Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

Thomas Lenz and Jochen W. Kleineke

Abteilung Klinische Biochemie, Zentrum Innere Medizin, University of Göttingen, 37075 Göttingen, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Calcium entry in nonexcitable cells occurs through Ca2+-selective channels activated secondarily to store depletion and/or through receptor- or second messenger-operated channels. In amphibian liver, hormones that stimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in the plasma membrane, which allows a noncapacitative inflow of Ca2+. To characterize this Ca2+ channel, we studied the effects of inhibitors of voltage-dependent Ca2+ channels and of nonselective cation channels on 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP)-dependent Ca2+ entry in single axolotl hepatocytes. Ca2+ entry provoked by 8-BrcAMP in the presence of physiological Ca2+ followed first-order kinetics (apparent Michaelis constant = 43 µM at the cell surface). Maximal values of cytosolic Ca2+ (increment ~300%) were reached within 15 s, and the effect was transient (half time of 56 s). We report a strong inhibition of cAMP-dependent Ca2+ entry by nifedipine [half-maximal inhibitory concentration (IC50) = 0.8 µM], by verapamil (IC50 = 22 µM), and by SK&F-96365 (IC50 = 1.8 µM). Depolarizing concentrations of K+ were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca2+ influx. This "profile" indicates a novel mechanism of Ca2+ entry in nonexcitable cells.

adenosine 3',5'-cyclic monophosphate; second messenger-operated calcium channel; calcium channel pharmacology; SK&F-96365; fenamates

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE REGULATION OF GLYCOGEN breakdown in mammalian liver by alpha -adrenergic agonists and vasoactive peptides has been extensively studied. These hormones generate as second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3); the latter mobilizes Ca2+ from the endoplasmic reticulum and in addition triggers Ca2+ entry into the cell (5). In most cells, including hepatocytes (18, 22, 26), the rate of Ca2+ influx after hormonal stimulation seems to be controlled by the filling state of internal InsP3-sensitive Ca2+ stores (34). When such stores are depleted, an inflow of Ca2+ is triggered by a mechanism that may depend on the presence of Ca2+, InsP3, and/or inositol 1,3,4,5-tetrakisphosphate, or other yet to be defined diffusible factors (reviewed in Ref. 6). In rat liver, the rate of Ca2+ entry into cells via store-operated channels may be enhanced if glucagon or other adenosine 3',5'-cyclic monophosphate (cAMP)-generating hormones are present during a challenge with Ca2+-dependent hormones (7, 28). The nature of this mechanism is obscure at the present.

Very recently, investigations on Drosophila melanogaster have drawn attention to certain proteins (trp, trpl) with an apparent capacity of both channel forming and the sensing of the filling state of the endoplasmic reticulum Ca2+ store (32, 33). Hence, the Drosophila store-operated channel has been put forward as a model for capacitative Ca2+ entry. Analogous proteins, however, have not been detected in liver (43).

In variance, in fish and amphibian liver, the effect of adrenergic agonists and vasotocin is mediated via the generation of cAMP (19, 20, 42), and not via InsP3. Yet, in parenchymal liver cells from axolotl (Ambystoma mexicanum), hormones that stimulated cAMP formation (the order of efficacy was glucagon > isoprenaline > epinephrine >=  arginine vasotocin) also provoked a pronounced increase in cytosolic Ca2+, which was not due to a mobilization of the cation from internal stores by InsP3/thapsigargin, but to an increased inflow from the extracellular medium. Thus, in axolotl liver, in contrast to rat liver, hormones that stimulate the production of cAMP also regulate the opening of an ion gate in the plasma membrane, which allows an inflow of Ca2+ (and Mn2+). The effect is rather specific, since guanosine 3',5'-cyclic monophosphate (cGMP) failed to induce Ca2+ entry (23). We have proposed that this channel could belong to the category of second messenger-operated Ca2+ channels, as defined by Meldolesi and Pozzan (29). In nonexcitable tissues, such channels have so far only been found in blood cells (10, 27, 36).

