-2 somehow might 12864 Ca2+ Oscillationsin Isolated Hepatocytes TABLEI1 Effects of varying K+ depolarizationson averageparameters of Ca2+oscillations induced by 0.5 p~ phenylephrine The percentate of oscillating cells and themean and standard deviation of their amplitude and frequency were defined as in Table I. Membrane potential estimates are from Ref. 19 except for the value in 5.9 mM, the normal extracellular K+ concentration in KH buffer, determined in this study. Membrane potential Cells treated Oscillating cells Oscillation amplitude Oscillation frequency Extracellular K+ (mM) 5.9 15 49.4 15 -19 71.3 136.8 mV -69 -30 2-8 % nM min" 100 324 f 149 1.43 f 1.71 15 80 271 ? 60 0.65 f 0.31 60 300 f 141 0.54 f 0.14 6 50 0.39 327 f 70 & 0.14 Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 0.d 0 . IO Time (min) I 20 FIG. 11. Effect of gramicidin on [Ca2+]ioscillations induced by phenylephrine. Oscillations were started with 0.5 p~ phenylephrine, and then5.3 p~ of gramicidin D, a pore-forming antibiotic, was added where indicated. Time (mi") Time lrnin) 0.154 ' 0 ,', 1 20 Time (mi") I 40 FIG. 12. Effect of removing extracellular Ca2+ on [Ca2+]i oscillations. After oscillations were begun with 0.5 p~ phenyleph- rine in normal KH buffer, the medium was changed to Ca2+-freeKH buffer containing 1 mM EGTA and 0.5 p~ phenylephrine. Finally normal KH buffer with the same drug concentration was restored. FIG. 13. Comparison of removal of extracellular Ca2+before uersus after phenylephrine stimulationa., the medium wasfirst changed to Ca2+-freeKH buffer containing 1mM EGTA. 1min later, 1p~ phenylephrine was added. After the [Ca2+],transient due to release from internal stores, the medium waschanged to normal KH buffer not containingphenylephrine. After a 15-min recovery period, 1p~ phenylephrine was added again (b). Finally, the medium was replaced with Caz+-free KH buffer with 1 mM EGTA and 1 pM phenylephrine. interfere with the feedback mechanisms generating oscilla- tions or that aequorin was seeing localized [Ca2+]; changes. However, our fura-2 loadings were higher (average 250-350 FM)than thoseof Monck et al. (12) (25-100 PM), a difference inconsistent with fura-i2nterferencweith oscillations. Though considerable variations in dye brightness were seen from cell to cell, there was no obvious tendency for the brighter cells (presumably containing more dye) to oscillate differently from the dimmer cells. No dramatic spatial gradients of [Ca2+Iiwere observed, though one cannot rule out the possibility that future technologies with yet better temporal and spatialresolution may detect such gradients. Gray (32) has noted that carbachol-stimulated parotid acinacrells FIG. 14. Effect of graded readmission of extracellular Ca" on repetitive spiking. 0.5 p~ phenylephrine was added and main- tained at that level during the replacement of normal KH buffer by Ca2+-free Lnedium containing 1 mM EGTA, replacement by KH medium with 20 p~ added Ca2+,and then readdition of increasing tend to give [Ca2+], oscillations only below 30 "C. However, concentrations of Ca2+. suchtemperature effects donotexplainthe difference in hepatocyte behavior, since Monck et al. (12) saw little differ- more than 4 min and may have missed subsequent oscilla- ence in results between 28 and 37 "C, whereas our cells were tions. It should also be noted that in many experiments we at 32 "C. Onedifference in protocol is that we monitored also obtainedsmall non-oscillatoryresponses (e.g. Fig. lb) [Ca"]; for tens of minutes, because in many cells the initial somewhat similar to those inFig. 