The aim of this investigation was to further characterize the nature of this cAMP-activated Ca2+ channel of axolotl liver cells, using a variety of compounds that influence Ca2+ entry in excitable and nonexcitable cells: the phenylalkylamine verapamil, the dihydropyridine nifedipine, both potent inhibitors of Ca2+ entry in heart and skeletal muscle, and the imidazole derivative SK&F-96365, which inhibited receptor-mediated Ca2+ entry (as compared with receptor-mediated Ca2+ release) in nonexcitable cells (human platelets, neutrophils, and endothelial cells) and which has been used as a tool to discriminate between voltage-gated Ca2+ entry and receptor-mediated Ca2+ entry in GH3 and artery smooth muscle cells (30). Because lanthanides (10-7 to 10-5 M) block stretch- and receptor-activated nonselective cation channels, but also Ca2+ entry through voltage-dependent channels (15), we investigated the effect of Gd3+ on cAMP-dependent Ca2+ entry. As an additional inhibitor of nonselectivc cation channels in membranes, we examined the effect of niflumate, a nonsteroidal anti-inflammatory drug (12, 15).

Using single-cell dual-wavelength epifluorescence measurements of cytosolic Ca2+ in amphibian hepatocytes, we report a strong inhibition of cAMP-dependent Ca2+ entry by SK&F-96365 [half-maximal inhibition concentration (IC50) = 1.8 × 10-6 M], by the dihydropyridine nifedipine (IC50 = 8 × 10-7 M), and by verapamil. Furthermore, the lanthanide Gd3+ and niflumate, both potent inhibitors of nonselective cation channels, suppressed Ca2+ influx. It is concluded that in axolotl hepatocytes the rise in intracellular Ca2+ after hormonal stimulation is due to a Ca2+ inflow via a novel dihydropyridine- and SK&F-96365-sensitive nonselective cation channel.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Fura 2 acetoxymethyl ester (AM) was purchased from Molecular Probes (Eugene, OR). SK&F-96365 {1-(beta -[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride} was a kind gift from SmithKline Beecham Pharmaceuticals (Welwyn, UK). BAY K 8644 was from Bayer. Collagenase ("Worthington" type CLS II, 206 U/mg) came from Biochrom (Berlin, Germany). 3-Aminobenzoic acid ethyl ester (MS-222) was from Sigma (Munich, Germany). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).

Isolation of hepatocytes. Axolotls (A. mexicanum) were maintained in aerated water tanks at 20°C. The animals were fed twice weekly on fish pellets (Fisch-Fit, Interquell Stärke, Wehringen, Germany) and had a body weight of 60-80 g when used. Results from both males and females are presented together, because there were no differences observed between sexes (19).

The animals were anesthetized by immersion in 0.05% (wt /vol) MS-222. The cannulation and extirpation of the liver were as described previously (19, 23).

Hepatocytes were prepared using Ca2+-free amphibian Krebs-Ringer bicarbonate buffer (aKRB) (80 mM NaCl, 3 mM KCl, 0.6 mM KH2PO4, 0.8 mM MgSO4, and 16 mM NaHCO3, pH 7.4) as the perfusate. Briefly, the liver was perfused via the portal vein for 15 min with the above medium in an open perfusion, and then after readdition of CaCl2 (1 mM) and collagenase (0.05 g/100 ml), the perfusion was continued for 40-50 min in a recirculating system. After this step, the liver was minced, and the disintegrating tissue fragments as well as separated single cells were collected and passed through a double layer of cheesecloth. This suspension was washed three times with aKRB by centrifugation (100 g for 1 min). Usually >85% of the cells were viable as judged by trypan blue exclusion (0.2% trypan blue, 1% bovine serum albumin in aKRB).

Measurement of cytosolic Ca2+. The cells were washed once (100 g for 1 min) and resuspended in a medium containing 80 mM NaCl, 3.2 mM KCl, 0.8 mM MgSO4, 1 mM CaCl2, 10 mM D-glucose, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 (medium A) to give a concentration of ~60 mg wet wt /ml. They were incubated with fura 2-AM (5 µM) for 30 min at 25°C in a shaking water bath (100 cycles/min). After this, the cells were spun down (1 min, 100 g), the supernatant was discarded, and the pellet was resuspended in the same volume of medium A and further incubated for up to 30 min at 4°C. Thereafter, the cells were washed twice with medium A (100 g, 1 min) and resuspended in medium A at a concentration of 80-100 mg wet wt /ml. This suspension was kept for up to 30 min at room temperature to allow deesterification of fura 2-AM. The latter was controlled during this period by monitoring the epifluorescence of single hepatocytes at 360-nm excitation. Ca2+ concentration was calculated from the fluorescence ratio 360/380 nm (31). Hepatocytes (1-2 mg wet wt) were suspended in 2 ml medium A (plus additions as specified) in a petri dish (Falcon 3001) with a central quartz window (diameter = 15 mm). Water-insoluble compounds were prepared as concentrated stock solutions in dimethyl sulfoxide (DMSO). The concentration of DMSO in the petri dish never exceeded 1% (vol/vol). The same amount of DMSO was added to control incubations.

Ca2+ measurements were performed on single hepatocytes using a fura 2 data aquisition system (Luigs and Neumann, Ratingen, Germany) mounted to an inverted microscope (Zeiss IM 35) equipped with epifluorescence, a xenon lamp (Osram, XBO 75 W/2), a rotating filter wheel (357/380- to 390-nm excitation, 480- to 540-nm emission), and a photomultiplier (Hamamatsu 928 SF). The sampling rate was 2/s. For a more detailed description and evaluation of the equipment, see Neher (31). Calibration of the system was done using fluorescent beads.

Application of agonists. Application of agonist [8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP)] was done using a microcapillary to direct a flow of solution of agonist under constant pressure (1,000 hPa) from a distance of ~30 µm for 5-10 s onto the equatorial surface of the single cell under investigation. The capillary (2-3 µm diameter) was positioned using an Eppendorf ECET 5170 micromanipulator, and an ECET microinjection system (Eppendorf 5242) coupled to the capillary was activated for the time and pressure specified to generate the flow of agonist. All other compounds were dissolved in medium A and were present in the "bath" (petri dish) at concentrations given in Figs. 1-5.

The rate of Ca2+ increase (nM/s) and the maximum level in cytosolic Ca2+ (Delta Ca2+) were calculated from fura 2 recordings of individual hepatocytes. The initial rate of Ca2+ increase reflects the rate of Ca2+ entry into single hepatocytes and is related to the proportion of active ("open") channels in the membrane. The initial rate is largely independent of signal distortion by compensating mechanisms (desensitization). At high concentrations of inhibitor(s), the determination of this value was more reliable.

If not otherwise stated, values given are means ± SE from 7-10 single cells exposed and stimulated individually under identical conditions per dish. The experiments were repeated at least three times with independent cell preparations.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Kinetics of cAMP-dependent Ca2+ uptake. 8-BrcAMP (1 mM) when applied from the outside using a microinjection glass capillary for 5 s from a distance of ~30 µm onto the surface of single axolotl hepatocyte led after a short delay to an increased influx of Ca2+, as shown for six of seven individual hepatocytes in the same petri dish (Fig. 1). In most cells, the increase of cytosolic Ca2+ was transient with a half-life of decay of ~1 min (57 ± 4 s, n = 6). Some cells however exhibited longer-lasting responses, some also with superimposed oscillations (not shown). Maximum levels of cytosolic Ca2+ (Delta Ca2+: 211 ± 20 nM; n = 5) were obtained within 15 s (rate: 13 ± 1.5 nM/s; n = 6).


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Fig. 1.   Stimulation of Ca2+ entry in presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) in single hepatocytes. Isolated hepatocytes from axolotl were loaded with fura 2 acetoxymethyl ester as given in MATERIALS AND METHODS. Hepatocytes equivalent to 1-2 mg wet wt were added to a petri dish filled with 2 ml of a medium containing 80 mM NaCl, 3.2 mM KCl, 0.8 mM MgSO4, 1 mM CaCl2, 10 mM D-glucose, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 (medium A). Cells were allowed to settle for 5 min. A microcapillary was filled with 1 mM 8-BrcAMP from which agonist was applied for 5 s from a distance of 30 µm on surface of a single hepatocyte (marked in graph by small vertical lines). Epifluorescence recorded at 360 and 390 nm and corresponding Ca2+ trace of 7 individual cells exposed and stimulated in succession are depicted.