4, A and C, of their work, Ca2+transient lasted several minutes, and spikingdeveloped but in our hands sucrhesponses were correlated with batches only later. Monck et al. (12)did not report observations lastingof fura-2/AM containing impurities detectable by high per- Ca2+Oscillations i n Isolated Hepatocytes 12865 Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 formance liquid chromatography (Fig. la). Scanlon et al. (20) Since Ca2+ activationof phosphorylase b kinase is a nonlinear have also noted that difficulties with fura-2IAM loading can function showing strong positive cooperativity (421, occa- be batch-dependent. In a preliminary report, Thomas et al. sional spikes that contribute little to the mean of the asyn- (33) have also observed rhythmic [Ca2+Iioscillations in fura- chronized population may be highly effective in stimulating 2-loaded hepatocytes, with indications of spatial propagation. the enzyme. Recently, we found (10)that in REF52 fibroblasts, depolar- In Ca2+-freesolution only a small number of Ca2+spikes ization-induced Ca2+influx synergized with mitogenic hor- were observed and for a short time (Figs. 12 and 13). The mones to generate Ca2+oscillations. Neither mitogenic hor- frequency of Ca2+spikes was strongly dependent on the con- mones nor depolarizations elicited rhythmic spikes in the centration of extracellularCa2+ (Fig. 14). This frequency absence of the other. Gramicidin, ouabain, and high extra- dependence is similar to that of REF52 fibroblasts (10) but cellular K+ all worked with mitogenic hormone to induce much different from that of BC3H-1 cells derived from [Ca2+Ioi scillations, though K+was somewhat less consistently smooth muscle (43). Because the amplitude and waveform of effective. Inthe presentstudy, however, we find that K+ the [Ca2+]s; pikes are little affected by extracellular Ca2+, each depolarization of hepatocytes decreases the frequency of Ca2+ individual spike is probably governed not by influx of extra- oscillations or stops them. The effects of gramicidin are less cellular Ca2+but by the release from intracellular Ca2+stores. consistent, althoughhigh doses of gramicidin do stop theCa2+ Probably Ca2+influx affects the frequency of the oscillations oscillations. Therefore, the effect of depolarizing agents and via the rateof Ca2+resupply to theintracellular store.Putney their relative efficacy on Ca2+oscillations appear to be oppo- has suggested (44) that such resupply might occur by a mys- site in hepatocytes from that in REF52 cells. In addition, terious pathway that lets Ca2+bypass the main part of the gramicidin and K+ depolarization had no effect on the resting cytosol. This hypothesis would explain why we generally do level of intracellular Ca2+concentration in hepatocytes (data not observe a pacemaker ramp of [Ca2+Ipi receding each spike. not shown).On the otherhand, inREF52 cells (10)gramicidin If replenishment of internal Ca2+stores were controlled by or or K+ depolarization by themselves produced one or occasion- drawn from cytosolic Ca2+,one might expect to observe such ally two Ca2+spikes followed bya plateauelevation. We have ramps, and indeed they have been reportedinendothelial considered the possibility that gramicidin seems to have little cells (45). effect on hepatocyte [Ca2+];merely because the plasma mem- Gelfand et al. (46) reported that in lymphocytes the mito- brane of isolated hepatocytes just afterisolation may already gen-induced uptake of Ca2+is depressed by K+depolarization. be injured, perhaps due to thecollagenase required to disperse They suggested that thereceptor-operated Ca2+permeability the cells. If the membrane potential were decreased in injured is an electrogenic pathway, though K+ depolarization only cells and intracellular Na+ concentration were already high, slightly reduces the huge electrochemical driving force favor- the addition of gramicidin would have no apparent effect. ing Ca2+entry. Roughly similar inhibitionof [Ca2+]ei levations However, this explanation is unlikely for the following rea- by depolarization have been seen in many nonexcitable cell sons: (a) Direct microelectrode measurements of our hepato- types (Ref. 47 and references therein). Ishida et al. (48) cyte membrane potentials give readings averaging -69mV, observed that even ionomycin-induced [Ca2+Iriises are dimin- in good agreement with the most recent literaturevalues (19). ished by depolarization. Because ionomycin is believed to be ( b ) High external K+ is effective but inhibits [Ca2+Iioscilla- an electroneutral ionophore, they concluded that enhance- tions. ( c ) Measurements of intracellular free Na+ with SBFI, ment of Ca2+pump activity by depolarization was the major a new fluorescent indicator for Na' (34) show