The rate of Ca2+ influx and the maximum increase of cytosolic Ca2+ of cells treated as defined above were dependent on the concentration of 8-BrcAMP (Fig. 2, A and B). The influx of Ca2+ followed first-order kinetics with an apparent affinity constant of 8.6 × 10-4 M. Maximal levels of cytosolic Ca2+ were observed at a 8-BrcAMP concentration of 1-2 mM. These data compare favorably with earlier measurements of hepatocytes in suspension (23). Because of the experimental topology (distance between cell and mound of the capillary, pressure, and time), the effective concentration of agonists is ~20-fold more diluted at the cell surface compared with the concentration in the capillary. Hence, an apparent minimal effective concentration of 25 µM (Fig. 2A, inset, intersection with the abscissa) is equivalent to a concentration of 1-2 × 10-6 M at the cell surface. The resting Ca2+ concentration of axolotl hepatocytes was 85 nM (Fig. 2B, inset, intersection with the ordinate). At saturating concentrations of agonist, a rise in cytosolic Ca2+ by ~300% is observed. 8-BrcAMP at a concentration of 1 mM in the micropipette (~50 µM at the cell surface) was nearly maximally effective in most cell preparations under these conditions. This effect of 8-BrcAMP was dependent on the availability of extracellular Ca2+, as has been reported earlier (23).


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Fig. 2.   Effect of 8-BrcAMP on rate of Ca2+ entry and cytosolic Ca2+ concentration in single hepatocytes. Conditions were as given in Fig. 1 except that 8-BrcAMP was applied at concentrations given on abscissa. Rate of Ca2+ increase (A) and maximal increment (B) in cytosolic Ca2+ (Delta Ca2+) were calculated from fluorescence recorded. Note: inset in B shows initial changes in cytosolic Ca2+. All other conditions were as given in MATERIALS AND METHODS.

Effect of SK&F-96365 on cAMP-dependent Ca2+ entry. The imidazole derivative SK&F-96365 has been introduced as a tool to discriminate between voltage-gated Ca2+ entry and receptor-mediated Ca2+ entry (30). SK&F-96365 inhibited cAMP-dependent Ca2+ inflow in axolotl hepatocytes in a dose-dependent manner. The dose-response curves for the rate of Ca2+ entry and for the maximal increase are shown in Fig. 3. The IC50 values for SK&F-96365 were 1.4 and 1.7 × 10-6 M for the rate of Ca2+ entry and maximal increase (Delta Ca2+), respectively.


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Fig. 3.   Inhibition of 8-BrcAMP-dependent Ca2+ entry by SK&F-96365. Conditions were as given in Fig. 1, except that SK&F-96365 was present in bath at concentrations indicated. 8-BrcAMP (1 mM) was applied from a microcapillary. Rate of Ca2+ increase (A) and maximal increment (B) in cytosolic Ca2+ (Delta Ca2+) were calculated from fluorescence recorded. For better comparison, data from 6 independent cell preparations were normalized. Values for 100% were as follows: A, 14 ± 3 nM/s (n = 6); B, 180 ± 19 nM (n = 6).

Effect of dihydropyridines and verapamil on cAMP-dependent Ca2+ entry. The dihydropyridine (8, 9) nifedipine, when tested under comparable conditions, inhibited markedly the cAMP-dependent Ca2+ influx (IC50 = 8 × 10-7 M). This sensitivity is 20-50 times more pronounced than that reported for liver by others (18, 26). BAY K 8644, an agonistic dihydropyridine, which binds during the open state of L-type Ca2+ channel and prolong their open time (24), when present in equimolar concentration had no additional effect (Fig. 4A, open square). BAY K 8644 at 2.5 µM on its own, however, increased the basal Ca2+ by 17% and cAMP (1 mM)-dependent Delta Ca2+ by 47%.


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Fig. 4.   Inhibition of 8-BrcAMP-dependent Ca2+ entry by nifedipine and verapamil. Nifedipine (bullet ) or verapamil (black-triangle) was present in bath at concentrations indicated (abscissa). BAY K 8644 plus nifedipine was present in equimolar concentration (square ). Other conditions were as given in Fig. 3. For better comparison, data from 6 independent cell preparations were normalized. Values for 100% were as follows: A, 14 ± 2 nM/s (n = 6); B, 170 ± 18 nM (n = 6).

The potency of the phenylalkylamine verapamil to block Ca2+ entry was about one order of magnitude lower (IC50 = 2.2 × 10-5 M) than that of nifedipine.

Dihydropyridines are the "classical" inhibitors of L-type Ca2+ channels that are abundant in electrically excitable tissues, like muscle and brain cells. These cells are depolarized in the presence of K+. In experiments in which KCl was applied at a concentration of 100 mM onto single axolotl hepatocytes, we could not detect any effect of such depolarizing concentrations of KCl on intracellular Ca2+ (data not shown).

Effect of Gd3+ on cAMP-dependent Ca2+ entry. The lanthanide Gd3+ inhibited cAMP-dependent Ca2+ entry very efficiently. A 50% inhibition of the rate of Ca2+ entry and of the maximal increase of cytosolic Ca2+ was observed at a concentrations of 2.5 × 10-6 M (Fig. 5).


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Fig. 5.   Inhibition of 8-BrcAMP-dependent Ca2+ entry by Gd3+. Conditions were as given in legend to Fig. 3, except that GdCl3 was present in bath at concentrations indicated. Values for 100% were 13 ± 2 nM/s (n = 2) (solid bars) and 176 ± 19 nM (n = 2) (open bars) for rate and increment, respectively.

Effect of niflumate on cAMP-dependent Ca2+ entry. Niflumate inhibited cAMP-dependent Ca2+ entry by ~90% (rate: 9.6 and 7.2%; Delta Ca2+: 13.6 and 13.2% of control at 1 or 5 × 10-4 M niflumate, respectively).

    DISCUSSION
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Using dual-wavelength excitation epifluorescence measurements of Ca2+ on single hepatocytes, we demonstrate here unique properties of this Ca2+-conducting channel in axolotl hepatocytes (a nonexcitable splanchnic cell).

The entry of Ca2+ evoked by cAMP was strongly inhibited by the imidazole derivative SK&F-96365 (IC50 ~2 × 10-6 M), whereas that after microinjection of InsP3 was inhibited only at concentrations >10-4 M (data not shown). This inhibition is about one order of magnitude more effective than that described for a variety of different cells including rat hepatocytes (0.8-3 × 10-5 M, see Refs. 10, 11, 25, 30, 40). SK&F-96365, which belongs to a group of imidazole antimycotics that have been originally used to block cytochrome P-450 but also Ca2+ and Ca2+-dependent K+ channels (3), was introduced as a novel inhibitor of receptor-mediated Ca2+ entry into cells (30). In addition, inhibition of voltage-gated Ca2+ entry in GH3 and rabbit ear artery smooth muscle cells by SK&F-96365 has been observed (30). The mechanism of this inhibition is still elusive. The proposal, however, that cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane influx (2) has been questioned by others (36). In axolotl liver, we could exclude a participation of intracellular, capacitative stores in cAMP-dependent Ca2+ influx, which is in support of a cytochrome P-450-independent interaction (23).

Opposing effects of SK&F-96365 on HL-60 cells have been recently reported by Leung et al. (25). At low concentrations (<16 µM), SK&F-96365 inhibited Ca2+ entry, whereas at higher concentrations (16-100 µM), it provoked release of intracellular Ca2+, and by this promoted even Ca2+ entry (30-100 µM). The latter could be inhibited by La3+, but not by nifedipine.

The comparably sensitive inhibition of cAMP-dependent Ca2+ entry observed in the presence of the dihydropyridine derivative nifedipine was not expected. Dihydropyridines are known to block rather selectively L-type voltage-dependent Ca2+ channels of excitable tissues (9), a channel type which is absent in hepatocytes, as judged by electrophysiological measurements (37) or Northern analysis (17). This is confirmed by our failure to demonstrate Ca2+ entry after membrane depolarization in the presence of K+ (100 mM), which reveals that the channel decribed here although sensitive to dihydropyridines lacks certain properties of a classical L-type channel of excitable cells, in particular, the ability of voltage sensing, a property which is thought to be located on the S4 segment of the alpha 1-subunit (9).

Ca2+ influx channels of nonexcitable cells sharing these properties have been recently found in B lymphocytes from rat, which showed dihydropyridine but no voltage sensitivity (1), and in an erythroleukemia cell line from mouse, where a truncated alpha 1-subunit lacking the first four transmembrane segments was expressed (27). Furthermore, the trp/trpl gene product from Drosophila that forms a nonselective cation channel presumably involved in capacitative Ca2+ entry in invertebrates and vertebrates shows some sequence homology to the voltage-operated Ca2+ channel alpha 1-subunit, but lacks arginine residues of the S4 region (33, 41).