values of 10- contributor. We now observe that K+ depolarization, like 20 mM in these hepatocytes; these values do indeed rise as removal of external Ca2+, mainly slows the frequency of expected in response to gramicidin or ouabain. For these [Ca2+];oscillations with little effect on their amplitude. This reasons, it is unlikely that [Ca2+];oscillations in hepatocytes finding explains and extends previous reports that K+ depo- are anartifact of membrane damage or depolarization. larization reduces hormone-stimulated phosphorylase activa- A t present there is much evidence that Ca2+-dependent tion (49, 50) and net Ca2+uptake by isolated hepatocytes or hormones initially raise cytosolic Ca2+mainly by releasing perfused livers in which non-parenchymal cells have been Ca2+ from nonmitochondrial stores, whichmay be either inhibited. However, our evidence does not yet say whether components of endoplasmic reticulum (1-3, 35, 36) or newly inhibition of entry orstimulation of pumping is predominant. described organelles called calciosomes (37).In previous stud- In conclusion, the present data show that in a large per- ies on large populations of liver cells, eitherthe perfused centage of fura-2-loadedisolated hepatocytes, hormones stim- organ or cuvet suspensions of isolated hepatocytes, such mo- ulaterhythmic Ca2+ spikes under reasonably physiological bilization was associated with an initial large efflux and loss conditions. The frequency but not the amplitude of the oscil- of total cell calcium over about 5 min (38, 39). However, in lation increases with extracellular Ca2+concentration, sug- low Ca2+ media (30 p ~ ) ,the cytosolic Ca2+ rise declined gesting that the amount of Ca2+influx is closely related to rapidly. Therefore, the influx of Ca2+was considered neces- the frequency. Depolarization causes no Ca2+rise of its own sary to maintain an elevated Ca2+concentration (39). Our and slows or inhibits the oscillations in a manner similar to results show how this population response is composed of the removal of extracellular Ca2+.These resultssuggest that volt- more complex and often oscillatory behavior of individual age-operated Ca2+ channelsare unimportant and that depo- cells. The continued occurrence of bursts of high [Ca2+Imi ay larization may inhibit receptor-mediated influx or stimulate help explain the previously puzzling observation (41) that efflux. At present we do not know the reason why Ca2+ phosphorylase activation can persist at a high level when oscillations are observed in some cells but not in other adja- average [Ca2+l,in the population is only slightly elevated cent cells. However, it is possible that Ca2+oscillations do not above resting. Individual cells are now seen to spike to at occur in those hepatocytes where Ca2+influx is very large or least many hundreds of nanomolar [Ca"];, and the trupeeaks very small: if Ca2+influx is large, the first transient willbe might be yet higher in unloaded cells, since dye buffering of followed by asustained phase. If Ca2+ influx is very low, [Ca2+];,partial AM ester hydrolysis, compartmentalization [Ca2+]m; ay fail to rise high enough to engagepositive feedback into nonresponsive organelles, and imperfect spatial and tem- mechanisms such as Ca2+-inducedCa2+release (8) or Ca2+ poral resolution would all tend to underestimate the peaks. stimulation of phospholipase C (11). 12866 Ca2+OscillationsHinepIasotolacyteteds Downloaded from www.jbc.org at HAM-TMC Library, on April 20, 2010 Acknowledgments-We thank Drs. Donato DiMonte and Martha Sandy for their kind help in making isolated hepatocytes, Dr. S. R. Adams for help with the high performance liquid chromatography, Dr. Joseph P. Y. Kao for help in measuring the fluorescence spectra of fura-2 in lysates, and Prof. J. Exton for helpful discussion on the effects of hepatocyte depolarization. REFERENCES 1. Campbell,A. K. (1983) Intracellular Calcium,John Wiley & Sons, New York 2. Carafoli, E. (1987) Annu. Rev. Biochem. 56,395-433 3. Exton, J. H. (1985) Am. J.Physiol. 248, E633-E647 4. Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 319,600-602 5. Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1987) Cell Calcium 8 , 79-100 6. Graf, P., vom Dahl, S., and Sies, H. (1987) Biochem. J.241,933- 936 7. Capiod, T.,Field, A. C., Ogden, D. C., and Sandford, C. A. (1987) FEBS Lett. 217,247-252 8. Berridge, M. J., Cobbold, P. H., and Cuthbertson, K. S. R. (1988) Phil. Trans. R. SOCh. nd.B 3 2 0 , 325-343 9. Rink, T.J., and Jacob, R. (1989) Trends Neurosci. 12,43-46 10. Harootunian, A. T., Kao, J. P. Y., and Tsien, R. Y. (1988) Cold Spring Harbor Symp. Quant.Biol. 53,945-953 11. Meyer, T., and Stryer, L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5051-5055 12. Monck, J. R., Reynolds, E. E., Thomas, A. P., and Williamson, J. R. (1988) J. Biol. Chem. 263,4569-4575 13. Moldeus, P., Hogberg, J., and Orrenius, S. (1978) Methods Enzymol. 52,60-71 14. Poenie, M., Alderton, J., Steinhardt,R., and Tsien, R. Y. (1986) Science 233,886-889 15. Poenie, M., Tsien, R.Y., and Schmitt-Verhulst, A. M. (1987) EMBO J.6,2223-2232 16. Tsien, R. Y. (1989) Cell Calcium,in press 17. Malgaroli, A., Milani, D., Meldolesi, J., and Pozzan, T. (1987) J. Cell Biol. 105,2145-2155 18. Johnson, L. V., Walsh, M. L., and Chen, L.B. (1980) Proc. Natl. Acad. Sci. U.S. A. 77,990-994 19. Petzinger, E., and Bigalke, H. (1986) Biochim.Biophys.Acta 863,318-324 20. Scanlon, M., Williams, D. A., andFay, F. S. (1986)J.Bwl. Chem. 262,6308-6312 21. Oakes, S. G., Martin, W. J., 11, Lisek, C. A., and Powis, G. (1988) Anal. Biochem. 169,159-166 22. Gunter, T. E., Restrepo, D., and Gunter, K. K. (1988) Am. J. Physiol. 2 5 6 , C304-C310 23. Davis, M. H., Altschuld, R. A., Jung, D. W., and Brierley, G. P. (1987) Biochem. Biophys. Res. Commun. 149,40-45 24. Tsien, R. Y. (1989) Annu. Reu. Neurobiol. 12,227-253 25. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440-3450 26. Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) J.Biol. Chem. 258,8769-8773 27. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J. Biol. Chem. 269,5574-5584 28. Binet, A., Berthon, B., and Claret, M. (1985) Biochem. J. 2 2 8 , 565-574 29. Lemasters, J. J., DiGiuseppi, J., Nieminen, A.-L., and Herman, B. (1987) Nature 3 2 6 , 78-81 30. Kongsamut, S., Kamp, T. J., Miller, R. J., and Sanguinetti, M. C.(1985) Biochem. Biophys. Res. Commun. 130,141-148 31. Kokubun, S., Prod'hom, B., Becker, C., Porzig, H., and Reuter, H. (1986) Mol. Phurmacol. 30,571-584 32. Gray, P. T. A. (1988) J. Physiol. (Lond.) 406,35-53 33. Thomas, A. P., Rooney, T. A., and Sass, E. J. (1988) J.Cell Biol. 107, 445 (abstr.) 34. Minta, A., Harootunian, A. T., Kao, J. P. Y., and Tsien, R. Y. (1987) J. Cell Biol. 1 0 5 , 89a 35. Burgess, G. M., Godfrey, P. P., McKinney, J. S., Berridge, M. J., Irvine, R. F., and Putney, J. W., Jr. (1984) Nature 309,63-66 36. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and Williamson, J. R. (1984) J.Bwl. Chem.2 5 9 , 3077-3081 37. Volpe, P., Krause, K.-H., Hashimoto, S., Zorzato, F., Pozzan, T., Meldolesi, J., and Lew,D. P. (1988) Proc.Natl.Acad.Sci. U. S. A. 86,1091-1095 38. Exton, J. H. (1984) in Calcium Regulation in Biological Systems (Ebashi, S., Endo, M., Imahori, K., Kakiuchi, A., and Nishizuka, Y., eds) pp. 141-156, Academic Press, Tokyo 39. Charest, R., Prpib, V., Exton, J. H., and Blackmore, P. F. (1985) Biochem. J.227.79-90 40. Reinhart. P. H.. Tavlor, W. M., and Bymave, F. L. (1984) " Biocheh. J. 223,1113. 41. JoseDh. S. K.. Coll. K. E., Thomas, A. P., Rubin, R., and William- son, j.R. (1985 j J.Biol. Chem. $60, i2508-i25i5 42. Burger. D.. Stein. E. A.. and Cox. J. A. (1983) J. Biol. Chem. 258,' 14733-14739 ' 43. Ambler. S. K.. Poenie. M.. Tsien. R. Y.. and Tavlor. P. (1988) J. ~ ~~ Biol.~Chem.*263,1952-1959 ' 44. Putney, J. W., Jr. (1986) Cell Calcium 7 , 1-12 45. Jacob, R., Merritt, J. E., Hallam, T. J., and Rink, T. J. (1988) Nature 336.40-45 46. Gelfand, E. W., Cheung, R. K., and Grinstein, S. (1984) J. Cell Physiol. 121,533-539 47. Penner, R., Matthews, G., and Neher, E. (1988) Nature 3 3 4 , 499-504 48. Ishida, Y., and Chused, T. M. (1988) J.Exp. Med. 168,839-852 49. Van der Werve, G., Hue, L., and Hers, H.-G. (1977) Biochem. J. 162,135-142 50. Altin, J. G., Biden, T. J., Karjalainen, A,, and Bygrave,F. L. (1988) Biochem. Biophys. Res. Commun. 1 5 3 , 1282-1289