The phenylalkylamine verapamil inhibited Ca2+ entry in axolotl hepatocytes (50% effective concentration = 22 µM) but in comparison with nifedipine with lower sensitivity. In contrast to dihydropyridines, phenylalkylamines enter the cell to interact with a high-affinity binding protein on the endoplasmic reticulum, which has been identified in guinea pig and human liver (14). As for nifedipine, the effects of verapamil reported so far for liver (and hepatocytes) are rather controversial. Studying capacitative Ca2+ entry, Llopis et al. (26) failed to see effects of verapamil or nifedipine (up to 50 µM), whereas Striggow and Bohnensack (38) observed an incomplete inhibition of this Ca2+ entry mechanism at verapamil or diltiazem concentrations between 200 and 400 µM. Others have reported complete inhibition of 45Ca2+ exchange across the liver cell plasma membrane in the presence of 50-100 µM nifedipine or verapamil (18). A stretch-activated nonselective cation channel found in rat hepatocytes and rat hepatoma cells was not affected by nifedipine, verapamil, or La3+ (4).

The effect of nifedipine (or verapamil) shown here on axolotl hepatocytes appears to be more specific, since the effective concentration of nifedipine (1-5 µM) is the order of magnitude used to block voltage-dependent L-type Ca2+ channels of excitable cells in vitro, i.e., 1-10 µM.

The inhibition of cAMP-dependent Ca2+ entry observed in axolotl hepatocytes in the presence of niflumate or the lanthanide Gd3+ was not surprising. Both compounds are potent inhibitors of Ca2+ entry through nonselective cation channels (Ref. 15 and references therein). Because of an ionic radius close to that of Na+ and Ca2+, Gd3+ (0.2-100 µM) can block efficiently stretch- or receptor-activated nonselective cation channels (4, 11, 40) but, like La3+, also voltage-dependent channels (39).

The nonsteroidal anti-inflammatory fenamates have been applied to block Ca2+ entry via nonselective cation channels in cells from rat exocrine pancreas (12), in human polynuclear leukocytes (21), and in mucosa-type mast cells (35). Apart from nonselective cation channels, Cl- channels are blocked by fenamates (13, 35). In mucosa-type mast cells, the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, however, fully obstructed Cl- currents without affecting Ca2+ influx, thus indicating that the effect of niflumate on Ca2+ influx may be dissociated from that on Cl- channels (35).

As discussed above, all compounds used here, except for nifedipine and verapamil, are to a variable degree inhibitory on capacitative Ca2+ entry (11, 16, 21, 25, 26, 40).

The agonist-induced Ca2+ influx in axolotl hepatocytes may be characterized as follows. The influx depends totally on the generation of cAMP, which in turn acts indirectly via protein phosphorylation catalyzed by protein kinase A (23). The influx of Ca2+ measured in the presence of 8-BrcAMP as a surrogate follows first-order kinetics, with a maximal rate of ~60 nM/s and an apparent Michaelis constant of ~5 × 10-6 M 8-BrcAMP, as calculated for the concentration present on the cell surface. Protein phosphorylation(s) could be coupled to and/or modulate the open state of an ion-gating channel in the membrane, as demonstrated for voltage-gated ion channels (8, 9). The pharmacological profile of the Ca2+ influx channel in amphibian hepatocytes reveals certain relationships to these channels, as well as to nonselective cation channels. The Ca2+ entry shows a remarkable dihydropyridine sensitivity but lacks the ability of voltage sensing, indicating certain homologies to the dihydropyridine binding site of the alpha 1-subunit, but apparently differences in the S4 segment. Examples of other nonexcitable cells sharing these properties have been discussed above. This relationship is reinforced by the distinct effects of verapamil and SK&F-96365 or Gd3+, because the selectivity of the latter compounds for voltage-gated Ca2+ entry and nonselective cation channels appears to be low (30, 39). The sensitivity to SK&F-96365, Gd3+, and niflumate, all of which act by different mechanisms (15), discloses the properties of a receptor-activated nonselective cation channel.

This novel dihydropyridine-sensitive channel, which to our knowledge is absent in rodent liver, could serve as an example for a diversity of types and subtypes of channels in various tissues and species.

    ACKNOWLEDGEMENTS

The generous gift of axolotls by Prof. Dr. W. Hanke, Karlsruhe, Germany, is gratefully acknowledged.

    FOOTNOTES

Address for reprint requests: J. W. Kleineke, Abt. Klin. Biochemie Zentrum Innere Medizin, Univ. of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.

Received 4 April 1997; accepted in final form 7 July 1997.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1526-C1532
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